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0003082 | Section title: Introduction Educational score: 4.3566813468933105 Domain: biomedical Document type: Study Language: en The functioning of the nervous system depends upon an immensely complex and precise network of neurons. Cell adhesion is thought to play a critical role in the formation and maintenance of synaptic contacts between neurons . Among different classes of molecules involved in cell adhesion, there is evidence that heparan sulfate proteoglycans (HSPGs) are present in central synapses and neuromuscular junctions . Furthermore, HSPGs are suggested to play a role in regulating synaptic strength, thereby acting as key molecules for synaptic stabilization that underlie neural plasticity . Based on these premises, we previously examined the expression of HSPGs in cultured rat hippocampal neurons. We demonstrated that syndecan-2, a member of the syndecan family of HSPGs, is concentrated in dendritic spines. Dendritic spines are numerous small membrane appendages protruding from dendritic surfaces that consist of specialized postsynaptic structures for the vast majority of excitatory synapses . Moreover, we showed that forced expression of syndecan-2 cDNA in young (1 wk in vitro) hippocampal neurons induces the formation of morphologically mature dendritic spines, which are normally seen after 3 wk in vitro . Section title: Introduction Educational score: 4.468814849853516 Domain: biomedical Document type: Study Language: en Syndecans are a major class of membrane-spanning HSPGs . There is increasing evidence that syndecans play significant roles in the organization of specific cell-surface structures, such as focal adhesions, through their interactions with cytoskeletal and signaling molecules , and thereby provide an important linkage between extracellular event and intracellular signaling. The cytoplasmic domains of syndecans have structural features that strongly suggest their involvement in intracellular signaling and cytoskeletal interactions. These domains consist of a 13-residue juxtamembrane segment highly conserved among the members (C1 region), a variable segment, and another highly conserved 7–9-residue segment (C2 region) at the COOH terminus of the molecule . Most importantly, the Glu-Phe-Tyr-Ala (EFYA) sequence located at the COOH terminus is identical in all syndecans. Moreover, our previous studies have shown that a syndecan-2 mutant in which the EFYA sequence was deleted lacks the ability to induce the formation of dendritic spines, suggesting that the molecular interactions involving this motif are crucial for this phenomenon . Section title: Introduction Educational score: 4.256311893463135 Domain: biomedical Document type: Study Language: en It has been shown that syndecans interact with two PDZ domain–containing proteins, syntenin and CASK/LIN2A , through the EFYA motif. We previously proposed a model in which these PDZ domain–containing molecules are potential downstream effectors of syndecan-2 in dendritic spines . In this model, we suggested that the spine formation is mediated by the cytoskeletal reorganization initiated by the syndecan-2–dependent clustering of PDZ domain proteins. In this paper, we report a novel syndecan-binding protein, synbindin. Unexpectedly, the structural property and the localization of synbindin suggest an entirely different mechanism for syndecan-2–induced spine formation. Section title: Introduction Educational score: 4.574228763580322 Domain: biomedical Document type: Study Language: en Synbindin is a neuronal cytoplasmic protein identified by yeast two-hybrid screening using the syndecan-2 cytoplasmic domain as a bait. Although it bears homologies with several PDZ domain proteins and binds to the COOH-terminal EFYA tail of syndecan-2 (as do CASK and syntenin), synbindin does not contain any classical PDZ domains. Rather, synbindin shares homologies with yeast proteins involved in membrane trafficking and vesicle transport acting upstream of the soluble NSF attachment protein receptor (SNARE) complex. There is increasing evidence that functional postsynaptic maturation involves the regulation of vesicle trafficking in postsynaptic sites . Dendritic spines contain intracellular membrane-bound cisterns, known as spine apparatus , which are frequently seen in the vicinity of the postsynaptic density . Synbindin is identified in vesicles in dendritic spines as well as in synapses. Furthermore, synbindin forms clusters in dendritic spines when syndecan-2 is coexpressed in neurons. Our observations suggest a role for cell-surface syndecan-2 in the translocation of postsynaptic vesicular compartments through the cytoplasmic interaction with this novel syndecan-2–binding molecule. Section title: Yeast Two-Hybrid Assays Educational score: 4.314328193664551 Domain: biomedical Document type: Study Language: en Yeast two-hybrid screens were performed using the L40 yeast strain, where the expression of both the reporter genes HIS3 and LacZ are driven by minimal GAL1 promoters fused to LexA-binding sites. A bait consisting of the entire cytoplasmic domain of the syndecan-2 fused in-frame to the LexA DNA–binding domain was constructed by PCR with the BTM116 vector. An embryonic mouse cDNA library constructed into the NotI site of pVP16 containing the Leu2 activation domain was screened with the syndecan-2 bait. Positive clones were selected by His prototrophy and assayed for β-galactosidase activity. Double-positive clones were isolated and characterized by sequencing. A double-positive clone (clone 28), which encodes a putative cytoplasmic protein with similarities to several PDZ domain–containing proteins (see Results), was further investigated as described in this paper. We named this protein synbindin. The specificity of the interaction between synbindin and the cytoplasmic domain of syndecan-2 was analyzed by two-hybrid assays . For this, we generated (by PCR) the following additional baits of the syndecan cytoplasmic domains: (1) a syndecan-2 deletion mutant lacking the COOH-terminal EFYA sequence (syndecan-2ΔEFYA); (2) a syndecan-4 deletion mutant lacking the COOH-terminal EFYA sequence (syndecan-4ΔEFYA); and (3) a bait with reverse sequence of syndecan-2 cytoplasmic domain. To determine the syndecan-2–binding site in synbindin, we generated by PCR four Leu2 fusion constructs representing the NH 2 -terminal half of synbindin (N-Sbd), the PDZ-related domain (P-Sbd), the COOH-terminal half (C-Sbd), and the PDZ-related domain plus the COOH-terminal half (P/C-Sbd) . Two-hybrid assays were performed as described above using HIS3 and LacZ as reporter genes. Section title: Cloning of the Full-length Synbindin cDNA Educational score: 4.1921844482421875 Domain: biomedical Document type: Study Language: en The full-length synbindin cDNA was isolated from a mouse brain λZAP cDNA library (Stratagene) with clone 28 as a probe. Four cDNA clones were isolated. One of the isolated clones contained an entire open reading frame encoding 219 amino acid residues. Full-length sequence of mouse synbindin cDNA was determined from this clone. Human and Caenorhabditis elegans synbindin homologues were identified in EST database by a BLAST search, and their entire sequences were reconstituted from overlapping EST clones. Section title: Production of Glutathione-S-Transferase (GST) Fusion Proteins Educational score: 4.208036422729492 Domain: biomedical Document type: Study Language: en A 663-bp EcoRI-XhoI fragment containing the entire coding region of mouse synbindin was amplified by PCR with the following primers and ligated into pGEX-4T-1 (Amersham Pharmacia Biotech): 5′ primer, ACCCGGAATTCATGGCGATTTTTACCGTGTAC; and 3′ primer, CGGCCGCTCGAGCTATGACCCAGGTCCAAAAGT. The GST-synbindin expression plasmid as well as insertless pGEX-4T-1 were transfected into BL21 Escherichia coli strains according to the manufacturer's instructions. BL21 cells were lysed by sonication in 20 mM Tris-HCl containing 0.15 M NaCl, 1 mM EDTA, 1 mM PMSF, 10 μg/ml pepstatin, 10 μg/ml aprotinin, and 2 μg/ml leupeptin. Sarkosyl was added to lysates to a final concentration of 1.5%, and the lysates were gently mixed for 15 min. After centrifugation, supernatants were adjusted to 2% Triton X-100 and 1 mM CaCl 2 , and GST-synbindin was purified with glutathione-agarose. Section title: Antibodies Educational score: 4.051045894622803 Domain: biomedical Document type: Study Language: en Two polyclonal antibodies against mouse synbindin were generated for this study. Rabbit anti-synbindin peptide antibody was raised against a synthetic peptide acetyl-CELFDQNLKLALELAEKV-amide (corresponding to amino acids 195–213 of mouse synbindin) and affinity-purified on amino-link/agarose beads coupled with the synthetic peptide (Quality Controlled Biochemicals). The other polyclonal antibody (No. 157) was raised against the bacterially produced recombinant synbindin protein released from GST-synbindin fusion protein by proteolytic cleavage and affinity-purified using synbindin-GST fusion protein coupled to glutathione-agarose. Other antibodies used in this study were as follows: anti–c-Myc rabbit polyclonal antibody A14 (Santa Cruz Biotechnology, Inc.); anti–syndecan-2 mAb 6G12 ; anti–syndecan-2 polyclonal antibody ; anti–PSD-95 mAb 6G6 (Affinity Bioreagents, Inc.); antisynaptophysin and anti-MAP2 mAbs (Sigma Chemical Co.); and anti-CASK polyclonal antibody . Section title: Transfection of 293 Cells, GST Pull-down, and Coimmunoprecipitation Experiments Educational score: 4.243962287902832 Domain: biomedical Document type: Study Language: en Human 293 cells were grown in DME supplemented with 10% FCS and antibiotics. Approximately 70% confluent 293 cells in 10-cm dishes were transfected with 20 μg of an expression vector for Myc-tagged full-length syndecan-2 or a control vector using the calcium phosphate method . 1 d after transfection, transfected cells were treated with or without heparitinase (Seikagaku America), and then sonicated in 25 mM Tris-HCl, pH 8.0, containing 0.15 M NaCl, 1% Triton X-100, 5 mM EDTA, 1 mM PMSF, 5 mM DTT, 10 μg/ml pepstatin, 10 μg/ml aprotinin, and 2 μg/ml leupeptin (lysis buffer). Heparitinase treatment was performed in 20 mM Hepes, pH 7.0, containing 0.15 M NaCl and 1 mM calcium acetate for 1 h at 37°C. After sonication, cell lysates were cleared by centrifugation at 14,000 rpm in a microcentrifuge. For pull-down assays, cleared lysates were incubated with glutathione-agarose beads charged with unfused GST or GST-synbindin fusion protein for 1 h at 4°C. After incubation, beads were washed once with lysis buffer and five times with 25 mM Tris-HCl, pH 7.4, containing 0.5 M NaCl and 0.2% Triton X-100 at room temperature. The materials retained on the beads were eluted with SDS-PAGE sample buffer and detected by SDS-PAGE and immunoblotting as described previously . The Myc-tagged syndecan-2 pulled down by GST-synbindin was detected with either anti–syndecan-2 mAb (clone 6G12; a gift from Dr. Guido David; 1:1,000 dilution) or anti-Myc polyclonal antibody (A14; Santa Cruz Biotechnology; 1:1,000 dilution). Section title: Transfection of 293 Cells, GST Pull-down, and Coimmunoprecipitation Experiments Educational score: 4.193101406097412 Domain: biomedical Document type: Study Language: en For coimmunoprecipitation assays, we generated intact and ΔEFYA syndecan-2 cDNAs that are epitope-tagged with the FLAG sequence (designated as FLAG-syndecan-2 and FLAG-syndecan-2ΔEFYA, respectively). A FLAG tag (DYKDDDDK) was inserted at the unique SpeI site in the ectodomain of syndecan-2. These FLAG-tagged syndecan-2 constructs were transfected into 293 cells with or without cotransfection of a synbindin expression construct in which the c-Myc epitope was added to the NH 2 terminus of synbindin (Myc-synbindin). Cell lysates from these transfectants were prepared as described above without heparitinase treatment. The lysates were incubated with the A14 anti-Myc polyclonal antibody for 2 h at 4°C, followed by an incubation with protein A–Sepharose for 1 h. Bound materials were eluted with SDS-PAGE sample buffer, separated on an 8–16% gel, and immunoblotted with the M2 anti-FLAG mAb (Sigma Chemical Co.; 1:1,000 dilution) to detect coprecipitated FLAG-tagged syndecan-2. Section title: Production of Hexahistidine (His)-tagged Syndecan-2 Cytoplasmic Domains and Overlay Assays Educational score: 4.208644866943359 Domain: biomedical Document type: Study Language: en For preparation of His-tagged syndecan-2 cytoplasmic domains, a DNA fragment encoding the entire syndecan-2 cytoplasmic domain was amplified by PCR and inserted into EcoRI-XhoI–linearized pET-30a (Novagen). The construct for His-tagged ΔEFYA syndecan-2 cytoplasmic domain was then generated by point mutagenesis of the AAG codon for the lysine residue preceding the EFYA sequence to a stop codon (TAG). These His-tagged cytoplasmic domains (intact and ΔEFYA) were expressed in BL21 (DE3) cells and purified on ProBond resin (Invitrogen). For overlay assays, equal amounts of the intact and ΔEFYA cytoplasmic domains were loaded on a 10–20% tricine gel and blotted onto a nitrocellulose membrane. The membrane was stained with Ponceau S to ascertain that equivalent amounts of the intact and ΔEFYA cytoplasmic domains were applied. The membrane was blocked with 5% nonfat dry milk in 25 mM Tris-HCl, pH 7.4, containing 0.15 M NaCl and 0.3% Tween 20 (TBS-Tween) overnight at 4°C, and incubated with 50 μg/ml of purified GST-synbindin in TBS-Tween containing 1 mM DTT and 2% BSA for 2 h at room temperature. Bound GST-synbindin was detected with goat anti-GST antibody (Amersham Pharmacia Biotech; 1:3,000 dilution) and HRP-conjugated anti-goat IgG (Sigma Chemical Co.; 1:5,000 dilution). Section title: Primary Cultures and Transfection of Hippocampal Neurons Educational score: 4.165201663970947 Domain: biomedical Document type: Study Language: en Cultures of rat hippocampal neurons were prepared as described previously from E17 embryos . Transient transfection of hippocampal neurons was performed at 1 d in vitro by the calcium phosphate coprecipitation method . For the analysis of coclustering of syndecan-2 and synbindin, hippocampal neurons were transfected with an expression vector containing synbindin-green fluorescent protein (GFP) fusion protein alone, or together with either the full-length syndecan-2 expression vector or the syndecan-2ΔEFYA expression vector . Cotransfection of the synbindin-GFP and the syndecan-2/syndecan-2ΔEFYA expression vectors were performed at the ratio of 1:10 . We confirmed that, under this condition, essentially every GFP-positive cell coexpresses syndecan-2/syndecan-2ΔEFYA as demonstrated by immunostaining for syndecan-2. The frequency of synbindin clustering in these cotransfected cultures was determined as the percentage of cells that show >10 synbindin clusters in their dendrites to the total GFP-positive cells. A total of 40 neurons were scored in four sampling windows for each culture. Section title: Immunofluorescence and Confocal Imaging Educational score: 4.11439323425293 Domain: biomedical Document type: Study Language: en Hippocampal neurons transfected with synbindin-GFP alone or together with syndecan-2 were examined 8 d after transfection (9 DIV) by a confocal microscopy as previously described . Synbindin localization was identified by GFP fluorescence. Transfected neurons were also immunostained with either anti–synapsin I polyclonal antibody (1:100 dilution; a gift from Dr. Andrew Czernik), anti–syndecan-2 polyclonal antibody (1:100 dilution; a gift from Dr. Merton Bernfield) or anti-MAP2 mAb (1:100 dilution; Sigma Chemical Co.). Rhodamine-conjugated anti-rabbit IgG (1:100 dilution; Chemicon International) or rhodamine-conjugated anti-mouse IgG (1: 50 dilution; Cappel Laboratories) were used as secondary antibodies. Section title: Immunofluorescence and Confocal Imaging Educational score: 4.131025314331055 Domain: biomedical Document type: Study Language: en To localize endogenous synbindin, hippocampal neurons at 1, 2, 3, and 4 wk in vitro were double-immunostained with affinity-purified antisynbindin polyclonal antibody (No. 157; 1:100 dilution) and antisynaptophysin mAb (1:100 dilution; Sigma Chemical Co.) or antisynbindin polyclonal antibody and anti-MAP2 mAb (1:100 dilution; Sigma Chemical Co.). FITC-conjugated anti-rabbit IgG (1:100 dilution; Chemicon International) and rhodamine-conjugated anti-mouse IgG (1:50 dilution; Cappel Laboratories) were used as secondary antibodies. After staining, cells were mounted with fluorescence H-1000 medium (Vector Laboratories) and analyzed on a confocal laser scanning microscope . Section title: RNA Purification, Reverse Transcriptase (RT)-PCR, and Northern Blot Analysis Educational score: 4.154760360717773 Domain: biomedical Document type: Study Language: en Total RNA was extracted from cultures of rat hippocampal neurons at different time points by using Trizol reagents (Life Technologies, Inc.). RT-PCR was performed with 2 μg of the total RNA with the following primer pair: 5′ primer, GAGGCTGAGAAGACTTTCAG; 3′ primer, AACATCGAGGCCAGCATAAG (corresponding to nucleotide numbers 291–311 and 537–557 of mouse synbindin, respectively). PCR products were analyzed on an agarose gel. The identity of amplified bands as synbindin was confirmed by sequencing. For Northern analysis, a mouse multiple tissue Northern blot (CLONTECH Laboratories, Inc.) was hybridized with a 32 P-labeled RNA probe of mouse synbindin, which was synthesized with T3 RNA polymerase (Promega) using pBluescript II SK+ containing mouse synbindin cDNA as a template. Hybridization was performed according to the manufacturer's instructions. Section title: In Situ Hybridization Educational score: 4.1482038497924805 Domain: biomedical Document type: Study Language: en Adult C57BL/6 mice were anesthetized and perfused transcardially with 4% paraformaldehyde in PBS. Whole brains were dissected, immersed in 30% sucrose in PBS overnight at 4°C, and embedded in OCT compound (Miles). Cryostat sections (20-μm thick) were hybridized with digoxigenin-labeled RNA probes as described previously . Antisense and sense RNA probes for mouse synbindin and syndecan-2 were in vitro transcribed from pBluescript II SK+ containing full-length synbindin or syndecan-2 cDNA using either T3 or T7 RNA polymerase (Promega) and the digoxigenin RNA labeling kit (Boehringer Mannheim). Section title: Subcellular Fractionation and Coimmunoprecipitation of Endogenous Proteins Educational score: 4.228954315185547 Domain: biomedical Document type: Study Language: en Subcellular fractions of adult mouse cortex or hippocampus were prepared as described by Li et al. 1996 and Torres et al. 1998 . In brief, adult mouse cortex was homogenized in 4 mM Hepes, pH 7.4, containing 0.32 M sucrose, 1 mM PMSF, 10 μg/ml pepstatin, 10 μg/ml aprotinin, and 2 μg/ml leupeptin with a Teflon head tissue grinder. Homogenates were centrifuged at 1,000 g for 10 min. The supernatant was collected and centrifuged at 12,000 g for 15 min, yielding a pellet (P2) and supernatant (S2). Supernatant (S2) was further centrifuged at 100,000 g for 90 min. The resulting supernatant represents the soluble fraction. The P2 pellet was resuspended in homogenization buffer and represented crude synaptosomes fraction. The crude synaptosome fraction was lysed by osmotic shock by adding 10 vol of ice-cold water containing protease inhibitors and homogenized. The synaptic membrane fraction was pelletted from this homogenate by centrifugation at 33,000 g for 20 min, and the supernatant contained synaptic vesicles. Equal amounts of proteins (50 μg per lane) from each fraction were resolved by SDS-PAGE on an 8–16% gradient gel, transferred to a nitrocellulose membrane, and analyzed by immunoblotting. The subcellular fractions were examined by immunoblotting with anti–PSD-95 (a marker for postsynaptic density), antisynaptophysin (a marker for synaptic vesicles), and anti-CASK antibodies. To identify subcellular localization of synbindin, the subcellular fractions were probed with antisynbindin peptide antibody at 1:500 dilution. As a control for specificity of antisynbindin antibody, blots were reprobed with preimmune serum. Section title: Subcellular Fractionation and Coimmunoprecipitation of Endogenous Proteins Educational score: 4.158234596252441 Domain: biomedical Document type: Study Language: en For coimmunoprecipitation of synbindin and syndecan-2 from the brain, the synaptic membrane fraction was solubilized with 1% CHAPS in TBS containing a protease inhibitor cocktail (Sigma Chemical Co.). After removing insoluble materials by centrifugation, supernatants were incubated with protein G–Sepharose charged with anti–syndecan-2 mAb (6G12) or uncharged protein G–Sepharose at 4°C for 2 h. Bound materials were eluted with SDS-PAGE sample buffer, separated on a 8–16% gel, and immunoblotted with antisynbindin polyclonal antibody (No. 157). Section title: Immunohistochemistry and Immunoelectron Microscopy Educational score: 4.182937145233154 Domain: biomedical Document type: Study Language: en Immunohistochemistry of synbindin was performed with free-floating sections. In brief, adult mice were perfused transcardially with ice-cold 0.9% saline followed by fixation in 4% paraformaldehyde. Free-floating Vibratome sections (50–100-mm thick) of the whole brain were first washed in 0.1 M Tris-HCl, pH 7.6, incubated with 1% hydrogen peroxide for 30 min, and washed again with 50 mM Tris-HCl, pH 7.6, containing 0.15 M NaCl (TBS). Sections were blocked for 30 min with TBS containing 0.1% Triton X-100, 3% normal goat serum and 0.1% BSA (blocking buffer), and then with the avidin-biotin blocking kit (Vector Laboratories). Blocked sections were incubated with affinity-purified antisynbindin antibody (No. 157), which was diluted in the blocking buffer at 2 μg/ml at room temperature overnight. After washing with TBS containing 0.1% Triton X-100 (TBS-T), sections were incubated with biotinylated goat anti–rabbit antibody (Vector Laboratories) at a dilution of 1:200 for 2 h, washed with TBS-T, and incubated with avidin-biotin HRP complex (Vectastain Elite; Vector Laboratories) for 2 h at room temperature. After washing with TBS-T and with 50 mM Tris-HCl, pH 7.6, sections were reacted with 0.05% diaminobenzidine and 0.001% hydrogen peroxide in TBS. Section title: Immunohistochemistry and Immunoelectron Microscopy Educational score: 4.302219390869141 Domain: biomedical Document type: Study Language: en For electron microscopy, adult mice were perfused through the aorta with ice-cold 0.9% NaCl and then with 4% glutaraldehyde and 0.1% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The brain was dissected and postfixed in the same fixative for 1 h at room temperature, and coronal sections (100 μm) of the cerebral cortex were cut by Vibratome. Preembedding immunostaining was performed as follows: the sections were cut into small pieces and incubated in PBS containing 50 mM glycine for 5 min, washed three times with PBS, and blocked with PBS containing 20% normal goat serum and 0.05% Triton X-100 for 1 h. Blocked sections were incubated with affinity-purified antisynbindin antibody (No. 157) or normal rabbit IgG (as a negative control) diluted in PBS containing 3% normal goat serum, 0.05% Triton X-100 (PBS-T) overnight. The next day, sections were washed three times with PBS-T and incubated with anti-rabbit nanogold conjugates (1.4 nm; Nanoprobes Inc.) at a dilution of 1:100 in PBS-T for 90 min at room temperature. After four washes with PBS-T, the sections were further washed four times with deionized water and treated for 7 min with gold enhancer-EM formulation (Nanoprobes Inc.) to enhance immunoreactive signals. The sections were postfixed in 1% osmium in PBS for 30 min, washed with PBS, dehydrated through graded ethanol solutions, and infiltrated with propylene oxide to Epon resin (Ted Pella). The sections were sandwiched between strips of ACLAR plastic film (Ted Pella), flattened between glass sheets, and polymerized in Epon Araldite at 60°C for 24 h. Epon-embedded samples were glued onto resin blocks, and ultrathin sections were cut and collected on 200-mesh collodion-coated copper grids (Electron Microscopy Sciences). The grids were poststained with uranyl acetate and lead citrate, and examined on a Hitachi 600E transmission electron microscope. The distribution of gold particles was analyzed by counting particles using NIH Image software as described in Srivastava et al. 1998 . Section title: Identification and Cloning of a Novel Syndecan-2–binding Protein, Synbindin Educational score: 4.289270401000977 Domain: biomedical Document type: Study Language: en To identify novel syndecan-2–binding proteins, we performed a yeast two-hybrid screen using the syndecan-2 cytoplasmic domain as bait. Screening of a mouse embryo library resulted in isolation of positive colonies selected by HIS prototrophy and β-galactosidase activity. One of the isolates (clone 28) showed a strictly specific interaction pattern with a series of control baits. The specificity of the interaction between clone 28 and the syndecan cytoplasmic domain was studied further by two-hybrid assays . This study demonstrated that clone 28 recognizes the COOH-terminal EFYA sequence of syndecans. As shown in Fig. 1 , clone 28 binds not only the syndecan-2 domain, but also the syndecan-4 cytoplasmic domain, suggesting that it recognizes a region conserved in both syndecans. Because syndecan-2 and syndecan-4 share the COOH-terminal EFYA motif, we deleted this motif from baits (designated as syndecan-2ΔEFYA and syndecan-4ΔEFYA, respectively). Both syndecan-2ΔEFYA and syndecan-4ΔEFYA mutants failed to bind clone 28 . These results indicate that it binds to the COOH terminus of syndecans, as do syntenin and CASK. Section title: Identification and Cloning of a Novel Syndecan-2–binding Protein, Synbindin Educational score: 4.496675491333008 Domain: biomedical Document type: Study Language: en A full-length cDNA for this gene was isolated from a mouse brain library. The entire open reading frame encodes a protein consisting of 219 amino acid residues with a predicted molecular mass of ∼24 kD . The clones isolated by two-hybrid screening corresponded to amino acid residues 18–182 of this protein. No signal sequence or transmembrane domain was identified in the protein. We named this protein synbindin. Synbindin has 48% amino acid identity with the C . elegans F36D4.2 gene and 29% identity with a 23-kD yeast protein, tentatively called p23 . These C. elegans and yeast proteins are likely to be orthologues to mouse synbindin, suggesting that synbindin is highly conserved across species. While no functional data are available for the C. elegans gene, the yeast p23 is a component of a multiprotein complex (transport protein particle [TRAPP]) involved in vesicle docking and fusion . This suggests that synbindin may be involved in vesicular transport in mammalian cells. Section title: Identification and Cloning of a Novel Syndecan-2–binding Protein, Synbindin Educational score: 4.730725288391113 Domain: biomedical Document type: Study Language: en A homology search further identified three segments of substantial homology with proteins known to be involved in vesicular transport as well as synaptic functions . First, an ∼60-amino acid residue segment in the NH 2 -terminal part of synbindin bears homologies with several PDZ domains. Weak but significant homologies (20–30% identity; 40–50% similarity) were identified with the following: the sixth PDZ domain of glutamate receptor–interacting protein (GRIP) 1 and GRIP2 ; the seventh PDZ domain of the 5-HT 2C receptor–binding protein MUPP1 ; the fourth PDZ domain of human INAD-like protein ; and the second PDZ domains of Mint-1, -2, and -3 . All of these proteins have been implicated in synaptic functions. The homology with these PDZ domains does not encompass the entire PDZ domain, but is restricted to the COOH-terminal part of the domain. Therefore, this segment of synbindin does not constitute a classical PDZ domain. Second, a 76-amino acid residue segment in the middle of the molecule shows a strong similarity with the yeast gene BET5 . BET5 is a gene involved in vesicle transport in yeast. A null mutation of BET5 blocks ER-to-Golgi transport . Moreover, like p23, the protein product of BET5 (Bet5p) is a component of the yeast TRAPP complex . Considering the phylogenetic distance between mouse and yeast, the homology between synbindin and BET5 in this segment is quite high (30% identity; 50% similarity). These observations further supports the notion that synbindin is involved in vesicular transport. Finally, the 35 residue COOH-terminal segment shows 45% identity (52% similarity) to a short cytoplasmic segment of the cardiac muscle ryanodine receptor (RyR-2), which is a calcium release channel on the membrane of intracellular Ca 2+ stores . Taken together, synbindin consists of four regions: the NH 2 -terminal region, which has no significant homology with any known proteins; a PDZ-related region; a BET5-related region; and a COOH-terminal tail similar to a short segment of RyR-2 Section title: Characterization of the Synbindin–Syndecan-2 Interaction Educational score: 4.194828987121582 Domain: biomedical Document type: Study Language: en To further assess the specificity of the synbindin–syndecan-2 interaction, we performed pull-down and coimmunoprecipitation assays. For pull-down assays, glutathione-agarose beads charged with synbindin-GST fusion protein or nonfused GST were incubated with lysates of 293 cells expressing Myc-tagged syndecan-2. Bound materials were probed by immunoblotting with anti–syndecan-2 mAb or anti-Myc polyclonal antibody . As shown in Fig. 2 , GST-synbindin brought down syndecan-2 from the cell lysates . No syndecan-2 was brought down with nonfused GST or from control transfected 293 cells . To examine coimmunoprecipitation of synbindin and syndecan-2, 293 cells were cotransfected with FLAG-tagged syndecan-2 (either FLAG-syndecan-2 or FLAG-syndecan-2ΔEFYA) and Myc-tagged synbindin. The FLAG-syndecan-2 was precipitated from the lysate of cotransfected cells with anti-Myc antibody . No coimmunoprecipitation was observed when synbindin was not introduced into cells . The syndecan-2ΔEFYA mutant was not coprecipitated with synbindin . Section title: Characterization of the Synbindin–Syndecan-2 Interaction Educational score: 4.13165283203125 Domain: biomedical Document type: Study Language: en Direct binding between synbindin and the syndecan-2 cytoplasmic domain was examined using purified His-tagged syndecan-2 cytoplasmic domains and GST-synbindin. GST-synbindin bound to His-tagged syndecan-2 cytoplasmic domain in the overlay assay . Consistent with pull-down and coimmunoprecipitation assays, the ΔEFYA cytoplasmic domain failed to bind GST-synbindin. Section title: Characterization of the Synbindin–Syndecan-2 Interaction Educational score: 4.276078224182129 Domain: biomedical Document type: Study Language: en Finally, we analyzed the region of synbindin that interacts with syndecan-2 by using two-hybrid assays. The full-length synbindin and four truncated forms were tested with baits of the intact and ΔEFYA syndecan-2 cytoplasmic domains . The three fragments containing the PDZ-like segment showed a strong interaction with the intact syndecan-2 cytoplasmic domain. The strongest of the three was the P-Sbd fragment, which consists only of the PDZ-like segment followed by the N-Sbd and P/C synbindin fragments. The fragment without the PDZ-like segment (C-Sbd) did not display a signal above background. The ΔEFYA bait did not interact with any fragments. Taken together, these binding studies establish that synbindin directly interacts with syndecan-2 through the EFYA tail of syndecan-2, and that the PDZ-like segment of synbindin is involved primarily in the interaction with syndecan-2. Section title: Syndecan-2–dependent Translocation of Synbindin in Hippocampal Neurons Educational score: 4.325884819030762 Domain: biomedical Document type: Study Language: en To examine further the physiological significance of the synbindin–syndecan-2 interaction in neurons, we tested whether these two proteins interact in the cytoplasmic environment of cultured hippocampal neurons. For this, hippocampal neurons at 1 DIV were transfected with synbindin-GFP fusion protein alone or together with syndecan-2. The localization of synbindin and syndecan-2 was examined at 8 DIV by GFP signals and immunofluorescence with anti–syndecan-2 antibody, respectively. At this stage in culture, endogenous syndecan-2 is not yet expressed by hippocampal neurons. Therefore, the syndecan-2 immunoreactivities detected in these experiments represent transfected syndecan-2 . We found that the distribution of synbindin changes dependent on cotransfection of syndecan-2. In neurons transfected with synbindin-GFP alone, synbindin was distributed diffusely in cytoplasm . In contrast, when neurons were transfected with synbindin-GFP together with intact syndecan-2, synbindin formed clusters along dendrites . Immunostaining of these double-transfected neurons with anti–syndecan-2 antibody demonstrated colocalization of synbindin and syndecan-2 . These results demonstrate that not only does synbindin colocalize with syndecan-2 in transfected hippocampal neurons, but also that syndecan-2 induces synbindin clustering in dendrites. Section title: Syndecan-2–dependent Translocation of Synbindin in Hippocampal Neurons Educational score: 4.145970344543457 Domain: biomedical Document type: Study Language: en To determine the localization of the synbindin clusters, we performed a series of double labeling experiments. These experiments demonstrated that the synbindin clusters observed in synbindin/syndecan-2 double-transfected neurons are localized in dendritic spines. As shown in Fig. 3 , double staining with anti-MAP2 antibody demonstrated that the clusters of synbindin-GFP are localized in small protrusions along dendrites . Double staining with the anti–synapsin I antibody revealed that synbindin clusters exhibit partial overlap with synapsin I immunoreactivities , a pattern typically seen for postsynaptic proteins . Section title: Syndecan-2–dependent Translocation of Synbindin in Hippocampal Neurons Educational score: 4.23838472366333 Domain: biomedical Document type: Study Language: en To examine if this clustering of synbindin is mediated by the interaction with the EFYA tail of syndecan-2, as demonstrated by biochemical binding studies, the same double transfection experiments were performed with the syndecan-2ΔEFYA deletion mutant. As shown in Fig. 4 , synbindin failed to cluster in neurons double-transfected with synbindin-GFP and the syndecan-2ΔEFYA mutant . In the syndecan-2/synbindin-GFP–cotransfected cultures, the majority (83 ± 7%) of GFP-positive neurons showed extensive clustering (>10 clusters per cell) of synbindin-GFP, whereas no cells (0%) were found to contain >3 GFP clusters in the syndecan-2ΔEFYA/synbindin-GFP–cotransfected cultures. We previously demonstrated that the syndecan-2ΔEFYA mutant is expressed and forms clusters in the same manner as the intact syndecan-2 in hippocampal neurons . Therefore, the lack of synbindin clustering in syndecan-2ΔEFYA/synbindin-GFP–cotransfected neurons is not due to the aberrant expression of the syndecan-2ΔEFYA mutant. Taken together, these transfection experiments provide further evidence for the synbindin–syndecan-2 interaction through the EFYA tail of syndecan-2, and suggest that syndecan-2 induces the clustering of synbindin in dendrites. Section title: Expression of Synbindin mRNA in the Nervous System Educational score: 4.5595502853393555 Domain: biomedical Document type: Study Language: en Northern blotting revealed a 4.4-kb transcript of synbindin that is widely expressed in adult mouse tissues . Such an expression pattern is similar to those of syntenin and CASK . RT-PCR and in situ hybridization studies demonstrated that synbindin is strongly expressed in neurons. RT-PCR analysis demonstrated that synbindin mRNA is expressed in cultured hippocampal neurons at 14 and 30 DIV , as was previously shown for syndecan-2 . In situ hybridization in the adult mouse central nervous system (CNS) showed that synbindin is expressed predominantly in large neurons. In the cerebrum, strong synbindin expression was observed in pyramidal neurons in the CA1-CA3 regions of hippocampus , pyramidal neurons in the cortex , and large neurons in the caudate putamen . In the cerebellum, Purkinje cells and neurons in the deep cerebellar nuclei exhibited strong synbindin expression . Motor neurons in the spinal cord also showed strong synbindin expression . In contrast, synbindin expression in smaller, granule-type neurons was much weaker or undetectable, including granule neurons in the dentate gyrus and those in the granular layer of the cerebellar cortex . Throughout the CNS, no synbindin expression was detected in glial cell types. Taken together, these observations suggest that, in the nervous system, synbindin is expressed predominantly by large, pyramidal-type neurons, which tend to have highly developed dendritic arbors. Section title: Expression of Synbindin mRNA in the Nervous System Educational score: 4.2014055252075195 Domain: biomedical Document type: Study Language: en We compared spatial expression patterns of synbindin and syndecan-2 by in situ hybridization in serial sections. This analysis revealed that these two molecules exhibit almost identical expression patterns in the adult mouse brain. In the hippocampus, syndecan-2 mRNA was detected in pyramidal cells, whereas granule cells of the dentate gyrus exhibited much weaker signals . In the cerebellum, Purkinje cells express syndecan-2, but little syndecan-2 expression was observed in granule neurons . This spatial expression pattern is very similar to that of synbindin . Section title: Immunohistochemical Identification of Synbindin In Vivo Educational score: 4.265843391418457 Domain: biomedical Document type: Study Language: en To investigate the localization of endogenous synbindin in vivo, we generated polyclonal antisera to synbindin. Antibodies affinity-purified from this antiserum reacted with a 24-kD band in adult mouse brain extracts , which comigrate with synbindin released from GST-synbindin (lane 2). In tissue sections, synbindin immunoreactivities were observed in various neurons in the adult mouse brain. Strong immunoreactivities were found in pyramidal neurons of the cerebral cortex , pyramidal neurons of the hippocampus , Purkinje cells of the cerebellum , and neurons of the deep cerebellar nucleus . In general, this result is consistent with the expression pattern of synbindin mRNA demonstrated by in situ hybridization . Synbindin immunoreactivities were mainly associated with the soma and the apical dendrites of these neurons. In the hippocampus, strong immunoreactivities were observed in the stratum lucidum where the dendrites of CA3 pyramidal neurons are present . In the cerebellum, the cell bodies and apical dendrites of Purkinje neurons were intensely labeled . Section title: Accumulation of Endogenous Synbindin in Spines of Cultured Hippocampal Neurons Educational score: 4.533939361572266 Domain: biomedical Document type: Study Language: en We have shown previously that endogenous syndecan-2 is concentrated in dendritic spines of cultured hippocampal neurons after 2 wk in vitro . Immunocytochemistry revealed that synbindin exhibits a similar temporal expression pattern in dendritic spines . At 7 DIV, synbindin immunoreactivity was detected diffusely in the cytoplasm of the soma . No punctate immunoreactivity was detected at this stage. At 14 DIV, punctate immunoreactivity became apparent along the dendrites . The intensity of the punctate staining grew stronger at 21 and 28 DIV . This time course is similar to that of syndecan-2 expression in dendritic spines and that of the spine morphogenesis in this culture system . Double immunostaining of 30 DIV hippocampal neurons confirmed the presence of endogenous synbindin in dendritic spines . Synbindin immunoreactivities correspond to small protrusions along MAP2-positive dendritic shafts . Double staining with antisynbindin and antisynaptophysin antibodies showed a partial overlapping pattern , as observed in transfected neurons . At high magnification , synbindin puncta (green) are in close apposition with synaptophysin puncta (red) but do not completely overlap, which is a typical distribution pattern for postsynaptic proteins. Taken together, these results demonstrate that endogenous synbindin molecules in mature neurons exist in dendritic spines, which is consistent with in vitro transfection studies . Section title: Accumulation of Endogenous Synbindin in Spines of Cultured Hippocampal Neurons Educational score: 4.197091102600098 Domain: biomedical Document type: Study Language: en Unlike syndecan-2, which exhibits essentially no immunoreactivity on dendritic shafts in mature (30 DIV) neurons , synbindin was detected within dendritic shafts and neuronal soma , even at 30 DIV. This is consistent with the immunohistochemical results in which synbindin immunoreactivities were observed along dendrites and in the soma . Considering that synbindin has similarities with proteins involved in membrane trafficking, it is suspected that synbindin may also associate with intracellular membrane compartments within dendrites and cell bodies. Section title: Endogenous Synbindin Is Enriched in Synaptic Membranes and Associated with Syndecan-2 Educational score: 4.166804313659668 Domain: biomedical Document type: Study Language: en To characterize the subcellular localization of synbindin, we prepared subcellular fractions from adult mouse cortex using a differential centrifugation procedure . Two marker proteins, PSD-95 (postsynaptic marker) and synaptophysin (synaptic vesicle marker), were used as controls. Synbindin was fractionated into the crude synaptosome fraction and further enriched in the synaptic membrane fraction . The fractionation pattern of synbindin was similar to that of PSD-95. These results indicate that synbindin is associated with synapses. Section title: Endogenous Synbindin Is Enriched in Synaptic Membranes and Associated with Syndecan-2 Educational score: 4.124731063842773 Domain: biomedical Document type: Study Language: en To obtain in vivo biochemical evidence for the synbindin–syndecan-2 interaction, we examined whether endogenous synbindin and syndecan-2 coprecipitate from brain extracts. As shown in Fig. 9 B, anti–syndecan-2 mAb precipitated synbindin from the CHAPS extracts of the synaptic membrane fraction. Taken together, these results provide further evidence for the synbindin-syndecan-2 interaction in vivo. Section title: Ultrastructural Localization of Synbindin in Synapses and Intracellular Organelles Educational score: 4.258475303649902 Domain: biomedical Document type: Study Language: en To determine the precise intracellular localization of synbindin in vivo, we performed immunogold electron microscopy in the adult mouse cerebral cortex. This analysis demonstrated synbindin immunolabeling in two distinct structures in neurons, synapses, and intracellular membrane organelles. Immunogold labeling for synbindin was concentrated at asymmetric synapses . Labeling was seen on both pre- and postsynaptic membranes, but the majority of the labeling was on the postsynaptic side in close association with postsynaptic membranes. Quantitative analysis of the distribution of gold particles confirmed that synbindin is concentrated on the postsynaptic side of synapses . Such a distribution pattern of synbindin is consistent with that of syndecan-2, as determined by immunogold labeling . Section title: Ultrastructural Localization of Synbindin in Synapses and Intracellular Organelles Educational score: 4.378398895263672 Domain: biomedical Document type: Study Language: en Immunogold labeling demonstrated that synbindin is also associated with membrane-bound compartments within neurons . Consistent with the light microscopic findings , immunogold labeling for synbindin was observed in the soma and dendritic shafts of cortical neurons. In these sites, labeling was associated predominantly with various cisterns and vesicles. Dense labeling was observed on the Golgi apparatus and unidentified vesicles in the dendritic shaft . Membrane compartments within dendritic spines were also labeled . These observations demonstrate that synbindin is indeed associated with intracellular cisterns and vesicles, supporting the notion that synbindin is involved in vesicular transport and membrane trafficking in dendrites. Thus, these immunoelectron microscopy results indicate that synbindin is localized in two distinct subcellular structures in neurons: (1) synapses (mainly postsynaptic membranes and PSD) and (2) intracellular membrane cisterns. Similar dual localization has been reported for other cytoplasmic proteins implicated in synaptic functions, including GRIP1 and α-actinin-2 . Section title: Discussion Educational score: 4.2852325439453125 Domain: biomedical Document type: Study Language: en In previous work, we demonstrated that syndecan-2 induces the formation of morphologically mature dendritic spines, and that this effect is mediated by the cytoplasmic interaction through the COOH terminus of syndecan-2 . We report here a novel syndecan-2-binding protein, synbindin. Our results indicate that synbindin is a postsynaptic protein that is likely to be involved in intracellular vesicle transport in dendritic spines. This suggests a possibility that syndecan-2 exerts its effects on spine morphogenesis by recruiting synbindin-positive membrane cisterns toward postsynaptic sites. Section title: Synbindin, A Novel Syndecan-2–binding Protein in Neurons Educational score: 4.390294075012207 Domain: biomedical Document type: Study Language: en Synbindin is the third molecule, after syntenin and CASK , that has been shown to bind the COOH terminus of syndecan-2. Both syntenin and CASK contain PDZ domains and bind syndecan-2 through these domains. Synbindin has a segment that shares homologies with PDZ domains, and this segment is primarily involved in binding to syndecan-2. However, this segment does not constitute a classical PDZ domain. It remains to be determined whether the synbindin–syndecan-2 interaction can be explained according to the well-established paradigm of the recognition of protein COOH termini by PDZ domains. Yet, the fact that proteins that bear homology with this segment of synbindin are predominantly PDZ domain proteins suggests that synbindin has some remote structural relationships with this family of proteins. Section title: Synbindin, A Novel Syndecan-2–binding Protein in Neurons Educational score: 4.423370361328125 Domain: biomedical Document type: Study Language: en On the other hand, synbindin has clear structural relationships with proteins involved in vesicular transport and membrane trafficking. Synbindin exhibits 43% homology with a yeast protein, tentatively named p23 , which has been identified as a component of multiprotein complex called TRAPP . In yeast cells, the TRAPP complex is thought to mediate the docking and fusion of ER-to-Golgi transport vesicles. Bet5p, which has sequence homology with synbindin , is also a component of the TRAPP complex . These observations suggest that synbindin is involved in membrane trafficking in neurons. Consistent with this possibility, immunoelectron microscopy demonstrated that synbindin is present on membrane-bound cisterns and vesicles within the soma and dendrites of cortical neurons. Thus, it is likely that synbindin is involved in membrane trafficking in neurons. Section title: Physiological Significance of the Interaction Educational score: 4.655678749084473 Domain: biomedical Document type: Study Language: en Despite the fact that the synbindin-syndecan-2 interaction may not be explained by the well-established paradigm of the interaction between PDZ domains and protein COOH termini, there is ample evidence for the physiological significance of the interaction. In situ hybridization and immunohistochemistry demonstrated that, in the CNS, synbindin is expressed by certain neuronal populations, most notably large, pyramidal-type neurons. In contrast, synbindin expression is weak in granular-type neurons, such as the neurons of the dentate gyrus and the granular layer of the cerebellum. This pattern of expression is very similar to that of syndecan-2 . In mature hippocampal neurons in culture, synbindin is concentrated in dendritic spines, as previously shown for syndecan-2 . Cotransfection experiments showed that syndecan-2 causes the clustering of synbindin in dendritic spines, and that this syndecan-2–dependent clustering of synbindin occurs through the EFYA tail of syndecan-2. These results indicate that the synbindin–syndecan-2 interaction occurs in the cytoplasmic environment of hippocampal neurons with the same specificity as observed in two-hybrid assays. By immunogold electron microscopy and biochemical fractionation, we demonstrated that synbindin is associated with synaptic membranes, mainly at the postsynaptic side, where the existence of syndecan-2 has been reported previously . Finally, synbindin coimmunoprecipitates with syndecan-2 from the synaptic membrane fractions. These results strongly suggest that synbindin and syndecan-2 interact physiologically in dendritic spines of certain populations of neurons. Section title: Clustering of Synbindin in Dendritic Spines Requires Syndecan-2 Educational score: 4.371192455291748 Domain: biomedical Document type: Study Language: en In the previous study, we showed that the interaction of the syndecan-2 and its cytoplasmic ligands is essential for transfected syndecan-2 to induce spine formation . Furthermore, our work demonstrated that the cytoplasmic syndecan-2 ligands essential for spine formation are recruited by syndecan-2 rather than the cytoplasmic ligands recruit syndecan-2 . The sorting behavior of transfected synbindin in hippocampal neurons is consistent with the following observation: the clustering of synbindin in spines requires syndecan-2 expression . Moreover, the clustering of endogenous synbindin during the course of hippocampal cultures is also consistent with this notion: the clustering of synbindin in spines occurs after 2 wk in vitro, coinciding with the accumulation of syndecan-2 in spines . These results indicate that syndecan-2 recruits cytoplasmic proteins to the membrane sites that later become dendritic spines. Thus, syndecan-2 may act as a mediator of extracellular signals, which specify the site of prospective dendritic spines, though we have not determined if any extracellular ligands are involved in the syndecan-2 action on spine morphogenesis. In this vein, it is interesting to examine whether other intracellular syndecan-2 ligands, namely CASK and syntenin, are also translocated to spines dependent on the expression of syndecan-2. If so, the role of syndecan-2 may be a more general one. Section title: Possible Role of Synbindin in Dendritic Spine Morphogenesis Educational score: 4.379213333129883 Domain: biomedical Document type: Study Language: en Our previous work showed that a molecule that binds to the syndecan-2 EFYA tail plays a crucial role in the process of syndecan-2–induced spine formation as a downstream effector ; however, it is not known which molecule among the three known EFYA ligands (syntenin, CASK, and synbindin) is relevant to this process. Some of these interactions may turn out to be irrelevant to spine formation. On the other hand, it is possible that all three interactions occur in neurons, as they are all expressed in neurons . It is also possible that these molecules play different roles within neurons. Having PDZ domains, CASK and syntenin are likely to be involved in the assembly of the cytoskeletal scaffold. Lacking the classical PDZ domain and other cytoskeletal motifs, synbindin is unlikely to be involved in the assembly of the postsynaptic cytoskeletal scaffold. On the other hand, the homology with the components of the yeast TRAPP complex suggests synbindin is involved in vesicle transport in neurons. Section title: Possible Role of Synbindin in Dendritic Spine Morphogenesis Educational score: 4.417280197143555 Domain: biomedical Document type: Study Language: en There is increasing evidence that membrane trafficking plays an important role in functional maturation of postsynaptic structures. For instance, Lledo et al. 1998 showed that inhibition of membrane trafficking with the botulinum toxin suppresses LTP. In dendritic spines, AMPA receptors are present in association with intracellular vesicles, and upon stimulation of synapses, these AMPA-containing vesicles dock and fuse with the postsynaptic plasma membrane . Membrane fusion in postsynaptic sites is thought to be mediated by N -ethylmaleimide-sensitive fusion protein and SNAREs . Interestingly, there is evidence that the TRAPP complex acts upstream of the SNARE complex assembly. Rossi et al. 1995 showed that the SNARE complex does not form in the yeast BET3 mutant, indicating that BET3 genetically interacts with the SNARE pathway. As discussed above, both BET3 and p23, the putative yeast homologue of synbindin, are components of the TRAPP complex . This suggests that synbindin is involved in membrane trafficking in postsynaptic sites. Section title: Possible Role of Synbindin in Dendritic Spine Morphogenesis Educational score: 4.722250461578369 Domain: biomedical Document type: Study Language: en At present, we do not know whether synbindin acts as a downstream effector in the syndecan-2–induced spine formation. Yet, there are a few possible scenarios that synbindin is involved in the process of spine formation. Dendritic spines contain membrane-bound organelles . These organelles, or spine apparatus, appear throughout the spine and are sometimes seen around the lateral margins of the postsynaptic density . These membrane-bound organelles are thought to play important roles in local synthesis and posttranslational modification of neurotransmitter receptors. Therefore, synbindin clustering induced by syndecan-2 expression may facilitate local synthesis and transport of neurotransmitter receptors. This may, in turn, result in an increase in synaptic efficiency and early maturation of postsynaptic structures. Another possibility, which we currently favor, is that the synbindin–syndecan-2 interaction promotes the recruitment of Ca 2+ -storing membrane compartments toward synapses, and the Ca 2+ mobilization from these compartments induces morphological changes of spines. Membrane cisterns immunoreactive for inositol trisphosphate and/or ryanodine receptors, which identify intracellular Ca 2+ stores, have been shown in dendritic spines, sometimes in the close vicinity of postsynaptic membranes . Moreover, a moderate and transient increase in [Ca 2+ ] due to release from these Ca 2+ stores has been shown to cause elongation of existing spines and the formation of new ones . These observations suggest that morphogenesis of dendritic spines involves the recruitment of Ca 2+ -storing vesicles in the vicinity of synapses. Therefore, the induction of spine formation by syndecan-2 may be due to the recruitment of synbindin-coated Ca 2+ -storing membrane compartments toward subsynaptic locations. In any event, the identification of a protein that is involved in vesicle transport as a ligand for a cell-surface proteoglycan suggests that extracellular cues may have a role in determining the destination of these vesicles over dendritic surfaces, which in turn promotes the formation of postsynaptic specialization at such a destination. | Study | biomedical | en | 0.999997 |
0003113 | Section title: Introduction Educational score: 4.189830303192139 Domain: biomedical Document type: Study Language: en Many pathologic stimuli induce the heart to undergo adaptive hypertrophic growth that temporarily augments cardiac function. Although the initial hypertrophic response may be beneficial, sustained hypertrophy often undergoes a transition to heart failure, which is a leading cause of mortality and morbidity worldwide, and is characterized by a progressive deterioration in cardiac function. Intensive investigation over the last several years has led to the identification of intracellular signaling pathways that are believed to transduce prohypertrophic signals. However, the nature of the cross-talk between these pathways remains unclear and, more importantly, negative regulators of the hypertrophic response have not been identified. Section title: Introduction Educational score: 4.189929008483887 Domain: biomedical Document type: Study Language: en Glycogen synthase kinase-3 (GSK-3) 1 is a highly conserved protein kinase that is believed to play a critical role in development as a component of the Wnt/wingless pathway, and in a number of human disease states including tumorigenesis, diabetes, and Alzheimer's disease . Unlike most protein kinases, GSK-3 is active in the unstimulated cell and becomes inactivated when cells are stimulated by a variety of mitogens or by the Wnt/wingless pathway . Section title: Introduction Educational score: 4.46332311630249 Domain: biomedical Document type: Study Language: en Many of the targets of GSK-3 that have been identified to date, including c-Jun, cyclin D1, several metabolic enzymes, β-catenin, and at least two nuclear factors of activated T cells (NF-ATs), are repressed by the action of GSK-3, and inactivation of GSK-3 relieves the repression . The list of putative substrates of GSK-3 suggested that this kinase might also play a negative modulatory role in hypertrophy since it negatively regulates the actions of major targets of two cytosolic signaling pathways that have been implicated in the hypertrophic response to pressure overload in the intact animal. These pathways, the calcineurin pathway and a pathway culminating in activation of the stress-activated protein kinases (SAPKs, also known as c-Jun NH 2 -terminal kinases or JNKs), activate NF-ATs and c-Jun, respectively . Section title: Introduction Educational score: 4.517978191375732 Domain: biomedical Document type: Study Language: en Calcineurin, activated by calmodulin binding in the presence of elevated cytosolic free [Ca 2+ ], dephosphorylates NF-ATs, exposing the nuclear localization signals . NF-ATs then translocate to the nucleus and activate transcription of a number of genes involved in a variety of responses, including the immune response . When calcium levels return to normal and calcineurin is inactivated, phosphorylation of NF-ATs leads to their rapid export from the nucleus, terminating the signal . Although the role of NF-ATs in the hypertrophic response of cardiomyocytes to physiologically relevant stimuli is not clear, Molkentin et al. 1998 were able to induce hypertrophy in transgenic mice by expressing activated NF-ATc4. These data suggest that calcineurin's prohypertrophic effects are mediated, at least in part, via activation of one or more NF-ATs. More recently, calcineurin-induced activation of NF-ATc1 (NF-AT2/c) was shown to play a role in skeletal myocyte hypertrophy . Section title: Introduction Educational score: 4.217308044433594 Domain: biomedical Document type: Study Language: en GSK-3β has been reported to regulate nuclear/cytoplasmic partitioning of various NF-ATs. GSK-3β has been shown to induce nuclear export of transfected NF-ATc1 in COS cells, and of transfected NF-ATc4 (NF-AT3) in hippocampal neurons . Although several other kinases also have been implicated in regulation of NF-AT subcellular localization , these data raise the possibility that GSK-3β could exert an antihypertrophic effect in the heart by affecting nuclear/cytoplasmic partitioning of endogenous NF-ATs in cardiac myocytes. Section title: Introduction Educational score: 4.399102687835693 Domain: biomedical Document type: Study Language: en c-Jun, a major SAPK target , is also negatively regulated by phosphorylation by GSK-3 . Phosphorylation reduces the DNA binding activity of c-Jun, and thus the activity of AP-1 (a heterodimer of c-Jun and c-Fos family members). Since the SAPKs recently have been shown to be necessary for the hypertrophic response of neonatal cardiomyocytes to endothelin-1 (ET-1) and for the development of pressure overload-induced hypertrophy in the intact rat , inhibition of activity of one of the primary targets of the SAPKs, AP-1, could be another mechanism whereby GSK-3 might negatively regulate hypertrophy. Section title: Introduction Educational score: 4.031991004943848 Domain: biomedical Document type: Study Language: en Herein, we explore the role of GSK-3β in the hypertrophic response of cardiomyocytes. Our data indicate that inhibition of GSK-3β is a critical step in the development of a cardiac hypertrophic response. Section title: Materials Educational score: 2.2458508014678955 Domain: biomedical Document type: Other Language: en Antibodies used were: anti-GSK-3β mAb (Transduction Laboratories), antiatrial natriuretic factor (anti-ANF; Peninsula Laboratories), anti-α-actinin mAb (Sigma-Aldrich), rabbit anti-NF-ATc1 (K-18), which recognizes all NF-AT family members (Santa Cruz Biotechnology), antiphospho Ser 9 GSK-3β that specifically recognizes Ser 9 phosphorylated GSK-3β (New England Biolabs), and Cy3-conjugated anti-rabbit and anti-mouse antibodies (BioRad). Other reagents included glycogen synthase peptide-2 (Upstate Biotechnology, Inc.), ET-1 and phenylephrine (PE; Sigma-Aldrich), and insulin-like growth factor-1 (IGF-I; Calbiochem). Section title: Cell Culture Educational score: 4.0358381271362305 Domain: biomedical Document type: Study Language: en Spontaneously beating neonatal myocytes were prepared from 1–2-d-old rats and cultured in F-10 medium in the presence of 5% FBS and 10% horse serum as previously described . Section title: Construction of Recombinant Adenoviral Vector Carrying the GSK-3βA9 cDNA. Educational score: 4.220794677734375 Domain: biomedical Document type: Study Language: en The cDNA encoding GSK-3βA9, carrying a Ser-to-Ala substitution at Ser 9 in the NH 2 -terminal region of GSK-3β, and an HA epitope tag at the COOH terminus was created by PCR as described . The cDNA was subcloned into the pAdTRACK-CMV shuttle vector (obtained from B. Vogelstein, Johns Hopkins University, Baltimore, MD) that encodes green fluorescent protein (GFP) from one CMV promoter and the gene of interest from a second CMV promoter . AdGSK-3βA9, the recombinant adenovirus, was prepared using the AdEASY system as described . The recombinant virus was propagated in 293 cells and high titer stocks (≥10 12 particles/ml) were purified by CsCl density gradient centrifugation. Section title: Other Adenoviral Vectors. Educational score: 4.1967058181762695 Domain: biomedical Document type: Study Language: en AdβgalEGFP (herein referred to as AdGFP), carrying the Escherichia coli LacZ gene in addition to the GFP gene, was used as a control virus. AdBD110, which encodes the 110-kD catalytic subunit of phosphoinositide 3-kinase (PI3-K), rendered constitutively active by including in-frame the p110-binding domain of human p85 (amino acids 474–552), has been previously described in detail . When cardiomyocytes are infected with AdBD110, they have constitutively elevated levels of 3-phosphorylated phosphoinositides and increased activity of PKB/Akt . AdPKB/Akt, encoding protein kinase B (PKB)/Akt made constitutively active by the addition of a myristylation signal at the NH 2 terminus of the kinase, was kindly provided by Dr. Thomas Franke (Columbia University, New York, NY) and has been described in detail . Section title: Cell Fractionation Educational score: 4.128717422485352 Domain: biomedical Document type: Study Language: en Cells were fractionated by hypotonic lysis. In brief, cells were suspended in lysis buffer containing Hepes (20 mM, pH 7.5) and NaCl (10 mM) with phosphatase and protease inhibitors. After 15 min on ice, lysates were spun at 2,500 rpm for 5 min in an Eppendorf centrifuge. The pellet (nuclear fraction) was washed twice in lysis buffer, and then the supernatant and pellet were spun at 14,000 rpm for 10 min. Protein concentrations of the cytosolic and nuclear fractions were equalized, and then SDS sample buffer was added to a final concentration of 1×. Section title: Immunoblot Analysis Educational score: 4.023358345031738 Domain: biomedical Document type: Study Language: en For Western blot analysis, cell lysates were matched for protein concentration and were then separated by SDS-PAGE and transfered to Hybond-C extra (Amersham Pharmacia Biotech). The membranes were blocked in 5% nonfat milk and then incubated with the indicated antibodies for 1 h at room temperature. Antibody binding was detected with a peroxidase-conjugated goat anti–rabbit or anti–mouse IgG and chemiluminescence. Section title: Immune Complex Kinase Assay of GSK-3β Educational score: 4.213712692260742 Domain: biomedical Document type: Study Language: en For the studies of GSK-3β activity in aortic banded hearts, the left ventricle was pulverized under liquid nitrogen, homogenized with a polytron in lysis buffer containing protease and phosphatase inhibitors , and then briefly sonicated. After 15 min on ice with vortexing, the samples were centrifuged at 100,000 g for 1 h at 4°C. Supernatants from heart lysates, or from lysates of neonatal cardiomyocytes in culture, were matched for protein concentration, and were incubated with anti-GSK-3β mAb or anti-HA mAb for 2 h, and then complexes were collected with protein G–Sepharose beads for an additional 1 h. Beads were washed six times in lysis buffer and three times in assay buffer, and then were incubated for 20 min at 30°C with glycogen synthase peptide-2 (50 μM) and 100 μM γ[ 32 P]ATP (3,000–4,000 cpm/pmol) in the presence of 10 mM MgCl 2 . Contents of the assays were spotted onto P81 phosphocellulose papers that were washed and then subjected to liquid scintillation counting. Kinase activity was reduced to background levels when 10 mM LiCl was included in the reaction mix, suggesting the activity measured was GSK-3β and not a contaminating kinase. Section title: [ 3 H]-leucine Incorporation Educational score: 4.099077224731445 Domain: biomedical Document type: Study Language: en Neonatal cardiomyocytes were infected with AdGSK-3βA9 or AdGFP in F-10 medium containing 0.1% FCS. 36 h later, cells in triplicate wells of 12-well plates were stimulated with ET-1 (100 nM) for 36 h in serum-free F-10 medium and then incubated in the same medium with 1.0 μCi/ml [ 3 H]-leucine for an additional 12 h. The cells were processed as described , and [ 3 H]-leucine incorporation was determined by liquid scintillation counting. Section title: Immunocytochemistry Educational score: 4.127936363220215 Domain: biomedical Document type: Study Language: en Cardiomyocytes, grown on laminin-coated plastic coverslips, were infected with either AdGFP or AdGSK-3βA9. 36 h later, they were exposed to ET-1 or PE in serum free medium. For assessment of sarcomere organization and ANF expression, the cells were fixed 48 h later for 10 min with 4% parformaldehyde/5% sucrose in PBS. Coverslips were processed as described . For sarcomere staining, coverslips were incubated in a 1:400 dilution of anti–α-actinin mAb and for ANF staining in a 1:400 dilution of anti-ANF antibody in blocking solution. Coverslips were then incubated in a Cy3-conjugated secondary antibody diluted 1:800 in blocking solution for 1 h at room temperature. Cells were photographed using a Nikon FXA photomicroscope. Figures were prepared using Canvas 6.0.1 (Deneba Systems, Inc.) and were then transferred to Adobe Photoshop 5.5 for printing. Section title: Statistical Analysis Educational score: 2.7975122928619385 Domain: biomedical Document type: Study Language: en Data are expressed and presented in the figures as mean ± SEM. A t test was used to compare the means of normally distributed continuous variables. A value of P < 0.05 was chosen as the limit of statistical significance. Section title: GSK-3β Is Inhibited by Hypertrophic Stimuli Educational score: 4.266287803649902 Domain: biomedical Document type: Study Language: en If GSK-3β plays an important role in the hypertrophic response, its activity should be inhibited by hypertrophic stimuli. Therefore, we exposed neonatal rat cardiomyocytes to ET-1 or, as a positive control, IGF-1, another hypertrophic agent known to inhibit GSK-3β. Cell lysates were prepared and subjected to immunoprecipitation with an anti-GSK-3β antibody, followed by assay with the glycogen synthase-2 peptide as substrate. IGF-1 produced a 45% decrease in GSK-3β activity, consistent with prior observations using insulin in various cell lines . In response to ET-1 (100 nM), GSK-3β was inhibited by as much as 60% at 40 min. Thereafter, GSK-3β activity returned toward control levels, but some inhibition persisted for at least 90 min . Inhibition with PE (20–30%) was not as marked as with ET-1, but was significant (data not shown). Although PE-induced inhibition is less than the inhibition seen with IGF-1, this percent inhibition is equivalent to that seen with NGF and HGF (20–30% inhibition), and this degree of inhibition is believed to play an important role in the antiapoptotic effect of NGF in PC12 cells and in the HGF-induced accumulation of β-catenin in mouse mammary epithelial cells . These data suggest that the degree of inhibition of GSK-3β by ET-1 and PE in cardiomyocytes is sufficient to have potentially important biological effects. Section title: GSK-3β Is Inhibited by Hypertrophic Stimuli Educational score: 4.177126884460449 Domain: biomedical Document type: Study Language: en To determine whether GSK-3β might play a role in the hypertrophic response to a physiologically relevant stimulus in vivo, rats were subjected to aortic banding or sham banding as described , and GSK-3β immune complex kinase assays were performed on myocardial lysates at various times after banding. We found that GSK-3β activity was significantly reduced in response to pressure overload and inhibition persisted for 24 h . Taken together, these data are consistent with a possible role for GSK-3β in the hypertrophic response of cardiomyocytes in culture and in pressure overload-induced hypertrophy in vivo. Section title: Mechanism of Inhibition of GSK-3β by Hypertrophic Stimuli Educational score: 4.494082450866699 Domain: biomedical Document type: Study Language: en Several mechanisms of inhibition of GSK-3 have been described. Insulin and IGF-1 inactivate GSK-3 via phosphorylation of a serine residue in the NH 2 -terminal region of the kinase . This is mediated by a PI3-K-dependent kinase, possibly either PKB/Akt or the integrin-linked kinase . Other mechanisms, including one mediated by Ca 2+ and a Ca 2+ /calmodulin-dependent protein kinase kinase , and an ill-defined mechanism employed by the Wnt/wingless pathway, possibly involving protein kinase C , also inactivate GSK-3, but these pathways are not PI3-K-dependent and do not result in phosphorylation of Ser 9. Therefore, we determined the mechanism of inhibition of GSK-3β by hypertrophic stimuli. We found that ET-1 induced pronounced phosphorylation of GSK-3β on Ser 9, and that this phosphorylation was blocked by the PI3-K inhibitors, wortmannin or LY294002 . The effect of the PI3-K inhibition on Ser 9 phosphorylation exactly correlated with the effect on GSK-3β kinase activity, since wortmannin prevented the ET-1–induced inactivation of GSK-3β . These data strongly suggest that the ET-1–induced inhibition of GSK-3β is mediated via phosphorylation of Ser 9 by a PI3-K-dependent kinase. Section title: Mechanism of Inhibition of GSK-3β by Hypertrophic Stimuli Educational score: 4.171180725097656 Domain: biomedical Document type: Study Language: en To confirm that phosphorylation of Ser 9 was the mechanism of inactivation of GSK-3β, we created an adenovirus encoding GSK-3β with a Ser 9 to Ala mutation that renders the kinase resistant to inhibition by Ser 9 kinases, and then determined whether GSK-3βA9 was inhibited in response to ET-1. Cells were transduced with AdGSK-3βA9, AdGFP (as a control), or no virus. 36 h later, cells were exposed to ET-1 for 40 min, followed by immunoprecipitation with anti-HA mAb and immune complex kinase assay. In contrast to endogenous GSK-3β , GSK-3βA9 was not inhibited by ET-1 . These data confirmed a critical role for Ser 9 phosphorylation in the ET-1–induced inactivation of GSK-3β. In addition, they demonstrated that we could employ the virus to study the role of GSK-3 inhibition in the hypertrophic response to ET-1. Section title: Inhibitors of GSK-3 Induce Hypertrophic Responses in Cardiac Myocytes Educational score: 4.29798698425293 Domain: biomedical Document type: Study Language: en If the ET-1–induced inhibition of GSK-3 were mediated by activation of the PI3-K/PKB pathway, and if this inhibition were important in the hypertrophic response, then directly activating the PI3-K/PKB pathway should induce hypertrophic responses. To initially explore this question, we induced inhibition of GSK-3 by adenovirus-mediated gene transfer of either the constitutively active mutant of PKB/Akt (AdPKB/Akt), or the constitutively active PI3-K (AdBD110), which produces persistent activation of endogenous PKB/Akt , and determined their effects on protein accumulation in neonatal myocytes. Adenoviral gene transfer of either BD110 or activated PKB/Akt significantly increased protein accumulation, demonstrating that activation of the PI3-kinase/Akt pathway is sufficient to induce hypertrophic responses . In addition, we employed LiCl, which has been used to directly inhibit GSK-3 in many contexts (see below). Even in the absence of a hypertrophic stimulus, LiCl was sufficient to induce protein accumulation in cardiac myocytes . Whereas BD110, PKB/Akt, and LiCl have effects in cells in addition to inhibiting GSK-3, the data are consistent with a possible role for the PI3-K/Akt/GSK-3 pathway in the hypertrophic response. Section title: Expression of GSK-3βA9. Educational score: 4.2112345695495605 Domain: biomedical Document type: Study Language: en To address our primary question, whether inactivation of GSK-3β is necessary for the hypertrophic response to physiologically relevant stimuli, we expressed GSK-3βA9 in neonatal cardiomyocytes via adenoviral gene transfer and determined its effect on the hypertrophic response. GSK-3βA9 was readily expressed in culture using multiplicities of infection (MOIs) of 50–125 pfu/cell. At an MOI of 100 pfu/cell, expression levels of GSK-3βA9 were only slightly greater than levels of endogenous GSK-3β , and transduction efficiency was ∼85% (not shown). It is important to note that the expression levels that we achieved at an MOI of 100 pfu/cell did not produce marked elevations in total cellular GSK-3β activity. When we measured total GSK-3β activity in cells infected with AdGSK-3βA9, activity was increased only 1.8 ± 0.2-fold over cells infected with control virus (AdGFP). This level of activity is in distinct contrast to activity levels seen after gene transfer or transfection of constitutively active kinases that are normally off in the resting cell. In these cells, total kinase activity is often many fold greater than endogenous activity. Section title: Effect of GSK-3 β A9 on Hypertrophic Responses. Educational score: 4.124980926513672 Domain: biomedical Document type: Study Language: en If inhibition of GSK-3β is important in the hypertrophic response of cardiomyocytes, preventing inactivation of GSK-3 should block the hypertrophic response. Therefore, we determined whether gene transfer of GSK-3βA9 blocked the hypertrophic response to ET-1 and PE. For these studies, we took advantage of the fact that we made AdGSK-3βA9 with the pAdTRACK/pAdEASY system , which allows expression of GFP from a separate promoter in the virus. We identified transduced cells by their green fluorescence, and could then compare hypertrophic responses in cells that were successfully transduced with those that were not transduced. To summarize the data presented below, we found that expression of GSK-3βA9 significantly inhibited ET-1– and PE-induced hypertrophy and, importantly, that LiCl, which inhibits endogenous GSK-3 and GSK-3βA9, reversed the effects of gene transfer of GSK-3βA9. Section title: Effect of GSK-3 β A9 on Hypertrophic Responses. Educational score: 4.229623794555664 Domain: biomedical Document type: Study Language: en Enhanced organization of sarcomeres, which characterizes the hypertrophic response of neonatal cardiomyocytes, was markedly inhibited by expression of GSK-3βA9 . However, in myocytes expressing GSK-3βA9 that were also treated with LiCl (10 mM), ET-1– and PE-induced sarcomere organization was restored . Of note, treatment of myocytes with LiCl alone, in the absence of ET-1 or PE, induced only moderate sarcomere organization , suggesting that inhibition of GSK-3β is necessary, but is not sufficient for the full expression of this relatively complex component of the hypertrophic response that requires the coordinate expression of a number of genes. Section title: Effect of GSK-3 β A9 on Hypertrophic Responses. Educational score: 4.168491840362549 Domain: biomedical Document type: Study Language: en Next we examined the role of the GSK-3β pathway in the induction of ANF, one of the marker genes that is upregulated in response to most hypertrophic stimuli. We found that ET-1 induced expression of ANF , but that gene transfer of GSK-3βA9 markedly inhibited the ET-1–induced expression of ANF . Again, LiCl completely reversed the effects of expression of GSK-3βA9. Although LiCl alone was not sufficient to induce full sarcomere organization , LiCl was sufficient, even in the presence of GSK-3βA9, to induce ANF expression, suggesting that inhibition of GSK-3β is not only necessary, but may also be sufficient for this component of the response . Section title: Effect of GSK-3 β A9 on Hypertrophic Responses. Educational score: 4.106477737426758 Domain: biomedical Document type: Study Language: en To determine whether inhibition of GSK-3β is necessary for the enhanced protein accumulation that is characteristic of the hypertrophic response, we measured ET-1–induced [ 3 H]-leucine incorporation in cells transduced with AdGSK-3βA9. Expression of GSK-3βA9 significantly reduced ET-1–induced [ 3 H]-leucine incorporation . Section title: Mechanisms of Action of GSK-3β Educational score: 4.220718860626221 Domain: biomedical Document type: Study Language: en The hypertrophic response is an enormously complex response that is regulated by multiple transcription factors acting on multiple genes. This complexity makes it difficult to identify critical roles for individual transcription factors. However, several lines of evidence, in addition to the findings described above with the transgenic mouse expressing an activated mutant of NF-ATc4, suggest NF-ATs play a role in the response. In neonatal rat cardiomyocytes in culture, cyclosporin A blocks ANF induction by angiotensin II and PE , and by ET-1 (data not shown). These data confirm that calcineurin is critical to this component of the response and are compatible with a role for one or more NF-ATs in ANF induction. Furthermore, adenovirus-mediated gene transfer of activated NF-ATc4 , in the absence of hypertrophic stimuli, induces ANF expression and sarcomere organization (data not shown). Section title: Mechanisms of Action of GSK-3β Educational score: 4.250330924987793 Domain: biomedical Document type: Study Language: en We found that ET-1 induced marked nuclear translocation of an endogenous NF-AT of molecular mass ∼95 kD . Specific antibodies to the various NF-ATs are not adequate for use in the rat. However, of the three NF-ATs expressed in the heart, NF-ATc1 (NF-ATc), NF-ATc3 (NF-AT4/x), and NF-ATc4 (NF-AT3; J. Molkentin, manuscript in preparation), this molecular mass is most compatible with NF-ATc1. NF-AT first appeared in the nucleus at ∼30 min after ET-1 , corresponding to the time of maximal inhibition of GSK-3β activity . Nuclear NF-AT levels declined after 120 min, a time when GSK-3β activity was returning toward normal . Thus, the time courses of endogenous NF-AT nuclear localization and GSK-3β activity are compatible with a role for GSK-3β in regulating nuclear/cytoplasmic partitioning of NF-AT. Section title: Mechanisms of Action of GSK-3β Educational score: 4.202406883239746 Domain: biomedical Document type: Study Language: en Prior studies examining the role of GSK-3β in the nuclear export of NF-ATs have employed overexpression of GSK-3β. In these studies, GSK-3β is found in the nucleus and induces export of NF-ATs . However, it is not clear whether endogenous GSK-3β also translocates to the nucleus in a stimulus-dependent manner. We found that in the unstimulated cardiomyocyte, little GSK-3β is nuclear localized . ET-1 induced a pronounced translocation of GSK-3β to the nucleus . This translocation was evident as early as 30 min and persisted until after 90 min. These data confirm that endogenous GSK-3β translocates to the nucleus in response to hypertrophic stimuli, colocalizing with its putative target, NF-AT. Section title: Mechanisms of Action of GSK-3β Educational score: 4.466760635375977 Domain: biomedical Document type: Study Language: en Thus, the time courses of GSK-3β activity and nuclear localization, and the time course of NF-AT nuclear localization, were consistent with a role for GSK-3β in regulating NF-AT nuclear/cytoplasmic partitioning, following hypertrophic stimuli. To directly address whether GSK-3β modulated NF-AT activity in response to hypertrophic stimuli, we examined the effects of expressing GSK-3βA9 on the nuclear/cytoplasmic partitioning of NF-AT in cardiomyocytes exposed to ET-1. As noted above, NF-AT first appeared in the nucleus at ∼30 min after ET-1 in control and AdGFP-infected cells . Expression of GSK-3βA9 significantly delayed the appearance of NF-AT in the nucleus, compatible with retardation of entry by cytosolic GSK-3β . By 60 min, however, the amount of intranuclear NF-AT was equivalent in cells infected with AdGSK-3βA9 and AdGFP , suggesting the inhibitory effect of GSK-3βA9 was overcome by activated calcineurin. A significant fraction of the NF-AT remained intranuclear at 120 min after ET-1 in control and AdGFP-infected cells , but expression of GSK-3βA9 accelerated its export from the nucleus such that little remained intranuclear at 120 min . Thus, expression of GSK-3βA9 significantly reduced the duration of NF-AT nuclear localization, both by retarding entry into and enhancing exit from the nucleus. These data suggest GSK-3β modulates the hypertrophic response of cardiac myocytes in part by regulating the nuclear/cytoplasmic partitioning of NF-AT. Section title: Discussion Educational score: 4.301590442657471 Domain: biomedical Document type: Study Language: en In this manuscript, we identify a novel function of GSK-3β. We demonstrate that GSK-3β plays a critical role in the hypertrophic response of cardiomyocytes by showing that GSK-3β kinase activity is inhibited by hypertrophic stimuli both in vitro and in vivo, and that inhibition of GSK-3β activity is essential for all three components of the hypertrophic response of cardiomyocytes enhanced protein accumulation and sarcomere organization, and reexpression of fetal genes. Furthermore, we show that GSK-3β likely limits the hypertrophic response, at least in part, by negatively regulating ET-1–induced nuclear localization of NF-AT. Section title: Discussion Educational score: 4.085503101348877 Domain: biomedical Document type: Review Language: en Numerous signaling molecules have been identified, which, when activated, transduce prohypertrophic signals, and some, such as the SAPK/JNKs, calcineurin, and the α subunit of Gq heterotrimeric G proteins, have been shown to be essential for the development of cardiac hypertrophy in vivo to physiologically relevant stimuli . To date, few studies have identified pathways that negatively regulate the hypertrophic response, yet these may be equally attractive targets for therapies designed to block the progression of hypertrophy and the transition to heart failure. Section title: Discussion Educational score: 4.247636795043945 Domain: biomedical Document type: Study Language: en GSK-3 is normally active in unstimulated cells, and is inactivated in response to growth factors, especially insulin and IGF-1, which activate the PI3-K pathway . We now demonstrate that GSK-3β is potently inhibited by hypertrophic agonists with receptors coupled to heterotrimeric G proteins of the Gq class. Morisco et al. 2000 recently showed that stimulation of β-adrenergic receptors, which are coupled to Gs proteins, also inhibited GSK-3β, suggesting that inhibition of this kinase may be a generalized phenomenon of hypertrophic signaling in cells in culture. We also showed that GSK-3β is markedly inhibited in the intact animal exposed to pressure overload induced by aortic banding, a stress that mimics severe valvular or hypertensive disease. Section title: Discussion Educational score: 4.181814670562744 Domain: biomedical Document type: Study Language: en Recently, Rezvani and Liew 2000 reported that human hypertrophy is associated with elevated levels of β-catenin protein, a transcriptional activator involved in embryonic development and tumorigenesis, not previously known to play a role in hypertrophy. Since GSK-3β, when active, phosphorylates β-catenin, targeting it for ubiquitination and degradation, inhibition of GSK-3β may account, in part, for the increased expression of β-catenin. We believe β-catenin may be an additional target of GSK-3β that is involved in the hypertrophic response and studies evaluating this hypothesis are in progress. Section title: Discussion Educational score: 4.2861833572387695 Domain: biomedical Document type: Study Language: en GSK-3β activity can be inhibited by a number of mechanisms, but our findings suggest that hypertrophic agonists utilize phosphorylation of Ser 9 by a PI3-K–dependent protein kinase. This phosphorylation can be catalyzed by at least two protein kinases, PKB/Akt and ILK, both of which are activated by phosphatidylinositol 3 phosphates . It is unclear which of the two is the physiologically relevant kinase that inactivates GSK-3 in response to hypertrophic stimuli. The data confirm that the inhibition of GSK-3β is not mediated via recruitment of the Wnt pathway by hypertrophic agonists since Wnt-induced inhibition of GSK-3β appears to be PI3-K-independent . Section title: Discussion Educational score: 4.419844627380371 Domain: biomedical Document type: Study Language: en Ultimately, cytosolic signaling pathways must modulate the activity of various transcription factors to direct the complex reprogramming of gene expression required to express the full hypertrophic phenotype. Our data suggest that one critical target of GSK-3 that likely plays a role in the hypertrophic response of cardiac myocytes is a member of the NF-AT family of transcription factors. NF-AT family members have been implicated in both cardiac hypertrophy and IGF-1–induced skeletal myocyte hypertrophy . NF-AT activity is largely controlled at the level of nuclear localization since the cytoplasmic forms are competent for both DNA binding and transcriptional activation . NF-ATs appear to be held in the cytoplasm by the masking of two nuclear localization signals by the intramolecular interaction of several phosphorylated serine residues with a second serine-rich region . Dephosphorylation of the serine residues by calcineurin exposes the nuclear localization signals leading to nuclear import. Section title: Discussion Educational score: 4.395697116851807 Domain: biomedical Document type: Study Language: en Several protein kinases in addition to GSK-3β have been implicated in the nuclear export and/or cytosolic anchoring of NF-AT family members. Three MAP kinases (SAPKs, ERKs, and p38), protein kinase A, casein kinase Iα in cooperation with MAP kinase/ERK kinase-1, and casein kinase 2 have been reported to phosphorylate critical residues in the SerPro-rich domain of one or more NF-ATs, blocking nuclear import and/or enhancing export . The role of these kinases has been examined primarily in T cells, hippocampal neurons, or transformed cell lines commonly used in studies using transfection (e.g., COS cells), and few studies have focused on the regulation of endogenous NF-ATs. It appears from these studies that the relevant kinase(s) regulating NF-AT nuclear/cytosolic partitioning depends on the NF-AT, the cell type, and, possibly, the stimulus. For our purposes, Beals et al. 1997b have clearly shown that GSK-3 regulates nuclear export of NF-ATc1, the NF-AT we believe to be most highly expressed in neonatal cardiomyocytes. More recently, Porter et al. 2000 proposed that casein kinase 2, which is constitutively nuclear localized, may serve as a priming kinase that phosphorylates residues of NF-ATc1, allowing more efficient phosphorylation by GSK-3β. They noted however, that GSK-3β could, itself, also serve as the priming kinase. Section title: Discussion Educational score: 4.23875093460083 Domain: biomedical Document type: Study Language: en Although the role of the other putative NF-AT kinases in cardiomyocytes is not clear, out data confirm a critical role for GSK-3β. We found that expression of GSK-3βA9 delayed the initial ET-1–induced import of NF-AT into the nucleus, and, subsequently, enhanced nuclear export, resulting in a markedly reduced duration of NF-AT nuclear localization. These data are compatible with an important role for GSK-3β in nuclear/cytoplasmic partitioning of NF-AT after stimulation by hypertrophic agonists. In addition, we found that treatment of cells with LiCl in the absence of ET-1 induced marked translocation of NF-AT to the nucleus (data not shown), suggesting that GSK-3β may not only retard stimulus-induced entry of NF-AT into the nucleus, but also may be the dominant mechanism for maintaining NF-AT in the cytosol in the unstimulated or resting cardiac myocyte. Section title: Discussion Educational score: 4.222992897033691 Domain: biomedical Document type: Study Language: en An alternative approach to studying the role of GSK-3β (and NF-ATs) in the hypertrophic response would be to create mice deleted for one or more of these genes. However, the GSK-3β deletion is embryonic lethal. Furthermore, the molecular mass of the dominant NF-AT in neonatal rat cardiomyocytes is most compatible with NF-ATc1, and mouse embryos lacking NF-ATc1 die at day 11 from congestive heart failure due to improper formation of the cardiac valves . Mice deleted for the other NF-ATs expressed in the heart are viable, but since cardiac myocytes contain more than one NF-AT, deletion of one may be compensated for by the others. Cross-breeding viable knockouts could clarify the role of the NF-ATs in hypertrophy, but increases the probability of embryonic lethality. Section title: Discussion Educational score: 4.688225746154785 Domain: biomedical Document type: Study Language: en Although we believe NF-ATs are important in the hypertrophic response, several pieces of evidence suggest that inhibition of NF-ATs is not the only mechanism by which GSK-3 attenuates the hypertrophic response. For example, in contrast to the marked hypertrophy seen when an activated mutant of NF-ATc4, NF-ATc4Δ317, is expressed in the hearts of transgenic mice , expression of NF-ATc4Δ317 in neonatal rat cardiomyocytes in culture induces a definite, but modest hypertrophic response. Therefore, either NF-ATs are necessary, but not sufficient for the full expression of the hypertrophic phenotype, or, more likely, there are additional targets activated by ET-1 and PE (and inhibited by GSK-3β) that play a role in the hypertrophic response. In support of the latter possibility, preliminary experiments suggest that expression of NF-ATc4Δ317 partially, but not completely, overcomes the inhibitory effect of GSK-3βA9 on the hypertrophic response. In this regard, GSK-3 has another target, c-Jun, which has been implicated in the hypertrophic response. c-Jun was the first transcription factor identified as a substrate of GSK-3 . GSK-3 phosphorylates several residues near the DNA binding domain of c-Jun, and this negatively regulates the DNA binding activity of the transcription factor. We have previously shown that the SAPKs/JNKs, which increase the transcriptional activating activity of c-Jun, are necessary for the hypertrophic response of cardiomyocytes both in vitro and in vivo , and, given the number of hypertrophic response genes that appear to be regulated, at least in part, by AP-1, a heterodimer of c-Jun and c-Fos , it is likely that c-Jun plays an important role in hypertrophy. Therefore, inhibition of c-Jun and, as a result, AP-1, may be a mechanism in addition to inhibition of NF-ATs, whereby GSK-3 signals to blunt the hypertrophic response. In addition to the effects of AP-1 itself on gene expression, AP-1 is also required for efficient binding of NF-ATs to DNA , suggesting an additional mechanism whereby GSK-3β, via inactivation of AP-1, could block NF-AT–dependent gene expression. Section title: Discussion Educational score: 4.304520606994629 Domain: biomedical Document type: Study Language: en We employed LiCl to inhibit activity of GSK-3βA9 and endogenous GSK-3. LiCl has been employed to this end to study the role of GSK-3 in embryonic development in organisms as diverse as Dictyostelium , Xenopus laevis , sea urchins, and zebrafish, and to study numerous processes in mammalian cells . LiCl has no known effects on other protein kinases, but does have effects on other enzymes, including inhibiting the inositol monophosphatase and adenylyl cyclase. Whereas LiCl may have other less well-described ancillary effects, the direct reversal by LiCl of the effects of GSK-3βA9 on sarcomere organization, ANF expression, and protein synthesis suggests Li + mediated its actions primarily via inhibition of GSK-3. An alternative strategy would have been to use kinase-inactive GSK-3β. In our hands and others, kinase-inactive GSK-3β is not an adequate dominant inhibitory mutant in mammalian cells , and needs to be expressed at high levels to function as an inhibitor of GSK-3 signaling. This may lead to nonspecific effects and requires infection at high MOIs that can be toxic to cardiac myocytes. Whereas this might not adversely affect activity of reporter constructs , it does disrupt the complex and highly coordinated responses required to produce the hypertrophic phenotype. Section title: Discussion Educational score: 4.227816581726074 Domain: biomedical Document type: Study Language: en In summary, we have identified a novel role for GSK-3 as a critical negative modulator of cardiomyocyte hypertrophy. Our data suggest a model whereby GSK-3 directly antagonizes the prohypertrophic effects of activated calcineurin by inhibiting activity of one of its primary targets, NF-AT. The elucidation of a central role for GSK-3 in hypertrophy identifies not only GSK-3 and its downstream effectors, but also a large number of signaling molecules upstream of GSK-3, including PI3-Ks, polyphosphatidylinositide-dependent protein kinases (PDKs), PKB/Akt, and ILK, as potential therapeutic targets for drugs to alter the natural history of hypertrophy and heart failure. | Study | biomedical | en | 0.999998 |
0003137 | Section title: Introduction Educational score: 4.197197914123535 Domain: biomedical Document type: Other Language: en A particular shape and a well-defined polarity are characteristic of many cell types, as illustrated by the extended morphology of differentiated nerve cells or the asymmetric organization of polarized epithelia. The components of the cytoskeleton play an essential role in establishing and maintaining the morphology of interphase eukaryotic cells . The microtubule cytoskeleton participates in this general function by providing an intracellular framework for vesicle transport, by contributing to the internal organization of the cell, and by aiding in the organization and function of the cell cortex. Section title: Introduction Educational score: 4.213120937347412 Domain: biomedical Document type: Study Language: en The interphase microtubule network is generally dynamic, with the addition and loss of tubulin subunits occurring mostly at one microtubule end, the “plus” end , whereas a microtubule's “minus” end is less dynamic and is usually associated with the centrosome or “spindle pole body” (SPB), as it is termed in yeasts. The stability and length of microtubules are governed primarily by the rates of subunit addition and loss and by the frequency of transitions between phases of growth and shrinkage. Section title: Introduction Educational score: 4.4774298667907715 Domain: biomedical Document type: Study Language: en Many proteins affect microtubule stability and length, including microtubule-associated proteins (MAPs), kinesin-like proteins (klps), and microtubule-severing enzymes . The klps comprise a superfamily of microtubule-based motor enzymes found in all eukaryotes; they share a conserved motor domain that is responsible for translocation of the enzyme along microtubules. Kinesin itself, the founding member of the superfamily, moves toward the microtubule plus end through the interactions of its NH 2 -terminal motor domain , whereas the COOH-terminal or tail region is thought to interact with cargo . Additional functions proposed for kinesins include the production of opposing inward and outward forces on the mitotic spindle and the destabilization of microtubules . Section title: Introduction Educational score: 4.209996223449707 Domain: biomedical Document type: Study Language: en The unicellular fission yeast Schizosaccharomyces pombe offers a useful model system in which to study the molecular mechanisms that control eukaryotic cellular morphology because it is amenable to detailed morphological, genetic, and molecular analyses. After cell division, growth begins only at the old end of the cell. Early in G2, growth is also initiated from the new end of the cell , and it continues from both tips until the cell enters mitosis and stops further elongation. Both cytoskeletal and regulatory components that control these events have been identified and characterized . Section title: Introduction Educational score: 4.406361103057861 Domain: biomedical Document type: Study Language: en Although the actin cytoskeleton appears to be needed for the actual deposition of growth material , the cytoplasmic microtubule network has been shown in several studies to play a role in defining the site of growth extension . Treatment with a drug that destabilizes microtubules or incubation of temperature-sensitive tubulin mutants at their restrictive temperature results in the formation of branched cells . Genetic screens for mutants with altered polarity have identified mutant alleles of the tubulin genes and tubulin-folding cofactors . Moreover, many mutant strains with altered morphology contain abnormal arrays of cytoplasmic microtubules . Cytoplasmic microtubules are also important for the localization of at least two cell tip–specific proteins, Tea1p and Pom1p . Mutations in these genes result in defects in cell morphology and/or bipolar growth . Section title: Introduction Educational score: 4.255669593811035 Domain: biomedical Document type: Study Language: en We have sought cellular components that work in conjunction with the microtubule cytoskeleton to establish and maintain cellular polarity. Through the molecular identification of tea2 + , a gene identified in a screen for morphology mutants and shown to be required for normal behavior of the cell's growing tip , we have demonstrated that a klp is required for normal cellular morphology. As described for the mutant alleles of this gene , the deletion results in defects in the cytoplasmic microtubule array and in cell shape. During the transition out of stationary phase growth, both tea2 Δ and tea2-1 cells often establish an ectopic growth site resulting in the formation of T-shaped cells. Likewise, long cells are particularly sensitive to the loss of tea2 + . Tea2p localizes to cell tips and is also often seen as dots coincident with cytoplasmic microtubule ends. Section title: Strains and Cell Culture Educational score: 2.957113742828369 Domain: biomedical Document type: Study Language: en All strains used are shown in Table . Strains were constructed and maintained as described in Moreno et al., 1991. Cultures were grown in rich medium containing yeast extract plus supplements (YES) or a Edinburgh minimal medium . Section title: PCR Screen for klps Educational score: 4.263922691345215 Domain: biomedical Document type: Study Language: en Primers to conserved portions of the kinesin motor domain were used to amplify genomic DNA. Genomic DNA was prepared as described in Moreno et al., 1991. The 5′ primers were TAC/TGGNCAA/GACNGG (corresponding toYGQTGSGK) or TAC/TGGNCAA/GACNGG (corresponding to YGQTGTGK), and the 3′ primer was C/TTCNG/CA/TNCCNG (corresponding to DLAGSE). PCR amplifications were performed on three different samples of DNA: (a) genomic DNA from wild-type cells; (b) genomic DNA from wild-type cells digested with XbaI, which restricts within pkl1 and klp2 + ; and (c) genomic DNA from klp2 Δ cells. Reaction conditions were 30 cycles of 95°C for 30 s, 40 or 45°C for 30 s, and 72°C for 30 s, generally followed by a single 5-min incubation at 72°C. PCR products were subcloned and analyzed by colony PCR. To identify clones that represent previously identified klps, colony PCR products were digested with enzymes that cut within cut7 + , pkl1 + , and klp2 + . Potentially novel products were size-fractionated on low melting point (LMP) agarose to purify the products from PCR primers, and then were sequenced directly in the gel using vector-specific primers. A total of 163 clones with inserts were analyzed, and two novel kinesin-like genes, designated klp3 + and klp4 + , were identified; klp3 + has recently been described by others , and will be further characterized elsewhere. klp4 + was shown by the work described below to be identical to tea2 + , so that name will be used henceforth. Section title: Cloning of tea2 + by Mapping and Complementation Educational score: 4.240900039672852 Domain: biomedical Document type: Study Language: en tea2 + was cloned by positional mapping and complementation . tea2-1 was mapped to within 0.1 centimorgan (cM) of orb2ts . The loci were shown to be separate genes by complementation in a tea2-1 / orb2ts heterozygous diploid. The XhoI-BstEII fragment of pak1 + was used to probe the Cold Spring Harbor and Imperial Cancer Research Fund cosmid libraries, provided by The Sanger Centre (Cambridge, England). Hybridizing cosmids were tagged with the his7 + selectable marker and retransformed into a tea2-1 his7-36 strain; one cosmid, c1604, was able to rescue the mutant phenotype. This cosmid was used to prepare a SauIIIA partial library in pIRT2 , which was transformed into tea2-1 leu1-32 . Rescuing clones were selected by replica plating to 36°C and examining the cells over the next 2–3 h. tea2-1 cells normally form a high percentage of T shapes upon regrowth from nutrient starvation. Plasmids were recovered from clones that did not form T shapes under these conditions. Four overlapping clones were obtained, and clone 14T was used for further analyses. Section title: Cloning of tea2 + by Mapping and Complementation Educational score: 4.160114288330078 Domain: biomedical Document type: Study Language: en To show that 14T contained tea2 + and not an extragenic suppressor, the clone was integrated into the genome by homologous recombination, and this strain was crossed to leu1-32 and tea2-1 leu1-32 strains. 14T was mapped to within 0.3 cM of tea2-1 and the rescuing activity to within 0.5 cM of LEU2 , demonstrating that the tea2-1 rescuing activity is linked to 14T and that the site of integration is very close to tea2-1 . Section title: Molecular Characterization of the tea2 + Region Educational score: 4.193696022033691 Domain: biomedical Document type: Study Language: en Unless otherwise specified, molecular biology techniques are essentially as described in Sambrook et al., 1989, and sequencing was performed at the University of Colorado automated DNA sequencing facility. A genomic library, provided by A. Carr , was screened using the PCR-generated clone of tea2 + . Of 20,000 clones screened, two unique clones were identified: 11B, which contained a 4.6-kb insert, and a second clone that contained only part of the tea2 + open reading frame (ORF). Subclones of 11B were constructed in pSPORT : the 4.6-kb BamHI (from vector multicloning site) to HindIII fragment was cloned into the BamHI/HindIII sites of pSPORT, creating 11–24; the 2.8-kb BglII fragment spanning the motor domain was inserted into the BamHI site of pSPORT, creating 8–24; and the 1.4-kb AvaI-HindIII fragment was inserted into the AvaI/HindIII sites of pSPORT, creating 4–24. Subclones of 11B were sequenced. Section title: Molecular Characterization of the tea2 + Region Educational score: 4.143623352050781 Domain: biomedical Document type: Study Language: en To isolate DNA further 3′ of the tea2 + ORF, the 11-kb XhoI fragment extending 3′ from the tea2 + ORF was cloned by constructing and screening an XhoI genomic library of 11-kb XhoI genomic fragments cloned into pBluescript. This cloned region, cloneX/X, was digested with XhoI and BamHI, and the 4.3-kb XhoI-BamHI fragment was cloned into the XhoI/BamHI sites of pBluescript. This clone, X/B, was used for sequencing and for Northern blot analysis. Section title: Molecular Characterization of the tea2 + Region Educational score: 4.1234965324401855 Domain: biomedical Document type: Study Language: en A 1.4-kb AvaI/ HindIII fragment containing the 3′ half of tea2 + ORF was used to screen a cDNA library (provided by F. LaCroute, Centre de Genetique Moleculare Gif sur Yvette, France). Approximately 60,000 clones were screened, and one cDNA was identified. The cDNA was excised using NotI and were inserted into the NotI site of pSPORT to construct pSPORT tea2 + cDNA, and this clone was sequenced. Section title: Molecular Characterization of the tea2 + Region Educational score: 4.101587772369385 Domain: biomedical Document type: Study Language: en Total RNA was isolated from wild-type S . pombe cells as described in Moreno et al., 1991. Poly(A + ) RNA was isolated using GIBCO BRL oligo(dT) cellulose columns according to the manufacturer's recommendations. Northern blot analyses were performed as described in Browning and Strome 1996 . Probes were labeled by random priming using 32 P-labeled dATP from Amersham Pharmacia Biotech or NEN Life Science Products. Section title: Molecular Characterization of the tea2 + Region Educational score: 4.046452522277832 Domain: biomedical Document type: Study Language: en Reverse transcription followed by PCR (RT-PCR) was performed using the Promega Access RT-PCR kit. Total RNA was used as template with the primer 5′-CGTAGTATATGATTGTAGCAGGTCGTC-3′ for reverse transcription and the primer combination 5′-CGTAGTATATGATTGTAGCAGGTCGTC-3′ and 5′-CTGTGACTCAGGAAACGCAACTTC-3′ for PCR. Section title: Computer-aided Sequence Analysis Educational score: 4.188765048980713 Domain: biomedical Document type: Study Language: en The BLAST program available at http://www.ncbi.nlm.nih.gov/BLAST/ was used for sequence searches. The BestFit program from the GCG sequence analysis package was used for direct sequence comparison. For phylogenetic analysis, the ∼340–amino acid (aa) motor domains of Tea2p and 42 other klps were aligned using the ClustalW program available at http://dot.imgen.bcm.tmc.edu:9331/multi-align/multi-align.html. This alignment was analyzed with the phylogenetic program PAUP version 4.0 (Sinauer Associates, Inc.), assuming maximum parsimony and using a heuristic search method with stepwise addition. 100 bootstrap replicas were performed. For coiled coil predictions, the Coils program available at http://www.ch.embnet.org/software/COILS_form. html was used . Both matrices (MTK and MTIDK) were tested, with and without the weighting option. Section title: Construction of Knockout Strain Educational score: 4.381296157836914 Domain: biomedical Document type: Study Language: en A null allele of tea2 + was constructed by single-step gene replacement protocol, replacing the tea2 + ORF with his3 + by homologous recombination. To construct the integration plasmid, clone 11–24 was digested with BbsI, blunt ended with Klenow, then digested with HindIII, and the 423-bp [BbsI]-HindIII fragment beginning 14 bp 3′ of tea2 + ORF was isolated. A SalI/SmaI fragment containing his3 + was isolated from pAFI . These two fragments were simultaneously ligated into the SalI and HindIII sites in pSPORT to create an intermediate integration construct. To place tea2 + 5′ flanking DNA into this plasmid, clone 8–24 was digested with PflmI, DNA ends were blunt ended with T4 DNA polymerase, the plasmid was further digested with KpnI, and the fragment from KpnI (in the vector multicloning site) to [PflmI], which contains the tea2 + 5′ flanking DNA, was ligated into the KpnI/SmaI sites of the intermediate integration construct. The final integration plasmid contained 1,063 bp of 5′ and 423 bp of 3′ DNA from the region flanking the tea2 + ORF placed on the 5′ and 3′ sides of his3 + , respectively. For transformation, the tea2-his3 + cassette was excised by digestion with PvuII. Diploids ( his3-D1/his3-D1, ura4-D18/ura4-D18, ade6-M210/ade6-M216, leu1-32/leu1-32 , h + /h − ) were transformed with this cassette using the PLATE method . Homologous integrants were identified by PCR and confirmed by Southern blot analysis. Section title: Identification of tea2 + and klp4 + as the Same Gene Educational score: 4.15227746963501 Domain: biomedical Document type: Study Language: en The gene initially characterized as klp4 + was found to be entirely contained within the 14T plasmid using PCR primers specific to klp4 + . Clone 14T was used to construct 5′ and 3′ deletions to further define the rescuing region . 14B and 14H are 3′ truncations with deletions extending to the BamHI and HindIII sites, respectively. 14X is a deletion with 5′ sequences removed to the XhoI site. The tea2-1 allele was sequenced by PCR amplification of tea2-1 genomic DNA using primers specific to the tea2 + region followed by sequencing of the PCR products. Section title: Inducing Cells to Exit from Stationary Phase Educational score: 4.153585910797119 Domain: biomedical Document type: Study Language: en Cells were grown in YES or EMM at 32°C until they reached stationary phase growth, generally 1 d beyond logarithmic growth in YES or 2 d after logarithmic growth in EMM. Cells were then diluted 1:10 or 1:25 in fresh medium and examined by microscopy at various times after dilution. For tea2Δ complementation tests, cells were grown to saturation in EMM with appropriate supplements. For lineage analysis, cells were grown to saturation in YES, placed on a YES agar pad (YES medium with 2% agar) on a microscope slide, and examined by differential interference contrast (DIC) microscopy using a Zeiss microscope. The slide was warmed to 32°C with an air curtain incubator (Sage Instruments) or a heatlamp. The temperature was controlled using a CN76000 microprocessor-based temperature and process controller from Omega Engineering. Images were captured using an Empix charge-coupled device camera and Metamorph software (Universal Imaging). Section title: Production of Antibodies Educational score: 4.145937919616699 Domain: biomedical Document type: Study Language: en For protein expression in Escherichia coli , a construct was made by digestion of clone 4–24 with BsmI and BbsI and generation of blunt ends with T4 DNA polymerase and Klenow. The 440-bp [BsmI-BbsI] fragment corresponding to the COOH-terminal region of Tea2p (which lacks motor sequences) was cloned into the EcoRI site of pGEXKG , which had been blunt ended with Klenow. Inclusion bodies were purified from cells expressing this construct, pGEXKG tea2 s/t, by the method of Lin and Cheng 1991 . The fusion protein was further purified by SDS-PAGE. Fusion protein was electroeluted from the gel, dialyzed against 1X PBS, and sent to Strategic Biosolutions for the immunization of two rabbits. Section title: Production of Antibodies Educational score: 4.153805732727051 Domain: biomedical Document type: Study Language: en A second fusion protein was constructed for antibody purification. The 440-bp [BsmI-BbsI] tea2 + fragment described above was cloned into the PvuII site of pRSETc (Invitrogen) and the fusion protein expressed in the BL21(DE3) E . coli strain. The fusion protein was solubilized by denaturization and then purified by chromatography on nickel columns according to the procedure recommended by Invitrogen. A column for affinity purification was made with purified fusion protein covalently cross-linked to cyanogen bromide–activated sepharose 4B (Sigma-Aldrich) as recommended by Amersham Pharmacia Biotech. The serum was purified on the column essentially as described in Harlow and Lane 1988 , except 1× PBS was substituted for 10 mM Tris (pH 7.5 and 8.8), and the unbound antibody was washed off the column with 5 bed volumes 1× PBS, 10 bed volumes 3× PBS, and 15 bed volumes 1× PBS. Section title: Immunofluorescence Microscopy Educational score: 4.1156134605407715 Domain: biomedical Document type: Study Language: en Cells were prepared for immunofluorescence staining by aldehyde or cold methanol fixation as described in Hagan and Hyams, 1988. For tubulin staining, a mouse moncolonal antibody against Drosophila α-tubulin was used (provided by M.T. Fuller, Stanford University, Stanford, CA) or tat1 with goat anti–mouse Alexa secondary antibody (Molecular Probes). Tea2p–green fluorescence protein (GFP) was visualized using a rabbit polyclonal antibody at 1:200 (a gift from Ken Sawin, Imperial Cancer Research Fund, London, UK) and Alexa 488 (Molecular Probes) as the secondary antibody. For Tea2p antibody staining, secondary antibodies were fluorescein-labeled goat anti–rabbit immunoglobulin (Jackson ImmunoResearch Laboratories). Tea1p staining was as described in Mata and Nurse, 1997. Tea2p staining was performed on methanol-fixed cells. Cells were mounted in Citifluor Mountant Media No. 0 (Ted Pella, Inc.). Section title: Immunofluorescence Microscopy Educational score: 3.276186466217041 Domain: biomedical Document type: Other Language: en Immunofluorescence microscopy was performed on a Leica DMRXA/RF4/V automated universal microscope, and images were acquired with a Cooke SensiCam high performance digital camera using the Slidebook software package (Intelligent Imaging Innovations, Inc.) or a Zeiss LSM510 Confocal microscope. In all cases, images were exported to Adobe Photoshop for figure preparation. Section title: Immunoblot Analysis Educational score: 4.189243316650391 Domain: biomedical Document type: Study Language: en pREP3X tea2 + cDNA was constructed by cloning the BamHI/SmaI 4.6-kb fragment from pSPORT tea2 + cDNA into the BamH1 and SmaI sites of pREP3X . The truncated cDNA construct was made by digesting pREP3X tea2 + cDNA with BbsI, filling in the 5′ overhang with Klenow, and then digesting with BamHI. The BamHI/[BbsI] fragment containing the entire ORF of tea2 + was then cloned into the SmaI and BamHI sites of pREP3X. tea2 Δ cells transformed with these constructs were grown in EMM with appropriate supplements and 5 μg/ml thiamine (Sigma-Aldrich), and cells were washed three times with thiamine-free medium then grown overnight in thiamine-free medium. Cells were harvested, and protein extracts prepared by vortexing cells with glass beads in sample buffer. Section title: Immunoblot Analysis Educational score: 3.1899945735931396 Domain: biomedical Document type: Study Language: en Western blot analysis was performed as described in Towbin et al. 1979 . 4% nonfat dry milk was used as a blocking agent. Blots were developed using enhanced chemiluminescence (ECL) reagents from Amersham Pharmacia Biotech. Section title: Construction of Tea2-GFP Homologous Integration Strain Educational score: 4.16079568862915 Domain: biomedical Document type: Study Language: en Tea2 was tagged with GFP at the COOH terminus as described in Bahler et al. 1998 using the forward primer 5′GAAACTAAAACTGAAATTTTGCCAGACGATCAACAGCAATCGAAAAAGGATTCTGTG-ACTCAGGAAACGCAACTTCTTTCTCGGATCCCCGGGTTAATTAA 3′ and the reverse primer 3′AATTTAAGGAGACATACAGGTT-GAATGGGTATAAAATTGTAAACAAGGTTGATGAGAGACG-CCTATAATTAAACAAGGTAGAATTCGAGCTCGTTTAAAC 3′. 1–2 μg of PCR product was transformed into ade6-M210/216 leu1-32/1-32 h + /h + cells and G418-resistant clones were selected and then sporulated. G418-resistant haploids were screened by PCR for homologous integration at the tea2 + locus. The morphology of one strain was tested upon recovery from nutrient starvation and in exponential growth. The strain was also tested for Tea1p localization and microtubule length. In all conditions tested, the Tea2p-GFP–tagged strain behaved as wild-type cells. Section title: Isolation of the Kinesin-like Gene, tea2 + , by PCR and Phenotype Rescue Educational score: 4.267438888549805 Domain: biomedical Document type: Study Language: en To understand the roles of klps in S . pombe , we carried out a PCR screen using degenerate primers to highly conserved regions in the motor domain of the kinesin superfamily. Two new S . pombe klps were identified, klp3 + and klp4 + . The PCR-generated clone for klp4 + was used to identify a genomic clone, 11B, containing the klp4 + ORF , and a fragment of this genomic region was used to identify a cDNA clone . Sequence analysis revealed that the 4583-bp cDNA contained the entire klp4 + ORF with a stop codon at the same position as predicted from the genomic sequence, as well as 2.6 kb of additional 3′ sequence that unexpectedly contained a second ORF of 658 aa . A fragment containing the downstream genomic region was cloned, clone X/X, and both this clone and clone 14T (described below) were used to sequence the genomic region corresponding to the cDNA clone . Comparison of the genomic and cDNA sequences indicated that there are no introns in klp4 + . Section title: Isolation of the Kinesin-like Gene, tea2 + , by PCR and Phenotype Rescue Educational score: 4.136662483215332 Domain: biomedical Document type: Study Language: en The PCR-generated clone also was used to map klp4 + by hybridization to a cosmid filter of the S . pombe genome to chromosome 2 between puc1 + and nda3 + . This region has since been sequenced by the S . pombe Sanger Centre genome project, and is located on cosmid c1604 with EMBL/GenBank/DDBJ accession nos. AL034433 and PID g4376084. Section title: Isolation of the Kinesin-like Gene, tea2 + , by PCR and Phenotype Rescue Educational score: 4.071043491363525 Domain: biomedical Document type: Study Language: en In an independent, parallel series of experiments, we were investigating the localization of Tea1p in tea2-1 cells and found that Tea1p was mostly delocalized from the cell tips compared with wild-type cells and was found along the microtubules and in the cytoplasm (data not shown). This result suggests that Tea2p might be required to transport Tea1p to the cell tips. To investigate this possibility, tea2 + was mapped by positional cloning to cosmid c1604 (described in Materials and Methods). This cosmid was subcloned, and the 14T plasmid, a subclone capable of rescuing the tea2-1 morphology defects, was used for further analyses. Section title: Isolation of the Kinesin-like Gene, tea2 + , by PCR and Phenotype Rescue Educational score: 4.278570175170898 Domain: biomedical Document type: Study Language: en The similarity of the phenotypes of the knockout of klp4 + (described below) and mutant alleles of tea2 + , together with the mapping data described above and in Materials and Methods, suggested that these two independently identified genes might be the same genetic locus. To explore this possibility, three sets of PCR primers covering the klp4 + region were used to amplify DNA from the 14T plasmid, and all three gave bands of the expected size, indicating that the 14T plasmid contained the entire klp4 + ORF. Transformation of the 14T plasmid into a strain deleted for klp4 + (described below) resulted in rescue of the klp4 Δ phenotype ( Table ), and the plasmids 14B and 14H also rescued both the klp4 Δ phenotype ( Table ) and the tea2-1 phenotype. However, neither the deletion nor the tea2-1 mutant was rescued by the 5′ truncation construct, 14X, that lacks 896 bp of the klp4 + ORF but leaves the downstream ORF intact . The 14T plasmid was also integrated into the genome, and the site of integration was genetically mapped to the tea2 + locus (described in Materials and Methods). In addition, the region corresponding to the klp4 + ORF was sequenced in DNA isolated from tea2-1 cells and shown to have a serine to phenylalanine transition at aa 384. This serine residue is in the motor domain and is a highly conserved aa found in nearly all klps. Section title: Isolation of the Kinesin-like Gene, tea2 + , by PCR and Phenotype Rescue Educational score: 3.066655397415161 Domain: biomedical Document type: Study Language: en These results establish that tea2 + and klp4 + encode the same gene; from this point on, the gene will be referred to as tea2 + and its protein product as Tea2p. Section title: Characterization of the tea2 + Transcript and Protein Product Educational score: 4.339448928833008 Domain: biomedical Document type: Study Language: en Sequence analysis of the genomic and cDNA clones indicated that tea2 + potentially encodes a 628-aa protein that is expressed from a transcript of at least 4.6 kb and that contains 2.6 kb of 3′ sequence. This 3′ region contains a second ORF of 658 aa . Because this is an unusual structure for an S . pombe gene, we sought additional evidence for the gene structure of tea2 + . The region corresponding to the tea2 + ORF hybridized to a ∼5-kb transcript on Northern blots . Northern blot analyses using probes further 3′ indicate that the ∼5-kb tea2 + transcript extends in this direction, and that a second transcript of 2.5 kb is present in this region . This smaller transcript presumably codes for the ORF in this region that is predicted from the genomic sequence. Section title: Characterization of the tea2 + Transcript and Protein Product Educational score: 4.1702423095703125 Domain: biomedical Document type: Study Language: en The junction between the two ORFs was confirmed by RT-PCR performed on RNA from wild-type cells. Primer B at the predicted 5′ end of the downstream gene was used for the reverse transcriptase reaction, and this primer in combination with primer A corresponding to the 3′ end of the tea2 + ORF was used for PCR. An RT-PCR product of 315 nucleotides was produced, indicating that this region is uninterrupted by introns (not shown). Section title: Characterization of the tea2 + Transcript and Protein Product Educational score: 4.218271255493164 Domain: biomedical Document type: Study Language: en Analysis of the protein product further supports the proposed genomic structure of the region. Affinity-purified antibodies generated to the COOH-terminal region of Tea2p reacted with an ∼70-kD protein in wild-type cells, which was absent in cells deleted for the tea2 + ORF . Furthermore, tea2Δ cells expressing just the tea2 + ORF or the entire tea2 + cDNA under the control of the inducible nmt + promoter produced a protein of the expected size for Tea2p . Section title: Characterization of the tea2 + Transcript and Protein Product Educational score: 3.889214038848877 Domain: biomedical Document type: Study Language: en Finally, the 2.6-kb 3′ region of the tea2 + transcript is not essential for rescue of the mutant phenotype. A multicopy plasmid containing the tea2 + ORF, 14H, rescued the phenotype of both tea2Δ and tea2-1 cells . Section title: Sequence Comparison Analysis Educational score: 4.496944427490234 Domain: biomedical Document type: Study Language: en Sequence searches using the motor domain of Tea2p revealed that of all the klps that have been characterized (beyond mere identification in a genome project), it is most similar to Saccharomyces cerevisiae Kip2p. Direct comparison of the motor domains, using the BestFit program from the GCG sequence analysis package, demonstrated that Tea2p and Kip2p are 58% similar and 51% identical over 332 aa. The motor domains of both proteins lie roughly in the middle of the proteins: the Kip2p motor domain extends from residues 97 to 500 within the 706-aa protein, and the Tea2p motor domain runs from residues 129 to 467 within the 628-aa protein. Outside the motor domain, the sequences are 35% similar and 26% identical over an 83-aa stretch in the NH 2 -terminal region and 46% similar and 32% identical over an 87-aa stretch in the COOH-terminal region . In the COOH terminus, Tea2p is predicted to contain one or two coiled coil regions of 28–41 aa, depending on the matrix employed and whether the weighting option was used . Kip2p also contains regions in the COOH terminus predicted to form a coiled coil, suggesting that both these motors are capable of self-association. Section title: Sequence Comparison Analysis Educational score: 4.176340103149414 Domain: biomedical Document type: Study Language: en An alignment containing Tea2p, Kip2p, and 41 other kinesin family members was analyzed with the phylogenetic program PAUP (version 4.0), assuming maximum parsimony and using a heuristic search method with stepwise addition (described in Materials and Methods). This analysis revealed that of 100 bootstrap replicas, 93 grouped Kip2p, Tea2p, and CaKrp together . A value of >90 strongly supports a phylogenetic relationship on statistical grounds . These klps represent a new subfamily, which we will refer to as the Kip2p subfamily after its founding member. Section title: Defects in Cell Morphology and in the Microtubule Cytoskeleton Educational score: 4.1472272872924805 Domain: biomedical Document type: Study Language: en To investigate further the cellular roles of Tea2p in S . pombe , a deletion allele was constructed by replacing the tea2 + ORF with his3 + . Transformants were screened by PCR, and homologous integration was confirmed by Southern blot analysis (not shown). At 32°C, tea2 Δ cells grow at rates similar to wild-type cells. These cells were examined by DIC microscopy to see if the deletion had an effect on the morphology of the cells. Cultures of exponentially growing cells contain ∼18% ( n = 117) obviously bent cells, whereas wild-type cells were generally straight cylinders, 0% bent . At 37°C, tea2Δ cells grew more slowly than wild-type cells, and a high percentage of T shaped cells were seen in the culture (up to 9%). These defects in cell morphology are similar to the tea2-1 mutant . Section title: Defects in Cell Morphology and in the Microtubule Cytoskeleton Educational score: 4.141900062561035 Domain: biomedical Document type: Study Language: en Because defects in cell shape may be related to defects in the cytoskeleton and because tea2 mutant alleles have short cytoplasmic microtubules , we examined the microtubule cytoskeleton in tea2Δ cells . Exponentially growing cells were stained with antibodies to tubulin, and the cytoplasmic microtubule network was found to be severely reduced . The defects appeared to be more severe when the cells were fixed with aldehyde rather than methanol, perhaps because of a difference in microtubule stability that is reflected by sensitivity to fixation. Astral microtubules were examined in tea2Δ cells using a tubulin-gfp construct , and were found to be much shorter than those seen in wild-type cells (data not shown). Section title: Defects in Cell Morphology and in the Microtubule Cytoskeleton Educational score: 4.471168041229248 Domain: biomedical Document type: Study Language: en It seemed possible that a transition from a phase of nongrowth to a phase of growth might involve an extensive reestablishment of cell polarity and therefore necessitate a relocalization of tip-defining components. This possibility was supported by an observation made during the cloning of tea2 + : tea2-1 cells had a more severe phenotype upon recovery from nutrient starvation. In addition, colonies of tea2Δ cells had variable percentages of T-shaped cells, perhaps caused by nutrient variations in the colony . To investigate these observations in more detail, we examined polarity reestablishment in tea2Δ cells as they emerged from stationary phase at 32°C. After tea2 Δ cells were grown to saturation in liquid rich medium and then diluted into fresh medium, 75% ( n = 700) acquired a T-shaped morphology. (Hundreds of wild-type cells examined all maintained their cylindical shape upon exit from stationary phase.) To more fully examine this defect in tea2Δ cells, 107 individual cells were followed by DIC microscopy through the first few divisions after release from stationary phase . 63 of the cells developed a T shape, 7 developed an L shape, and 6 developed other abnormal morphologies, whereas 31 developed relatively normally. T-shaped cells were tracked through their second division, and 36/39 of these cells grew again from the same ectopic site in the next division . In contrast, 34/34 of the normal shaped cells produced from the first division of the T-shaped cells underwent a normal subsequent division . This lineage analysis suggests that upon exit from stationary phase, a cell that intiates growth from an ectopic site generally continues to use that ectopic site in the subsequent division. Furthermore, once a cell acquires a nonbranched morphology (i.e., the daughter cell formed from the base of the T), this cell is able to maintain a relatively normal morphology in the following divisions. This latter point is further supported by the absence of T-shaped cells in exponentially growing cultures at 32°C. Section title: Enhancement of Morphological Defects with Increases in Cell Length Educational score: 4.212069511413574 Domain: biomedical Document type: Study Language: en The short cytoplasmic microtubules in tea2Δ cells were generally clustered around the nucleus. This arrangement of the cytoskeleton might be especially detrimental in long cells because they may require a more extensive microtubule transport system for tip specification. To test this idea, the phenotype of tea2 Δ in genetic backgrounds that result in long cells was examined. Entry into mitosis is delayed in cdc25-22 cells even at permissive temperature, so these cells are 54% longer than wild-type cells at the time of division . tea2 Δ cdc25-22 cells grown at permissive temperature formed microcolonies of very long and often branched cells , indicating that the extra length of cdc25-22 cells cannot be tolerated in a tea2 Δ background. This interpretation was supported by similar observations in diploid cells, which are 85% longer than haploid cells . Homozygous diploid tea2 Δ cells grew poorly; they are very unstable, haploidize at a high frequency, and are often bent or branched . Section title: Examination of Essential Functions among klps Educational score: 4.12399959564209 Domain: biomedical Document type: Study Language: en To investigate redundancy for essential functions of tea2 + and other klps, double, triple, and quadruple mutants were constructed with pkl1Δ , klp2Δ (C. Troxell and J.R. McIntosh, personal communication), klp3Δ , and tea2 Δ. All possible mutant combinations were constructed. Deletions were monitored by the auxotrophic markers used to delete each gene and by colony PCR using primers specific for each deletion. All combinations were viable at 32°C. To test for temperature sensitivity, each strain was streaked on a YES agar plate and grown at 32°C. These plates were replica plated to EMM agar with appropriate supplements and YES agar plates, and were incubated at 20°C, 25°C, 32°C, and 35.5°C. All mutant combinations were able to grow at these temperatures, suggesting that there are no redundancies of essential functions between Tea2p and these other klps. Section title: Localization of Tea2p Educational score: 4.274023532867432 Domain: biomedical Document type: Study Language: en The cellular localization of Tea2p was determined by fusing the endogenous tea2 + at its 3′ end with the gene for GFP by homologous recombination. Exponentially growing cells were examined by epifluorescence microscopy, and Tea2p-GFP was seen concentrated at the cell tips with some fluorescence throughout the cytoplasm, particularly as cytoplasmic dots . Because the fluorescence from Tea2p-GFP was faint, some cells were fixed and stained with antibodies to GFP in an effort to enhance the signal . As in live cells, Tea2p-GFP was seen at the cell tips, but the signal to noise ratio of the overall cytoplasmic pattern was enhanced in fixed cells. Punctate staining throughout the cytoplasm was observed in these cells. This is likely to be a combined result of signal enhancement, due to the use of antibodies, and some delocalization caused by fixation. Costaining with antibodies to GFP and microtubules revealed that the most intense cytoplasmic dots generally colocalized with the interphase microtubules and were sometimes at the microtubules' ends . In mitotic cells, Tea2p-GFP was less concentrated at the cell tips . Section title: Localization of Tea2p Educational score: 4.130755424499512 Domain: biomedical Document type: Study Language: en The localization of the GFP tagged allele was confirmed using antibodies generated against a fusion protein containing GST and the stalk/tail region of Tea2p. The resulting immune sera were affinity purified against a second fusion protein that contained the Tea2p stalk/tail region tagged with six histidines, and the purified antibodies were used to examine the localization of Tea2p in exponentially growing cells. Staining of tea2 Δ cells showed very faint cytoplasmic background fluorescence . In exponentially growing wild-type cells, Tea2p was detected at the cell tips and often at the end of cytoplasmic microtubules , whereas in mitotic cells Tea2p was less concentrated at the cell tips (not shown). Section title: Localization of Tea2p Educational score: 4.095922946929932 Domain: biomedical Document type: Study Language: en Because of the severity of the morphological defects observed as cells emerged from stationary phase, the localization of Tea2p was examined in stationary phase cells and in cells as they were released from growth arrest. In fixed cells, Tea2p was more concentrated at the cell tips in stationary phase cells and in cells released from stationary phase than in exponentially growing cultures . This could be a reflection of increased resistance to delocalization by fixation, as well as to a change in distribution. Both in stationary phase and in cells exiting stationary phase, non–cell tip staining was often found to be coincident with microtubules or microtubule ends . Section title: Tea2p Dependence on Microtubules for Localization Educational score: 4.268233299255371 Domain: biomedical Document type: Study Language: en To determine whether microtubules are required for Tea2p localization, the position of Tea2p was determined in the presence of the microtubule poison methyl 2-benzimidazolecarbamate (MBC). Because Tea2p is concentrated at the cell tips in cells exiting from stationary phase, this transition was used to characterize the need for microtubules for Tea2p tip localization. Wild-type cells were grown to stationary phase, diluted into fresh medium, and allowed to grow for 25 min. MBC (25 μg/ml) or DMSO (control cells) was then added to the culture, and the cells were further incubated with aliquots removed at 5, 8, and 20 min for staining with antibodies to Tea2p and microtubules. Although the cytoplasmic microtubule network was severely reduced at the 5- and 8-min time points, Tea2p remained concentrated at the cell tips . By 20 min, Tea2p was no longer concentrated at the cell tips, but after the drug was washed out and the microtubules were allowed to repolymerize, Tea2p relocalized to the cell tips . These results suggest that microtubules are required for transporting Tea2p to the tip but not for the short-term maintenance of this localization. Treatment with the microtubule poison thiabendazole (TBZ) or cold shock also resulted in the delocalization of Tea2p (data not shown). Section title: Localization of Tea1p in tea2-1 and tea2Δ Cells and Tea2p in tea1Δ Cells Educational score: 4.154662609100342 Domain: biomedical Document type: Study Language: en Tea1p is proposed to be an end marker that directs the growth machinery to the cell tip . It localizes to the cell tips throughout the cell cycle, and this localization is dependent on microtubules. To investigate the possible role of Tea2p in the localization of Tea1p, exponentially growing tea2-1 , tea2 Δ, and wild-type cells were stained with antibodies to Tea1p . In wild-type cells, Tea1p localized to the cell tips , whereas in the tea2-1 and tea2 Δ mutant cells, Tea1p localized primarily to the short cytoplasmic microtubules . Finally, to investigate whether Tea1p had an effect on Tea2p localization, we examined the localization of Tea2p-GFP in a tea1Δ strain. Tea2p-GFP was still localized at the cell tips, but was more extended in distribution along the microtubules compared with a wild- type strain . Section title: Tea2p Affects Cellular Morphology through an Interaction with Microtubules Educational score: 4.515308380126953 Domain: biomedical Document type: Study Language: en We have shown that tea2 + encodes a klp that is required to establish proper cellular morphology in the fission yeast. Mutant alleles of tea2 + , including its complete deletion, result in cytoplasmic microtubules of reduced length. Because microtubules are required for proper cellular morphology in S . pombe , the abnormal microtubule cytoskeleton is likely to contribute to the morphological abnormalities observed in tea2 Δ cells. These shape abnormalities are most severe in long cells, either diploids or mutants that are longer than haploid wild-type cells, and in cells progressing from a phase of nongrowth to a phase of growth. These results suggest that the importance of microtubules for normal cell growth varies with cell length and growth stage. Tea2p localizes to the cell tips and often to the ends of cytoplasmic microtubules including microtubules that do not reach the cell tip; its localization at cell tips is dependent on cytoplasmic microtubules. Analysis of microtubule dynamics in wild-type cells suggests that microtubules extend from the cell center out to the cell tips, with the minus ends located near the nucleus and the plus ends at the cell tips . Microtubules seen in fixed cells to extend from cell tip to tip probably represent two interphase arrays with minus ends overlapping near the cell equator . Thus, the localization of Tea2p at the cell tips suggests that, if this kinesin has motor activity, it is plus end directed. Section title: Tea2p Affects Cellular Morphology through an Interaction with Microtubules Educational score: 4.266143321990967 Domain: biomedical Document type: Study Language: en The localization of Tea2p and the phenotype of tea2 Δ and tea2-1 mutants are consistent with two mechanisms by which Tea2p might function: Tea2p may affect the length of microtubules through a direct interaction with the microtubules, or it could act indirectly by transporting one or more proteins to the plus end of microtubules, which in turn results in microtubule stabilization. In either case, because there is a high concentration of microtubule plus ends at the cell tips , the concentration of Tea2p at the cell tips supports the hypothesis that the Tea2p-mediated stabilization of microtubules is occurring at the plus ends of the microtubules. Section title: Tea2p Affects Cellular Morphology through an Interaction with Microtubules Educational score: 4.374798774719238 Domain: biomedical Document type: Study Language: en In the first model, Tea2p could act directly on the end of a microtubule to affect the rate of polymerization or depolymerization, or the frequency of rescue or catastrophy. Previous analyses have revealed that klps can affect the dynamic stability of microtubules in vitro . For example, Kar3p, a klp from S . cerevisiae , induces depolymerization from microtubule minus ends in vitro , and XKCM1 and XKIF2 from Xenopus destabilize both microtubule ends in vitro . Several microtubule-based motor proteins affect the lengths of the spindle and/or cytoplasmic microtubules of S . cerevisiae in vivo: deletion of KAR3 , DYN1 , or KIP3 results in longer cytoplasmic microtubules or spindles, whereas deletion or mutation of KIP2 , CIN8 , or KIP1 result in shorter cytoplasmic microtubules or spindles . Section title: Tea2p Affects Cellular Morphology through an Interaction with Microtubules Educational score: 4.721529006958008 Domain: biomedical Document type: Study Language: en In the second model, Tea2p would bind tip-specific protein(s) and transport them along microtubules to their plus ends. The cargo proteins could then modulate microtubule stability, promoting growth. As the microtubules elongate, by either the direct or indirect mechanism, they would be expected to reach the cell tip; interaction there with the cell cortex could provide additional regulation of the length and stability of the microtubule. Kirschner and Mitchison 1986 proposed a similar model to explain the reorganization of the microtubule cytoskeleton observed during polarization of various cell types. They suggested that the asymmetric reorganization of the microtubule cytoskeleton could be controlled at the cell periphery by a localized stabilization of the microtubule ends. Cortical complexes that interact with microtubules have been described previously. For example, in several organisms, alignment of the mitotic spindle is thought to occur through an interaction of the astral microtubules and the cell cortex, resulting in the rotation or movement of the centrosome–nuclear complex or mitotic spindle toward the cortical site . In S . cerevisiae , capture of astral microtubules at the cortical site appears to occur through an interaction between the EB1 homologue Bim1p and the cortical marker protein Kar9p . Section title: Comparison of Tea2p with S. cerevisiae klps Educational score: 4.501986503601074 Domain: biomedical Document type: Study Language: en The analyses of klps in S . cerevisiae have provided a wealth of information about the roles of these enzymes in cell behavior, but none of the mutations in budding yeast has the effect described here for the deletion of tea2 + in fission yeast. This is likely to be due to the observation that S . cerevisiae , in contrast to S . pombe , does not require cytoplasmic microtubules for morphological decisions and development . Instead, microtubules and microtubule motors are required for the proper positioning of the nucleus, spindle formation and function, mating, and karyogamy . Tea2p is most similar in sequence to the S . cerevisiae Kip2p and to a klp identified by the Candida albicans genome project. Phylogenetic analysis illustrates that these enzymes represent a new subfamily of klps. Although deletion of KIP2 also results in short cytoplasmic microtubules, the consequence is a defect in nuclear migration rather than a change in cellular morphology . This difference in phenotypic effect could be a result of the different functions of cytoplasmic microtubules in these two yeasts rather than a divergence in the role of the related klps. Section title: Possible Cargoes of Tea2p: Tea1p Requires Tea2p for Proper Localization Educational score: 4.305203437805176 Domain: biomedical Document type: Study Language: en Tea2p may also transport proteins that help to define the cell's growing tip. Several proteins that are important for cell morphology are also localized to the cell tip including Tea1p and Pom1p . These two proteins are candidates for cargoes of Tea2p because disruption of microtubules disrupts their localization . Pom1p is a protein kinase required for the reinitiation of growth from the old end of the cell after cytokinesis, the switch to bipolar growth, and the positioning of the septum . The microtubule network in pom1 Δ cells appears normal , so this protein is more likely to be part of a tip-defining complex rather a microtubule-regulating complex. Section title: Possible Cargoes of Tea2p: Tea1p Requires Tea2p for Proper Localization Educational score: 4.293032646179199 Domain: biomedical Document type: Study Language: en Tea1p has been proposed to direct the cell growth machinery to the cell tip . Tea1p localizes to the cell tips throughout the cell cycle, and its localization is dependent on microtubules. In the absence of Tea1p, 30–35% of cells are obviously bent during phases of growth, suggesting that this protein is required for normal antipodal growth. Cells deleted for tea1 + can have unusually long cytoplasmic microtubules that curl around the end of the cell in 10–15% of the cells versus <0.5% in wild-type cells . This microtubule phenotype supports the hypothesis that Tea1p is part of a microtubule-controlling complex. Section title: Possible Cargoes of Tea2p: Tea1p Requires Tea2p for Proper Localization Educational score: 4.273104190826416 Domain: biomedical Document type: Study Language: en In the absence of Tea2p, Tea1p localizes along the short cytoplasmic microtubules characteristic of tea2 Δ cells. Therefore, although Tea1p has an affinity for microtubules in the absence of Tea2p, proper localization of Tea1p to the cell tip requires Tea2p. One possibility is that Tea2p transports Tea1p along microtubules and deposits it at the cell tip. A second possibility is that Tea1p uses another microtubule-mediated mechanism to get to the tip of the cell, and the absence of a normal array of cytoplasmic microtubules in tea2 Δ cells results in the mislocalization of Tea1p. Section title: Possible Cargoes of Tea2p: Tea1p Requires Tea2p for Proper Localization Educational score: 4.272542476654053 Domain: biomedical Document type: Study Language: en Tea1p may also have a direct effect on Tea2p localization. When tea1 + is deleted, Tea2p is distributed more broadly along the microtubules, possibly because it is moving more slowly or binding to microtubules less efficiently. Alternatively, Tea1p may be required for efficient anchoring of Tea2p to the cell tip. Interestingly, although both proteins require microtubules for tip-specific localization, they are able to remain at the cell tips for short periods in the absence of microtubules, suggesting that the requirement for microtubules is for transport but not for anchorage . Section title: Possible Cargoes of Tea2p: Tea1p Requires Tea2p for Proper Localization Educational score: 4.261119842529297 Domain: biomedical Document type: Study Language: en Our analyses of Tea2p and tea2 mutant cells provide new evidence for the role of microtubules in the proper positioning of the growth site in fission yeast. The involvement of the microtubule cytoskeleton in the control of cell shape is a widely observed phenomenon that is likely to have many conserved components. Determining whether the mechanism by which Tea2p functions is through the direct stabilization of microtubules or the transport of a microtubule-regulating complex will provide insight into the control of morphogenesis in S . pombe , and this mechanism may represent a more general function of klps in the morphology of eukaryotic cells. | Other | biomedical | en | 0.999998 |
0004087 | Section title: Introduction Educational score: 4.173458576202393 Domain: biomedical Document type: Study Language: en Dynamic regulation of the actin cytoskeleton plays a central role in a variety of cellular events, including adhesion, division, spreading, and motility . Engagement or activation of cell surface growth factors and adhesion receptors influences the assembly and arrangement of F-actin networks . Transmission of extracellular signals to the actin cytoskeleton is governed by small GTPases of the Rho family, as well as by the activity of numerous actin-binding proteins . Section title: Introduction Educational score: 4.572348594665527 Domain: biomedical Document type: Study Language: en The Rho family GTPases, Cdc42 and Rac, play a critical role in the formation and organization of cortical actin networks in mammalian cells. Treatment of cells with agents that increase GTP-bound Cdc42 stimulates filopodia formation , whereas activation of Rac leads to membrane ruffle and lamellipodia formation . Formation of cortical actin networks, resulting either from EGF stimulation or activated Rac , requires de novo formation of F-actin filaments, indicating that Cdc42 and Rac integrate signal pathways leading to actin polymerization. In addition, Rac activation is closely coupled to activation of Cdc42 , allowing for the coincident and coordinated formation of filopodia and lamellipodia that are often concurrently observed at the leading edge in motile cells . Actin polymerization at the leading edge provides the protrusive force required for the extension of lamellipodia observed during cell motility and spreading . Section title: Introduction Educational score: 4.553166389465332 Domain: biomedical Document type: Study Language: en Cortical actin polymerization initiated by Cdc42 and Rac requires the participation of members of the Wiskott-Aldrich Syndrome protein (WASp) superfamily . Binding of activated Cdc42 to N-WASp induces filopodia , whereas Rac-induced membrane ruffling utilizes the structurally related protein Scar1 (also known as WASp family verpolin-homologus protein [WAVE]) . All WASp/Scar family proteins contain a carboxyl-terminal motif rich in acidic residues, which is responsible for their activity and mediates binding to the actin-related protein (Arp) 2/3 complex . Arp2/3 complexes consist of the actin-related proteins Arp2 and Arp3 along with five other proteins designated p41-, p34-, p21-, p20-, and p16-Arc (also designated as ARPC1–5). Section title: Introduction Educational score: 4.524579048156738 Domain: biomedical Document type: Study Language: en Arp2/3 complex binds to the sides of preexisting actin filaments and stimulates new filament formation to create branched actin networks, a process termed the “dendritic nucleation” model of cortical actin assembly . As predicted by this model, the Arp2/3 complex is located at branch points of actin filament networks in lamellipodia, as seen by electron microscopy , and is localized to sites of dynamic actin assembly and motility in living cells . Arp2/3-induced actin nucleation and polymerization are greatly enhanced by the binding of WASp family acidic carboxyl-terminal domains to p21-Arc , thus providing a molecular link for Cdc42 and Rac leading to cortical actin polymerization . Section title: Introduction Educational score: 4.818421363830566 Domain: biomedical Document type: Study Language: en In addition to the Arp2/3 complex, several actin binding proteins are selectively recruited into cortical actin structures upon activation of Cdc42 or Rac , including the Src kinase substrate cortactin , an actin binding protein enriched within lamellipodia . The localization of cortactin within membrane ruffles and lamellipodia is controlled by activation of Rac . Cortactin possess a multidomain structure consisting of an acidic domain at the amino terminus, followed by 6 and 1/2 tandemly repeated 37–amino acid segments, an α helical region, a proline-rich segment, and a Src homology (SH) 3 domain located at the carboxyl terminus . The direct binding to F-actin is mediated through sequences within the tandem repeat region . The SH3 domain interacts with several postsynaptic density (PSD)95/dlg/ZO-1 (PDZ) domain–containing proteins, including cortactin-binding protein 1 (CortBP1) , SHANK3 , ZO-1 , and an unrelated protein cortactin-binding protein (CBP-90) . Tyrosine phosphorylation of cortactin occurs in response to a wide variety of cellular events, including v-Src transformation , growth factor treatment , bacterial invasion , osmotic stress , and integrin or syndecan-3 ligation with the extracellular matrix . Section title: Introduction Educational score: 4.364992141723633 Domain: biomedical Document type: Study Language: en These previous studies suggest that cortactin plays an important role in coupling tyrosine kinase–based signaling events to cortical cytoskeletal reorganization. We have investigated the molecular mechanism by which cortactin interacts with the dynamic actin cytoskeleton. In this report we provide evidence that the Rac-induced localization of cortactin to the cell periphery requires two separate regions within the amino terminus: the amino-terminal acidic domain (NTA) and the fourth tandem repeat. We find that the fourth repeat is necessary for the F-actin binding activity of cortactin and that the NTA region binds directly to the Arp2/3 complex. Cortactin colocalizes with Arp2/3 complex at sites of dynamic actin assembly in lamellipodia. We propose that one of the roles of cortactin is to link PDZ-containing scaffolding proteins to sites of Arp2/3-mediated cortical actin assembly. Section title: DNA Constructs Educational score: 4.233020782470703 Domain: biomedical Document type: Study Language: en Cortactin expression constructs were created by PCR amplification of mp85.L7 . For production of cytomegalovirus (CMV)-driven cortactin constructs containing the FLAG epitope, the following cDNA fragments were produced by PCR: N-term (codons 1–330), NTA (codons 1–84), repeats (codons 85–330), and C-term (codons 350–546). All 5′ primers contained a KpnI restriction endonuclease site and all 3′ primers contained a stop codon followed by an EcoRI site. Amplified products were subcloned into KpnI-EcoRI–digested pcDNA3FLAG2AB . In some cases, PCR fragments were first cloned into PCR-Script (Stratagene) before subcloning. For construction of full-length cortactin lacking the fourth repeat, a KpnI-BamHI PCR fragment spanning the amino terminus to the end of the third repeat (codons 1–195) was ligated in frame with a BamHI-EcoRI fragment encoding the start of the fifth repeat through the carboxyl terminus (codons 232–546) and KpnI-EcoRI–digested pcDNA3FLAG2AB. The full-length FLAG-tagged cortactin construct has been described previously . Section title: DNA Constructs Educational score: 4.184899806976318 Domain: biomedical Document type: Study Language: en CMV-driven myc-tagged cortactin expression constructs were produced by PCR as BamHI-EcoRI fragments and subcloned into BamHI-EcoRI–digested pRK5myc . Constructs produced were: full-length (codons 1–546), N-term (codons 1–330), N-repeat 5 (codons 1–269), N-repeat 4 (codons 1–232), N-repeat 3 (codons 1–195), N-repeat 2 (codons 1–158), N-repeat 1 (codons 1–121), repeat 3-C-term (codons 158–546), repeat 4-C-term (codons 195–546), repeat 5-C-term (codons 232–546), repeat 6-C-term (codons 269–546), and C-term (codons 350–546). FLAG-RacL61 was constructed by digestion of pRK5myc-RacL61 with BamHI and EcoRI, and the resultant Rac cDNA fragment was subcloned into BamHI-EcoRI–digested pcDNA3FLAG2AB. Expression and immunoreactivity of each cortactin variant were verified by Western blotting of whole cell lysates with either the antiepitope tag mAbs M5 (against FLAG) or 9E10 (against myc) and anticortactin antibodies (data not shown). Section title: DNA Constructs Educational score: 4.143178939819336 Domain: biomedical Document type: Study Language: en For production of the glutathione S -transferase (GST)-cortactin prokaryotic expression constructs, GST-N-term, GST-NTA, and GST-repeats, BamHI-EcoRI PCR fragments were produced encoding the appropriate codons and subcloned into BamHI-EcoRI–digested pGST-parallel 2 (a gift from P. Sheffield, University of Virginia), as described previously . For the GST-N-repeat 5 construct, pRK5myc-N-repeat 5 was digested with BamHI and EcoRI and the cortactin fragment was subcloned into pGST-parallel 2. The GST construct containing the verpolin homology, cofilin, and acidic (VCA) domains of N-WASp has been described previously . All PCR-generated constructs were verified by DNA sequencing. Section title: Antibodies and Western Blotting Educational score: 3.26123046875 Domain: biomedical Document type: Study Language: en The anticortactin antibodies anti-N-term, anti-C-term, and 4F11, have been described previously . The specificity of the anti-N-term and C-term cortactin antibodies was verified by Western blotting of transfected cell lysates . The 9E10 mAb against the cmyc epitope was purchased from Santa Cruz Biotechnology, Inc. Anti-FLAG mAb M5 was purchased from Sigma-Aldrich. Affinity-purified rabbit pAbs against p21-Arc and Arp3 (a gift from M. Welch, University of California at Berkley, Berkeley, CA) were used for immunofluorescence detection of the Arp2/3 complex; rabbit antisera against Arp3, Arp2, and p34-Arc were used for Western blotting (a gift from L. Machesky, University of Birmingham, Birmingham, England) . Fluorescently labeled secondary antibodies were purchased from Molecular Probes, Jackson ImmunoResearch Laboratories, and CHEMICON International, Inc. Secondary antibodies coupled to horseradish peroxidase were purchased from Amersham Pharmacia Biotech. Section title: Antibodies and Western Blotting Educational score: 4.036315441131592 Domain: biomedical Document type: Study Language: en Western blotting was performed as described previously . Primary antibodies were used at the following concentrations or dilutions: 9E10 (3 μg/ml), M5 (5 μg/ml), 4F11 (1 μg/ml), anti-N-term or anti-C-term (1 μg/ml), Arp3 (1:500), and p34-Arc (1:500). Primary antibodies were detected with the appropriate horseradish peroxidase–conjugated secondary antibody (1:2,000) and immunoreactive bands were visualized using enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech). Protein concentrations were determined using a bicinchonic acid assay kit (BCA; Pierce Chemical Co.). Section title: Cell Culture, Microinjection, and Transfection Educational score: 4.154550075531006 Domain: biomedical Document type: Study Language: en Swiss 3T3, C3H 10T1/2 (a gift from S. Parsons, University of Virginia), and PtK1 cells expressing a green fluorescent protein (GFP)-Arp3 fusion were cultured as described previously . Subconfluent, serum-starved Swiss 3T3 cells were prepared as described . Microinjection of myc-tagged cortactin constructs and membrane ruffling initiated by PDGF and PMA were performed as described . At least 50 microinjected cells expressed each construct as determined by immunofluorescence microscopy. For transfection experiments, C3H 10T1/2 cells were grown to 80% confluence in 100-mm dishes and transfected with 10 μg of each epitope-tagged cortactin construct using SuperFect™ (QIAGEN). Cotransfection experiments with epitope-tagged RacL61 and cortactin constructs were conducted at a 4:1 (wt/wt) Rac/cortactin ratio. For immunofluoresence studies, cells were cultured 18 h after transfection, detached with trypsin/EDTA, neutralized with soybean trypsin inhibitor (Sigma-Aldrich), plated onto fibronectin-coated coverslips, and allowed to spread for 1 h, after which cells were fixed and processed for immunofluorescence microscopy. A minimum of 75 cotransfected cells was evaluated for each cortactin construct. Section title: Immunofluoresence Microscopy Educational score: 4.135986804962158 Domain: biomedical Document type: Study Language: en Swiss 3T3 and C3H 10T1/2 cells were fixed and immunolabeled as described . Epitope-tagged constructs were detected with either M5 (5 μg/ml) or 9E10 (3 μg/ml). Endogenous cortactin was labeled with either anti-N-term or anti-C-term (2 μg/ml). PtK1 cells were immunolabeled as described . Cells were double labeled with the anticortactin mAb 4F11 (0.9 μg/ml) and anti-Arp3 (4 μg/ml) or p21-Arc (6 μg/ml). GFP-Arp3–expressing cells were labeled with 4F11. Section title: Actin Cosedimentation Assays Educational score: 4.238675117492676 Domain: biomedical Document type: Study Language: en F-actin binding assays were adapted from Fanning et al. 1998 . Confluent 100-mm dishes of transfected C3H 10T1/2 cells were rinsed twice with PBS and collected on ice by scraping into 0.5 ml of binding buffer (10 mM imidazole, pH 7.2, 75 mM KCl, 5 mM MgCl 2 , and 0.5 mM DTT) supplemented with 1 mM EGTA, 1 μg/ml leupeptin, and 1 μg/ml aprotinin. Cells were lysed with a Dounce homogenizer (50 strokes), and the lysate was centrifuged at 100,000 g for 1 h at 4°C. Rabbit muscle G-actin (Cytoskeleton) was diluted to 2.5 mg/ml in binding buffer and polymerized for 1 h at room temperature. F-actin (5.5 μM) was incubated with 60–80 μl of cell lysate (∼50 μg of protein) in a final volume of 200 μl for 30 min at room temperature. Samples were centrifuged at 100,000 g for 1 h at 20°C in a 42.2 Ti rotor (Beckman Instruments), the supernatant was removed, and the pellet was incubated for 1 h at 4°C with 50 μl of G buffer (5 mM Tris-HCl, pH 8.0, 0.2 mM ATP, 0.5 mM DTT, and 0.2 mM CaCl 2 ) . Supernatant and pellet fractions were normalized, and equal amounts were solublized in 2× SDS-PAGE sample buffer. Fractions were analyzed by SDS-PAGE and Western blotting with 4F11, M5, or 9E10 mAbs. Binding was quantitated by densitometric scans of the x-ray films shown in Fig. 3 and Fig. 4 for each immunoreactive band or an equivalent area from films exposed in a linear range using the program ImageQuant ® (v.1.2; Molecular Dynamics). Percent bound was calculated as: P / S + P × 100, where S and P are the amounts of cortactin immunoreactivity in the supernatant ( S ) and pellet ( P ) fraction. Section title: Protein Preparation and Purification Educational score: 4.265320777893066 Domain: biomedical Document type: Study Language: en GST-cortactin constructs were transformed into Escherichia coli strain DH5α, and fusion proteins were purified from isopropyl-1-thio-β- d -galactopyranoside–induced bacteria, as described . Fusion proteins were captured by incubation with glutathione–Sepharose 4B (5 ml/2 liters of culture; Amersham Pharmacia Biotech) and eluted with 10 mM reduced glutathione in suspension buffer (20 mM Tris-HCl, pH 8.0, 0.3 M NaCl, and 0.5 mM EDTA). TEV™ protease (300 U; GIBCO BRL) was added to the eluate, and the solution was dialyzed overnight at 4°C against 4 liters of suspension buffer containing 1 mM DTT. In cases of incomplete digestion, an additional 200 U of TEV™ was added, and the solution was incubated at 16°C overnight. Cleaved GST was removed by the addition of glutathione-Sepharose. TEV™ protease was removed by the addition of 200 μl of Ni-NTA™ agarose (50% slurry; QIAGEN). Arp2/3 complex was purified from bovine brain as described . Protein purity was monitored by Coomassie blue staining after SDS-PAGE, with all products estimated to be at least 90–95% pure. Section title: Affinity Chromatography and Capillary Mass Spectrometry Educational score: 4.086475372314453 Domain: biomedical Document type: Study Language: en Recombinant cortactin fragments (10 mg) were coupled to cyanogen bromide–activated Sepharose 4B (Amersham Pharmacia Biotech) according to the manufacturer's instructions. Resins were suspended in 1 ml of column buffer (20 mM Hepes-KOH, pH 7.8, 50 mM KCl, 1 mM EDTA, and 1% NP40) and stored at 4°C. Coupling efficiency was ∼90%. Section title: Affinity Chromatography and Capillary Mass Spectrometry Educational score: 4.212219715118408 Domain: biomedical Document type: Study Language: en For affinity purification of amino-terminal cortactin binding proteins, extracts were initially prepared from murine brain. Whole brain tissue from 11 5–6-wk-old NIH 3T3 mice was Dounce homogenized in 8 ml of column buffer containing 10 mg/ml leupeptin and 1 mM PMSF. The lysate was centrifuged at 1,900 g for 10 min, and the supernatant was stored at −80°C. Cortactin N-term–Sepharose beads (100 μl of a 50% slurry containing ∼1 mg of coupled protein) were incubated with 16 mg of brain lysate (4 mg/ml) for 30 min at 4°C. Beads were then washed with 4 ml of column buffer followed by 3 ml of column buffer containing 150 mM KCl. Bound proteins were eluted with 60 μl of SDS-PAGE sample buffer, separated by SDS-PAGE, and visualized by Coomassie blue staining or by Western blotting. Coomassie-stained bands representing proteins specifically associated with cortactin N-term were excised, trypsin digested, subjected to liquid chromatography mass spectrometry (LC-MS) analysis , and analyzed by searching the NCBI database using a computer-based algorithm . Section title: Affinity Chromatography and Capillary Mass Spectrometry Educational score: 4.098217487335205 Domain: biomedical Document type: Study Language: en Additional experiments were performed using C3H 10T1/2 fibroblast lysate with an N-repeat 5 resin, since this protein was purified at much higher yields than cortactin N-term. Cells were lysed in column buffer containing inhibitors and centrifuged at 5,000 g for 5 min, and 3 mg of lysates (6 mg/ml) were incubated with 80 μl of GST, N-repeat 5, NTA, or repeats-Sepharose for 2 h at 4°C. After incubation, beads were washed twice with lysis buffer, and bound proteins were analyzed by Western blotting with anti-Arp3 antibodies. Section title: Immunoprecipitation Educational score: 4.137409210205078 Domain: biomedical Document type: Study Language: en C3H 10T1/2 cells transfected with FLAG-tagged cortactin constructs were lysed in lysis buffer supplemented with protease and phosphatase inhibitors (10 μg/ml leupeptin, 1.0 U/ml aprotinin, 0.5 mM PMSF, 0.5 mM EDTA, 1 mM sodium vanadate, and 40 mM sodium fluoride) and centrifuged at 5,000 g for 5 min. Clarified lysates (700 μg) were incubated with 20 μl of FLAG M2 affinity resin (Sigma-Aldrich) for 2 h at 4°C. Immune complexes were collected by centrifugation, washed twice with 1.0 ml of lysis buffer, separated by SDS-PAGE, and Western blotted with anti-Arp2, -Arp3, and -M5 antibodies. Section title: Binding Assays Educational score: 4.146261215209961 Domain: biomedical Document type: Study Language: en Arp2/3 complex (700 ng) was incubated with Sepharose conjugated to GST, cortactin NTA, cortactin N-repeat 5 or cortactin repeats 1–5 in 200 μl of binding buffer (20 mM Hepes, pH 7.5, 50 mM KCl, 1 mM EDTA, 0.1 mM ATP, 1% NP40) for 30 min at 4°C. The Sepharose was collected by centrifugation, washed twice with binding buffer without ATP, and bound Arp2/3 was visualized by Western blotting with anti-Arp3 after SDS-PAGE. For affinity measurements, 2.1 nM Arp2/3 was incubated with 0.46, 1.2, 2.3, and 4.7 μM cortactin N-repeat 5, as described above. The amount of Arp2/3 bound to cortactin was quantitated by densitometry and ImageQuant ® as described above. Section title: Actin Polymerization Assays Educational score: 4.150547027587891 Domain: biomedical Document type: Study Language: en Actin polymerization assays were conducted essentially as described . In brief, recombinant cortactin proteins (0.2–2.0 μM) or the GST-VCA fragment of human N-WASp (a gift from Marie-France Carlier, Dynamique du Cytosquelette, Laboratoire d'Enzymologie et Biochemie Structurales, Gif-sur-Yvette, France) were incubated with 10 nM Arp2/3 complex in 20 mM imidazole, pH 7.0, 100 mM KCl, 2 mM MgCl 2 , and 1 mM EGTA at 25°C. Actin polymerization was initiated by the addition of monomeric actin (7.5% pyrene labeled) and monitored by continuous measurement of the emission at 386 nm in a SPEX FluoroMax fluorometer (JY, Inc.). The temperature was maintained at 25°C throughout the experiment. Section title: Growth Factor–induced Translocation of Cortactin into Membrane Ruffles Requires the Amino-terminal Domain Educational score: 4.214438438415527 Domain: biomedical Document type: Study Language: en In serum-starved cells, growth factor–mediated activation of Rac1 leads to the translocation of cortactin from cytoplasmic pools into lamellipodia, where it colocalizes with cortical F-actin . To identify the cortactin sequences necessary for lamellipodia localization, epitope-tagged cortactin expression constructs encoding various regions of the protein were introduced into mouse fibroblasts, and the Rac-induced translocation of the variant cortactin proteins to the cell cortex was evaluated . Section title: Growth Factor–induced Translocation of Cortactin into Membrane Ruffles Requires the Amino-terminal Domain Educational score: 4.218870162963867 Domain: biomedical Document type: Study Language: en Subconfluent, serum-starved Swiss 3T3 fibroblasts were microinjected with either myc-N-term or myc-C-term constructs, and the intracellular distribution of N-term and C-term cortactin was compared with that of endogenous cortactin . In serum-deprived cells, myc-N-term, myc-C-term, and endogenous cortactin were diffusely distributed throughout the cytoplasm . Treatment of cells with PDGF or PMA, agents that activate Rac , resulted in the accumulation of myc-N-term and endogenous cortactin within membrane ruffles at the cell cortex . Myc-C-term cortactin failed to translocate efficiently to the cell periphery . Therefore, the amino-terminal half of the cortactin molecule is necessary to target cortactin to the cell periphery. Section title: Rac-induced Translocation of Cortactin to the Cell Cortex Requires Both the Amino-terminal Acidic and Tandem Repeat Region Educational score: 4.169250011444092 Domain: biomedical Document type: Study Language: en To determine which region(s) within the amino-terminal domain are required for Rac-induced cortactin translocation, FLAG-tagged cortactin expression constructs were cotransfected with constitutively active myc-RacL61 into 10T1/2 fibroblasts. After plating on fibronectin for 1 h, cells expressing RacL61 were flat and round, with cortactin localized almost exclusively at the cell cortex . FLAG-full-length cortactin and FLAG-N-term showed peripheral localization, whereas FLAG-C-term did not localize to the cortex, although endogenous cortactin did localize correctly . Section title: Rac-induced Translocation of Cortactin to the Cell Cortex Requires Both the Amino-terminal Acidic and Tandem Repeat Region Educational score: 4.333667278289795 Domain: biomedical Document type: Study Language: en To further delineate the regions within the amino-terminal domain responsible for cortical targeting, FLAG-tagged constructs that encoded the NTA and the repeats domain (encompassing the six and one half tandem repeat sequences) were tested . Both FLAG-NTA and FLAG-repeats failed to accumulate at the cell cortex when coexpressed with myc-RacL61, in spite of the near complete translocation of endogenous cortactin in cells expressing these constructs . These data indicate that both the NTA and the repeat region are necessary for translocation of cortactin to the cell cortex, and that neither region alone is sufficient for translocation. Since the actin binding activity of cortactin is contained within the tandem repeat region , cells expressing various FLAG-tagged cortactin constructs were lysed and the actin binding activity of the tagged cortactin constructs was evaluated . FLAG-full-length and FLAG-N-term cosedimented with F-actin, whereas FLAG-C-term did not . FLAG-repeats, which lacks the NTA region, bound actin at levels comparable to FLAG-N-term . These data suggest that F-actin binding alone is insufficient for the peripheral localization of cortactin. Section title: The Fourth Cortactin Repeat Is Required for Cortical Localization and F-Actin Binding Educational score: 4.326407432556152 Domain: biomedical Document type: Study Language: en The sequences of the six 37–amino acid tandem repeats in the cortactin amino terminus are highly similar to each other . To determine whether one or more of the tandem repeats was involved in cortical targeting, a series of myc-tagged cortactin expression constructs was produced that retained the NTA, but in which the individual tandem repeats were truncated from the carboxyl terminus of the myc-N-term construct . Myc-N-term, Myc-N-repeat 5, and N-repeat 4 accumulated at the cell edge, but translocation of N-repeat 3, N-repeat 2, or N-repeat 1 (not shown) was significantly reduced . These results suggest that the fourth repeat is required for cortical targeting. To directly test this, a full-length cortactin construct was produced lacking the fourth repeat (FLAG-full-lengthΔ4) and assayed for cortical localization, where it also failed to accumulate at the cell periphery . Endogenous cortactin translocated normally in all experiments. Taken together with the data presented above, we conclude that sequence elements within the fourth tandem repeat of cortactin are necessary for localization to the cell cortex. Section title: The Fourth Cortactin Repeat Is Required for Cortical Localization and F-Actin Binding Educational score: 4.656986236572266 Domain: biomedical Document type: Study Language: en Cortactin binds directly to F-actin with a K d of 0.4 μM , suggesting that targeting of cortactin to the leading edge may involve direct binding to cortical actin filaments. To define the portion of the repeat region responsible for actin binding, myc-N-term constructs containing serial deletions of individual repeats were assayed for F-actin binding in cell lysates . Myc-N-term and myc-N-repeat 5 bound actin at comparable levels, whereas myc-N-repeat 4 bound actin at diminished levels . Myc-N-repeat 3, myc-N-repeat 2, and myc-N-repeat 1 all failed to bind F-actin . These data map the actin binding region of cortactin between the fourth complete and seventh partial tandem repeats. To further define which sequences within this region were required for actin binding, myc-tagged cortactin constructs were produced where the NTA and the individual tandem repeats were serially deleted from full-length cortactin beginning with the third repeat . Myc-repeat 3-C-term displayed strong binding to F-actin, whereas myc-repeat 4-C-term weakly associated with F-actin . Myc-repeat 5-C-term, myc-repeat 6-C-term, and myc-C-term all failed to significantly bind F-actin . These data indicate that repeat four is central to the actin binding activity of cortactin, with efficient binding requiring a single adjacent repeat, either repeat three or repeat five . The requirement for repeat four in actin binding was directly examined using FLAG-full-lengthΔ4, where removal of repeat four eliminated the binding of cortactin to F-actin . These data, combined with the requirement for repeat four in cortical targeting, suggest that the ability of cortactin to localize with cortical actin networks in vivo requires the actin binding site located within the fourth tandem repeat. Section title: The Cortactin Amino-terminal Domain Associates with the Arp2/3 Complex Educational score: 4.365251541137695 Domain: biomedical Document type: Study Language: en Although the actin binding domain in repeat four is essential for cortical localization, the inability of the repeats region alone to target the cell cortex indicated that actin binding alone is insufficient for localization at the cell periphery. The NTA alone also failed to target to the cortex, but the NTA plus the repeats (i.e., N-term) did target appropriately. Therefore, we searched for other proteins that might interact with the amino-terminal domain and contribute to cortactin translocation. The cortactin amino-terminal domain was expressed in E . coli as a GST fusion protein, and the purified protein was covalently coupled to agarose beads. After application of a mouse brain extract, the affinity matrix was washed extensively and bound proteins were subjected to SDS-PAGE . Based on a comparison with control lanes, 13 bands that appeared to represent proteins specifically associated with the cortactin amino-terminal domain were selected for LC-MS analysis. Sequence analysis and a search of the National Center for Biotechnology Information database revealed that four bands contained peptide sequences corresponding to five members of the Arp2/3 complex . The presence of Arp2/3 complex proteins associated with N-term, but not control beads, was confirmed by Western blotting with antisera specific for Arp3 and p34Arc . These data indicate that the Arp2/3 complex represents a subset of the proteins that interact with the amino terminus of cortactin. Section title: Cortactin Binds the Arp2/3 Complex through Sequences in the NTA Region Educational score: 4.150706768035889 Domain: biomedical Document type: Study Language: en To identify the region within the cortactin amino terminus responsible for associating with the Arp2/3 complex, Sepharose beads covalently coupled to the NTA and repeats regions were incubated with 10T1/2 fibroblast lysate, and Arp2/3 binding was assayed by immunoblotting . Arp3 specifically bound to N-repeat 5 (see Materials and Methods) and NTA beads, but not to GST and repeats beads , indicating that the NTA region is responsible for association with the Arp2/3 complex. Section title: Cortactin Binds the Arp2/3 Complex through Sequences in the NTA Region Educational score: 4.20320987701416 Domain: biomedical Document type: Study Language: en To confirm the interaction between cortactin and the Arp2/3 complex in vivo, 10T1/2 fibroblasts were transfected with FLAG-cortactin expression constructs, and overexpressed cortactin fusion proteins were immunoprecipitated with anti-FLAG. Coimmunoprecipitation of Arp2/3 complex proteins was assessed by immunoblotting. Both Arp3 and Arp2 (data not shown) coimmunoprecipitated with FLAG-full-length, FLAG-N-term, and FLAG-NTA. Precipitation of FLAG-repeats and FLAG-C-term fusion proteins failed to coprecipitate the Arp2/3 complex . Precipitation of each FLAG-cortactin peptide was verified by immunoblotting with the anti-FLAG mAb M5 . These data support the conclusion that cortactin interacts with the Arp2/3 complex through sequences within the 84–amino acid NTA domain. Section title: Cortactin Binds to Purified Arp2/3 Complex: Functional Analysis of the Interactions Educational score: 4.1842145919799805 Domain: biomedical Document type: Study Language: en To determine whether the association of cortactin with Arp2/3 was direct, affinity chromatography assays were performed with various Sepharose-conjugated amino-terminal cortactin proteins and purified Arp2/3 complex . Purified Arp2/3 complex bound to both NTA- and N-repeat 5–Sepharose but failed to bind to Sepharose alone, GST, or repeat 1–5–Sepharose . Furthermore, addition of increasing amounts of cortactin N-repeat 5-Sepharose to a fixed amount of Arp2/3 complex displayed saturable binding, with an estimated K d of 1.3 μM . These data strongly indicate that cortactin directly interacts with the Arp2/3 complex. Section title: Cortactin Binds to Purified Arp2/3 Complex: Functional Analysis of the Interactions Educational score: 4.197641849517822 Domain: biomedical Document type: Study Language: en Since cortactin binds directly to Arp2/3 complex, we hypothesized that cortactin might stimulate the actin nucleation activity of Arp2/3. Stimulation of Arp2/3 complex was measured in a pyrene actin polymerization assay . Nucleation of new actin filaments by Arp2/3 complex causes a rapid rise in pyrene actin fluorescence due to the increased number of free barbed ends available for polymerization . In the presence of 10 nM Arp2/3 complex and 2.5 μM actin, cortactin N-repeat 5 (curve b), repeat 1–5 (curve c), full-length (curve d), and NTA (curve f) (all at 0.5 μM) failed to stimulate Arp2/3 complex to nucleate new actin filaments . Concentrations of cortactin proteins up to 1 μM did not activate the Arp2/3 complex ( n = 3, data not shown). Addition of the N-WASp VCA domain (curve a) strongly promoted actin nucleation, indicating that the Arp2/3 complex was functional. Also, cortactin proteins, at concentrations up to 2 μM, did not inhibit the ability of the VCA fragment at threshold (0.5 nM) concentrations to activate Arp2/3 complex. When the concentration of the Arp2/3 complex was increased 10-fold to 100 nM, cortactin at 0.5 μM showed weak activation of nucleation. The nature of the weak cortactin-dependent nucleation activity is currently under study. Section title: Cortactin Localizes with the Arp2/3 Complex in Fibroblast Lamellipodia Educational score: 4.255051612854004 Domain: biomedical Document type: Study Language: en To determine if cortactin and the Arp2/3 complex were present within the same subcellular compartments, cortactin and Arp2/3 complex localization in fibroblast lamellipodia was determined by indirect immunofluoresence. Double staining of PtK1 cells with antibodies specific for Arp3 or p21-Arc and the anticortactin mAb 4F11 yielded very similar staining patterns, with both components being present at the leading edge and in peripheral spots . Merged images demonstrated a large degree of overlap in both subcellular regions. Colocalization of cortactin with Arp3 was also demonstrated in a PtK1 cell line stably expressing GFP-Arp3 . GFP-Arp3 and cortactin were both present at the leading edge and within peripheral spots, with significant overlap in merged images . Based on these data, we conclude that cortactin is a component of the Arp2/3-regulated cortical actin structures present in lamellipodia. Section title: Discussion Educational score: 4.689519882202148 Domain: biomedical Document type: Study Language: en Cortactin is involved in several protein tyrosine kinase–based signaling pathways, where its phosphorylation is coincident with reorganization of cortical actin cytoskeletal networks. The localization of cortactin with cortical F-actin within lamellipodia requires Rac activity . The sequences responsible for the selective targeting of cortactin to the cell periphery are unknown. In this report we provide evidence that Rac-induced localization of cortactin to the cell cortex requires two distinct sequence motifs within the amino-terminal half of the protein. One targeting motif encompasses the fourth 37–amino acid tandem repeat. Removal of repeat four abolishes the ability of cortactin to localize at the cell periphery in cells cotransfected with RacL61 and greatly diminishes binding of cortactin to F-actin in vitro. The second localization motif resides within the 84–amino acid NTA region and is also required for Rac-induced cortical localization. The NTA region binds to the Arp2/3 complex as determined by affinity chromatography and immunoprecipitation from cell lysates and by direct binding assays with purified cortactin and the Arp2/3 complex. Immunofluoresence localization experiments support a functional interaction between cortactin and the Arp2/3 complex in that the Arp2/3 complex and cortactin colocalize at the leading edge and within punctate spots within lamellipodia, both regions of active actin dynamics. Therefore, we conclude that the Rac-induced interaction of cortactin with cortical sites of actin assembly requires the bipartite interaction of cortactin with actin filaments and the Arp2/3 complex . Section title: Discussion Educational score: 4.285727024078369 Domain: biomedical Document type: Study Language: en Cortactin interacts with components of the cortical actin network in several cell types. In addition to the lamellipodia of cultured cells , cortactin is also found at sites of cortical F-actin reorganization in many differentiated cells, including growth cones and the PSD of neurons , the terminal web of polarized epithelia , and neuromuscular junctions . The biochemical mechanism discussed here, in which cortactin interacts with both F-actin and the Arp2/3 complex, may also be the basis for the interaction of cortactin with dynamic actin in these other specialized cellular compartments. Section title: Discussion Educational score: 4.5764031410217285 Domain: biomedical Document type: Study Language: en Regulation of the actin cytoskeleton plays an essential role in the organization of transmembrane receptor complexes within highly specialized cell structures . The data presented in this report, in conjunction with the characterization of several cortactin SH3 domain binding proteins, leads us to propose that cortactin functions to link a variety of cell surface receptors to sites of Arp2/3-directed actin polymerization . In neurons, the SH3 domain of cortactin interacts with members of the CortBP1/SHANK family of scaffolding proteins . CortBP1/SHANK proteins possess a single PDZ domain and are capable of interacting directly with the type 2 somatostatin receptor or indirectly through the scaffolding proteins guanylate kinase–associated protein (GKAP), PSD95, or Homer to the N -methyl- d -aspartate (NMDA), inositiol trisphosphate, or metabotropic glutamate receptors . Additionally, the SH3 domain of cortactin binds ZO-1, a component of tight junctions . Tight junctions occur in the terminal web of polarized epithelia , a region where ZO-1 and cortactin are colocalized . ZO-1 is structurally similar to PSD-95 and has been implicated in regulating tight junction assembly . ZO-1 binds to cytoplasmic motifs in occludin and claudin , two transmembrane proteins implicated in forming the paracellular seal between adjacent cells . Therefore, the coupling of ZO-1 to Arp2/3-mediated actin dynamics by cortactin may in part contribute to the organization and assembly of transmembrane networks within tight junctions as well as at the PSD . Section title: Discussion Educational score: 4.4303789138793945 Domain: biomedical Document type: Study Language: en Activation of the Arp2/3 complex has been suggested to be critical for protrusive-based actin locomotion . Colocalization of cortactin with the Arp2/3 complex at the leading edge and in protrusive distal lamellar regions , the direct binding of cortactin to Arp2/3 in vitro, and the tyrosine phosphorylation of cortactin in response to growth factor receptor activation suggest a role for cortactin in linking sites of actin polymerization to cell surface receptor complexes. The observation that cortactin does not efficiently stimulate Arp2/3-dependent actin nucleation (although low levels of nucleation are observed at higher concentrations of Arp2/3) is consistent with cortactin not being a direct activator of actin polymerization. However, it is possible that additional factors/proteins may cooperate to increase the effect of cortactin on Arp2/3 activation. Section title: Discussion Educational score: 4.03458309173584 Domain: biomedical Document type: Study Language: en Thus, possible functions of cortactin may be to bridge sites of dynamic actin reorganization with receptor signaling complexes and/or to recruit, via SH3 interactions, other proteins that may positively or negatively regulate Arp2/3 actin polymerization. Section title: Discussion Educational score: 4.4375739097595215 Domain: biomedical Document type: Study Language: en In addition to WASp/Scar family proteins, binding of ActA from Listeria monocytogenes or yeast Myo3p and 5p stimulates actin reorganization mediated entirely, or in part, by the Arp2/3 complex. Whereas carboxyl-terminal acidic regions from WASp and Scar bind to p21-Arc , Myo3p and 5p bind Arc40p (equivalent to mammalian p41-Arc) through the carboxyl-terminal tryptophan residue . The cortactin NTA region contains a sequence motif (DDW) that is identical to the last three amino acids in Myo3 and Myo5p, and homologous sequences are present in the carboxyl terminus of nearly all WASp/Scar family proteins . The ActA amino terminus contains a similar motif near sequence elements required for the maintenance of Listeria actin tail formation . This sequence in cortactin is conserved across species and is also present in the cortactin-related protein HS1 . We are currently testing if this motif is required for the binding of cortactin to the Arp2/3 complex. Section title: Discussion Educational score: 4.538822174072266 Domain: biomedical Document type: Study Language: en The data presented here indicate that the fourth tandem repeat is necessary for the F-actin binding activity of cortactin and for targeting cortactin to cortical actin structures. The sequence of repeat four is not homologous to other actin binding domains. Isoforms of cortactin are generated by alternative splicing, with cortactin A corresponding to the full-length cortactin form used in these studies. Alternative splicing removes repeat six (cortactin B) or repeats 5 and 6 (cortactin C); all of these isoforms bind F-actin. These results, coupled with the reported inability of the first three cortactin repeats to bind F-actin , support the conclusion that the F-actin binding domain is contained within repeat four. Cortactin variants containing the sixth repeat are reported to cross-link F-actin , an activity that can be downregulated by Src-mediated phosphorylation of cortactin . Furthermore, based on the analysis of the cortactin-related protein HS1 and HS1–cortactin hybrid proteins, the fourth cortactin tandem repeat has been suggested to interact with phosphatidylinositol 4,5-bisphosphate (PIP 2 ), which also downregulates F-actin cross-linking by cortactin . In spite of these data, the mechanism by which cortactin cross-links actin filaments is currently unknown. Section title: Discussion Educational score: 4.588883399963379 Domain: biomedical Document type: Study Language: en The association of cortactin with the Arp2/3 complex and F-actin is likely to impact multiple signaling pathways in a variety of cell types. In addition to linking scaffolding proteins in neuronal and epithelial systems, cortactin may also link the machinery for actin polymerization, Arp2/3 complexes, to signals emanating from the leading edge of motile cells. Ectopic overexpression of cortactin stimulates cell motility that is dependent on Src phosphorylation , suggesting that cortactin contributes to cell migration mediated by Src kinase activity . Cortactin is overexpressed in many cell lines derived from human tumors containing amplification of chromosome 11q13 that posses high metastatic potential and is present within invadopodia in invasive breast cancer cell lines . Invadopodia serve as sites of extracellular matrix degradation by sequestering or secreting various serine and metalloproteinases , and such structures directly correlate with invasive potential. How cortactin overexpression leads to increased motility and metastatic potential is currently unclear. Section title: Discussion Educational score: 4.91748046875 Domain: biomedical Document type: Study Language: en The proper spatial and temporal localization of cortactin with newly forming cortical actin networks is likely to be important for cortactin function. Recently, it was shown that the activity of Rac and the EGF receptor synergize to enhance signaling to the Raf-Mek-Erk pathway leading to cell motility . Therefore, Rac-induced localization of cortactin at sites of Arp2/3 activity may enhance the efficiency of signal transmission at the leading edge by linking growth factor–mediated receptor signals to downstream effector pathways . Activated Arp2/3 complex forms a polarized gradient during neutrophil chemotaxis, becoming concentrated at the migratory front , and is coincident with sites of activation of chemoattractant receptor signaling complexes . Organization of receptor signaling complexes at the leading edge may in part be controlled by Arp2/3 and/or cortactin. EGF-induced cell motility also requires the integration of multiple signaling pathways . Tyrosine phosphorylation of cortactin initiated by EGF , in conjunction with the distribution of cortactin with dynamic cortical actin structures, positions cortactin as a potential mediator of one or more of these signaling pathways. The possibility that cortactin plays a role in the regulation of cortical actin assembly and/or spatial organization of cell surface receptors suggests that cortactin may provide an important site of signal integration between protein tyrosine kinases and the actin cytoskeleton. | Study | biomedical | en | 0.999996 |
0004096 | Section title: Introduction Educational score: 4.406662464141846 Domain: biomedical Document type: Study Language: en Messenger RNA degradation is a mechanism by which eukaryotic cells regulate gene expression and influence cell growth and differentiation . It is dependent upon both cis-elements in the RNA and trans-acting factors. The best-characterized cis-element in mammalian messages is the AU-rich element (ARE) . AREs are present in the 3′ untranslated regions of many mRNAs, including those of proto-oncogenes, cytokines, and lymphokines, and target these RNAs for rapid degradation . Direct or indirect interactions of these sequences with specific protein factors are believed to govern mRNA half-life. Section title: Introduction Educational score: 4.748924255371094 Domain: biomedical Document type: Study Language: en The overexpression of HuR stabilizes messages containing AREs in transient transfection experiments . HuR (or HuA) is a ubiquitously expressed member of the ELAV (embryonic lethal abnormal vision) family of RNA binding proteins , originally identified in Drosophila as essential for neural development . There are three neural-specific Hu family members in mammals: HuB (or HelN1/N2) , HuC , and HuD . All four Hu proteins contain three RNA recognition motifs (RRMs). Both gel shift and UV–cross-linking experiments have provided evidence that HuR binding parallels the in vivo ability of ARE sequences to direct mRNA degradation. ARE recognition appears to be mediated by the first two RRMs of HuR; the third RRM has been suggested to bind the poly(A) tail . In transient transfection assays, deletion of RRM3 alone abolishes HuR's ability to stabilize ARE-containing reporter mRNAs . Although predominantly nuclear, HuR shuttles between the nucleus and the cytoplasm by virtue of a novel shuttling sequence, HNS, located in the hinge region between its second and third RRM . This has led to the suggestion that HuR may initially bind mRNAs in the nucleus and accompany them into the cytoplasm to provide ongoing protection from the degradation machinery. Recent in vivo cross-linking experiments and gradient analyses established that HuR can bind poly(A)+ RNA in both cellular compartments and that a substantial fraction of cytoplasmic HuR is found associated with polysomes . Section title: Introduction Educational score: 4.431858062744141 Domain: biomedical Document type: Study Language: en ARE-mediated mRNA stability is subject to regulation. Cell stress , stimulation , and transformation have all been shown to stabilize ARE-containing mRNAs. Several lines of evidence suggest the involvement of signal transduction pathways. Stimulation of quiescent primary T cells with antibodies directed against CD3/CD28 receptors stabilizes several mRNAs containing AREs . The stabilization of ARE-containing mRNA has been associated with the activation of c-jun NH 2 -terminal kinase, which is correlated with lower decay rates of IL-3 mRNA in mast cells . Stabilization has also been linked to the activation of MAP kinase-activated protein kinase 2 in HeLa cells. Phosphatases, like kinases, have been implicated in ARE-mediated stability. Cyclosporin A, an antagonist of calcineurin (protein phosphatase 2B), destabilizes IL-3 mRNA in autocrine tumor cell lines . However, the molecular details by which any of these pathways impact mRNA stability is not known. Considering the multiple players and their various cellular roles, the mechanisms are likely to be complex. Section title: Introduction Educational score: 4.200992584228516 Domain: biomedical Document type: Study Language: en To begin to understand molecular interactions underlying the regulation of ARE-mediated mRNA stability, we looked for HuR binding partners. Here, we use affinity chromatography to identify four protein ligands to HuR in HeLa cell extracts. All of these proteins contain unusually long acidic stretches at their COOH termini. Although initially recognized in other contexts, three of the ligands have been reported to be inhibitors of protein phosphatase 2A (PP2A) . We have examined the subcellular location and trafficking of these ligands and have delineated the nature of their interactions with HuR. We provide evidence for the in vivo association of HuR with these ligands, as well as data suggesting that their association modulates HuR interactions with ARE-containing mRNAs. Section title: Plasmid Constructions Educational score: 4.233493804931641 Domain: biomedical Document type: Study Language: en Plasmids described in this manuscript were synthesized using oligonucleotides containing restriction sites adjacent to the coding regions. Amplified products were digested with the appropriate enzymes and cloned into their respective vector(s). Two glutathione-S-transferase (GST)–HuR constructs were used. That encoding the protein used in the initial purification was created by amplifying human HuR cDNA from pcDNA3-HuR and inserting it into the BglII and EcoRI sites of pGEX-2TK (Amersham Pharmacia Biotech) to form an in-frame fusion with GST. The second GST-HuR construct and mutants 1–5, used in the in vitro binding assays, have been reported . Like these constructs, the remaining HuR mutants were created by amplifying portions of HuR from pcDNA3-HuR and inserting them into the EcoRI and NotI sites of pGEX-5X-2 (Amersham Pharmacia Biotech) to form in-frame fusions with GST. SETβ, pp32, and acidic protein rich in leucine (APRIL) cDNAs were amplified from a human kidney library (kindly provided by R. Lifton, Howard Hughes Medical Institute, Yale University, New Haven, CT). The cDNAs encoding SETβ and a truncated form of APRIL (amino acids 1–194) were inserted into the EcoRI and NotI sites of pGEX-5X-2. pp32 cDNA was inserted into the BamHI and NotI sites of the same vector. pp32-Flag and APRIL-Flag were created by amplifying their cDNAs with a downstream oligonucleotide containing the eight–amino acid Flag sequence and inserting them into the EcoRI and NotI, or BamHI and NotI sites of pcDNA3, respectively. Truncated portions of pp32 were created by PCR and inserted into the BamHI and NotI sites of pcDNA3. All oligonucleotide sequences can be obtained from the authors upon request. Section title: Recombinant Protein Expression, Purification, and In Vitro Binding Assay Educational score: 3.1307976245880127 Domain: biomedical Document type: Study Language: en Plasmids for bacterial expression were transformed into BL21 cells. The protocol for the growth, induction, and lysis of these cells has been reported . The purification of the GST fusion proteins followed the protocol supplied by pGEX manufacturer (Amersham Pharmacia Biotech). Section title: Recombinant Protein Expression, Purification, and In Vitro Binding Assay Educational score: 3.844750165939331 Domain: biomedical Document type: Study Language: en In vitro transcription and translation was performed using the TNT T7 Quick-Coupled Transcription/Translation System (Promega) in accordance with the protocol supplied by the manufacturer. Section title: Recombinant Protein Expression, Purification, and In Vitro Binding Assay Educational score: 4.134294509887695 Domain: biomedical Document type: Study Language: en For the in vitro binding assays, affinity matrices were prepared by saturating 10 μl of packed glutathione sepharose beads with one of the tagged versions of HuR. After purification, the matrix was reequilibrated with 20 mM Hepes, pH 7.9; 100 mM KCl, and incubated with either 200 μl of HeLa nuclear extract or equimolar amounts of the in vitro translated proteins (as judged by autoradiography) at 4°C for 1 h. The beads were then washed with two 1-ml volumes of the reequilibration buffer. Bound material was eluted with 20 mM Hepes, pH 7.9, and 1 M KCl, and TCA precipitated before loading on gels. Section title: Purification and Identification of HuR Binding Proteins Educational score: 4.233577251434326 Domain: biomedical Document type: Study Language: en After purifying GST-HuR in batch, ∼2.5 mg of the protein (as judged by SDS-PAGE analysis with reference to standards), bound to the glutathione sepharose, was packed into a column and re-equilibrated with 50 column volumes of 20 mM Hepes, pH 7.9, 100 mM KCl. Afterwards, RNase-treated nuclear extract derived from 5.5 × 10 9 HeLa cells was slowly passed over the column (5 ml/h) and the column was washed with reequilibration buffer. Interacting proteins were eluted with a 0.1–2 M KCl gradient. Protein identification was performed by the Keck Foundation Biotechnology Resource Laboratory at Yale University. The peptides recognized by mass spectrometry or Edman sequencing are as follows: SETα: 155–167; SETβ: 11–26, 12–26, 27–44, 45–55, 60–70, 65–70, 71–77, 110–119, 124–137, 142–154, 169–176; pp32: 6–12, 7–12, 21–28, 68–75, 69–75, 100–110, 100–111, 102–111, 111–116, 138–150, 138–153, 238–249; APRIL: 6–12, 7–12, 68–75, 69–75, 76–87, 87–99, 100–110, 100–111, 102–110, 102–111, 117–132, 138–150. Isoelectric points were calculated using the pI/Mw program, part of the Swiss Institute of Bioinformatics' Expert Protein Analysis System. Section title: Purification and Identification of HuR Binding Proteins Educational score: 4.061689376831055 Domain: biomedical Document type: Study Language: en The amount of RNase A used in treating the nuclear extract was determined by titration. Extract was incubated with varying amounts of RNase A at 25°C for 15 min. Subsequently, the RNA in each sample was pCp labeled and its degradation assessed by gel fractionation. 1 μg of enzyme was sufficient to digest the RNA from 10 6 HeLa cells. Section title: Antibodies, Immunoblotting, and Quantitation Educational score: 4.130557537078857 Domain: biomedical Document type: Study Language: en Recombinant GST-SETβ, GST-pp32, and a GST-tagged truncated form of APRIL (amino acids 1–194) were expressed and purified as described above, and dialyzed against PBS. Rabbits were injected with 500 μg of the proteins at 3-wk intervals by Yale University's Immunization Services. Antibodies were purified by affinity chromatography: serum was passed through two columns sequentially, the first containing GST (Amersham Pharmacia Biotech) to remove anti–GST antibodies and the second containing the antigen to obtain the purified antibody. Both columns were prepared by conjugating the proteins to cyanogen bromide–activated Sepharose (Amersham Pharmacia Biotech) following the manufacturer's protocol. The flow-through from the first column was applied to the second. The purified antibodies were obtained by eluting the second column with 100 mM Tris, pH 2.5 . Section title: Antibodies, Immunoblotting, and Quantitation Educational score: 4.0564775466918945 Domain: biomedical Document type: Study Language: en For immunoblot analysis, proteins were fractionated on 12% denaturing polyacrylamide gels and transferred to nitrocellulose. Proteins derived from human tissues were obtained from CLONTECH Laboratories, Inc. The blots were probed as described . The anti–ligand antibodies were used at 1:5,000; the anti–CRM1 (chromosomal region maintenance protein 1; kindly provided by G. Grosveld, St. Jude Children's Research Hospital, Memphis, TN) was used at 1:1,000; the 4B10 anti–hnRNP A1 (kindly provided by S. Pinol-Roma, Mount Sinai School of Medicine; New York, NY) was used at 1:1,000; the 3A2 anti–HuR antibody was used at 1:30,000. The secondary antibody was either HRP-conjugated donkey anti–rabbit or HRP-conjugated donkey anti–mouse. The blots were developed using the ECL system (Amersham Pharmacia Biotech) according to the manufacturer's directions. Section title: Antibodies, Immunoblotting, and Quantitation Educational score: 1.5880295038223267 Domain: biomedical Document type: Other Language: en All quantitations were performed using the National Institutes of Health image 1.62 program. Section title: Cell Lysate, Glycerol Gradient, and Coimmunoprecipitation Assays Educational score: 4.130889415740967 Domain: biomedical Document type: Study Language: en HeLa whole-cell lysate known to preserve complexes was prepared at 1.5 × 10 8 cells/ml as described and was clarified by centrifugation at 40,000 rpm for 40 min. The supernatant was loaded onto 4 ml 5–20% glycerol gradients made with 10 mM triethanolamine, pH 7.9, 100 mM KCl, 5 mM MgCl 2 , and 1 mM DTT, and centrifuged at 40,000 rpm (SW50.1 rotor; Beckman Coulter) for 12 h. Immunoprecipitations were performed from gradient fractions as described , except that each fraction was diluted threefold in lysis buffer before incubation with the antibody. Section title: Cell Culture and Transient Transfections Educational score: 3.4446961879730225 Domain: biomedical Document type: Study Language: en Suspension HeLa cells were obtained from the National Cell Culture Center. Adherent HeLa cells and murine L929 cells were maintained in MEM medium (GIBCO BRL) supplemented with 10% fetal bovine serum (GIBCO BRL). Section title: Cell Culture and Transient Transfections Educational score: 3.682709217071533 Domain: biomedical Document type: Study Language: en Adherent HeLa cells were transfected by incubating them with a calcium phosphate precipitate of the plasmid of interest . Section title: Heterokaryon Formation, Immunofluorescence Microscopy, In Situ Hybridization, and In Vivo Cross-linking Educational score: 3.887817859649658 Domain: biomedical Document type: Study Language: en The immunofluorescence protocol has been reported . The anti–Flag monoclonal antibody M2 (Sigma-Aldrich) was diluted to 10 μg/ml. The anti–Myc monoclonal antibody (Sigma-Aldrich) was used at a 1:1,000 dilution. The polyclonal antibodies were used at ∼5 μg/ml. Two secondary antibodies were used, both at concentrations of 6–8 μg/ml: Alexa 488 conjugated anti–mouse (Molecular Probes) and Texas red conjugated anti–rabbit (Molecular Probes). Hoechst dye 33258 (Sigma-Aldrich) was included with the secondary antibodies at 1 μg/ml. Section title: Heterokaryon Formation, Immunofluorescence Microscopy, In Situ Hybridization, and In Vivo Cross-linking Educational score: 3.1741364002227783 Domain: biomedical Document type: Study Language: en HeLa and L929 fusions were formed as described . Leptomycin B (kindly supplied by M. Yoshida, The University of Tokyo, Tokyo, Japan) was used at a concentration of 10 ng/ml. Section title: Heterokaryon Formation, Immunofluorescence Microscopy, In Situ Hybridization, and In Vivo Cross-linking Educational score: 4.140923500061035 Domain: biomedical Document type: Study Language: en In situ localization of poly(A)+ RNA has been described . For in situ localization of specific mRNAs, the cells were serum starved for 24 h and leptomycin B (LMB) was added (5 ng/μl) 8 h before serum stimulation. 30 min after serum stimulation, the cells were fixed and permeabilized . 5 ng/μl of message-specific oligonucleotide probes [two for c- fos (Calbiochem and gift of J.-L. Veyrune, Institut de Genetique Moleculaire de Montpellier, CNRS, Montpellier, France) and one for GAPDH (Calbiochem)] were used to establish the c- fos and GAPDH mRNA cellular distribution. The probes had been previously 3′-end labeled with digoxigenin according to the protocol supplied by Boehringer. In situ hybridization was performed according to Gallouzi et al. 2000 . Section title: Heterokaryon Formation, Immunofluorescence Microscopy, In Situ Hybridization, and In Vivo Cross-linking Educational score: 2.5990512371063232 Domain: biomedical Document type: Study Language: en In vivo cross-linking was performed as described . Section title: Affinity Purification of Four Previously Identified Proteins as HuR Ligands Educational score: 4.1461076736450195 Domain: biomedical Document type: Study Language: en Potential complexes containing HuR were examined by density gradient centrifugation. HeLa whole-cell extract was prepared in a manner known to preserve complexes , but treated with ribonuclease A (RNase A, see Materials and Methods) to prevent RNA from tethering HuR to other RNA binding proteins. After fractionation on a 5–20% glycerol gradient, immunoblots were probed with the 3A2 anti–HuR monoclonal antibody . This analysis revealed that endogenous HuR (36 kD) is dispersed throughout the gradient , in contrast to the discrete profile of His-tagged recombinant HuR (bottom). These data suggest that HuR in HeLa cells may engage in several complexes not mediated by RNA. Section title: Affinity Purification of Four Previously Identified Proteins as HuR Ligands Educational score: 4.1661834716796875 Domain: biomedical Document type: Study Language: en We used affinity chromatography to identify HuR binding partners. Human HuR cDNA was subcloned downstream of the glutathione-S-transferase coding region to form an in-frame fusion. This fusion protein, which mimics HuR binding to RNA in UV–cross-linking and competition experiments (data not shown), was expressed in bacteria and purified on glutathione sepharose. RNase A–treated HeLa nuclear extract from 5.5 × 10 9 cells was passed over the GST-HuR column and, after washing, bound proteins were eluted with a 0.1–2 M KCl gradient. 1/10 of each fraction was analyzed by SDS-PAGE followed by silver staining , showing several proteins that peak at ∼250 mM KCl. None of these proteins bound to either the GST protein alone or the column matrix (data not shown). The remainder of fractions 5–10 were pooled, run on a second gel, and stained with Coomassie brilliant blue. The four proteins denoted in Fig. 1 , ranging from ∼28 to ∼45 kD, were excised from the gel, treated with trypsin, and analyzed by a combination of mass spectrometry and Edman degradation sequencing (see Materials and Methods). Section title: Affinity Purification of Four Previously Identified Proteins as HuR Ligands Educational score: 3.338799238204956 Domain: biomedical Document type: Study Language: en Each of the four proteins had been previously described and three had been given several different names. We use either the protein's original name or that most commonly seen in the literature: SETα/β , pp32 , and acidic protein rich in leucine . Three of these proteins (SETα, SETβ, and pp32) had been identified as inhibitors of PP2A . Their other reported activities are detailed in the Discussion. Section title: Affinity Purification of Four Previously Identified Proteins as HuR Ligands Educational score: 4.568732738494873 Domain: biomedical Document type: Study Language: en The four HuR binding proteins exhibit striking structural similarity . All contain a long, acidic COOH-terminal tail: residues 242–290 of SETα, 222–277 of SETβ, 164–249 of pp32, and 162–251 of APRIL. These tails probably contribute to the proteins' mobilities in SDS-polyacrylamide gels , which are slower than predicted from their molecular weights . The calculated isoelectric points for SETα, SETβ, pp32, and APRIL are accordingly low: 4.23, 4.12, 3.99, and 3.94, respectively. Beyond their common acidic regions, the HuR binding proteins divide into two subsets: SETα and SETβ, and pp32 and APRIL. SETα and SETβ are identical over their 253 COOH-terminal amino acids, diverging only for the first 37 amino acids of SETα and the first 24 amino acids of SETβ; thus, these proteins are probably splice variants of one another, as previously noted . In contrast, pp32 and APRIL exhibit 71% sequence identity and 81% sequence similarity (gray boxes) and are clearly products of separate genes. They contain a second structural similarity: rev-like leucine-rich repeats in their NH 2 -terminal regions . Section title: Affinity Purification of Four Previously Identified Proteins as HuR Ligands Educational score: 4.099078178405762 Domain: biomedical Document type: Study Language: en We examined the tissue distribution of the four HuR binding proteins in various human tissues by immunoblotting (see Materials and Methods) and found, in agreement with reports on several mammalian species, that each is selectively expressed in certain tissues. The SET proteins are present in brain, heart, lung, spleen, and kidney (data not shown) . pp32 is expressed in brain and kidney (data not shown), as well as in cells capable of self-renewal, such as those of the intestinal crypts and prostate . APRIL is present in brain, kidney, liver, skeletal muscle, and testis (data not shown) . It has been previously observed that HuR is several-fold more abundant in mammalian tissue culture cells that divide most rapidly . This is also true of all four HuR binding proteins (data not shown). Section title: Detection of Complexes between HuR and Its Ligands in Cell Extract Educational score: 4.070183277130127 Domain: biomedical Document type: Study Language: en Polyclonal antibodies were raised against GST-tagged versions of SETβ, pp32, and a portion of APRIL (see Materials and Methods). These antibodies were affinity purified (Materials and Methods) and used to probe the gradient-fractionated, RNase-treated cell extract analyzed in Fig. 1 A. The four ligands are dispersed throughout the gradient , suggesting that they, like HuR, are involved in multiple complexes. As anticipated, polyclonal antibodies directed against SETβ cross-react with SETα. Section title: Detection of Complexes between HuR and Its Ligands in Cell Extract Educational score: 4.122401714324951 Domain: biomedical Document type: Study Language: en The interaction of HuR with its ligands in these gradients was confirmed by immunoprecipitating each fraction with antisera to the various ligands, followed by probing immunoblots of the precipitates with the monoclonal 3A2 anti–HuR antibody . Fig. 3A–C (bottom), reveals that SETα and/or SETβ interact with HuR in complexes with a range of sizes that might include additional proteins. In contrast, pp32- and APRIL-containing complexes are smaller and more discrete (peaking at 50–60 kD), suggestive of simple heterodimeric interactions with HuR. Section title: Detection of Complexes between HuR and Its Ligands in Cell Extract Educational score: 4.143823146820068 Domain: biomedical Document type: Study Language: en Immunoprecipitations performed on unfractionated HeLa whole-cell extract revealed that ∼5% of total cellular HuR binds to each of these four ligands (data not shown). Since no hnRNP A1, another RNA-binding protein that is at least 100-fold more abundant than HuR , was detected in these same precipitates (data not shown), we conclude that HuR's interactions with SETα/β, pp32, and APRIL are highly specific. The converse immunoprecipitation experiments (in which HuR was quantitatively immunoprecipitated and the precipitates probed for the four binding proteins) indicated that SETα, SETβ, pp32, and APRIL are at least 10-fold more abundant than HuR in HeLa cells (data not shown); thus, only a small fraction of each ligand is associated with HuR. Section title: RRM3 and the Hinge Region of HuR Are Required for Ligand Binding Educational score: 4.173435211181641 Domain: biomedical Document type: Study Language: en Previous studies have shown that RRM3 is crucial for HuR's ability to stabilize ARE-containing mRNAs and that its hinge region contains a nucleocytoplasmic shuttling sequence . To ask whether interactions between HuR and SETα, SETβ, pp32, and APRIL involve these important regions, the ligands' binding sites on HuR were mapped by deletion analysis . Eight HuR mutants, designed to preserve conserved domains , were subcloned downstream of GST. The resulting fusion proteins were immobilized on glutathione sepharose, incubated with RNase A–treated HeLa nuclear extract, and their ability to bind the HuR ligands examined by immunoblotting with affinity purified anti–ligand antibodies. Section title: RRM3 and the Hinge Region of HuR Are Required for Ligand Binding Educational score: 4.159020900726318 Domain: biomedical Document type: Study Language: en Fig. 4A and Fig. B , shows that while all four HuR binding proteins interact robustly with GST-tagged full-length HuR (lane 1), removal of HuR's third RRM (lane 2), or longer sequences from the COOH terminus (lanes 3 and 4) completely abolishes ligand binding. This is not the case for deletions that remove the NH 2 terminus or the first RRM of HuR (lanes 5 and 6). More detailed studies revealed that RRM3 alone retains some ability to bind SETα/β and APRIL (lane 8), while the hinge region alone binds reduced amounts of pp32 (lane 9). However, both RRM3 and the hinge are required for efficient retention of all four ligands (lane 7). Section title: RRM3 and the Hinge Region of HuR Are Required for Ligand Binding Educational score: 4.124353408813477 Domain: biomedical Document type: Study Language: en We also investigated which regions of the ligands are necessary for interaction with HuR . Here, in vitro translated ( 35 S-methionine labeled) full-length and NH 2 - and COOH-terminal portions of pp32 (lanes 1–3) were incubated with immobilized GST-HuR, and the bound material was gel fractionated and detected by autoradiography (lanes 4–6). The pattern of retained polypeptides compared with the input reveals that the acidic tail of pp32 (amino acids 168–249) accounts for its interaction with GST-HuR (compare lanes 3 and 6), whereas the NH 2 -terminal portion (amino acids 1–167) does not bind (lane 2 vs. 5). Although we were unable to produce comparable 35 S-labeled subfragments of SET or APRIL for study, it seems probable that these proteins likewise bind HuR via their acidic COOH-terminal tails. Section title: pp32 and APRIL Appear Nuclear, but Shuttle between the Nucleus and the Cytoplasm Educational score: 4.115819454193115 Domain: biomedical Document type: Study Language: en To determine where HuR interactions with its ligands might occur in the cell, immunofluorescence experiments were performed. Confocal microscopy using affinity-purified antibodies showed that the four HuR binding proteins differ in their subcellular location in HeLa cells. Antibodies directed against SETα/β produced nuclear as well as cytoplasmic staining , while pp32 and APRIL appeared strictly nuclear (panels 6 and 8, and 10 and 12, respectively). Two other laboratories have reported that pp32 is nuclear , whereas two have observed cytoplasmic presence as well . HA-tagged versions of both SETα and SETβ have previously been observed to be nuclear when overexpressed in HeLa cells . Section title: pp32 and APRIL Appear Nuclear, but Shuttle between the Nucleus and the Cytoplasm Educational score: 4.124325752258301 Domain: biomedical Document type: Study Language: en Because pp32 and APRIL are nuclear proteins, it was possible to employ the heterokaryon assay to determine whether they, like HuR, shuttle between the nucleus and the cytoplasm. pp32 and APRIL were tagged with the eight amino-acid Flag epitopes at their COOH termini, expressed in HeLa cells, and shown to retain their nuclear location by immunofluorescence . To produce heterokaryons, transfected cells were incubated for 3 h with mouse L929 cells in the presence of cycloheximide (to block translation) and fused using polyethylene glycol. After an additional 3-h incubation period (also in the presence of cycloheximide), the coculture was fixed and immunofluorescence was performed . To distinguish the human and mouse nuclei, the heterokaryons were treated with Hoechst 33258, which stains the human nucleus uniformly and the mouse nucleus in a spotted fashion . Section title: pp32 and APRIL Appear Nuclear, but Shuttle between the Nucleus and the Cytoplasm Educational score: 3.9886655807495117 Domain: biomedical Document type: Study Language: en Like HuR , pp32 (panel 6) and APRIL (panel 9) exhibited shuttling behavior, appearing in both the human and mouse nuclei of heterokaryons. Myc-epitope–tagged hnRNP C1 , a nonshuttling hnRNP protein, provided a negative control (panel 3) . Shuttling may explain the appearance of pp32 in the cytoplasm of some cell types . Section title: pp32 and APRIL Bind CRM1 and Their Nuclear Export Is Inhibited by Leptomycin B Educational score: 4.399850845336914 Domain: biomedical Document type: Study Language: en pp32 and APRIL contain 2 and 3 leucine-rich repeats in the NH 2 -terminal portions of their sequences , respectively. For rev and several other proteins, this motif constitutes a nuclear export signal (NES) that binds to CRM1, a nuclear export receptor . CRM1, in turn, binds Ran GTP and several nuclear pore and nucleoporin-like proteins [e.g., RIP , the CAN/Nup88 complex , NLP-1 ]. LMB, an antifungal and antitumor agent , inhibits nuclear export by covalently modifying a critical cysteine residue in CRM1 , thereby preventing the formation of the trimeric NES-CRM1-RanGTP complex . Experiments performed both in Saccharomyces cerevisiae and with HeLa extract have shown the reaction of leptomycin B with CRM1 to be extremely specific and efficient. Section title: pp32 and APRIL Bind CRM1 and Their Nuclear Export Is Inhibited by Leptomycin B Educational score: 4.174116134643555 Domain: biomedical Document type: Study Language: en To determine whether their leucine-rich repeats enable pp32 and APRIL to interact with CRM1, coimmunoprecipitation experiments were performed . HeLa whole-cell extract was precipitated with polyclonal anti–pp32 or –APRIL antiserum, and immunoblots of the precipitates were probed with a polyclonal anti–CRM1 antibody . Indeed, CRM1 can be detected in the immunoprecipitates . Moreover, complex formation was stimulated in cells treated with LMB (lanes 2 and 4). While only ∼1% of CRM1 was immunoprecipitated by anti–pp32 or –APRIL serum when extract was prepared from cells grown in the absence LMB, ∼5% of CRM1 was coimmunoprecipitated with pp32 and APRIL in extracts derived from cells treated with LMB (see Materials and Methods). Immunoprecipitations performed using the 3A2 anti–HuR and the 4B10 anti–hnRNP A1 monoclonal antibodies provided negative controls. As suggested by their sequences, neither HuR nor hnRNP A1 (data not shown) detectably interacted with CRM1 in extracts prepared from cells grown in the absence or presence of LMB. The converse experiments, in which extracts were immunoprecipitated with an anti–CRM1 antibody and immunoblots of these precipitates were probed with the 3A2 anti–HuR and 4B10 anti–hnRNP A1 antibodies, confirm these results (data not shown). Section title: pp32 and APRIL Bind CRM1 and Their Nuclear Export Is Inhibited by Leptomycin B Educational score: 4.249107360839844 Domain: biomedical Document type: Study Language: en We next asked whether leptomycin B inhibits the nuclear export of pp32 and APRIL, using HuR and hnRNP A1, which lack leucine-rich repeats, as controls. Heterokaryon fusion experiments were performed as above, except that for 12 h before incubation of the coculture, HeLa cells transfected with plasmids encoding pp32-Flag, APRIL-Flag, HuR-Flag , or Myc-hnRNP A1 were incubated with leptomycin B . LMB treatment continued during the coculture and after fusion. Finally, the cells were fixed and immunofluorescence was performed. As expected, the nuclear export of pp32- and APRIL-Flag is significantly inhibited by leptomycin B. While these proteins migrate from the human nucleus to the mouse nucleus in untreated cells , they are restricted to the human nucleus in the presence of LMB . In contrast, Myc-hnRNP A1 and HuR-Flag are capable of shuttling between the nuclei under either condition , confirming that LMB is specific in targeting CRM1-mediated protein export. We obtained the same results when HeLa cells were incubated with LMB for just 3 h before coculturing (data not shown). These data strongly suggest that CRM1 governs the nuclear export of both pp32 and APRIL. Section title: Leptomycin B Increases HuR's Interaction with pp32 and APRIL and Alters HuR's RNA-binding Activity Educational score: 4.1687188148498535 Domain: biomedical Document type: Study Language: en Although LMB does not interfere with the nuclear export of HuR, as judged by heterokaryon fusion assays , we reasoned that the abnormal retention of pp32 and APRIL in the nucleus might nevertheless impact their interaction with HuR. To test this, we examined complex formation between HuR and its ligands after treatment of cells with LMB. HeLa cells were grown in the presence of leptomycin B, whole-cell extract was prepared, and immunoprecipitations were performed with anti–pp32, –APRIL, or –SET antisera. Subsequently, the precipitates were probed on immunoblots with the 3A2 monoclonal anti–HuR antibody . Fig. 8 A shows that, after LMB treatment, the association of HuR with pp32 and with APRIL increases . While only a small amount of HuR coimmunoprecipitated with pp32 and APRIL in extract prepared from cells not treated with LMB (∼5%), six- and fivefold more HuR associated with these ligands in extract derived from LMB-treated cells, respectively (see Materials and Methods). This is not true of the interaction between SETα/β and HuR, as the same amount of HuR was coimmunoprecipitated in the presence and absence of LMB . The cellular levels of pp32, APRIL, and HuR do not change after LMB treatment (data not shown). Section title: Leptomycin B Increases HuR's Interaction with pp32 and APRIL and Alters HuR's RNA-binding Activity Educational score: 4.255188941955566 Domain: biomedical Document type: Study Language: en To ask whether increased interaction with its ligands might alter HuR binding to its target mRNAs, we performed in vivo UV cross-linking in HeLa cells in both the presence and absence of LMB. Subsequently, nuclear and cytoplasmic fractions were prepared and poly(A)+ RNA was purified from each fraction by oligo(dT)-cellulose chromatography using conditions that release noncovalently attached proteins from the RNA . Cross-linked, poly(A)+ RNA/protein complexes were eluted, treated with RNase, and the presence of HuR was detected by probing immunoblots with the 3A2 monoclonal anti–HuR antibody. In the absence of leptomycin B, HuR cross-linked to only cytoplasmic poly(A)+ RNA , as previously observed . We have interpreted this result to mean that HuR binds nuclear mRNA shortly before its export. However, in cells treated with leptomycin B, HuR's RNA binding profile was altered: HuR cross-linked equally to poly(A)+ RNA in both the nucleus and the cytoplasm (lanes 6 and 7, respectively). This is not true of hnRNP A1, which cross-linked to nuclear poly(A)+ RNA in cells grown in both the absence and presence (data not shown) of LMB. Section title: Leptomycin B Increases HuR's Interaction with pp32 and APRIL and Alters HuR's RNA-binding Activity Educational score: 4.484355926513672 Domain: biomedical Document type: Study Language: en Recently, several laboratories have suggested that CRM1 plays a role in mRNA export in S . cerevisiae , Xenopus oocytes, and rat 3Y1 cells . Therefore, it might be argued that the change in HuR's cross-linking profile after LMB treatment simply represents the interaction of HuR with higher levels of poly(A)+ RNA now in the nucleus. However, Fig. 9 A shows that the treatment of HeLa cells with leptomycin B, using the conditions described, does not significantly affect the cellular distribution of total poly(A)+ RNA, as judged by in situ hybridization using a digoxigenin-labeled oligo(dT) probe and a rhodamine-conjugated antidigoxigenin antibody (panels 1–4). Quantitation of these results revealed that nuclear retention of poly(A)+ RNA was only marginally increased (∼3%) after LMB treatment (see Materials and Methods), similar to data reported by other laboratories for S . cerevisiae or the Xenopus oocyte system . We conclude that the nuclear cross-linking of HuR to poly(A)+ RNA in cells treated with LMB represents an in vivo perturbation of HuR's activity, apparently through its interaction with pp32 and/or APRIL. Importantly, while LMB does not significantly alter the distribution of poly(A)+ RNA in HeLa cells, it does alter the localization of an ARE-containing, c- fos mRNA . Cells were serum stimulated to increase the production of c- fos mRNA to detectable levels in the presence or absence of leptomycin B, fixed, and assayed by in situ hybridization using digoxigenin-labeled complementary oligonucleotides (see Materials and Methods). Unlike GAPDH mRNA (compare panel 3 with 4), c- fos mRNA is selectively retained in the nuclei of LMB-treated cells (compare panel 1 with 2). This result was obtained using two different probes to c- fos mRNA, one spanning the 5′ untranslated region (UTR) and proximal coding sequence and one complementary to a region within the 3′ UTR . The somewhat spotted staining pattern of c- fos mRNA has been observed previously for another short-lived mRNA . These data reveal that CRM1 is instrumental in the nuclear export of particular mRNAs (perhaps all ARE-containing mRNAs) at least under certain conditions. Section title: Leptomycin B Increases HuR's Interaction with pp32 and APRIL and Alters HuR's RNA-binding Activity Educational score: 4.037694931030273 Domain: biomedical Document type: Study Language: en Collectively, these data argue that increased in vivo interactions with pp32 and APRIL are associated with a change in HuR's binding to ARE-containing mRNAs in cells treated with leptomycin B. Thus, the HuR ligands we have identified may also modulate HuR function in cells under normal conditions. Section title: Discussion Educational score: 4.288625717163086 Domain: biomedical Document type: Study Language: en We have isolated and characterized four abundant HeLa cell proteins that specifically associate with HuR: SETα, SETβ, pp32, and APRIL. Coimmunoprecipitation of binding protein/HuR complexes from glycerol gradient-fractionated, RNase-treated cell extracts suggests that the aggregates are of relatively low molecular weight, arguing that these proteins (particularly pp32 and APRIL) interact directly with HuR. All four ligands contain highly acidic COOH-terminal tails. Since other human proteins possessing equally long acidic regions exist [e.g., nucleolin , nucleosome assembly protein , transcription factor UBF ], the selection of these proteins argues that their association with HuR reflects more than simply the affinity of an RNA binding protein for a negatively charged polymer. Only the extreme NH 2 -terminal sequences vary between SETα and SETβ, indicating that they are splice variants of one another. pp32 and APRIL exhibit a high degree of sequence identity (71%) and similarity (81%), and both contain several rev-like leucine-rich motifs. Section title: Discussion Educational score: 4.44826602935791 Domain: biomedical Document type: Study Language: en Like HuR, pp32 and APRIL are nucleocytoplasmic shuttling proteins. The leucine-rich repeats present in both proteins hinted that their nuclear export might occur through binding of the nuclear export receptor, CRM1. Coimmunoprecipitation of CRM1 with pp32 and APRIL and the observation that leptomycin B inhibits their shuttling provides confirmation of this export pathway. Interestingly, LMB significantly increases the interaction of pp32 and APRIL not only with CRM1 , but also with HuR. The concomitant increased cross-linking of HuR to nuclear poly(A)+ RNA suggests that the association of HuR with its ligands may modulate HuR's interaction with its target ARE-containing mRNAs in vivo. At least under these conditions, CRM1 appears to be instrumental in the export of an ARE-containing mRNA (c- fos ). Section title: Discussion Educational score: 4.515640735626221 Domain: biomedical Document type: Study Language: en The HuR binding proteins that we have identified appear in the literature in a remarkable diversity of contexts. However, a common finding is that SETα, SETβ, and pp32 are inhibitors of protein phosphatase 2A . Another shared property of the HuR ligands is their association with cell growth or differentiation. The SET proteins have long been implicated as players in leukemogenesis. The SET locus was first identified as a partner in chromosomal translocations with the CAN/Nup214 locus in patients with acute undifferentiated leukemia . Subsequently, SETβ was observed in a complex with HRX leukemic fusion protein and PP2A in myeloid leukemic cell extracts . SETα and SETβ have been shown to associate with histone/DNA complexes (as template activating factor 1, TAF1, α and β) and to remodel chromatin structure in vitro . Both SETβ and pp32 were purified as HLA class II–associated proteins from human lymphoblastoid cells . pp32 is expressed at high levels in neoplastic cells, as well as in normal tissues competent for self-renewal . The pattern of expression of pp32 (LANP) during postnatal development suggested a role in the differentiation of cerebellar neurons . More recently, pp32 has been observed to inhibit the oncogene-mediated transformation of rat embryo fibroblasts and to be present in intranuclear aggregates of mutant ataxin-1 that are associated with , but not essential for , neural degeneration. Alternatively, pp32 has been observed to associate with microtubule-associated proteins, including MAP2, MAP4, and tau in vitro . APRIL was only recently identified and therefore has not been well studied. Section title: Discussion Educational score: 4.462613105773926 Domain: biomedical Document type: Study Language: en Our localization of the protein domains responsible for intermolecular contacts with HuR suggests that the ligands' common acidic tails are critical . In the case of pp32, both its acidic region (amino acids 168–249) and other portions of the molecule have been previously demarcated as functionally important. Amino acids 150–174 of pp32 are absolutely required for inhibiting the appearance of transformed foci elicited by oncogene pairs , whereas the first 147 amino acids of pp32 (as LANP) interact with ataxin-1 . pp32's ability to modulate the interaction of microtubule-associated proteins with microtubules (as mapmodulin) has also been assigned to its acidic, COOH-terminal domain . In SET, it is the NH 2 -terminal region that is required for PP2A inhibitor activity, whereas the acidic tail contributes to chromatin remodeling in vitro . The acidic tail of SET is included in the SET-CAN fusion implicated in leukemogenesis . Thus, some of these ligand interactions with other cellular molecules would be predicted to be compatible with, while others would be expected to compete with, their binding to HuR. This remains to be investigated. Section title: Discussion Educational score: 4.687420845031738 Domain: biomedical Document type: Study Language: en In HuR, deletion of the third RRM alone abrogates recognition by SETα, SETβ, pp32, and APRIL, whereas the hinge region and RRM3 comprise a minimal substrate for efficient interaction with these ligands . Previously, these two domains have been characterized as being important for HuR's ability to shuttle between the nucleus and the cytoplasm and to bind and stabilize ARE-containing mRNAs , respectively. Interestingly, the third RRM has been shown to be important for the functioning of other Hu-family proteins. For instance, RRM3 is not only essential for the full activity of HuB and HuC in inducing a neuronal phenotype upon overexpression in PC12 cells, but it also acted as a dominant negative protein when cotransfected with wild-type HuB or HuC, and even when expressed in vivo in cells of the embryonic central nervous system . Similarly, RRM3 and the hinge region of HuD were observed to be important for neurite-inducing activity in PC12 cells . While it remains to be shown that HuB, HuC, and HuD associate with the HuR ligands we have characterized, the high degree of conservation among the Hu proteins suggests that interaction is likely. Perhaps by binding to HuR and the other Hu proteins, the HuR ligands facilitate the stabilization of ARE-containing mRNAs and promote cellular differentiation. Section title: Discussion Educational score: 4.304643630981445 Domain: biomedical Document type: Study Language: en It was surprising to find that leptomycin B produces greater cross-linking of HuR to nuclear poly(A)+ RNA , even though CRM1 does not directly bind HuR, and LMB fails to inhibit HuR shuttling . Specifically, increased ligand binding induced by LMB might have been expected to decrease HuR shuttling (perhaps by sequestering HNS), leading to greater retention of HuR in the nucleus and therefore greater cross-linking to nuclear RNA. However, it is not clear from our experiments whether interaction with its ligands does not in fact alter the nucleocytoplasmic trafficking of HuR: even though ∼50% of HuR is bound by pp32 and APRIL in the presence of leptomycin B , shuttling of the remaining free HuR could generate the positive result obtained in the heterokaryon experiment of Fig. 7 . Rather, our observation of HuR shuttling in the presence of LMB can only be interpreted to indicate that the HNS of HuR (which contains no leucine-rich repeats) is not recognized by CRM1 , but instead by some other export receptor. Section title: Discussion Educational score: 4.397200584411621 Domain: biomedical Document type: Study Language: en An alternative explanation for the LMB-induced increase in the cross-linking of HuR to nuclear poly(A)+ RNA is that ligand binding stabilizes HuR's interaction with ARE-containing mRNAs. Since in vivo cross-linking experiments like those in Fig. 8 B have demonstrated that SETα/β, pp32, and APRIL do not detectably bind RNA directly (data not shown), the change is likely to be propagated through RRM3 of HuR. Conceivably, the acidic tail of a ligand could occupy the RNA binding site of RRM3 and thereby displace the poly(A) tail of the mRNA. It is not clear whether this would decrease or increase HuR binding to ARE-containing mRNAs since the other two RRMs of HuR have been reported to have much higher affinity and to be specific for ARE sequences . Further analyses are required. It will also be important to establish whether interaction with its ligands under normal conditions enhances HuR's binding to ARE-containing mRNAs, as it appears to do in the presence of leptomycin B . Section title: Discussion Educational score: 4.313434600830078 Domain: biomedical Document type: Study Language: en Whatever the mechanism, LMB clearly causes the selective retention of c- fos , an ARE-containing mRNA, in the nucleus . Such abnormal nuclear retention could also be the basis for numerous reports of ARE-containing mRNA stabilization in cells subjected to various stress conditions . Clearly, it is most important to investigate in each case whether the mRNA has been transported to the cytoplasm and is undergoing translation. Although it is possible that leptomycin B inhibits other components of the nucleocytoplasmic trafficking machinery, the most straightforward interpretation of the results in Fig. 9 B is that CRM1 is involved in c- fos mRNA export via HuR interaction with its ligands. Thus, CRM1 should be considered a potential nuclear export receptor for certain cellular mRNAs (perhaps all ARE-containing messages). The possibility that the dominant export pathway for a particular mRNA may switch, subject to changes in cellular physiology, is also important to consider. Section title: Discussion Educational score: 4.581909656524658 Domain: biomedical Document type: Study Language: en Our discovery of three HuR ligands (SETα, SETβ, and pp32) that were previously identified as inhibitors of protein phosphatase 2A suggests that a regulatory cascade involving this enzyme influences ARE-mediated mRNA stability. PP2A is a highly conserved multimeric serine/threonine phosphatase, affecting a wide variety of cellular functions, including cell cycle progression, DNA replication, transcription, splicing, development, and morphogenesis . It is dynamic, capable of reversible interconversion between holoenzyme forms in response to stimulation . PP2A's ability to dephosphorylate both the substrates of kinases and kinases themselves appears to be an important aspect of its function. This phosphatase regulates the activities of several major protein-kinase families, including those of the AGC subgroup [e.g., protein kinase B , protein kinase C , and p70 S6 kinase ], the calmodulin-dependent kinases , and members of the ERK MAP-kinase pathway . The recent demonstration that expression of MAP kinase–activated protein kinase 2 leads to stabilization of ARE-containing mRNAs highlights the importance of particular kinases. Our data argue that in these signaling cascades, the HuR binding proteins act directly on HuR, with PP2A placed farther upstream in the pathway(s) that regulate ARE-mediated mRNA stability. Section title: Discussion Educational score: 3.776688814163208 Domain: biomedical Document type: Study Language: en Since we and others have observed that pp32 and APRIL are phosphoproteins (data not shown) , it is tempting to speculate that their shuttling, or their interactions with HuR, could be regulated by phosphorylation. This idea is particularly attractive since our attempts to identify a phosphorylated form of HuR have been unsuccessful (data not shown). Experiments are currently underway to determine whether particular phosphorylated forms of pp32 and APRIL selectively bind and influence the activity of HuR. | Study | biomedical | en | 0.999997 |
0004111 | Section title: Introduction Educational score: 4.75501823425293 Domain: biomedical Document type: Study Language: en Receptor-mediated endocytosis for recycling receptors is a process in which a ligand binds to its cell surface receptor and is internalized via clathrin-coated vesicle formation . After uncoating, this vesicle acidifies , leading to dissociation of ligand from receptor . Subsequently, the endocytic vesicle undergoes a series of fissions, resulting in the eventual segregation of ligand from receptor . After segregation, daughter vesicles destined to recycle to the cell surface contain a majority of receptor and a reduced content of ligand. Other vesicles, which have much less receptor in proportion to the content of ligand , traffic through the cell to the lysosome, where ligand is degraded . Little is known about the mechanism by which endocytic vesicles undergo fission and sort ligand from receptor, although previous studies suggested a role for microtubules (MTs) and molecular motors in this process and/or in movement of ligand-enriched late endosomes toward lysosomes . Here, we provide the first in vitro reconstitution of the process of segregation of ligand and receptor in early endocytic vesicles. We show that it is necessary for presegregation vesicles to bind to and move along MTs to undergo fission. This results in a daughter vesicle enriched in ligand relative to receptor and a second daughter with a majority of receptor. Motility and fission are established by the addition of ATP. Although vesicle motility both before and after fission can be either toward the plus or minus end of the MT, these processes are prevented by addition of 5′-adenylylimido-diphosphate (AMP-PNP), an inhibitor of kinesins, or by incubation with kinesin antibody, but not by addition of vanadate, an inhibitor of cytoplasmic dynein, or dynein antibody. Additionally, kinesins are immunolocalized to many of the presegregation vesicles bound to MTs, whereas dynein is rarely seen in this vesicle population. Fission under these conditions evidently requires MTs and kinesin-based motors; whether other motors and related proteins are necessary is not known. However, these observations establish a well-defined system in which additional vesicle-associated and cellular factors that are required for segregation can be identified and their functional roles dissected. Section title: Reagents Educational score: 3.49408221244812 Domain: biomedical Document type: Study Language: en Mouse mAbs (IgM) against the dynein intermediate chain (clone 70.1) and the kinesin I heavy chain (clone IBII) were obtained from Sigma-Aldrich. Mouse mAbs (IgG) against the amino termini of the dynein intermediate chain and the kinesin I heavy chain were obtained from Chemicon International, Inc. Specificities were confirmed by immunoblot. Nonimmune mouse IgG was obtained from Sigma-Aldrich. Affinity purified rabbit IgG was prepared against a KLH-linked peptide (VNRWACERKRDITYC) corresponding to a sequence on the cytoplasmic tail of the rat asialoglycoprotein receptor (ASGPR) H 2 subunit . All reagents were from Sigma-Aldrich unless otherwise noted. Section title: Motility Assay Educational score: 4.094700813293457 Domain: biomedical Document type: Study Language: en Texas red–labeled early endocytic vesicles were prepared from livers that were removed from male Sprague-Dawley rats (200–250 g; Taconic Farms) 5 min after i.v. injection of Texas red asialoorosomucoid (ASOR) . All procedures were approved by the University Animal Use Committee. After Dounce homogenization, a postnuclear supernatant was prepared and chromatographed on a Sephacryl S200 column. Vesicle-enriched peaks were pooled and subjected to centrifugation (200,000 g for 135 min) on a sucrose step gradient consisting of 1.4, 1.2, and 0.25 M sucrose. Vesicles were harvested from the 1.2/0.25 M sucrose interface and stored at −80°C until used. Details of these procedures have been published recently . Section title: Motility Assay Educational score: 4.133301734924316 Domain: biomedical Document type: Study Language: en Motility assays were performed in a chamber consisting of two pieces of double-stick tape sandwiched between optical glass; the internal volume was ∼3 μl. The chamber was coated with an affinity-purified mixture of liver motor proteins as described previously . After three 15-μl washes with modified motility buffer (35 mM Pipes, 5 mM MgCl 2 , 1 mM EGTA, 0.5 mM EDTA, 4 mM DTT, 20 μM taxol, 2 mg/ml BSA, pH 7.4, containing an oxygen scavenging system), taxol-stabilized MTs were added and incubated for 3 min. After another three washes with motility buffer containing 5 mg/ml casein, endocytic vesicles were added to the chamber, incubated for 10 min, and washed. In some experiments, as indicated below. 0.02 mg/ml DEAE-dextran (Amersham Pharmacia Biotech), rather than motor proteins, was used to adhere MTs to glass, as described previously . Motility was initiated with the addition of 50 μM ATP in the absence of a regenerating system. In some experiments, as indicated below, 4 mM ATP was used. Section title: Image Analysis Educational score: 4.064186096191406 Domain: biomedical Document type: Study Language: en Imaging was performed at the Analytical Imaging Facility of the Albert Einstein College of Medicine. A 60×, 1.4 numerical aperture planapo objective was used on an Olympus 1X70 inverted microscope, containing automatic excitation and emission filter wheels connected to a Photometrics charge-coupled device camera run by IPLab Spectrum software (Scanalytics) running on a Power Macintosh. IPLab Spectrum scripting software was used to collect images rapidly and to switch between fluorescence channels. Images were also recorded directly on videotape. In all experiments the microscope stage was maintained at 35°C. Videos were digitized with the use of the Scion Image (Scion Corporation) movie-making macro (1 frame/s) and saved as tiff files. Integrative density of fluorescence was determined using the density slice and wand option of Scion Image. Each quantification of fluorescence density was the average of three determinations performed on each vesicle. Section title: Preparation of Early Endocytic Vesicles Educational score: 4.128167152404785 Domain: biomedical Document type: Study Language: en Fluorescent endocytic vesicles were prepared from rat liver that was surgically removed 5 min after intravenous injection of 3 mg of Texas red–labeled ASOR . This short time period was chosen so that events early in the endocytic pathway could be examined. As anticipated, ASGPR was present in >95% of the ASOR-containing vesicles, as determined by fluorescence microscopy using anti-ASGPR IgG to localize receptor . This observation indicates that these ligand-containing vesicles represent a population of early (i.e., presegregation) endosomes. Partial purification of vesicles was performed to minimize nonspecific ATPase activity as described previously . Section title: Assay of Vesicle Motility and Fission Educational score: 4.369037628173828 Domain: biomedical Document type: Study Language: en Labeled endocytic vesicles were drawn into a 3-μl microscopy chamber in which taxol-stabilized, rhodamine-labeled MTs had been attached to the glass surface after coating of the glass with an affinity-purified mixture of liver motor proteins, as described previously . Many vesicles bound to the MTs. Upon addition of 50 μM ATP, examination of >800 MT-attached vesicles revealed that 25% immediately became motile . These vesicles moved along the MTs at ∼0.7 μm/s, as described previously . Vesicles that were bound to the glass surface and not to MTs did not move upon ATP addition. Of the motile vesicles, 13% underwent fission, resulting in two daughter vesicles. None of the nonmotile vesicles underwent fission. During fission, one pole of the vesicle remained attached to the MT, whereas the opposite end advanced. Fission occurred over the next 10–15 s. In companion experiments , vesicles were incubated for 1 min at 4°C with affinity-purified anti-ASGPR IgG or with nonimmune IgG. Cy2-labeled secondary antibody was then added, and incubation was continued for an additional 6 min. These vesicles were then drawn into a microscopy chamber as above. IgG addition nonspecifically reduced motility slightly, but had no effect on the ability of moving vesicles to undergo fission. In vesicles to which nonimmune IgG was added, motility was reduced to 19%, but 17% of these vesicles underwent fission. Of >1,200 MT-bound, receptor-tagged vesicles that were examined, 16% moved upon addition of ATP and 14% of these vesicles underwent fission . Similar to results in the absence of receptor antibody, only MT-bound vesicles moved upon ATP addition, and none of the nonmotile vesicles underwent fission. Representative frames from one of these studies are shown in Fig. 3 . Before addition of ATP (0 s), the arrowhead points to a single spherical vesicle that is attached to an MT and contains ligand and receptor. Within 8 s of ATP addition, this vesicle elongates along the MT and forms a constriction in the middle. Although there is substantial ligand in both portions of this vesicle, ∼97% of receptor is present in only one (arrowhead). By 19 s, the original vesicle has clearly separated into two vesicles of near equal size, with segregation of receptor to the vesicle on the right. In this case, the receptor-containing vesicle is motile, whereas the vesicle containing essentially only ligand appears to be fixed to the MT. Section title: Quantification of Vesicle Fission Educational score: 4.125581741333008 Domain: biomedical Document type: Study Language: en Distribution of fluorescence was quantified in vesicles before and after fission. In nonmotile vesicles that did not undergo fission, recovery of fluorescence was essentially unchanged over time. When a group of 30 nonmotile vesicles was examined before and 10 s after ATP addition, recovery of ligand fluorescence was 105 ± 4.5% (mean ± SEM) of the initial level. In a group of 20 nonmotile vesicles labeled with fluorescent antibody to the receptor, recovery of fluorescence was 99.3 ± 1.1%. Similarly, fluorescence due to ligand or receptor in motile vesicles before fission was recovered completely in the daughter vesicles. In motile vesicles that underwent fission, as seen in Table , ligand was distributed in the daughter vesicles after each fission event. In most cases, the distribution was uneven. On average, the more fluorescent of the two vesicles contained ∼67% of the ligand in the original vesicle. In a few cases, ligand was about equally distributed. In this series of experiments, distribution of receptor was not monitored. Section title: Quantification of Segregation of Ligand and Receptor Educational score: 4.111327171325684 Domain: biomedical Document type: Study Language: en To examine whether sorting of ligand from receptor occurred in this in vitro system, similar experiments were performed in which the receptor was tagged with a fluorescent antibody. In these experiments, in motile vesicles that underwent fission, on average ∼90% of receptor was segregated to one of the two daughter vesicles ( Table ). In approximately half of these fission events, sorting of receptor into one daughter was virtually complete. The other daughter vesicle with ∼10% of the receptor contained almost 40% of the ligand ( Table ). Section title: Quantification of Segregation of Ligand and Receptor Educational score: 4.0055999755859375 Domain: biomedical Document type: Study Language: en From a consideration of the geometry of vesicle–MT interaction, the possibility that two overlapping vesicles move to produce a spurious fission seems remote. To test this, we diluted the vesicle population by 50% before binding to MTs. This would be expected to reduce considerably the possibility of overlap. As seen in Table and Fig. 4 , there was no reduction in motility and fission in experiments in which diluted vesicles were used. Section title: Roles of Molecular Motors in Endocytic Vesicle Motility and Fission Educational score: 4.112279415130615 Domain: biomedical Document type: Study Language: en Experiments were performed to determine whether kinesins or cytoplasmic dynein were associated with endocytic vesicles. In these studies, rhodamine-labeled MTs were attached to the chamber surface using DEAE-dextran as we have described previously . Vesicles were incubated for 3 min at 4°C with mouse mAb IgM against kinesin or cytoplasmic dynein. FITC-labeled goat anti–mouse IgM was then added and incubation was continued for an additional 6 min. These vesicles were then drawn into a microscopy chamber as above. As seen in Fig. 5 , kinesin antibody colocalized with endocytic vesicles that contained Texas red–ASOR; many of these vesicles appear to be bound to MTs. Although antibody to cytoplasmic dynein was clearly associated with vesicles, most of which were attached to MTs, few of these vesicles contained ASOR; those containing ASOR apparently colocalize with MTs . Section title: Roles of Molecular Motors in Endocytic Vesicle Motility and Fission Educational score: 4.16389274597168 Domain: biomedical Document type: Study Language: en We also examined the effects of antikinesin and antidynein IgG and IgM antibodies on endocytic vesicle motility and fission . Motility and fission in the presence of either IgG or IgM antibodies to dynein were identical to controls. In contrast, in vesicles treated with IgG or IgM antibodies to kinesin, motility and fission were markedly reduced. There was no motility of vesicles that had been treated with antikinesin IgM, and only 4% of vesicles were motile when treated with antikinesin IgG compared with 19% of vesicles that had been treated with nonimmune IgG. Of the small number (26) of motile vesicles in this group, only 3 (11%) underwent fission. Although this rate of fission is similar to that seen in control studies, the actual number of fission events is markedly reduced. The finding of a small residual amount of fission suggests either that inhibition of kinesin function is incomplete in this experiment or that a nonkinesin motor is functional in this small population of vesicles. Section title: Roles of Molecular Motors in Endocytic Vesicle Motility and Fission Educational score: 4.124066352844238 Domain: biomedical Document type: Study Language: en Studies were also performed to examine the effect of vanadate, an inhibitor of cytoplasmic dynein , and AMP-PNP, an inhibitor of kinesins , on vesicle motility and fission. Polarity-marked MTs were used in these studies. In previous studies performed in an MT gliding assay, we found that 1 mM AMP-PNP inhibited 80% of plus end–directed movement and only 20% of minus end–directed movement; 5 μM vanadate had the opposite effect. As seen in Table , upon addition to the microscopy chamber of 50 μM ATP in the absence of inhibitor, there were approximately equal amounts of minus and plus end–directed vesicle movements. Simultaneous addition of 50 μM ATP and 5 μM vanadate had no effect on the number or direction of motile vesicles. Vesicle fission and directional movement of daughter vesicles were also unaffected by vanadate inclusion. In contrast, simultaneous addition of 50 μM ATP and 1 mM AMP-PNP eliminated vesicle motility and fission ( Table ). Section title: Roles of Molecular Motors in Endocytic Vesicle Motility and Fission Educational score: 4.217449188232422 Domain: biomedical Document type: Study Language: en There is a possibility that proteins in the motor fraction that were used to coat the glass to which MTs were attached could bind substantial amounts of vanadate, thus falsely suggesting that it is without effect in the endosomal motility and fission assays. For this reason, a series of experiments was performed using MTs that were bound to glass via DEAE-dextran . In addition, studies were performed at ATP concentrations of either 50 μM or 4 mM, in the absence of a regenerating system. As seen in Fig. 6 , vanadate at concentrations as high as 20 μM in the presence of 4 mM ATP had no effect on vesicle motility or fission. Under these conditions, vanadate should preferentially inhibit dynein function. At concentrations of 50 μM or above, motility was reduced. Although the fraction of fission events in motile vesicles remained unchanged , the absolute number of fissions was reduced in proportion to the reduction in motility. At these high concentrations, vanadate is known to inhibit many ATPases, including kinesins . Vesicle motility was markedly reduced by 1 mM AMP-PNP even at high (4 mM) concentrations of ATP, and no fission events were seen. Under these conditions, AMP-PNP should preferentially inhibit function of kinesins. Section title: Discussion Educational score: 4.433035850524902 Domain: biomedical Document type: Study Language: en These studies indicate that early endocytic vesicles have the capacity to undergo fission and receptor–ligand segregation in vitro, which is dependent on their binding to MTs and on their motility in the presence of ATP. Previous studies suggested that molecular motors might be involved in the interaction of endocytic vesicles with MTs . However, a system for direct examination of the role of molecular motors in endocytic vesicle segregation and fission has been lacking. Here we show that early endocytic vesicles, as defined by the presence of both ligand and receptor in the same vesicle, are associated with kinesins, but not cytoplasmic dynein, and are capable of movement, receptor–ligand sorting, and fission in vitro. ATP-mediated motility of these vesicles is inhibited by antibodies to kinesins, but not by antibodies to cytoplasmic dynein. In addition, inclusion of vanadate at concentrations as high as 20 μM does not interfere with vesicle motility or fission, whereas inclusion of 1 mM AMP-PNP prevents both of these processes, even when ATP is in molar excess. When motility of vesicles was quantified on polarity-marked MTs, vesicles were seen to move approximately equally towards plus and minus ends. AMP-PNP inhibited motility in both directions, consistent with the possibility that plus and minus end–directed kinesins mediate vesicle motility and fission . Each fission event was associated with movement of only one daughter vesicle after fission along the MT. This could be an attribute peculiar to the method of preparing the endosomes. Section title: Discussion Educational score: 4.401381015777588 Domain: biomedical Document type: Study Language: en Several previous investigations indicated that segregation of ligand from the ASGPR is incomplete, and that ∼20% of internalized ligand returns with the receptor back to the cell surface (diacytosis) . However, here, substantial amounts of ligand remained associated with receptor in one daughter vesicle after fission ( Table ). We recently demonstrated that movement of early endocytic vesicles along MTs is oscillatory, with frequent stops and changes in direction . This could facilitate the distribution of these vesicles throughout the cytoplasm and may aid in the separation of ligand from receptor by allowing receptor-enriched daughter vesicles to undergo subsequent rounds of fission. Specifically, a more complete segregation of ligand from receptor, as occurs in vivo before recycling, could be achieved by an iterative process whereby the receptor-enriched daughter vesicles undergo multiple rounds of fission, as has been suggested by Maxfield and colleagues . Consistent with our in vitro data, each event would generate a new complementary daughter vesicle containing ∼40% of the ligand of the parent vesicle with little receptor. Eventually, this vesicle would move toward and fuse with lysosomes where ligand would be degraded. Section title: Discussion Educational score: 4.423311233520508 Domain: biomedical Document type: Study Language: en This in vitro system has been optimized to study components that are required for processing of endocytic vesicles. Nevertheless, the percentage of MT-attached vesicles that become motile in our studies is somewhat less than might be predicted, which suggests that critical factors required for reconstitution of motility may be lost or that conditions of reconstitution are still suboptimal . An important component of this system is the use of a liver-derived endocytic vesicle preparation that is depleted of contaminating ATPase activity . This has permitted reconstitution of vesicle motility and sorting at ATP concentrations as low as 50 μM, as used here. Although these low levels of ATP suggest that this nucleotide would not normally be limiting or regulatory in these processes, it is possible that higher levels are needed in vivo due to competition for ATP with other cellular reactions, as we have hypothesized previously . Our earlier studies suggested that late endocytic vesicles are associated with cytoplasmic dynein as they traffic to the lysosome along MTs. Another recent publication suggests a role for dynein in movement of late endocytic vesicles and in the transition of early to late endocytic vesicles, but not in early endosomal vesicle trafficking or receptor recycling . When proteins were examined in endosomal populations that were isolated from rat liver, it was found that early and recycling endosomes were enriched in kinesin and dynein, whereas late endosomes were enriched in dynein only . It should be noted that none of these studies preclude functional association of other proteins and multiple motors with endocytic vesicles as well. The immunomicroscopic technology described here may allow determination of a point in which motility of endocytic vesicles undergoes a switch from kinesin to dynein dependency and should provide a means to identify and characterize other proteins that may be involved in vesicle movement and processing. | Study | biomedical | en | 0.999998 |
0005018 | Section title: Introduction Educational score: 4.580744743347168 Domain: biomedical Document type: Study Language: en The ends of eukaryotic chromosomes (telomeres) consist of simple DNA repeats associated with proteins and play important roles in replication, malignant transformation, and cellular aging . Besides the vital functions telomeres perform in vegetative cells, they have been implicated as key players in the chromosome pairing process during meiosis . The pairing of homologues and their recombination during meiotic prophase is critical for reduction of DNA content from diploid to haploid before fertilization . In many organisms, the premeiotic (vegetative) telomere distribution is altered during the onset of prophase I such that telomeres attach to the inner nuclear membrane, and then move along it to cluster in a limited nuclear envelope sector at the onset of zygotene . This bouquet formation is thought to contribute to homologue alignment and pairing during first meiotic prophase . Section title: Introduction Educational score: 4.484771728515625 Domain: biomedical Document type: Study Language: en In Schizosaccharomyces pombe , loss of the telomere protein taz1(+) has been shown to abrogate meiotic telomere clustering and to virtually eliminate recombination and ordered disjunction of chromosomes at metaphase I in this asynaptic organism . Furthermore, abrogation of nuclear movements during and after telomere clustering impairs chromosome pairing at meiosis . An essential role for telomeres in the reductional division is underlined by the observation that deletion of telomeres and the resulting rearrangement of linear chromosomes into rings is compatible with vegetative growth, but obstructs meiosis and hence sexual reproduction . Section title: Introduction Educational score: 4.512756824493408 Domain: biomedical Document type: Study Language: en Numerous cytogenetic studies have shown that a congregation of telomeres occurs in the vicinity of the centrosome or spindle pole body (SPB), during the leptotene/zygotene transition . Genetic and cytological analyses of the meiotic prophase of Saccharomyces cerevisiae have suggested that the formation of a chromosomal bouquet in the synaptic meiosis of budding yeast occurs independent of homologous recombination and synapsis . In the vegetative (premeiotic) yeast nucleus, telomeres associate in a few aggregates at the nuclear periphery , while centromeres form a single cluster near the SPB . During the leptotene/zygotene equivalent stage of yeast meiosis, centromeres disperse from the SPB and telomeres cluster de novo at this location . Hence, the bouquet stage of yeast resembles the classical bouquet arrangement formed during prophase I of multicellular eukaryotes . Section title: Introduction Educational score: 4.333212852478027 Domain: biomedical Document type: Study Language: en Genetic analysis of disomic S . cerevisiae strains has indicated that telomeres play an important role for homologue search and alignment in synaptic meiosis . The only telomeric protein that has been shown to be involved exclusively in meiosis of budding yeast is Ndj1p/Tam1p . NDJ1 is expressed specifically after induction of meiosis. Ndj1p-deficient cells show reduced efficacy in homologue disjunction, increased occurrence of nonrecombinant chromosomes (a defect in crossover interference), and a delayed progression through meiotic prophase . Furthermore, spread meiocytes show a disordered distribution of telomeric Rap1p . In the present study, we investigate the extent to which bouquet formation, telomere positioning, and chromosome pairing are affected in ndj1Δ meiosis and cytologically identify Ndj1p as the first telomeric protein demonstrated to be required for bouquet formation in a synaptic organism. Section title: Yeast Strains Educational score: 4.182132720947266 Domain: biomedical Document type: Study Language: en To exploit the relatively synchronous progress of SK1 meiosis , the NDJ1 locus was disrupted in the S . cerevisiae SK1 strain background by replacing the sequence coding for amino acids 13–252 with the KanMX4 cassette . SK1 yeast transformants resistant to G418 were isolated by A. Goldman and M. Lichten and were assigned the strain name MDY1490 (MATa ura3 lys2 leu2 ho::LYS2 ndj1::KanMX6) and MDY 1493 (MATα ura3 lys2 trp1 ho::LYS2 ndj1::KanMX6). These strains were mated and diploids were selected on the appropriate media (trp−;leu−). Wild-type SK1 haploids were mated to result in a diploid wild-type control strain. These strains were used in all fluorescence in situ hybridization (FISH) experiments. Section title: Yeast Strains Educational score: 3.643089771270752 Domain: biomedical Document type: Study Language: en Immunolocalization of telomeres by immunofluorescence to hemagglutinin (HA)-tagged Ndj1p was done in a diploid strain derived from MDY431 and MDY 433 . Section title: Cell Culture and Preparation Educational score: 4.184884548187256 Domain: biomedical Document type: Study Language: en For nuclear preparation, cultures were grown in presporulation medium to a density of 2 × 10 7 cells/ml, and then transferred to sporulation medium (2% KAc) at a density of 4 × 10 7 cells/ml . Aliquots from the sporulating cultures were obtained during time course experiments at induction of meiosis (transfer to sporulation medium = 0 min) and from 180 to 420 min at 10- or 20-min intervals. Aliquots were immediately transferred to tubes on ice containing 1/10 vol acid-free 37% formaldehyde (Merck). After 30 min, cells were removed from fixative, washed with 1× SSC and spheroplasted with Zymolyase 100T (100 μg/ml; Seikagaku) in 0.8 M sorbitol, 2% KAc, and 10 mM dithiothreitol. Spheroplasting was terminated by adding 10 vol ice-cold 1 M sorbitol. To allow for the delay in meiotic prophase progression in ndj1Δ , we additionally sampled at 5, 6, and 7 h. Spheroplasts were subdivided in two aliquots and subjected either to nuclear spreading or to preparation of structurally preserved nuclei. The latter were obtained as described by Trelles-Sticken et al. 1999 and subjected to FISH. Section title: DNA Probes and Labeling Educational score: 4.347146511077881 Domain: biomedical Document type: Study Language: en A composite pancentromeric DNA probe was used to delineate all yeast centromeres . One plasmid containing a conserved core fragment of the subtelomeric X element and one containing the Y′ element were used to probe all yeast telomeres . The following cosmid probes were used to determine the pairing of homologous chromosome regions: the telomere-adjacent region on the right arm of XI was probed with a cosmid (cos) located at 628.5–665.8 (cos l ; pUKG066). The smaller chromosomes IX and III were tagged with cosmid probes hybridizing internally on the left arm of chromosomes IX and at HML near the left telomere of chromosomes III . An internal chromosomes XI cosmid mapping 231.8–264.9 on the left arm of chromosomes XI was used to monitor meiotic pairing at a telomere-distant chromosomal region . Chromosome condensation was measured by determining the distance between cosmid b (pUKG 040) and cos h (pEKG 011) on the left arm of XI . Chromosome morphology was monitored by FISH with a painting probe for chromosomes XI , which covers 338 of the 666 kbp large chromosomes XI . Section title: DNA Probes and Labeling Educational score: 3.7035276889801025 Domain: biomedical Document type: Study Language: en Probes were labeled either with dig-11-dUTP (Roche Biochemicals) or with biotin-14-dCTP (Life Technologies) using a nick translation kit, according to the supplier's instructions (Life Technologies). Section title: Fluorescence In Situ Hybridization Educational score: 4.187044620513916 Domain: biomedical Document type: Study Language: en All preparations were subjected to two-color FISH as described previously . The hybridization solution contained various differentially labeled probe combinations. It contained the yeast pan-telomere probe, which delineates all telomeres in the SK1 strain (2n = 32) investigated and one of the following probes: (a) chromosome-specific cosmid probes (not shown), (b) a chromosomes XI paint probe, and (c) a pan-centromeric DNA probe . Analysis of pairing of homologous regions was done by two-color FISH to spread nuclei using pairwise combinations of differentially labeled cosmid probes. Immunofluorescent detection of hybrid molecules was carried out with Avidin-FITC (Sigma-Aldrich) and rhodamine-conjugated sheep anti–dig Fab fragments (Roche Biochemicals) . Before microscopic inspection, preparations were embedded in antifade medium (Vector Laboratories) containing 0.5 μg/ml DAPI (4′6-diamidino-2-phenylindole) as DNA-specific counterstain. Section title: Immunostaining Educational score: 4.1427507400512695 Domain: biomedical Document type: Study Language: en A polyclonal antiserum against a S . cerevisiae spindle pole body component was used to stain the SPB in conjunction with telomeres . A rabbit antiserum against Zip1p transverse filament protein of the yeast synaptonemal complex was applied to identify nuclei with synapsis in progress. Ndj1p was stained in freshly prepared, mildly spread nuclei obtained from a strain that expresses HA-tagged Ndj1p using a monoclonal anti-HA–tag antibody (Biotec Santa Cruz) and secondary anti–mouse Cy3-conjugated antibodies (Dianova). Section title: Immunostaining Educational score: 4.187415599822998 Domain: biomedical Document type: Study Language: en Rap1p immunostaining of wild-type and ndj1Δ diploid cells in the MDY strain background was performed as follows: wild-type and mutant cells were harvested at 4, 7, and 12 h after the shift into sporulation, fixed, spheroplasted, adhered to poly- l -lysine–coated coverslips, and then prepared for immunolabeling by blocking with PBS/0.05% Tween 20/4% nonfat dry milk. Preparations of three-dimensionally preserved cells were labeled with a rabbit anti–Rap1p serum (gift of J. Berman, University of Minnesota, St. Paul, MN) diluted 1/10,000, followed by Oregon green-labeled anti–rabbit secondary antibodies (Jackson ImmunoResearch Laboratories), and then mounted with Citifluor anti-fade solution (Ted Pella, Inc.) containing 0.5 μg/ml DAPI under a coverslip. Section title: Microscopic Evaluation Educational score: 4.1151018142700195 Domain: biomedical Document type: Study Language: en Preparations were evaluated using an epifluorescence microscope (Axioskop; Carl Zeiss, Inc.) equipped with a double band-pass filter for simultaneous excitation of red and green fluorescence, and single band-pass filters for excitation of red, green, and blue (Chroma Technologies). To allow for the evaluation of a large number of nuclei from the time course experiments, investigation of structurally preserved in situ – hybridized nuclei was performed by careful focusing through the nuclei using a 100× plan-neofluoar lens (Carl Zeiss, Inc.). Signal patterns in spread nuclei were investigated with the same set up. Digital images were obtained using a cooled grey-scale CCD camera (Hamamatsu) controlled by the ISIS fluorescence image analysis system (MetaSystems). More than 100 nuclei were scored for each time point and probe combination. Fluorescence signal patterns were analyzed in nuclei with an undisrupted, homogeneous appearance in the DAPI-image. Section title: Confocal Microscopy and Quantitative 3-D Distribution Analysis of Telomeres Educational score: 4.184864521026611 Domain: biomedical Document type: Study Language: en Three-dimensionally preserved, Rap1p-stained nuclei were subjected to 3-D microscopy using a Meridian Ultima Z laser-scanning confocal microscope equipped with lasers to image DAPI and fluorescein. Simultaneous two-color scans were made of 10 randomly selected meiotic nuclei from each time point, without regard to the distribution of Rap1p signal, at a pixel and slice step-size of 0.1 μm. An aperture of 100 μm was used with short exposure times to minimize fluorescence fading. Appropriate wavelength scans were made of four fluorescent point source beads (Molecular Probes, Inc.) using the same settings. To avoid any influence of nonrandom telomere clustering that occurs during the bouquet stage, nuclei with less than five signal spots upon visual inspection were excluded from scoring. Averaged point source images were used to deconvolve the experimental images using the freeware xcosm v2.1 EM routine , mainly to reduce noise in the images, before image analysis. Section title: Confocal Microscopy and Quantitative 3-D Distribution Analysis of Telomeres Educational score: 4.132030487060547 Domain: biomedical Document type: Study Language: en Image analysis employed Pascal macros running Image (The National Institutes of Health, Bethesda, MD) and free-standing C++ routines written by M.E. Dresser. To measure the distances between the Rap1p spots and the DAPI-defined nuclear periphery: (a) the nuclear periphery was defined by thresholding the DAPI images over a range of different values, where increasingly large values exclude an increasing number of Rap1p spots from the inclusion volume, (b) the coordinates of each Rap1p spot were defined as lying at the center of each distinct signal spot (scored manually using Image and blinded with respect to the location of the nuclear periphery), and (c) the distance between each spot and the threshold-defined periphery was determined by a program that scores the radius of the smallest sphere, centered on the spot, which intersects an “exterior” voxel for spots included in the volume, or which intersects an “interior” voxel for excluded spots; if adjacent, the distance is defined as 0. Section title: Confocal Microscopy and Quantitative 3-D Distribution Analysis of Telomeres Educational score: 4.067338943481445 Domain: biomedical Document type: Study Language: en To unequivocally determine the relation of the Rap1-FITC signals with the nuclear periphery, we determined the nearest distance from Rap1-FITC signal centers and the nuclear boundary. The latter was determined from the optical section of the DAPI-stained nucleus from the same image plane. All signal spots analyzed were requested to lie with their center in the equatorial plane (within ±0.1 μm of best focus, as determined from the confocal image stacks of sectioned nuclei). The distance of signal gravity centers to the nearest edge of the nucleus was measured using a dedicated program (M.E. Dresser, unpublished data). Section title: Results Educational score: 4.283600330352783 Domain: biomedical Document type: Study Language: en Induction of meiosis leads to a dramatic reorganization of yeast nuclear architecture. Centromeres that are tightly clustered at the SPB during vegetative growth disperse throughout the nuclear volume , while telomeres accumulate de novo at this location . To determine whether this dramatic reorganization of the early meiotic nucleus requires the Ndj1 protein , an NDJ1 null-mutation ( ndj1Δ ::KanMX4) was introduced into the SK1 strain background because the high synchrony in this strain background facilitates bouquet analysis . Telomere clustering, centromere distribution, and chromosome pairing were studied by FISH with telomere-, centromere-, and chromosome-specific probes in detergent spread and in three-dimensionally preserved nuclei from meiotic time-course experiments of diploid wild-type and ndj1Δ strains. Additionally, using anti–Rap1p signals as marker for meiotic telomeres , telomere positioning with respect to the nuclear periphery was examined by confocal microscopy in the MDY strain background. All experiments analyzed displayed sporulation rates ≥85%. Section title: Dissolution of Premeiotic Centromere and Telomere Clusters Does Not Require Ndj1p Educational score: 4.190037250518799 Domain: biomedical Document type: Study Language: en It has been shown that the nuclear organization of vegetative/premeiotic yeast cells is dominated by centromere clustering at the SPB , which resolves during the onset of meiotic prophase . When we determined centromere distribution by FISH to nuclei of SK1 strains, it was found that the frequency of nuclei with one centromere cluster diminished at similar rates after induction of meiosis, both in wild-type and in ndj1Δ meiotic time courses . This suggests that dissolution of the centromere cluster is not affected by the absence of Ndj1p. Section title: Dissolution of Premeiotic Centromere and Telomere Clusters Does Not Require Ndj1p Educational score: 4.348967552185059 Domain: biomedical Document type: Study Language: en Premeiotic (vegetative) yeast nuclei usually contain few (two to eight) perinuclear telomere clusters that are resolved at the onset of meiotic prophase . To monitor whether the absence of Ndj1p influences dissolution of vegetative telomere clusters, we determined the fraction of spread nuclei with two to eight telomere clusters at transfer to sporulation medium (0 min) and from 180–420 min, and in undisrupted nuclei at 0 and 180–480 min. Nuclei were also sampled at 530 and 590 min, following the shift into meiosis. It was found that the frequency of spreads with premeiotic telomere distribution (we consider nuclear topology at t = 0 to represent premeiotic/vegetative nuclear organization) diminished at similar rates in wild-type and ndj1Δ meioses . Similar results were obtained in repeated time courses and with undisrupted nuclei (not shown). These data suggest the dissolution of premeiotic nuclear architecture during the onset of sporulation occurs at wild-type rates in the absence of Ndj1p. To further determine whether the ndj1Δ mutation affects telomere distribution in vegetative nuclei, we compared telomere FISH signal numbers in 20 randomly selected nuclear spreads obtained during logarithmic vegetative growth in YPD. Spreading of vegetative nuclei dissociates the few clusters seen in intact nuclei to a mean of 22 ± 5 (SD) and to 21 ± 3 telomere signal spots/nucleoid in wild-type and ndj1Δ cells, respectively. Similar telomere signal numbers in spread premeiotic wild-type and ndj1Δ cells suggest that telomere distribution in vegetative cells is not affected by the ndj1Δ mutation, which contrasts with the situation in meiocytes (see below). Section title: NDJ1 Is Required for Meiosis-specific Telomere Distribution Educational score: 4.174890518188477 Domain: biomedical Document type: Study Language: en The induction of meiosis leads to the repositioning of telomeres over the nuclear periphery seen as peripheral dispersion of RAP1p-GFP foci in live cells and by telomere FISH signals in fixed nuclei . To determine whether the telomere distribution patterns obtained by FISH are representative for meiocytes and to see whether dispersion of meiotic telomeres over the nuclear periphery occurs during sporulation of our strains, we determined the telomere distribution patterns in spread and undisrupted nuclei from time-course experiments after induction of meiosis by FISH with the XY′ telomere probe and performed immunofluorescence (IF) staining of HA-tagged Ndj1p in spread nuclei at t = 240 min. Section title: NDJ1 Is Required for Meiosis-specific Telomere Distribution Educational score: 4.411101341247559 Domain: biomedical Document type: Study Language: en Ndj1p is expressed only during meiosis ; therefore, the distribution patterns detected with this probe exclusively represent meiotic telomere arrangements. The signal patterns obtained by Ndj1p IF matched the telomere distribution patterns revealed by telo-FISH . Costaining experiments revealed colocalization of HA-tagged Ndj1p and telomere-FISH signals, with the Ndj1-HA signals often extending beyond the XY′ FISH signals . This could relate to the abundance of Ndj1p at chromosome ends during meiosis and/or to slight swelling of the epitope-bearing chromatin during the FISH procedure. The lack of IF at some telo-FISH signals may relate to loss of protein during the denaturation and hybridization procedure. In any case, we observed that meiotic telomeres, as marked by FISH as well as with anti–HA-Ndj1p, do adopt a peripheral dispersed arrangement in early meiocytes, which is seen as a rim-like fluorescent signal in mild spreads and at the equatorial focus plane of undisrupted nuclei (not shown). At later time points, telomeres congregate to form a single large signal cluster at the SPB . This clustering is resolved as cells enter pachytene with telomere signals, again becoming dispersed . Section title: NDJ1 Is Required for Meiosis-specific Telomere Distribution Educational score: 4.165708541870117 Domain: biomedical Document type: Study Language: en Nuclei with a rim-like peripheral telomere signal distribution are rarely encountered at the induction of sporulation, but increase early after induction of meiosis in the wild type . In contrast, the frequency of spread ndj1Δ meiocytes with such a preleptotene-like perinuclear telomere distribution essentially remained at a vegetative background level during sporulation, well below the level seen in the wild type . Similar observations were obtained in time courses of undisrupted ndj1Δ nuclei (not shown). Section title: Perinuclear Distribution of Telomeres Is Altered in ndj1Δ Meiocytes Educational score: 4.232151031494141 Domain: biomedical Document type: Study Language: en To test whether the ndj1Δ mutation affects the meiosis-specific three-dimensional telomere distribution (i.e., peripheral location of dispersed telomeres in the meiocyte nucleus), we performed Rap1p staining with undisrupted nuclei and investigated the three-dimensional spot distribution by laser scanning microscopy and digital image analysis. Telomeric Rap1p signal spots and the corresponding DAPI-stained nuclei were obtained by light optical serial sectioning . Because nuclei in meiotic prophase are nonspherical and nuclear pore staining failed to reveal the typical rim staining seen in mitotic interphase (not shown), we determined the nuclear boundary in light optical sections of DAPI-stained nuclei. DAPI highly selectively stains DNA and renders the nuclear boundary at a high contrast. The number and relation of Rap1p signal centers to the nearest sector of the nuclear boundary was computed from 30 nuclei of uninucleate cells, 10 each at 4, 7, and 12 h after induction of meiosis from wild type and ndj1Δ . Bouquet nuclei, which rarely exceed 5–10% at any time point in this strain background, were excluded from this analysis (see Materials and Methods). The 30 prophase nuclei from wild-type and ndj1Δ cells were analyzed as a single class, where totals of 519 and of 729 Rap1p spots were scored, respectively. The difference in the number of total spots is consistent with the smaller number of larger Rap1p signals seen in spread meiotic prophase nuclei of wild type, compared with ndj1Δ cells . Section title: Perinuclear Distribution of Telomeres Is Altered in ndj1Δ Meiocytes Educational score: 4.353025436401367 Domain: biomedical Document type: Study Language: en The relation of the Rap1p telomere signals to the nuclear periphery was determined in the light optical section of the equator of each nucleus. The distance from Rap1/FITC signal centers to the nearest nuclear boundary of the DAPI image was calculated by a dedicated program. This two-dimensional procedure accounts for the small dimensions of the yeast nucleus and its irregular shape at meiosis , which precludes applying a distribution analysis of signal spots from a geometrically defined center. The data obtained are displayed in a bar graph according to frequency of signals at distance units of multiples of 0.2 μm from the nearest nuclear boundary segment . In wild-type cells, the vast majority of telomeric spots were found near the nuclear periphery . A deviation from this peripheral telomeric Rap1p distribution in wild-type meiocyte nuclei was apparent in ndj1Δ nuclei. In the latter, a significant fraction of signals (97.5% level by G test) located remote from the nuclear boundary (i.e., 34% of signal spots were located 0.6–1 μm from the nearest nuclear edge), while in the wild type only 18% of spots were found in this more internal nuclear compartment . These results suggest that the perinuclear localization of meiotic telomeres is disrupted in ndj1Δ meiosis. Section title: Ndj1p Is Essential for Formation of the Bouquet Educational score: 4.174123287200928 Domain: biomedical Document type: Study Language: en In the SK1 background, we observed that the premeiotic telomere distribution is resolved in diploid ndj1Δ cells at wild-type rates. However, a rim-like perinuclear telomere distribution was detected only at insignificant rates . This prompted us to also determine the frequency of meiocytes with a single telomere-FISH signal cluster, a telomere distribution diagnostic for a chromosomal bouquet that transiently forms at the leptotene/zygotene transition during prophase I of S . cerevisiae . Induction of meiosis in the wild type led to a 22-fold increase in the frequency of mildly spread nuclei with a single telomere cluster over premeiotic background at t = 240 min . In undisrupted nuclei, a 5.3-fold increase at 260 min (not shown) was noted, which is well in agreement with earlier observations in undisrupted wild-type SK1 meiocytes . Section title: Ndj1p Is Essential for Formation of the Bouquet Educational score: 4.200016498565674 Domain: biomedical Document type: Study Language: en A severe deviation from the wild-type situation was observed in ndj1Δ meiosis. The frequency of nuclei with a single telomere cluster never exceeded premeiotic levels, even in time courses with prolonged duration and in time-course experiments where we performed FISH to intact meiocyte nuclei . The defect in telomere clustering was also evident when telomere FISH signal numbers were compared in 20 randomly selected wild-type and mutant nuclei. Spread ndj1Δ meiocytes (obtained 300 min after induction of meiosis) contained a significantly increased spot number/nucleus as compared with wild-type meiocytes that were sampled at 200 min to compensate for the delay in mutant prophase I . Similar results were obtained using anti–Rap1p IF and confocal microscopy (data not shown). Section title: Ndj1p Is Essential for Formation of the Bouquet Educational score: 4.331822395324707 Domain: biomedical Document type: Study Language: en Since formation of a true bouquet involves telomere clustering at the SPB during the leptotene/zygotene transition stage , we determined whether SPB/telomere cluster association is absent in ndj1Δ meiocytes. To this end, we simultaneously immunostained for spindle pole body and telomeres by FISH in intact nuclei at 240 min in wild-type and mutant meiosis. We failed to detect a physical association of the SPB signal and XY′ telomeric signal accumulations seen in a few ndj1Δ nuclei (not shown). This and the constant and negligible frequency of nuclei with a single telomere-FISH signal cluster over mutant time courses corroborates that bouquet formation is impaired in ndj1Δ meiosis. To further investigate whether telomere clustering is defective during the leptotene/zygotene transition stage, we immunostained spread meiocytes with antibodies to Zip1p, a component of transverse filaments of the synaptonemal complex and determined the telomere distribution by FISH in SK1 meiocytes, obtained at t = 200 min with an incomplete speckled Zip1p distribution, which represent nuclei with synapsis in progress. It was found that telomere signals were scattered throughout mildly spread ndj1Δ nuclei ( n = 168; obtained at 300 min) with a fragmented Zip1p distribution , while in the wild type 20% of zygotene cells ( n = 166) contained a single telomere cluster . These data suggest that true bouquet formation is disrupted in ndj1Δ meiosis. Section title: Ndj1p Is Essential for Formation of the Bouquet Educational score: 4.332463264465332 Domain: biomedical Document type: Study Language: en Zip1p IF disclosed furthermore that ndj1Δ nuclei contained an unusually high frequency of intensely staining Zip1-positive structures that have been called “polycomplexes” . At 210 min after induction of meiosis, 81% of ndj1Δ nuclei contained predominantly one Zip1 polycomplex (PC) in the form of a brightly stained rod , while at this time point only 28% of wild-type nuclei contained a PC. This represents an approximately threefold increase of PCs in the mutant. At 330 min, 60% of ndj1Δ meiocytes still contained PCs, while these were absent in the wild type. In yeast meiosis, formation of Zip1 polycomplexes has been observed in a variety of conditions and strain backgrounds . An increased frequency of PCs is often seen in recombination mutants, which generally show defects in synapsis . Taken together, these data suggest that normal synaptic progression requires Ndj1p and bouquet formation. Section title: Chromosome Pairing Is Impeded in ndj1Δ Meiosis Educational score: 4.398725509643555 Domain: biomedical Document type: Study Language: en Since a mutant with disrupted bouquet formation offers the possibility to test for the impact of meiotic telomere clustering for the homologue pairing process, we investigated homologue pairing by FISH with cosmid probes to regions on chromosmes III , IX , and XI during wild-type and mutant sporulation. While chromosomes XI represents a large chromosome that is capable spanning the entire nuclear volume, IX is of intermediate size, and III is among the smallest yeast chromosomes. Due to the clustering of vegetative centromeres at the spindle pole body and other functions, the yeast nucleus displays a substantial amount of premeiotic homologue association . Pairing interactions are disrupted during premeiotic S-phase, and then return at meiosis-specific levels that well exceed vegetative levels . In the present investigation, pairing of homologous chromosome regions was determined by FISH of two differentially labeled cosmid probes to two-dimensional nuclear spreads that yields two signals pairs in the same focal plane. Spreading has been shown to enhance cytological resolution in the unfavorably small yeast nucleus and to dissociate weak chromosome interactions while leaving stable interactions intact . We regarded homologous regions paired when cosmid signals of the same color were touching each other or formed an enlarged confluent signal . To ensure saturated target regions, only nucleoids containing strong signals of both colors were analyzed. Two-color FISH with cosmid pairs f/l and m/p was performed on nuclear spreads obtained from meiotic time courses of ndj1Δ and wild-type cells. In initial experiments, we determined the frequencies of associations between heterologously colored signals, which provides a measure for accidental signal contacts. Both wild-type and mutant time courses displayed nearly identical frequencies of heterologous signal associations at 0, 3, 5, and 7 h in sporulation. Specifically, at all time points, the mean fraction of successfully hybridized spreads showing heterologous signal contacts for cosmid combinations f/l and m/p was 8.5% (range 7.3–8.9) and 9.3% (range 9–10.2), respectively. These values are well in agreement with estimations reported by others . To adjust for accidental contacts between cosmid signals, which may be influenced by a number of parameters (see above), we used centromere distant probes and furthermore subtracted 50% of the obtained fraction showing heterologous contacts from the obtained pairing values, since in two cosmid FISH experiments a signal has a two- to fourfold higher probability to be associated with a heterologous than with a homologous signal. It should be noted that the results obtained with and without such a correction remained essentially the same (not shown). Section title: Chromosome Pairing Is Impeded in ndj1Δ Meiosis Educational score: 4.383491039276123 Domain: biomedical Document type: Study Language: en In wild-type and ndj1Δ time courses, the fraction of nucleoids with FISH signals homologously paired increased well above premeiotic values . In ndj1Δ meiosis, all homologous regions investigated reached nearly wild-type levels of homologous signal pairing, but with a 2–3-h delay . This slowed progression of homologous pairing is mirrored by a 2-h delay in the appearance of anaphases I and II in ndj1Δ meiosis as compared with the wild type, where these first appeared at 300 and 320 min, respectively (not shown, consistent with earlier reports). In wild-type meiosis, the homologous region near the left telomere of the small chromosomes III paired most rapidly ( Table ). However, in the absence of Ndj1p and bouquet formation, the telomeric region of chromosomes III showed the most prominent retardation of homologous pairing, expressed as the difference in time required in wild-type and ndj1Δ meiosis to reach peak values in signal pairing . Pairing of the telomeric region of the right arm of the large chromosomes XI (cos l), in contrast, showed only a delay of 100 min in ndj1Δ meiosis. Pairing of large chromosomes may thus be less dependent on a catalytic action of telomere clustering on homologue pairing, since these may span the entire nucleus and therefore have a higher probability for chance encounters with their homologues in the absence of bouquet formation. Section title: Condensation of Chromosome Territories Occurs in the Absence of Ndj1p Educational score: 4.407194137573242 Domain: biomedical Document type: Study Language: en Previous chromosome painting studies have shown that yeast chromosomes extend, pair, and condense during the course of meiotic prophase . To test whether the ndj1Δ mutation influences this morphological change of yeast chromosomes during meiosis, we painted chromosomes XI in conjunction with telomere FISH in wild-type and ndj1Δ cells at 180, 240, 360, and 420 min after induction of meiosis. Nuclei with all aspects of meiotic chromosome morphology were detected in wild-type and ndj1Δ cells . The painting of yeast chromosomes also allows chromatin condensation to be assessed. The fraction of uninucleated meiocytes that exhibit clear and compact FISH signals represents the fraction of cells in which chromosomes are condensed . The frequency of condensed XI pachytene bivalents was determined in uninucleate mutant and wild-type SK1 meiocytes . In ndj1Δ meiosis, chromatin condensation was found to be significantly delayed with respect to wild type , although chromosome condensation per se is not affected by the ndj1Δ mutation. Thus, chromosome condensation was further monitored by FISH with differentially labeled cosmid probes b and l in uninucleated meiocytes obtained at t = 240 min in wild type and t = 360 min in ndj1Δ time courses. Cosmids b and l map to the proximal and distal part of the left (long) arm of XI . The distance between the centers of the paired and differentially colored cosmid signals on pachytene bivalents was determined in 30 randomly selected spread nuclei. The mean distance observed between the two cosmid signals was 1.1 ± 0.4 μm (±SD) in the wild type and 1.0 ± 0.4 μm in ndj1Δ nuclei. The difference between the two data sets did not differ significantly ( P = 0.23 at 0.01; Student's t test). This further suggests that chromosome condensation is not affected by the absence of Ndj1p. Section title: Ndj1p May Be Required for Peripheral Localization of Meiotic Telomeres Educational score: 4.322659969329834 Domain: biomedical Document type: Study Language: en Chromosome topology of the vegetatively growing yeast is dominated by tightly clustered centromeres next to the spindle body and by telomeres forming a few aggregates at the nuclear periphery . Upon induction of meiosis, nuclear topology is reorganized. Centromeres become dispersed throughout the nucleus, and telomeres spread over the nuclear periphery , except during an intermediate stage when telomeres form a tight cluster in the vicinity of the spindle pole body; i.e., at the bouquet stage . Section title: Ndj1p May Be Required for Peripheral Localization of Meiotic Telomeres Educational score: 4.663573265075684 Domain: biomedical Document type: Study Language: en Two of the early steps in the meiotic nuclear reorganization, dispersion of centromeres and telomeres, apparently are unaffected by deletion of NDJ1 . However, this close proximity of dispersed telomeres to the nuclear periphery, which is evident in wild-type cells does appear defective in ndj1Δ . During earliest meiotic prophase, telomeres dissociate from the vegetative aggregates and line the nuclear periphery , giving raise to a rim-like staining in ∼20% of wild-type nuclei, which resembles the telomere distribution seen in the late preleptotene/leptotene stage of mammalian prophase I . In mammals, the switch from premeiotic to meiotic telomere distribution is a two-step process, with telomeres first attaching at scattered points over the nuclear envelope, and then congregating in the bouquet to a limited sector of the nuclear envelope . In ndj1Δ meiosis of yeast, FISH and IF analyses of telomere distribution in undisrupted and mildly spread nuclei revealed a paucity of nuclei with telomeres lining the nuclear periphery and a concomitant increase in the frequency of nuclei with telomeres scattered throughout the nuclear lumen, as inferred from three-dimensional Rap1p analysis. These observations indicate that the absence of Ndj1p impairs the assembly of a meiosis-specific perinuclear telomere topology after dissolution of the few vegetative telomere clusters . Furthermore, these observations indicate that localization of telomeres to the nuclear periphery requires Ndj1p, in accord with speculation based on an earlier genetic analysis of NDJ1/TAM1 -deficient cells . Localization of the ends of yeast synaptonemal complexes to the nuclear envelope has been demonstrated by electron microscopy and a similar analysis of ndj1Δ is underway (Z. Zhang, M.N. Conrad, and M.E. Dresser). Section title: Bouquet Formation Facilitates Homologue Interaction Educational score: 4.493549823760986 Domain: biomedical Document type: Study Language: en Distribution of recombination events (crossover interference), chromosome segregation, and distributive disjunction are all affected by deletion of NDJ1 but, for the major part, the defects are relatively mild and may arise from a telomere-related delay in meiotic prophase . The delay in onset and completion of synapsis potentially is due to a defect in some aspect of chromosome pairing that normally brings homologues into close apposition for the initiation of homosynapsis. FISH in conjunction with Zip1p immunostaining showed that telomeres fail to cluster during the leptotene/zygotene-equivalent stages, when bouquet formation normally occurs . Moreover, the meiotic SPB failed to show an association with a telomere accumulation occasionally seen in Ndj1-deficient nuclei. This suggests that loss of bouquet formation is the underlying defect of the retarded synapsis progression and chromosome pairing in the ndj1Δ mutant (see below). Section title: Bouquet Formation Facilitates Homologue Interaction Educational score: 4.583267688751221 Domain: biomedical Document type: Study Language: en When we investigated homologue pairing at four regions on three chromosomes by two-color cosmid FISH, a highly significant reduction in chromosome pairing was noted at time points in mutant meiosis when, in the wild type, signal pairing had reached its maximum. Prolonged time courses in the ndj1Δ mutant showed that there is a 2–3-h delay in chromosome pairing. A similar delay was noted for the compaction of chromosomes XI bivalents, but, ultimately, chromosome condensation as measured by cosmid FISH appeared normal. When we compared the time required for the homologous regions assayed to reach meiosis-specific values in wild-type and ndj1Δ meiosis, it appeared that the pairing of the telomeric regions of the relatively small chromosomes III was most severely delayed. In Ndj1p-deficient meiosis, the left telomere region on chromosomes III took 60 min longer to reach peak-pairing values than the telomeric region of the right arm of the large chromosomes XI and on chromosomes IX . This may indicate that smaller chromosomes are more dependent on the action of telomere clustering to instigate homologue alignment and pairing. However, after a delay of ∼2–3 h, all homologous regions investigated reached approximately wild-type pairing values, which shows that telomere-independent routes of homologue pairing exist. Altogether, it appears that bouquet formation is not required for homologue recognition and synapsis per se, but mediates an important catalytic action on homologue pairing, which shortens the duration of sporulation, and thus may, in turn, confer selective advantages in a natural environment. This conclusion is in agreement with long-standing hypotheses that telomere clustering during the bouquet stage facilitates homologue interactions by aligning chromosome ends . Section title: Bouquet Formation Facilitates Homologue Interaction Educational score: 4.724947452545166 Domain: biomedical Document type: Study Language: en Furthermore, there is supporting experimental evidence from S . pombe where, in analogy to the situation in budding yeast ndj1Δ mutants, absence of the telomere protein Taz1p(+) abrogates telomere clustering and leads to reduced recombination and increased meiotic nondisjunction in fission yeast . Thus, the primary, important consequence of deletion of NDJ1 is likely to be the failure of bouquet formation, with the subsequent defects arising from distorted telomere distribution and the absence of bouquet-facilitated homologue alignment and pairing, as suggested based on genetic data from disomic synaptic meiosis . The ndj1Δ -related delay may result from overloading of the homology testing machinery due to disordered chromosome distribution in the absence of meiotic telomere clustering. Dissolution of premeiotic/vegetative centromere and telomere aggregates at the onset of meiosis may still create sufficient chromosome movement in the Ndj1p-deficient prophase nucleus for portions of chromosomes to make contact and, once partially aligned, to commence with homosynapsis. Particularly, larger-sized chromosomes may benefit from an increased probability of encountering a portion of its homologue and to initiate stable pairing earlier than smaller ones. Synapsis, which may initiate at a few interstitial sites seems to proceed at a slower rate in the ndj1Δ mutant, since Zip1p polycomplexes resolve more gradually. Jumbled telomere localization and nonsynchronized homologue pairing may be the cause for defective crossover interference in the absence of Ndj1p . However, at the present state, it cannot be excluded that the ndj1Δ phenotypes result from other so far unknown effects besides the telomeric ones. Section title: Bouquet Formation Facilitates Homologue Interaction Educational score: 4.20262336730957 Domain: biomedical Document type: Study Language: en An interesting possibility is that Ndj1p may link meiotic telomeres to motor proteins at the nuclear envelope, the action of which is thought to bring about telomere aggregation during the bouquet stage . Future research will have to show whether perinuclear filaments, like the Mlp proteins , which are involved in tethering vegetative telomeres to nuclear pore complexes , also play a role in telomere clustering during meiotic prophase. | Study | biomedical | en | 0.999997 |
0005026 | Section title: Introduction Educational score: 4.591047286987305 Domain: biomedical Document type: Study Language: en The congression of chromosomes to the metaphase plate and subsequent poleward movement at anaphase are complex processes that occur with remarkable accuracy during cell division. An important organelle in chromosome movement is the kinetochore, a protein complex that associates with centromeric DNA . Through the interaction with spindle microtubules, kinetochore proteins have direct roles in propelling chromosomes toward the equatorial plane at prometaphase , and subsequently away to opposite spindle poles at anaphase . In addition, a handful of kinetochore proteins participate in the spindle checkpoint pathway, which ensures that chromosomes align correctly at the metaphase plate before anaphase begins . Even a single unaligned chromosome can activate the spindle checkpoint and prohibit anaphase onset . It has been proposed that tension registered at the kinetochore is either directly or indirectly involved in the spindle checkpoint, at least in meiosis . Section title: Introduction Educational score: 4.5094709396362305 Domain: biomedical Document type: Study Language: en Normally, chromosomes possess either two sister kinetochores (mitosis/meiosis II) or the paired kinetochores from homologous chromosomes (meiosis I). A widely held view is that kinetochore pairs are required to ensure that sister/homologous chromosomes segregate to opposite poles. The natural polarity of opposed kinetochores matches the bipolarity of the spindle, allowing the chromosomes to adopt a stable position at the spindle midzone . However, the importance of paired kinetochores in chromosome congression was questioned by Khodjakov et al. 1997 , who used laser ablation to experimentally remove a kinetochore from each of 50 mammalian mitotic chromosomes. The remaining single kinetochores were sufficient to generate the congression of 38% of the chromosomes analyzed. Electron microscopy of three cells revealed that the single kinetochores were distorted and attached to microtubules from both poles. These, and similar data involving detached kinetochore fragments , suggest that mitotic mammalian kinetochores are composed of subunits that can interact with microtubules independently . In contrast, single kinetochore chromosomes failed to align at the spindle equator when the same technique was applied to African blood lily ( Haemanthus ) endosperm cells , suggesting that single kinetochores and/or their interactions with the spindle differ among species or cell types. Section title: Introduction Educational score: 4.280693054199219 Domain: biomedical Document type: Study Language: en Here, we extend the analysis of single kinetochores to maize meiotic cells. For a source of material, we exploit the phenotype of the maize meiotic mutant absence of first division 1 ( afd1 ) , which, as a result of premature sister kinetochore separation at meiosis I, produces cells at meiosis II that contain a complete set of single kinetochore chromosomes. By analyzing these single kinetochore chromosomes in detail, we demonstrate that they can align with ∼60% accuracy at metaphase II by interacting with kinetochore fibers from opposite spindle poles. During alignment, the single kinetochores appear to divide into halves that are capable of functioning independently. The connections established by half kinetochores are stable enough to dissociate/dephosphorylate two well-studied spindle checkpoint proteins. Finally, in anaphase, considerable poleward force was generated by the half kinetochores, stretching and nearly separating the kinetochores into two parts. Section title: Plant Materials Educational score: 3.385890007019043 Domain: biomedical Document type: Study Language: en The original stocks carrying the recessive afd1 mutation were provided by Inna Golubovskaya (N.I. Vavilov Institute of Plant Industry Research, St. Petersburg, Russia). This strain was crossed once to the inbred line KYS, and a single resulting Afd1 / afd1 plant was self-crossed to generate all of the material used here. Homozygous afd1/afd1 plants were identified cytologically in microsporocytes. Section title: Immunolocalization Educational score: 4.123284816741943 Domain: biomedical Document type: Study Language: en Meiocytes or anthers from both wild-type and mutant plants were fixed and processed as described previously . For the analysis of mitosis in afd1 plants, the tips of prop roots were excised, fixed, and sectioned on a cryostat . The maize centromere protein (CENPC) antibodies, maize MAD2 antibodies, 3F3/2 mAb , and mAb against α-tubulin were used as described previously . The CENPC and MAD2 antibodies were detected by rhodamine-conjugated goat anti–rabbit secondary antibodies, and the 3F3/2 and α-tubulin mAbs were detected by FITC-conjugated goat anti–mouse secondary antibodies (secondary antibodies were purchased from Jackson ImmunoResearch Laboratories). In double labeling studies, primary antibodies were incubated simultaneously. Chromosomal DNA was stained with diamino phenylindole (DAPI) at 0.1 μg/ml. Section title: In Situ Hybridization Educational score: 4.162813186645508 Domain: biomedical Document type: Study Language: en For in situ hybridization, a maize centromeric satellite tandem repeat called CentC was PCR amplified from genomic DNA derived from the inbred line W23 (primers were 5′-GATTGGGCATGTTCGTTGTG and 5′-CACTACTTTAGGTCCAAAAC). Two clones of the ∼155-bp PCR product were sequenced to verify their identity as CentC. Gel-purified PCR products were labeled with fluorescently tagged dUTP and used as probes for in situ hybridization as described previously , except that the denaturing temperature was reduced to 90°C. In experiments where CENPC and CentC were both labeled, immunolocalization of CENPC was performed first, followed by in situ hybridization. Section title: Microscopy and Data Analysis Educational score: 4.079026699066162 Domain: biomedical Document type: Study Language: en Except where specifically noted in the text, all data were collected using a DeltaVision SA3.1 three-dimensional (3D) light microscope workstation as described previously . The data were processed by constrained iterative deconvolution. For the analysis of meiosis in living cells, meiocytes were cultured in a synthetic culture medium supplemented with the vital DNA stain Syto12 . Cells regularly survive in this medium for >6 h. Time lapse 3D (4D) data were collected at intervals from 1 to 30 min depending on the experiment. Section title: Microscopy and Data Analysis Educational score: 4.127433776855469 Domain: biomedical Document type: Study Language: en To estimate the frequency of single kinetochore chromosome alignment in afd1 cells, we first determined that a rectangle with a width of 2 μm encompassed all the kinetochores in four wild-type metaphase II cells. Based on this estimate, a rectangle with a width of 2 μm was applied to the equator of the metaphase II spindles in six afd1 cells . The placement of the rectangle in afd1 cells was necessarily subjective, but in each case it was positioned roughly at the equator of the spindle and at right angles to the spindle axis. If a kinetochore was located within the rectangle, it was counted as aligned at the metaphase plate. Section title: Microscopy and Data Analysis Educational score: 4.1721954345703125 Domain: biomedical Document type: Study Language: en To evaluate the effect of tension on the dephosphorylation of the 3F3/2 antigen at the kinetochore, 3F3/2 staining was first normalized for kinetochore size by dividing it by the intensity of CENPC staining. This was done for all the kinetochores in two afd1 prometaphase II cells that did not overlap with another kinetochore or with the background 3F3/2 staining. A square composed of 10 × 10 pixels was used to cover the kinetochore. The gray level intensity of the CENPC and 3F3/2 staining within the square was obtained from three contiguous sections (section thickness, 0.25 μm), averaged, and subtracted from the background intensity. Kinetochore edges were identified as the position half way from the tip to the base of a one-dimensional plot profile drawn over the kinetochore. The longest axis of the kinetochore was used as the length, except when it was spherical, and the diameter was used. To analyze the relationship between staining intensity and kinetochore length, we tested linear, log linear, and power models using maximum coeefficient of determination ( R 2 ) as our optimality criterion (using SAS statistical analysis software at the University of Georgia Research Computing Resource Facility). Section title: Sister Kinetochores Separate Prematurely during Meiosis I in afd1 Meiocytes Educational score: 4.279297828674316 Domain: biomedical Document type: Study Language: en We have recently identified and characterized a maize homologue of CENPC, a constitutive kinetochore protein . Anti-CENPC antibodies effectively label each of the 20 chromosomes of a diploid maize cell at all stages of the cell cycle. As a first step in our study, we used affinity-purified anti-CENPC antibodies to confirm the phenotypic description of afd1 given by Golubovskaya and colleagues . Fig. 1 illustrates a comparison of kinetochores from wild-type (left) and sibling afd1 (right) plants at various stages of meiosis I. A complete description of kinetochore morphology in wild-type cells can also be found in our previous report . The earliest detectable prophase stage in afd1 plants is a diplotene-like stage, which in wild-type cells is typified by partially condensed and desynapsed chromosomes. All four (homologous and sister) kinetochores are usually associated at this stage in wild-type cells , such that only 10 CENPC-positive spots are usually observed. Consistent with the assertion that minimal chromosome pairing occurs in afd1 meiocytes , 20 CENPC-positive spots were generally observed at the diplotene-like stage of mutant cells . Section title: Sister Kinetochores Separate Prematurely during Meiosis I in afd1 Meiocytes Educational score: 4.247862815856934 Domain: biomedical Document type: Study Language: en After diplotene and prometaphase I, the sister kinetochores in wild-type cells stay conjoined but separate slightly, revealing a doublet structure . Approximately ten doublet kinetochores are the norm for each half spindle at metaphase I , anaphase I , and telophase I . However, in afd1 cells, ∼20 single kinetochores were observed in each half spindle at all 3 stages . These data confirm the conclusion, made by Golubovskaya and Mashnenkov 1975 , that sister kinetochores separate prematurely in afd1 plants to generate single kinetochore chromosomes before meiosis II. Our data also support the data of Chan and Cande 1998 , who demonstrated that meiosis I spindle formation is essentially unaltered by the afd1 mutation . Finally, we observed a low frequency of small chromosome fragments in afd1 plants that lacked visible CENPC staining . Section title: Sister Kinetochores Separate Prematurely during Meiosis I in afd1 Meiocytes Educational score: 4.230826377868652 Domain: biomedical Document type: Study Language: en To investigate the mitotic phenotype of the afd1 mutation, we extended our studies to somatic cells from afd1 plants. Data acquired from the cells in prop roots (aerial roots extending from the base of the stem) indicate that mitosis in mutant plants is essentially the same as was documented for normal maize mitosis . As shown in Fig. 2 , the sister kinetochores can be distinguished from each other at the earliest stages of mitotic prophase , and a complete separation of sister kinetochores occurs as early as prometaphase (not shown). Sister kinetochores then orient and segregate to opposite spindle poles. Acentric chromosome fragments were not observed in any of 16 anaphase/telophase cells from 2 mutant plants. Meiosis I and mitosis in the afd1 mutant can be distinguished from each other by several criteria . The distinct differences in the timing of kinetochore separation, chromosome condensation patterns, and spindle morphology suggest that the afd1 mutation does not substitute meiosis I with a mitotic division. Section title: 3D Analysis of Meiosis II in the afd1 Mutant Educational score: 4.213950157165527 Domain: biomedical Document type: Study Language: en To determine whether the single kinetochore chromosomes generated by the afd1 mutation align at the metaphase II plate, we first employed 3D time lapse (4D) microscopy. As described previously , live meiocytes were extruded into a culture medium and stained with the vital DNA stain Syto12. A total of 32 cells from afd1 plants was observed undergoing meiosis II. For 12 of the cells, data collection began before metaphase II and included all or part of prometaphase. As shown in Fig. 3A–D , a clearly identifiable metaphase plate was formed. Once at the metaphase plate , the single kinetochore chromosomes oscillated back and forth in a manner similar to wild-type cells . The full prophase–metaphase II alignment process was observed in two cells, where prometaphase lasted ∼1 h longer (a total of ∼150 min) than expected for a wild-type meiocyte . Section title: 3D Analysis of Meiosis II in the afd1 Mutant Educational score: 4.196465492248535 Domain: biomedical Document type: Study Language: en In an additional 6 wild-type cells and 20 cells from sibling afd1 plants, the earliest stages recorded were metaphase or early anaphase II. In each cell where the start of anaphase was documented, all of the chromosomes appeared to begin poleward movement together. The orderly chromosome segregation characteristic of a wild-type cell is shown in Fig. 4 . In mutant cells, however, normal chromosome segregation was not observed. Instead, the chromosomes demonstrated erratic behavior typified by irregular rates of movement and frequent changes in direction. The rates of chromosome movement for individual chromosomes varied from <0.4 to 1.4 μm/min . Anaphase II in mutant cells was typically three to four times longer than is characteristic for wild-type cells; the cell shown in Fig. 3 remained in anaphase for ∼100 min before the distinct nuclei structures characteristic of telophase were observed . Section title: 3D Analysis of Meiosis II in the afd1 Mutant Educational score: 4.185546875 Domain: biomedical Document type: Study Language: en The chromosome fragments generated during meiosis I were observed in living afd1 cells as small Syto12-stained structures. One such fragment was observed in the cell illustrated in Fig. 3 (arrows). The fragment moved slowly towards a spindle pole at 0.22 μm/min during prometaphase and remained suspended in the spindle throughout metaphase II. The same fragment moved rapidly poleward during mid-anaphase II at 0.71 μm/min . Section title: A Majority of Single Kinetochore Chromosomes, but Not Acentric Fragments, Aligns at the Spindle Midzone Educational score: 4.166075706481934 Domain: biomedical Document type: Study Language: en By treating fixed cells with anti-CENPC antibodies, we were able to view the position and morphology of the single kinetochores during meiosis II. These data are shown in Fig. 5 , with control meiocytes from wild-type plants (left) and meiocytes from sibling afd1 plants (right). The spindle in afd1 meiocytes was usually irregular in shape , though a basic bipolar structure was always observed. As in living cells, a majority of the single kinetochores chromosomes appeared to align at the spindle equator in metaphase II . We did not observe any examples of kinetochore-carrying chromosomes located at the spindle poles. Using the thickness of the metaphase II plate in wild-type cells as a standard (see Materials and Methods), we estimated from a sample of six afd1 cells that 60 ± 16% of the single kinetochore chromosomes congressed to the spindle equator . Those chromosomes that aligned at the plate frequently took on a stretched appearance , whereas those that failed to align at the plate usually appeared spherical . Section title: A Majority of Single Kinetochore Chromosomes, but Not Acentric Fragments, Aligns at the Spindle Midzone Educational score: 4.4433369636535645 Domain: biomedical Document type: Study Language: en Among 72 prometaphase–metaphase II afd1 cells that were analyzed in detail (from 6 plants), 32 possessed at least 1 acentric chromosome fragment, i.e., a small DAPI-stained body that lacked detectable CENPC staining. In contrast, a survey of 922 wild-type cells at the same stages revealed no visible fragments (these data were obtained by standard 2D microscopy from 3 wild-type siblings of mutant plants). The localization of acentric fragments in the spindle can be used to assess the direction of the forces prevailing on chromosome arms. Among the acentric fragments scored in afd1 plants, 4 were located in the vicinity of the spindle midzone, whereas a majority of 28 (88%) were located in a polar region. Acentric fragments that were intermingled among the chromosomes at the midzone would have been difficult to detect, so it is possible that we have overestimated the proportion of fragments that migrated to a pole. At a minimum, however, the data show that while no kinetochore carrying chromosomes was found near the spindle poles, a substantial number of acentric fragments was. The simplest interpretation is that kinetochores, not motile forces associated with chromosome arms, are responsible for the alignment of single kinetochore chromosome at the spindle midzone. Section title: Single Kinetochore Chromosome Alignment Occurs by Tension-sensitive Interactions with the Spindle Educational score: 4.196850299835205 Domain: biomedical Document type: Study Language: en In prior studies, we described the localization of two spindle checkpoint proteins on maize mitotic and meiotic kinetochores . MAD2 is a widely conserved checkpoint protein that binds specifically to unaligned kinetochores . The 3F3/2 antigen, also a presumed checkpoint protein, is a phosphoepitope that is sensitive to tension applied at the kinetochore . Antibodies to both proteins recognize an outer domain of the kinetochore in wild-type maize meiocytes . Similarly, single kinetochore chromosomes at prometaphase II stain brightly with both the 3F3/2 antibody and the MAD2 antibody. These data are shown in Fig. 6 . The single kinetochores progressively lost 3F3/2 and MAD2 staining as the cells proceeded through prometaphase and by anaphase II the staining was no longer detectable at kinetochores . The 3F3/2 antibody also stains nonkinetochore sites , which can be distinguished from kinetochore staining by double labeling with either the CENPC or MAD2 antibodies. Section title: Single Kinetochore Chromosome Alignment Occurs by Tension-sensitive Interactions with the Spindle Educational score: 4.181066513061523 Domain: biomedical Document type: Study Language: en We previously demonstrated in wild-type cells that MAD2 and 3F3/2 show nearly identical staining patterns both spatially and temporally . To test whether MAD2 and 3F3/2 are also colocalized on single kinetochores, we analyzed 20 double-labeled cells ranging from early to late prometaphase II. Each of the cells was fixed at a stage when the checkpoint proteins were detectable on some but not all of the kinetochores present (the average number of MAD2-stained kinetochores was 12.4). We found that 98.8% of the kinetochores that were MAD2-positive were also labeled with the 3F3/2 antibody (247/250 single kinetochores). These data lend strong support to the conclusion that 3F3/2 dephosphorylation and MAD2 dissociation are coincident , and indicate that 3F3/2 staining at kinetochores is a reliable marker for the presence of MAD2. Section title: Single Kinetochore Chromosome Alignment Occurs by Tension-sensitive Interactions with the Spindle Educational score: 4.272950172424316 Domain: biomedical Document type: Study Language: en Double labeling for the 3F3/2 antigen and CENPC and for the 3F3/2 antigen and MAD2 indicated that the 3F3/2 antigen lies outside the CENPC domain but in the same domain as MAD2. Since nearly identical results were obtained in wild-type cells , our analysis suggests that the premature disjunction caused by afd1 does not disrupt the basic composition and organization of the kinetochores. Interestingly, a bipolar staining pattern was clearly observed on several single kinetochores, with the 3F3/2 staining occupying opposite ends of stretched kinetochores . These data indicate that single kinetochores have the capacity to divide into half kinetochore units, with each end of the elongated kinetochore interacting independently with a pole. Section title: Single Kinetochore Chromosome Alignment Occurs by Tension-sensitive Interactions with the Spindle Educational score: 4.225070953369141 Domain: biomedical Document type: Study Language: en The results of our previous work suggest that the loss of MAD2 and 3F3/2 staining during meiosis is correlated with the level of tension applied to the kinetochores . Because chromatin is elastic , we were able to use the distance between paired homologous or sister kinetochores to estimate the tension between the kinetochores and their associated kinetochore fibers . We used a similar assay on the single kinetochore chromosomes in afd1 meiocytes. At mid-prometaphase II, the single kinetochores can be observed in a variety of states of alignment. Some show no evidence of a bipolar interaction with the spindle, others are stretched, indicating a bipolar interaction with the spindle, and others lie in between these two extremes. We chose two such mid-prometaphase II cells, and for each scorable kinetochore determined the 3F3/2 staining intensity (using CENPC staining to normalize for kinetochore volume) and the diameter/length of the single kinetochore. As shown in Fig. 7 , there was a strong negative correlation between the two. These data indicate that the loss of 3F3/2 staining on stretched kinetochores is a result of tension applied to the single kinetochore, not an inherent limitation on the duration of the spindle checkpoint (discussed below). Section title: Poleward Movement at Anaphase Causes Stretching of Single Kinetochores Educational score: 4.235264778137207 Domain: biomedical Document type: Study Language: en Anaphase II in afd1 meiocytes can be identified both by the absence of staining for checkpoint proteins and by the degree of kinetochore stretching that occurs during this stage. While kinetochore stretching during prometaphase and metaphase II was mild , in anaphase II it was frequently extreme . An average of 24% of the single kinetochores at anaphase II were stretched into cylindrical shapes ( n = 347 kinetochores in 18 cells), and in several cases the long axes exceeded 5 times the diameter of a normal anaphase II kinetochore . Section title: Poleward Movement at Anaphase Causes Stretching of Single Kinetochores Educational score: 4.185378551483154 Domain: biomedical Document type: Study Language: en In wild-type cells, the kinetochores appear to interact with microtubules in a tangential way early in prometaphase and then display distinct end-on interactions with kinetochores in late prometaphase through anaphase . To analyze the interaction of single kinetochores with microtubules, we analyzed a set of 153 chromosomes in 8 anaphase II cells. Of 36 stretched single kinetochores, 27 showed primarily tangential interactions with microtubules, while 9 others showed the distinct end-on interactions characteristic of wild-type cells. As shown in Fig. 8B–E , when end-on interactions were apparent, there were two kinetochore fibers from opposite poles interacting with the ends of the stretched kinetochore. It is likely that the microtubules that appeared to interact tangentially with stretched kinetochores also extended away from the kinetochores in both directions, but other possibilities cannot be excluded. Section title: Poleward Movement at Anaphase Causes Stretching of Single Kinetochores Educational score: 4.300446033477783 Domain: biomedical Document type: Study Language: en The observation that a single kinetochore can be stretched between two spindle poles suggests that the force may occasionally divide the kinetochore into two parts. That this is indeed the case was demonstrated by a combination of CENPC immunolocalization and in situ hybridization using an ∼155-bp centromeric DNA repeat called CentC . At prophase II, CentC colocalized well with the CENPC-stained kinetochores , although CentC staining varied considerably from chromosome to chromosome . However, on several anaphase chromosomes (eight chromosomes in five anaphase II cells), the kinetochores had clearly separated into two units that were joined by a thin thread of kinetochore material . In each case, the two kinetochore units were attached by centromeric DNA, ruling out the possibility that these were rare examples of normal (i.e., two-chromatid) chromosomes that may have segregated correctly in meiosis I (in fact, we have no evidence that such chromosomes are ever present at meiosis II in the afd1 mutant). We made an effort to quantify the frequency with which centromere/kinetochores were actually broken during anaphase II, i.e., misdivided , by counting the number of CENPC spots in 10 telophase II cells from afd1 plants. Misdivision would be expected to increase the number of kinetochores to a value significantly greater than the expected number of 20. The average kinetochore number in the 10 afd1 cells was 19.78 ± 1.20, which was not significantly different from the kinetochore number in 8 wild-type telophase II cells (19.80 ± 0.84). Section title: Discussion Educational score: 4.280753135681152 Domain: biomedical Document type: Study Language: en Here we demonstrate that meiotic kinetochores, which move to one spindle pole with high fidelity under normal circumstances, will regularly interact with two poles if they are denied a sister kinetochore. The evidence suggests that single kinetochore alignment involves the same basic processes employed during normal chromosome alignment, and that in anaphase, a significant fraction of the single kinetochores divides into half kinetochore units that interact independently with the spindle. In discussing our results, we first evaluate the afd1 phenotype in relation to other published data, and follow with our interpretations of how the single kinetochore chromosomes align, interact with spindle checkpoint proteins, and finally segregate in anaphase. Section title: afd1 Causes a Defect in Sister Chromatid Cohesion at Meiosis I Educational score: 4.29853630065918 Domain: biomedical Document type: Study Language: en Sister kinetochores are normally conjoined in meiosis I and then disjoin to move to opposite spindle poles in meiosis II. However, as described by Golubovskaya and colleagues, these events are significantly altered by the afd1 mutation . Plants homozygous for afd1 appear to skip the early prophase stages of meiosis I and then to prematurely segregate the sister kinetochores to opposite poles. The single kinetochore chromosomes released during meiosis I are then carried through to meiosis II, where they form a haphazard metaphase plate and then segregate randomly at anaphase II . Using antibodies to the recently identified maize CENPC protein , we were able to confirm this phenotype and demonstrate that the equational segregation of sister kinetochores at meiosis I is close to 100% . Section title: afd1 Causes a Defect in Sister Chromatid Cohesion at Meiosis I Educational score: 4.3531622886657715 Domain: biomedical Document type: Study Language: en To explain the afd1 phenotype, Golubovskaya and colleagues proposed that the mutation causes a substitution of meiosis I with mitosis-like division, i.e., that meiosis I is absent in afd1 plants . However, our immunocytochemical analysis suggests that the phenotype is more complex than this. Although some prophase I stages cannot be identified (roughly leptotene to pachytene), sister kinetochore separation in afd1 meiocytes starts after nuclear envelope breakdown as is characteristic of normal meiosis I cells . This contrasts with mitosis or normal meiosis II , where kinetochore separation is readily apparent at prophase. Further, the chromatin condensation patterns and spindle morphology of the first division more closely resemble meiosis I in wild-type cells than mitosis . We also show here that a low level of chromosome fragmentation occurs during meiosis I in afd1 plants and that many of these fragments lack kinetochores . Section title: afd1 Causes a Defect in Sister Chromatid Cohesion at Meiosis I Educational score: 4.412683486938477 Domain: biomedical Document type: Study Language: en The available data suggest that Afd1 encodes a cohesin such as budding yeast rec8p, a meiosis-specific rad21-like protein . Mutations in REC8 cause premature separation of sister chromatids and inhibit reciprocal recombination. The chromosome fragmentation caused by afd1 may be evidence of aborted recombination events, since meiotic recombination involves the formation of double-strand breaks . REC8 / RAD21 -like genes are widely conserved in eukaryotes , and mutants of a meiosis-specific RAD21 -like gene called SYN1/DIF1 have recently been described in Arabidopsis . Consistent with a homology between the two genes, there are clear similarities between the afd1 phenotype and the Arabidopsis syn1/dif1 phenotype . Section title: afd1 Causes a Defect in Sister Chromatid Cohesion at Meiosis I Educational score: 4.2096710205078125 Domain: biomedical Document type: Study Language: en Recent data from both Saccharomyces cerevisiae and Schizosaccharomyces pombe suggest that cohesin is not required to maintain the integrity of the mitotic kinetochore . These data are consistent with our own data, showing that at least three kinetochore proteins (CENPC, MAD2, and the 3F3/2 antigen) show the stage-specific and subkinetochore localization typical of wild-type kinetochores . Although we cannot rule out the possibility that afd1 has subtle effects on the morphology or makeup of the kinetochore, the available data suggest that the sister kinetochores are separated but otherwise undisturbed as they enter meiosis II. Section title: Single Meiotic Kinetochores Regularly Align at the Metaphase Plate Educational score: 4.203956604003906 Domain: biomedical Document type: Study Language: en Using both living and fixed specimens, we demonstrate that ∼60% of single kinetochore chromosomes align at the metaphase II plate , whereas ∼88% of acentric fragments move in the opposite direction towards a pole . These data support the observations of Khodjakov et al. 1997 , who, with data obtained from a different class of cell division (mitosis) and a distantly related species (rat kangaroo), showed that single kinetochores are capable of autoaligning at metaphase. Our data are also in agreement with earlier data from the same group showing that acentric fragments in Haemanthus mitotic cells move poleward during chromosome alignment . Section title: Single Meiotic Kinetochores Regularly Align at the Metaphase Plate Educational score: 4.387717247009277 Domain: biomedical Document type: Study Language: en There are also notable differences between our data and those published previously. Perhaps the most significant is the demonstration by Khodjakov et al. 1996 that Haemanthus single kinetochore chromosomes fail to align at the spindle midzone. An explanation for this may lie in the fact that meiotic kinetochores display morphological plasticity in the course of their normal function, first associating closely with a sister kinetochore in meiosis I and then subsequently changing their behavior to dissociate from the sister in meiosis II. The inherent capacity for remodeling may make the meiotic kinetochore especially susceptible to aberrant alignment during meiosis II. Another notable difference is that acentric chromosome fragments move plateward during prometaphase in mammalian mitotic cells , not poleward as in Haemanthus mitosis or maize meiosis. This difference is probably related to the function of centrosomes, which organize the spindle poles in animal mitotic cells, but which are absent in all higher plant cells and the meiotic cells of some animals . The active polymerization of microtubules outward from centrosomes is thought to be part of the force that drives chromosome fragments plateward . In cells that lack centrosomes, the dominant force affecting the movement of acentric fragments may be the poleward flux of tubulin monomers within the spindle . Section title: Single Kinetochores Interact Normally with Spindle Checkpoint Proteins and Demonstrate Anaphase Motility Educational score: 4.269735336303711 Domain: biomedical Document type: Study Language: en In normal cells, sister/homologous kinetochores orient towards opposite spindle poles and generate tension with attached kinetochore fibers . The kinetochores move farther away from each other as increasing tension is applied during prometaphase, such that there is a rough correlation between tension and kinetochore–kinetochore distance . MAD2 and 3F3/2 staining decrease as kinetochore–kinetochore distance increases during maize meiosis, suggesting that the release/dephosphorylation of these proteins is tension sensitive . Here we demonstrate the same effect on single kinetochore chromosomes. Single kinetochores showed variable degrees of stretching during prometaphase , and stretching was negatively correlated with 3F3/2 staining intensity . The double staining required for these experiments revealed that many of the single kinetochores had divided into half kinetochore units. A distinct bipolar staining pattern was observed, where 3F3/2 staining was fully divided and localized at the poles of the elongated kinetochores . Section title: Single Kinetochores Interact Normally with Spindle Checkpoint Proteins and Demonstrate Anaphase Motility Educational score: 4.385554790496826 Domain: biomedical Document type: Study Language: en It is known that tension at the kinetochore makes kinetochore fibers more stable and/or promotes microtubule bundling within the fiber . Therefore, it is likely that once bipolar connections are established by the single kinetochore, the resulting tension stabilizes the interaction and serves to further separate and define the half kinetochore units. For the numerous kinetochores that did not appear to adopt a bipolar interaction with the spindle , the loss of MAD2 and 3F3/2 staining may be the result of a normal “timing out” of the checkpoint. In the presence of microtubule-destabilizing drugs that activate the spindle checkpoint in budding yeast, the cell cycle is delayed for a period of ∼6 h, after which the cell cycle proceeds regardless of the state of the chromosomes . Similarly, Li and Nicklas 1995 demonstrated a 5–6-h delay when a single unaligned chromosome was present during insect meiosis. In our study of the afd1 mutant, the full prophase–metaphase II period was documented in only two cells. Prometaphase II extended for ∼1 h longer than expected in these cells, suggesting that the meiosis II spindle checkpoint times out relatively quickly in maize. Section title: Single Kinetochores Interact Normally with Spindle Checkpoint Proteins and Demonstrate Anaphase Motility Educational score: 4.307825088500977 Domain: biomedical Document type: Study Language: en Anaphase II in the afd1 mutant is marked by two events: the loss of staining for checkpoint proteins , and a significant increase in the stretching of many single kinetochores . In many cases, the kinetochore stretching appeared to be caused by prometaphase-like tangential interactions with microtubules , whereas in others, there were clearly identifiable kinetochore fibers interacting with each end of the stretched kinetochores . The type of end-on interactions shown in Fig. 8 B occurred on 25% of the stretched kinetochores and 6% of all single kinetochores. These data support the idea that single kinetochores can divide into half kinetochore units, and indicate that each unit can undergo poleward motility. Section title: Centromere/Kinetochore Redundancy Educational score: 4.426342964172363 Domain: biomedical Document type: Study Language: en A large body of literature suggests that the centromeric DNA, which underlies the kinetochore and may be entwined within it , has a redundant structure. The bulk of the cytological evidence for redundancy comes from experiments where centromeres are split by a process known as centromere misdivision . One method for detecting misdivision is to observe the behavior of univalents at meiosis I (unpaired chromosomes) or single kinetochore chromosomes at meiosis II. Such chromosomes often appear to break at the centromeres, in some species, such as wheat, at frequencies close to 40% . More recently, the molecular analysis of centromeres in Drosophila and Arabidopsis has revealed highly reiterated sequence elements . Maize centromeres appear to have a similar structure. Several abundant sequence repeats have been identified at maize centromeres , and the centromere of the B chromosome can be reduced by misdivision to ∼10% of its natural size and still retain function . Section title: Centromere/Kinetochore Redundancy Educational score: 4.411586761474609 Domain: biomedical Document type: Study Language: en Other evidence suggests that the centromere–kinetochore complex has a visibly redundant external structure. Lima-de-Faria 1958 reviewed this literature, which in some cases provides convincing descriptive evidence for half kinetochore units on single kinetochore chromosomes . More recently, Mole-Bajer et al. 1990 used kinetochore-specific human (calcinosis, Raynaud phenomenon, esophageal dismotility, sclerodactyly, telangiectasia [CREST]) autoantisera to show that, under enhanced immunogold detection conditions, the mitotic kinetochores in Haemanthus appear to be composed of two (and sometimes four) distinct units. Similarly, when mammalian mitotic kinetochores are artificially stretched, a repetitive staining pattern is observed . We show here using functional assays and specific antibodies to a kinetochore outer domain that the maize meiotic kinetochore is divisible into two parts. We consider it unlikely that kinetochores are especially prone or otherwise limited to being divided into two parts. As discussed above, it is probably the interaction with the spindle and the stabilizing properties of kinetochore–microtubule attachment that is responsible for the twofold redundancy we have observed. Section title: Centromere/Kinetochore Redundancy Educational score: 4.257602691650391 Domain: biomedical Document type: Study Language: en Östergren 1947 suggested that the half kinetochore units that were sometimes visible in the light microscope might independently orient towards different spindle poles and cause misdivision . Our data support this model for centromere misdivision. However, in maize the high frequency of single kinetochore alignment (∼60%) and stretching at anaphase (∼23%) does not result in a comparable level of centromere breakage. In our data set of 10 telophase II cells, we found no evidence for centromere–kinetochore misdivision. Therefore, the data suggest that while maize half kinetochore units frequently orient towards opposite spindle poles, this biorientation is usually resolved by one half of the kinetochore releasing its attachment. The frequency with which a bipolar kinetochore orientation results in centric misdivision is likely to be a species-specific parameter that depends on the size and sequence of the centromere as well as the molecular makeup of the kinetochore. | Study | biomedical | en | 0.999998 |
0005034 | Section title: Introduction Educational score: 3.892241954803467 Domain: biomedical Document type: Study Language: en Differentiated oligodendrocytes synthesize large amounts of myelin, a multilamellar membrane that ensheathes and insulates the axons of the central nervous system . The specific molecular organization of myelin is essential for the function of the nervous system, and the disturbance of this architecture leads to severe neurological symptoms observed in diseases such as multiple sclerosis. How oligodendrocytes form and maintain this specialized membrane is not known. Section title: Introduction Educational score: 4.348992824554443 Domain: biomedical Document type: Study Language: en In contrast to most plasma membranes, myelin has a specialized lipid composition and contains a restricted set of proteins. Two main proteins comprise the majority (∼85%) of the myelin protein: the cytoplasmic protein myelin basic protein (MBP) and the integral membrane protein proteolipid protein (PLP) . PLP and its alternatively spliced isoform DM20 are very hydrophobic proteins spanning the membrane four times with a molecular mass of 26 and 20 kD, respectively . The lipophilic character of both isoforms increases during transport through the cell due to the addition of several acyl chains during the final stages of biosynthesis . Section title: Introduction Educational score: 4.240111351013184 Domain: biomedical Document type: Study Language: en Myelin has an unusual lipid composition. In contrast to most plasma membranes, myelin is enriched in the glycosphingolipids, galactosylceramide and sulfatide. This high level of glycosphingolipids is accompanied by a low level of phosphatidylcholine, the main lipid of plasma membranes of most cells. The ratio of glycosphingolipid/phospholipid/cholesterol is very similar to that of the apical brush border membrane of epithelial cells . This led us to postulate that oligodendrocytes and epithelial cells may utilize common mechanisms to form these specialized membranes. Section title: Introduction Educational score: 4.945408821105957 Domain: biomedical Document type: Study Language: en Two major sorting principles have been described in epithelial cells. For transport to the basolateral surface, the interaction of a cytoplasmic protein machinery with transmembrane proteins is essential . In contrast, lipid–lipid and protein–lipid interactions play a major role in apical transport . According to this model, glycosphingolipid and cholesterol segregate from bulk lipids within the TGN and form so called lipid rafts that recruit a specific set of proteins, which are transported via vesicular carriers to the apical surface membrane. The formation of rafts is thought to occur by self-association of sphingolipids via their long saturated acyl chains . Hydrogen bonding between the glycosphingolipid headgroups of rafts is not required, but might additionally stabilize rafts . These interactions lead to the formation of a less fluid, liquid-ordered phase, separating from a phosphatidylcholine-rich liquid-disordered phase within the exoplasmic leaflet . It has been shown that molecules within the liquid-ordered phase remain as an insoluble membrane fraction after extraction with detergent under mild conditions . Due to their high lipid content, detergent-insoluble membrane fractions float in a low-density fraction after density gradient centrifugation and are sequestered from other detergent-insoluble material such as the majority of the cytoskeleton. An interesting feature of proteins that associate with rafts is the presence of specific posttranslational modifications. For example, glycosylphosphatidylinositol (GPI)-anchored proteins are anchored via a lipid moiety, whereas Src-family kinases, nitric oxide synthase, influenza virus hemagglutinin (HA), and caveolin are acylated . Cholesterol has been shown to be essential for association of these proteins with rafts . Thus, the behavior of proteins in response to cholesterol depletion and their resistance to extraction with mild detergent can be used as criteria for raft association. Section title: Introduction Educational score: 4.060626983642578 Domain: biomedical Document type: Study Language: en Rafts are thought to be small and highly dynamic and thus are not resolved on cultured cells by conventional microscopic techniques . However, the patching of membrane components of rafts with antibodies leads, to the coalescence of small rafts into larger domains that can be readily visualized . Proteins present in similar lipid phases tend to cocluster during patching experiments, while proteins present in immiscible lipid phase remain separate . Hence, the patching behavior of a protein can be used as a morphological criterion for raft association complementary to the biochemical criteria. Section title: Introduction Educational score: 3.628675699234009 Domain: biomedical Document type: Study Language: en Here, we have studied the mechanism of myelin assembly and asked the question whether lipid–protein interactions are involved in the generation of myelin. Section title: Reagents Educational score: 3.6160194873809814 Domain: biomedical Document type: Study Language: en Unless otherwise stated, all chemicals were obtained from the sources described previously by Krämer et al. 1997 . 3-[(3-cholamidopropyl) dimethylammonia]-1-propanesulfonate (CHAPS), fumonisin B1, and methyl-β-cyclodextrin (mβCD) were from Sigma-Aldrich; Optiprep was from Nycomed Pharma. The following rabbit pAbs were used: antibodies recognizing neural cell adhesion molecule (NCAM) , HA , E2, the COOH-terminal peptide of PLP , and myelin-associated glycoprotein (MAG; a gift from F. Kirchhoff, Max-Planck-Institute of Experimental Medicine, Göttinger, Germany). The following mAbs were used: murine mAb 27-11-111 made against chick F11 , provided by F. Rathjen (Max Delbrick Center for Molecular Medicine, Berlin-Buch, Germany), which cross-reacts with mouse F3; murine mAb against myelin oligodendrocyte glycoprotein (MOG) (clone 818C5) and mouse mAb against MBP provided by C. Linington (Max-Planck-Institute of Neurobiology, Planegg-Martinstied, Germany); murine mAb O10 , which recognizes PLP . Secondary antibodies were from Dianova. Section title: Cell Culture and Transfection Educational score: 4.145689964294434 Domain: biomedical Document type: Study Language: en Primary cultures of oligodendrocytes were prepared from embryonic day 14–16 mice as described . Oligodendrocytes growing on top of a layer of astrocytes were shaken off and cultured further in modified Sato medium supplemented with 1% horse serum (HS) on poly- l -lysine–coated dishes. The cells were fed with 10 ng/ml human recombinant PDGF and 5 ng/ml basic FGF immediately and 24 h after the shake. The cells were left for at least 5 d in culture before using in experiments. For inhibition of sphingomyelin synthesis, fumonisin B1 was added from a 1 mM stock in 20 mM Hepes, pH 7.4, to oligodendrocytes 24 h after plating at a final concentration of 50 μM. Fumonisin B1 was subsequently added every 24 h at a concentration of 50 μM for a total of 5 d. Section title: Cell Culture and Transfection Educational score: 3.8837475776672363 Domain: biomedical Document type: Study Language: en BHK-21 cells were maintained at 37°C in 5% CO 2 in a humidified atmosphere in G-MEM (GIBCO BRL), 10% FCS, supplemented with penicillin (100 U/ml)/streptomycin (100 μg/ml) and 2 mM glutamine (all from GIBCO BRL). Section title: Cell Culture and Transfection Educational score: 3.466891288757324 Domain: biomedical Document type: Study Language: en Transfection of BHK cells was performed with FuGENE Transfection reagents (Roche Molecular Biochemicals) according to the manufacturer's protocol. Section title: Myelin Preparation Educational score: 4.114459037780762 Domain: biomedical Document type: Study Language: en Myelin was prepared from adult mice as described previously . In brief, brains were homogenized in ice-cold 10.5% sucrose using an Ultra-Turrax T25 (IKA) and centrifuged in a sucrose step gradient. Myelin was recovered from the 10.5 and 30% interface and further purified by two rounds of hypoosmotic shock by resuspension in a large volume of ice-cold water, followed by a second round of centrifugation in a sucrose step gradient. Purified myelin was collected from the interface, washed twice with ice-cold water, resuspended in a small volume of water, and frozen in small aliquots at −80°C. The protein content of the myelin preparations was determined using a protein assay from BioRad Laboratories. Section title: Preparation of Detergent-insoluble Membrane Fractions and Total Membrane Fraction Educational score: 3.6402924060821533 Domain: biomedical Document type: Study Language: en Detergent extraction with CHAPS and Triton X-100 was performed as detailed by Fiedler et al. 1993 . Section title: Preparation of Detergent-insoluble Membrane Fractions and Total Membrane Fraction Educational score: 4.240913391113281 Domain: biomedical Document type: Study Language: en Primary oligodendrocytes cultured in a 5-cm dish were washed once in PBS and scraped into 300 μl 50 mM Tris-HCl, pH 7.4, 5 mM EDTA (TE), or 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA (TNE) buffer supplemented with protease inhibitors (Complete Mini; Roche Molecular Biochemicals). Cells were sheared by resuspending 20 times with a blue pipette tip and 25 times with a 25 G needle and centrifuged for 5 min at 3,000 rpm. The postnuclear supernatant was subjected to detergent extraction for 30 min at 4°C by adding CHAPS (20 mM final) to TE buffer or Triton X-100 (1% final) to TNE buffer. For extraction of myelin, 1–10 μg of myelin was added to 300 μl of 20 mM CHAPS in TE, or 1% Triton X-100 in TNE, supplemented with protease inhibitors, and incubation was performed for 30 min at 4°C. For density gradient centrifugation, we prepared a discontinous gradient with different dilutions of Iodixanol (Optiprep; Nycomed Pharma), which has a density of 1.32 g/ml. After the extraction, 250 μl of the lysates was adjusted to 40% Optiprep by adding 500 μl of Optiprep solution (60%), overlaid with 1.2 ml of 30% Optiprep in extraction buffer, and 200 μl of extraction buffer. The samples were centrifuged for 2 h at 55,000 rpm in a TLS 55 rotor (Beckman) and six fractions of equal volume were collected from the top. In some experiments, the top fraction was subjected to a second round of detergent extraction, adjusted to 40% Optiprep, and centrifuged in a density gradient as described above. Section title: Preparation of Detergent-insoluble Membrane Fractions and Total Membrane Fraction Educational score: 3.8934032917022705 Domain: biomedical Document type: Study Language: en An equal volume of each fraction was either directly processed further for SDS-PAGE, or fractions were concentrated by adding an equal volume of 20% TCA, centrifuging for 30 min at 15,000 g at 4°C, and washing the pellets with acetone at −20°C. Section title: Preparation of Detergent-insoluble Membrane Fractions and Total Membrane Fraction Educational score: 4.04641056060791 Domain: biomedical Document type: Study Language: en Total membranes were prepared as described above except that the extraction step was omitted and preparation of cleared lysates and flotations in density gradients were performed with a buffer containing 10 mM Hepes, pH 7.4, and 2 mM EGTA. Total cellular membranes were recovered from the 0–30% interface. Section title: Immunoblotting and Immunoprecipitation Educational score: 4.194966793060303 Domain: biomedical Document type: Study Language: en For immunoblotting, samples were incubated at 95°C for 5 min or at 55°C for 10 min in Laemmli sample buffer and proteins were resolved on SDS-PAGE and transferred to polyvinylidene difluoride. Membranes were blocked in PBS/4% milk powder/0.2% Tween and incubated overnight at 4°C with primary antibody diluted in blocking buffer. Blots were incubated with appropriate horseradish peroxidase–labeled secondary antibody for 1 h. The blots were developed by enhanced chemiluminescence (Amersham Pharmacia Biotech). For pulse-labeling experiments, oligodendrocytes were incubated with 1 mCi/ml [ 35 S]methionine/cysteine for 5 min and chased in the presence of 150 μg/ml unlabeled methionine and cysteine before detergent extraction and density gradient flotation. Immunoprecipitation was performed from the collected Optiprep fractions. The samples were incubated overnight at 4°C with anti-PLP antibody and immunocomplexes were recovered with protein A–Sepharose. Washed samples were solubilized in Laemmli buffer, incubated for 10 min at 55°C, subjected to SDS-PAGE, and followed by fluorography. Section title: Viral Infection and Antibody-induced Patching Educational score: 4.105593681335449 Domain: biomedical Document type: Study Language: en For infection with HA influenza virus or Semliki Forest virus (SFV), virus was diluted in culture medium and virus absorption was performed for 1 h. Infection was allowed to continue for a further 1–3 h. For antibody-induced patching, cells were incubated with polyclonal anti-HA or polyclonal anti-E2 antibodies in combination with monoclonal O10 anti-PLP antibody or monoclonal NCAM antibody BSP2 for 1 h at 12°C. After washing briefly in PBS, cells were incubated with the respective FITC- and Cy-3–coupled secondary antibodies for 1 h at 12°C. Cells were washed, then fixed for 4 min in 4% PFA at 8°C and for 5 min with methanol at −20°C. The coverslips were mounted in moviol, and immunofluorescence pictures of the FITC and rhodoamine stainings were taken separately using a Zeiss Axiophot microscope coupled to a Photonic Science 8-bit CCD color camera (Photonic Science Ltd.). Digital images were processed using Photoshop ® software (Adobe Systems). Section title: Cholesterol Depletion and Labeling with Photoactivable Lipids Educational score: 4.056851387023926 Domain: biomedical Document type: Study Language: en Cholesterol depletion was performed as detailed by Scheiffele et al. 1997 . In brief, oligodendrocytes were washed once with Sato medium, treated with 5 mM mβCD in Sato medium for 30 min at 37°C, and washed again with Sato medium before further processing. Photoaffinity labeling was performed exactly as described by Thiele et al. 1999 . Section title: Cholesterol Depletion and Labeling with Photoactivable Lipids Educational score: 4.136350631713867 Domain: biomedical Document type: Study Language: en Cells cultured in 5-cm dishes were washed with Sato supplemented with 1% lipid-free HS and incubated with 100 μl 3 H-photocholesterol–mβCD inclusion complex in 2 ml Sato/1% lipid-free HS for 16 h. Cells were washed with PBS/1% lipid-free HS and irradiated for 20 min with UV light at 4°C. The cells were washed, lysed in lysis buffer (1% NP-40, 50 mM Tris-HCl, pH 7.4, 5 mM EDTA, and protease inhibitors) and cleared of nuclei by a brief centrifugation. Lysates were either directly processed for SDS-PAGE or first subjected to immunoprecipitation with anti-PLP antibodies. Section title: Cholesterol Depletion and Labeling with Photoactivable Lipids Educational score: 4.100324630737305 Domain: biomedical Document type: Study Language: en For labeling with photophosphatidylcholine, cells (5-cm dish) were washed with labeling medium (phenol red–free DMEM, without choline, supplemented with 1% lipid-free HS) and incubated with 3 ml of labeling medium and 100 μl serum loaded with 10-ASA . After 20 min incubation, 200 μCi of [ 3 H]choline was added and incubation continued for 16 h. Cells were washed with labeling medium, irradiated for 20 min with UV light at 4°C, and further processed as described above. Section title: Lipid Analysis Educational score: 4.1831793785095215 Domain: biomedical Document type: Study Language: en Oligodendrocytes were labeled overnight with 50 μCi/ml [ 14 C]acetate and a total membrane fraction was prepared as described above. Floated membranes were split in two fractions, treated with detergent (either 20 mM CHAPS or 1% Triton X-100) or buffer alone, and floated in a density gradient. For analysis of lipids from myelin, 10 μg (protein weight) of myelin was extracted with 20 mM CHAPS, 1% Triton X-100, or buffer alone, before flotation in a density gradient. Lipids were extracted from the top fraction by the method of Bligh and Dyer 1959 . Dried samples were dissolved in 20 μl of chloroform/methanol (1:1, vol/vol) and spotted on activated Silica Gel 60 F 254 plates. After resolution of lipids in chloroform/methanol/water (65:25:4, by volume), the plates were air-dried and lipids were visualized either by autoradiography or by exposure of the plates to 20% sulfuric acid and charring. Section title: Lipid Analysis Educational score: 3.123737335205078 Domain: biomedical Document type: Study Language: en Quantification of lipids was performed by densitometric scanning (Arcus II; Agfa) using Adobe Photoshop ® and MacBAS. Results from three independent experiments are given as an average ± SD. Section title: Myelin Is Resistant to Extraction with CHAPS Educational score: 4.076902866363525 Domain: biomedical Document type: Study Language: en To investigate whether components of myelin associate to form rafts, we prepared myelin from adult mice and subjected it to extraction with various detergents. The sample was adjusted to 40% Optiprep and loaded at the bottom of a step gradient (40, 30, and 0%). After centrifugation, six fractions were collected starting at the top. Proteins were separated by SDS-PAGE followed by Western blotting using antibodies against specific myelin proteins. Section title: Myelin Is Resistant to Extraction with CHAPS Educational score: 4.193913459777832 Domain: biomedical Document type: Study Language: en As shown previously , extraction with 1% Triton X-100 leads to solubilization of the majority of PLP/DM20, MAG, MOG, and MBP . Only a minor fraction of these proteins was recovered from the low-density, detergent-insoluble membrane fraction (0–30% interface) . However, when myelin was extracted with 20 mM CHAPS, the difference compared with extraction with Triton X-100 was striking. PLP/DM20, MAG, and MOG were almost exclusively recovered from the detergent-resistant interface . In contrast, a prominent fraction of the major cytosolic protein, MBP, was found at the bottom of the gradient . Section title: Myelin Is Resistant to Extraction with CHAPS Educational score: 4.171888828277588 Domain: biomedical Document type: Study Language: en To determine the lipid composition of the detergent-insoluble membrane fractions, myelin was extracted with detergents, or was treated with buffer alone as a control, and centrifuged in a density gradient. Analysis of the lipid composition of the floating membrane fraction showed that Triton X-100– and CHAPS-extracted membranes were composed of similar lipids . While a fraction of the glycerophospholipids was solubilized, the majority of galactosylceramide and cholesterol was found to be resistant to extraction with Triton X-100 or CHAPS . Section title: Myelin Is Resistant to Extraction with CHAPS Educational score: 4.274023056030273 Domain: biomedical Document type: Study Language: en We investigated whether the CHAPS-insoluble membrane fraction (CIMF) was generated in primary cultures of oligodendrocytes. After several days in culture, these cells produce an extensive network of branching processes followed by the formation of myelin-like membrane sheets. The morphological changes are accompanied by the expression of the major components of myelin, such as the lipids galactosylceramide and sulfatide, and the proteins PLP, MBP, and MOG . Lysates of these cells were extracted with either 1% Triton X-100 or 20 mM CHAPS at 4°C, mixed with Optiprep, and subjected to centrifugation and SDS-PAGE. Western blotting showed that the majority of PLP/DM20 was recovered from the CIMF . When this fraction was reextracted and refloated in a density gradient, it remained completely associated with CIMF . In contrast, extraction with Triton X-100 leads to almost complete solubilization of PLP after two rounds of detergent extraction. MOG was also shown to be resistant to extraction with CHAPS, but not with Triton X-100, whereas MBP was solubilized by both Triton X-100 and CHAPS . Section title: Myelin Is Resistant to Extraction with CHAPS Educational score: 4.205735206604004 Domain: biomedical Document type: Study Language: en To determine the lipid composition of the CIMF from oligodendrocytes, cells were labeled with [ 14 C]acetate and the total membrane fraction was incubated either with 20 mM CHAPS or with buffer alone and centrifuged in a density gradient. Lipids were extracted from the gradient fractions and separated by TLC. We found that the majority of galactosylceramide (63 ± 3%) and cholesterol (63 ± 3%) was insoluble, whereas most of the glycerophospholipids were solubilized . For comparison, 83 ± 6% of galactosylceramide, 78 ± 5% of cholesterol, and 34 ± 3% of the glycerophospholipids remained insoluble when extractions were performed with Triton X-100 . Comparison of the ratio of cholesterol/phospholipid/glycosphingolipid of the detergent-insoluble membrane fractions (4:4:2) with the ratio of the respective lipids in myelin (4:4:2–4:3:2) reveals a striking similarity. Section title: GPI-anchored Proteins Resist Extraction with Triton X-100 but Are Largely Solubilized by CHAPS Educational score: 4.147673606872559 Domain: biomedical Document type: Study Language: en Having shown that the major components of myelin were resistant to extraction with CHAPS, it was important to rule out that the conditions for detergent extraction were so mild that large fragments of the plasma membrane remained completely intact. Oligodendrocytes were subjected to metabolic radiolabeling with 35 S-Translabel, and the total floated membrane fraction was either extracted with 20 mM CHAPS or left untreated; fractions were then separated on a density gradient. The majority of the radioactivity was recovered at the 0–30% interface from membranes that had not been incubated with CHAPS . In contrast, the radioactivity was mainly found in the bottom fractions (especially fraction 4) from membranes that had been incubated with CHAPS, demonstrating that CIMF at the 0–30% interface represents a subdomain of the oligodendrocyte membrane . Section title: GPI-anchored Proteins Resist Extraction with Triton X-100 but Are Largely Solubilized by CHAPS Educational score: 4.137144565582275 Domain: biomedical Document type: Study Language: en Subsequently, we further characterized the CIMF. The fact that GPI-anchored proteins are highly enriched in a Triton X-100–insoluble complex in oligodendrocytes prompted us to investigate if GPI-anchored proteins also associate with CIMF. To address this question, we compared the distribution of two major GPI-anchored proteins of oligodendrocytes, F3 and NCAM120, on density gradients from CHAPS- and Triton X-100–extracted cells. As shown previously, we found that F3 and NCAM120 were highly enriched in the Triton X-100–insoluble membrane, whereas a large fraction of F3 and NCAM120 was solubilized by CHAPS . The high molecular weight forms of NCAM, NCAM140, and NCAM180, which lack a GPI anchor, were exclusively found in the soluble fraction with both detergents . Section title: GPI-anchored Proteins Resist Extraction with Triton X-100 but Are Largely Solubilized by CHAPS Educational score: 3.8984246253967285 Domain: biomedical Document type: Study Language: en These results demonstrate that the CIMF represents a subdomain of the oligodendrocyte membrane, which is highly enriched in myelin components. Section title: PLP Associates with CIMF in the Golgi Apparatus Educational score: 4.138916015625 Domain: biomedical Document type: Study Language: en To determine if PLP associates with CIMF during the biosynthetic pathway, we subjected primary oligodendrocytes to a 5-min labeling “pulse” of 35 S-Translabel. After various times of “chase,” cells were lysed, incubated with CHAPS, and fractions were separated on a density gradient, as described previously. PLP/DM20 was immunoprecipitated from the low-density, detergent-insoluble fraction (fraction 1) and from the soluble fraction (fraction 4), which contains the majority of soluble PLP/DM20. PLP/DM20 was mainly found in the soluble fraction up to 30 min after the pulse, but started to become insoluble at later time points . Section title: PLP Associates with CIMF in the Golgi Apparatus Educational score: 4.120688438415527 Domain: biomedical Document type: Study Language: en Since PLP does not acquire carbohydrate modifications after translation, which could be used to trace the transit of the protein through the biosynthetic pathway, we took advantage of the fact that newly synthesized proteins accumulate in the ER at 15°C and in the Golgi apparatus at 20°C to determine where PLP/DM20 first associates with CIMF. Primary oligodendrocytes were labeled with a 10-min pulse and chased for 120 min at 15°C or 20°C. While PLP/DM20 remained soluble during the entire chase period at 15°C, CHAPS-insoluble complexes containing a fraction of PLP/DM20 could be isolated when the chase was performed at 20°C . Section title: PLP Associates with CIMF in the Golgi Apparatus Educational score: 4.038543224334717 Domain: biomedical Document type: Study Language: en These data show that PLP/DM20 becomes CHAPS insoluble during passage through the biosynthetic pathway and suggest that the insoluble complexes are generated after the protein has left the ER but before exit from the TGN. Section title: HA Colocalizes with PLP on the Surface of Oligodendrocytes Educational score: 4.18012809753418 Domain: biomedical Document type: Study Language: en Our biochemical data using detergents suggest that PLP/DM20 is present in a membrane fraction with properties similar to lipid rafts. If this were indeed the case, PLP should colocalize on the surface of oligodendrocytes with other components of CIMF and should segregate from components that are not part of CIMF. It has been shown previously that the influenza virus HA protein is recovered to a greater extent from CHAPS-insoluble membrane than from Triton X-100–insoluble membrane and thus might be a useful marker for CIMF. We confirmed that HA was also insoluble in CHAPS in primary oligodendrocytes . HA was largely insoluble in Triton X-100 (data not shown) as well. For a non-CIMF marker, we used the SFV spike protein, E2, which we found to be soluble in CHAPS in oligodendrocytes . Section title: HA Colocalizes with PLP on the Surface of Oligodendrocytes Educational score: 4.1609787940979 Domain: biomedical Document type: Study Language: en To determine whether the proteins are found in a similar environment on the surface of oligodendrocytes as PLP, we performed copatching experiments. Cells were infected either with influenza virus or SFV and subsequently incubated with anti-HA and O10 anti-PLP antibodies, or anti-E2 and O10 anti-PLP antibodies respectively, at 12°C. This was followed by incubation with the appropriate secondary antibodies for an additional 1 h at 12°C. Microscopic analysis showed that HA and PLP appeared in the same patches in the majority of cells, suggesting that both molecules are present in the same domains of the plasma membrane . In contrast, E2 and PLP only partially colocalized on the surface of oligo dendrocytes . When antibody against NCAM was used to localize the GPI-anchored protein NCAM120, the major NCAM isoform in more mature oligodendrocytes, it was apparent that HA and NCAM were largely colocalized (data not shown). Section title: HA Colocalizes with PLP on the Surface of Oligodendrocytes Educational score: 3.7709310054779053 Domain: biomedical Document type: Study Language: en Since proteins present in similar lipid phases tend to cocluster, while proteins present in immiscible lipid phases separate , these data support the premise that PLP is found in a similar lipid environment to HA at the surface of oligodendrocytes. Section title: Cholesterol Interacts with PLP/DM20 and Is Required for the Formation of CIMF Educational score: 4.140779495239258 Domain: biomedical Document type: Study Language: en To study the involvement of cholesterol in the formation of CIMF, we analyzed whether PLP/DM20 was still associated with CIMF in cholesterol-depleted membranes. To deplete oligodendrocytes of cholesterol, we used mβCD, which is known to extract cholesterol with a higher specificity than other lipids . Oligodendrocytes were cultured in the presence of 5 mM mβCD for 30 min, extracted with CHAPS, and run on a density gradient gel, where the distribution of PLP/DM20 was compared with untreated cells. After cholesterol depletion, PLP/DM20 was no longer associated with CIMF to the same extent as in control cells . These results demonstrate that cholesterol is an essential component of CIMF. Section title: Cholesterol Interacts with PLP/DM20 and Is Required for the Formation of CIMF Educational score: 4.178189277648926 Domain: biomedical Document type: Study Language: en To investigate whether PLP/DM20 interacts directly with cholesterol, we used a recently developed photocholesterol probe . In this compound a photoactivable diazirine ring replaces the Δ5 double bond and the hydrogen bond at C-6. Use of this probe has recently identified synaptophysin as a major cholesterol-interacting protein in neuroendocrine cells . To determine whether PLP/DM20 interacts with cholesterol, oligodendrocytes were cultured for 16 h in the presence of 3 H-photocholesterol complexed to mβCD. Subsequently, proteins interacting with cholesterol were cross-linked by irradiation with UV light. The autoradiography of proteins separated by SDS-PAGE shows a major radiolabeled low molecular weight protein with an apparent molecular mass of 25 kD . To determine if this protein is indeed PLP, the oligodendrocytes were immunoprecipitated with an anti-PLP antibody after the photolabeling. As shown in Fig. 6 B, immunoprecipitation clearly identifies the 25-kD photolabeled protein as PLP. Section title: Cholesterol Interacts with PLP/DM20 and Is Required for the Formation of CIMF Educational score: 4.295167446136475 Domain: biomedical Document type: Study Language: en To demonstrate that the interaction of PLP with cholesterol is specific and not merely due to the high abundance of these two components in the plasma membrane of oligodendrocytes, we compared the labeling of proteins by photocholesterol with the labeling of proteins by another lipid that is present at high levels in the plasma membrane. Photoactivable phosphatidylcholine is an ideal control; however, this lipid cannot be readily introduced into the membrane by exogenous addition. Thus, we used the nonradioactive fatty acid analogue 10-azisteric acid, which bears the same photoactivable carbene-generating azi group as photocholesterol, and [ 3 H]choline. Both compounds are taken up simultaneously and metabolized to photoactivable phosphatidycholine . Cells were cultured for 16 h in the presence of these two compounds and UV irradiated. Radiolabeled proteins were separated by SDS-PAGE and visualized by fluorography. When the pattern of labeled proteins was compared with those proteins labeled after cross-linking to photocholesterol, a specific photolabeling of different proteins with each compound was strikingly apparent . Whereas photocholesterol was largely cross-linked to PLP, photophosphatidylcholine was cross-linked to other proteins, but not to PLP . Additionally, immunoprecipitation experiments confirmed that phoshatidylcholine does not interact with PLP (data not shown). Together, our results identify PLP as a major cholesterol-interacting protein in oligodendrocytes. Section title: Association of PLP with CIMF Is Reduced in CGT Knockout Mice Educational score: 4.195917129516602 Domain: biomedical Document type: Study Language: en We have demonstrated that cholesterol interacts with PLP and is required for its association with CIMF. To determine whether sphingolipids are also required for CIMF formation, we cultured primary cultures of oligodendrocytes 1 d after plating for 5 d in the presence of 50 μM fumonisin B1 . This treatment completely abolished the association of PLP/DM20 with CIMF . To investigate more specifically the requirement of the major myelin glycosphingolipids, galactosylceramide and sulfatide, we took advantage of a ceramide galactosyl transferase (CGT) knockout mouse . Mice that lack CGT, the key enzyme of galactolipid biosynthesis, are completely deficient in galactosylceramide and sulfatide . Myelin from CGT knockout mice was prepared, extracted with 20 mM CHAPS, and floated in a density gradient. Section title: Association of PLP with CIMF Is Reduced in CGT Knockout Mice Educational score: 4.226835250854492 Domain: biomedical Document type: Study Language: en When the density gradient distribution of PLP/DM20 from CGT knockout mice was compared with PLP/DM20 from wild-type mice, the difference was striking. Whereas PLP/DM20 from wild-type mice was mainly found associated with CIMF, a significant fraction of PLP/DM20 from CGT knockout mice was no longer associated with CIMF . When extractions were performed with Triton X-100, no substantial differences were observed . Lipid analysis of myelin from CGT knockout mice showed the absence of galactosylceramide and sulfatide concomitant with an upregulation of glucosylceramide, which was mainly recovered from CIMF . In contrast, a large fraction of the myelin glycerophospholipids of CGT knockout mice was soluble in CHAPS . Section title: Association of PLP with CIMF Is Reduced in CGT Knockout Mice Educational score: 4.390724182128906 Domain: biomedical Document type: Study Language: en The above results suggest that galactosylceramide and/or sulfatide are required for the association of PLP with CIMF. We tested whether PLP is associated with CIMF in cells that generate rafts, but do not synthesize galactosylceramide and sulfatide as major sphingolipids . For this purpose, we transiently transfected BHK cells with PLP and prepared CIMF as described above. In contrast to oligodendrocytes, PLP was not associated with CIMF in BHK cells . Exogeneously expressed HA was recovered from CIMF in BHK cells , thus showing that this cell type does indeed generate rafts. In case the transport of PLP is dependent on the presence of the DM20 isoform, we cotransfected cells with both PLP and DM20. However, PLP still did not associate with the isolated CIMF . We ensured that exogenously expressed PLP had reached the plasma membrane surface by labeling living BHK cells at 4°C with O10 mAb, which recognizes an extracellular epitope of PLP . The positive staining with O10 of transfected BHK cells confirmed that PLP had reached the cell surface and suggests that the protein was correctly folded (data not shown). Hence, we conclude that association of PLP with CIMF is not an intrinsic property of PLP, but depends on the specialized lipid environment generated by oligodendrocytes. Section title: Discussion Educational score: 3.5595741271972656 Domain: biomedical Document type: Study Language: en The myelin sheath has a very restricted composition containing specific lipids and proteins. How myelin lipids and proteins are assembled to generate the myelin sheath in oligodendrocytes is not known. Here, we provide evidence that specific lipid–protein interactions are involved in the generation of myelin. Section title: Discussion Educational score: 4.295478343963623 Domain: biomedical Document type: Study Language: en We show that PLP/DM20, the major myelin protein, is recovered in a low-density CIMF enriched in myelin lipids from primary cultures of oligodendrocytes. Several lines of evidence led us to conclude that the association of PLP/DM20 with CIMF is specific and due to distinct lipid–protein interactions. First, we show that insolubility of PLP/DM20 in CHAPS is acquired during biosynthetic transport. When PLP in cultured oligodendrocytes was labeled with a short pulse of radioactivity and chased at a temperature (15°C) at which proteins are trapped in the ER , PLP was soluble in CHAPS. However, when the chase temperature was raised to 20°C, a temperature at which proteins accumulate in the Golgi complex where the majority of glycolipids are synthesized, PLP became insoluble. Hence, insolubility is not an inherent characteristic of PLP, but depends on the lipid environment. Section title: Discussion Educational score: 4.563204288482666 Domain: biomedical Document type: Study Language: en Second, removal of cholesterol or inhibition of sphingolipid synthesis of cultured oligodendrocytes renders PLP CHAPS-soluble. Cholesterol is thought to be essential for the generation and stability of sphingolipid–cholesterol rafts in biological membranes and enhances the detergent insolubility of sphingolipids in model membranes . Third, PLP was efficiently cross-linked to cholesterol, but not to another abundant lipid, phosphatidylcholine, which was not associated with the CHAPS-insoluble membrane fraction. Fourth, PLP clusters with the influenza HA on the surface of oligodendrocytes in overlapping patches more than with the SFV spike protein E2. The patching behavior of the two proteins is thought to be mediated by the miscibility of their lipid environments . Copatching of PLP and HA is probably caused by a preference of both proteins for similar lipid environments. Taken together, these results suggest that the association of PLP/DM20 with CIMF within the Golgi apparatus represent an important sorting step in the biogenesis of myelin. We propose that CIMF represents specialized lipid rafts, myelin rafts, generated by myelin-producing cells. Section title: Discussion Educational score: 4.2987470626831055 Domain: biomedical Document type: Study Language: en These results are at variance with a previous study using a different experimental strategy. The vesicular stomatitis virus G protein (VSV G), a marker of the basolateral pathway of vesicle traffic in epithelial cells, was found within myelin-like membrane extensions of cultured oligodendrocytes. HA in contrast, a marker of the apical pathway in epithelial cells, was excluded from this domain. These observations lead the authors to conclude that the myelin membrane is the target of a basolateral-type pathway . In contrast, we found that all exogenously expressed viral markers (SFV-E2, influenza-HA, and VSV G) were found within the membrane extensions of primary cultures of oligodendrocytes (unpublished observation). The reason why we did not observe differential sorting of HA and VSV G in our oligodendrocytic culture remains to be determined. We assume that primary oligodendrocytes in our cultures are not yet terminally differentiated and therefore the sorting processes that lead to the formation of mature myelin are not yet fully developed. This behavior is reminiscent of what has been observed in stage three cultured hippocampal neurons . In all likelihood, maturing oligodendrocytes require axonal contact to become fully polarized and to segregate markers into different domains. The solubility of the major myelin proteins in cold Triton X-100 has also argued against the association of the main myelin proteins with lipid rafts . Section title: Discussion Educational score: 4.574405670166016 Domain: biomedical Document type: Study Language: en The involvement of lipid rafts in the generation of membrane compartments has been demonstrated in many cell types . Our work suggests that oligodendrocytes employ a rafting process for myelin assembly. However, our results suggest that myelin rafts, represented by the CIMF in our study, are a specialized form of lipid rafts. When PLP was expressed in BHK cells, PLP was CHAPS soluble. This might be due to the lack of specific lipids in the rafts derived from BHK cells, which are present in oligodendrocytes. The most abundant sphingolipid in the CIMF in oligodendrocytes was galactosylceramide, whereas sphingomyelin represents the major sphingolipid in most cell types, including BHK cells. The requirement of galactosylceramide for association of PLP/DM20 with CIMF is supported by our finding that PLP/DM20 is recovered to a lesser extent from CIMF of myelin from CGT knockout mice than from myelin from wild-type mice. CGT knockout mice, are unable to synthesize galactosylceramide and sulfatide, but compensate partially by upregulating the synthesis of glucosylceramide . However, production of glucosylceramide in CGT knockout mice accounts for ∼30% of the galactosylceramide and sulfatide level in wild-type mice . Hence, the glucosylceramide level may not suffice to generate enough membrane in a raft phase to recruit all of the PLP, the major myelin protein. Another perhaps more likely explanation is that the lack of galactosylceramide and/or sulfatide in CGT knockout mice leads to generation of lipid rafts that are not fully competent to recruit PLP/DM20. Although CGT knockout mice produce myelin with apparently normal ultrastructure of compact myelin, alterations of the paranodal and periaxonal myelin domains are observed . It will be interesting to determine if the underlying cause may be the disturbance of the normal molecular organization of these domains by missorted PLP. Section title: Discussion Educational score: 4.230218887329102 Domain: biomedical Document type: Study Language: en Taken together, our results show that PLP requires a specific lipid matrix for raft association. The direct interaction of PLP with cholesterol and galactosylceramide in the Golgi complex may be a critical step in the sorting of components destined for the myelin membrane. Section title: Discussion Educational score: 4.459612846374512 Domain: biomedical Document type: Study Language: en The existence of several different myelin subdomains with different molecular composition (compact myelin, paranodal loop, and periaxonal and abaxonal membrane) adds an additional level of complexity to the sorting process. Segregation of these different subdomains is unlikely to be driven by the immiscibility of different lipid phases alone; additional sorting determinants (for example, defined protein–protein interactions) must also contribute. This is exemplified in caveolae, considered to be subsets of lipid rafts, which function as signal transduction platforms in many cell types . Homooligomeric interactions of caveolin proteins are intrinsic to the formation of this specific subdomain . In the case of myelin, it is important to address which protein and lipid molecules are involved in the assembly of the different myelin subdomains. Biochemical dissection of these subdomains, isolation of specific components, and their in vivo localization will yield information regarding molecular interactions driving the formation of compact myelin, paranodal loops, and periaxonal and abaxonal membranes. Section title: Discussion Educational score: 4.269371032714844 Domain: biomedical Document type: Study Language: en We have reported previously that a Triton X-100–insoluble membrane fraction from primary cultures of oligodendrocytes is enriched in molecules involved in signal transduction, such as GPI-anchored proteins and the nonreceptor tyrosine kinases of the Src family, Fyn and Lyn, but devoid of PLP and MAG . Interestingly, these kinases are maximally active, reflected by their phosphorylation status, during myelination and are downregulated in adult myelin . A dominant negative form of Fyn was shown to inhibit morphological differentiation, reflected by process outgrowth of oligodendrocytes . We have shown previously that these molecules are purified with myelin, but are minor components . The precise localization within the myelin membrane remains to be determined. However, we do not expect them to be located within compact myelin, which is not accessible to signals from neighboring cells. Section title: Discussion Educational score: 4.393561363220215 Domain: biomedical Document type: Study Language: en The different extractability of the above molecules and PLP by CHAPS- and Triton X-100 raises the question as to whether these detergents allow the isolation of different myelin subdomains. Fiedler et al. 1993 have shown previously that the CHAPS- and Triton X-100–insoluble membrane fractions in MDCK cells are qualitatively similar, but differ only quantitatively in their individual components. The CHAPS complex is depleted of GPI-anchored proteins and retains a minor fraction of lipids, the latter similar in composition to that of the Triton X-100–insoluble complex . Furthermore, we have shown that HA is insoluble in both Triton X-100 and CHAPS in oligodendrocytes (data not shown). HA copatches both with PLP (enriched in CIMF) and the GPI-anchored protein NCAM120 , demonstrating the location of these proteins in overlapping membrane domains. In individual cells, the GPI-anchored proteins may play a role in the early phases of axon–glial wrapping , whereas PLP is synthesized late in oligodendrocyte development and is located throughout the compact myelin sheath. Thus, the generation of rafts containing GPI-anchored proteins may be earlier and thus largely temporally separated from the synthesis of rafts containing PLP. Section title: Discussion Educational score: 4.270853519439697 Domain: biomedical Document type: Study Language: en Other experiments using lymphoid cells have shown that certain multichain recognition receptors, such as FcεRI or the T cell receptor, are highly sensitive to Triton X-100 extraction, but fulfill all other known criteria for raft association . In these cells the detergent Brij 58 appears to preserve proteins in lipid rafts better than Triton X-100 . The extractability of a protein by detergents presumably depends on the level and the specific content (e.g., acyl chain length) of sphingolipids and cholesterol in a given cell type. Detergents differ in their structure; this results in differential partitioning into the plasma membrane and the disruption of specific protein–lipid and protein-protein interactions in rafts by individual detergents. However, it is unlikely that specific subdomains of lipid rafts can be isolated based on differing extractability with individual detergents, unless these domains form different lipid phases. Section title: Discussion Educational score: 4.186999320983887 Domain: biomedical Document type: Study Language: en An interesting feature of the CIMF in oligodendrocytes is the high proportion of glycosphingolipids (63%) and cholesterol (60%). In BHK cells, for example, only 30% of the cholesterol and 40% of sphingomyelin (the major sphingolipid in these cells) were detergent insoluble . These differences probably reflect the abundance of raft lipids in oligodendrocytes. These cells are specialized to form myelin rafts. Section title: Discussion Educational score: 4.443957328796387 Domain: biomedical Document type: Study Language: en Recent experiments have demonstrated that in unpolarized BHK and PTK2 cells, rafts are small (50 nm), dispersed, liquid-ordered domains in an otherwise fluid (i.e., liquid-disordered phase) membrane . However, in the plasma membrane of oligodendrocytes, as the mass fraction of sphingolipids and cholesterol continuously increases, the liquid-ordered phase may become continuous. Therefore, the myelin membrane is likely to be a percolating raft domain . Generation of macrodomains is thought to be facilitated by trapping or clustering of microdomains by factors which are intrinsic or extrinsic to the cell . For example, clustering of caveolin-3–containing caveolae in developing T tubules promotes the formation of large raft macrodomains and thereby serves as a starting point for T tubule biogenesis . Ligation of myelin rafts by cytosolic components (for example, MBP) or via extracellular factors (axonal contact) may drive the formation of a continuous myelin raft membrane. Section title: Discussion Educational score: 4.304474830627441 Domain: biomedical Document type: Study Language: en Perturbation of the equilibrium of lipid phases might occur by an alteration in the turnover of components. In this regard, it is interesting to note that several lipidoses result in dysmyelination and demyelination with accumulation of specific lipids in oligodendrocytes . The extreme susceptibility of oligodendrocytes to accumulating lipids might be due to destabilization of the myelin raft phase equilibrium. Oligodendrocytes may be exquisitely sensitive to an imbalance in the synthesis and turnover rate of their protein and lipid components due to their long in vivo life and the very high rate of myelin synthesis. Section title: Discussion Educational score: 4.023362159729004 Domain: biomedical Document type: Study Language: en In summary, we show here that sorting of myelin proteins via myelin rafts is a major pathway of membrane assembly in oligodendrocytes and that lateral lipid–protein interactions represent an important mechanism in the assembly of myelin by these cells. The challenge for the future is to elucidate further components of this sorting machinery. | Study | biomedical | en | 0.999997 |
0006005 | Section title: Introduction Educational score: 4.429399490356445 Domain: biomedical Document type: Study Language: en A significant component of eukaryotic cellular protein degradation occurs at the ER. ER-associated degradation (ERAD) is widely employed in cellular quality control, resulting in the destruction of numerous misfolded or mutant proteins present in the lumen or at the surface of the organelle . ERAD continuously occurs in normal cells to minimize levels of unfolded proteins in the ER lumen . ERAD is also used to regulate the steady state levels of specific proteins in response to physiological cues . For instance, production of sterols by the mevalonate pathway is controlled, in part, by the regulated, ER-associated destruction of hydroxymethylglutaryl-coenzyme A reductase (HMGR), a key enzyme of the mevalonate pathway. Section title: Introduction Educational score: 4.277091979980469 Domain: biomedical Document type: Study Language: en Genes involved in ERAD have been identified by several genetic analyses in yeast . These studies indicate that the ER-associated ubiquitin-conjugating enzyme Ubc7p is centrally involved in the degradation of ER substrates , sometimes in conjunction with other ubiquitin-conjugating enzymes . These studies also revealed two previously unknown membrane proteins, Hrd1p (also isolated as Der3p) and Hrd3p, required for the ER degradation of numerous proteins . Unlike Ubc7p, the functions of Hrd1p and Hrd3p are not as clear. Our recent work demonstrates that Hrd1p provides the core ubiquitin ligase (E3) activity in ERAD (our unpublished results), recruiting Ubc7p for substrate ubiquitination. However, the interplay and specific actions of Hrd1p/Hrd3p are poorly defined in current models of ERAD. Section title: Introduction Educational score: 4.372811794281006 Domain: biomedical Document type: Study Language: en A key, but poorly understood feature of ERAD is the coordination of molecular processes on separate sides of the ER membrane. The degradation process begins in the interior of the ER, where ERAD substrates partially or completely present in the lumen are recognized for degradation by an unknown mechanism. Many proteins required for ERAD have critical determinants located in the lumen . However, ubiquitination and degradation of ERAD substrates occur on the cytosolic face of the ER by the action of ER-associated ubiquitin-conjugating enzymes and the 26S proteasome . How these membrane-separated events are coordinated is not clear. In particular, it is unknown if Hrd1p and Hrd3p function to allow communication across the ER membrane. Section title: Introduction Educational score: 4.073739528656006 Domain: biomedical Document type: Study Language: en Hrd3p is an ER resident glycoprotein with a single membrane span near the COOH terminus and a large NH 2 -terminal region in the lumen of the ER . Hrd3p has widely conserved sequence motifs and full-length orthologues in a variety of species , including the Caenorhabditis elegans SEL-1 protein, which may participate in membrane protein degradation . However, the exact role of Hrd3p in ER degradation has yet to be elucidated. Section title: Introduction Educational score: 4.485193729400635 Domain: biomedical Document type: Study Language: en Hrd1p is also an ER-associated protein with two distinct domains: an NH 2 -terminal, hydrophobic region with multiple predicted transmembrane spans and a COOH-terminal, hydrophilic region containing a RING-H2 motif required for the function of Hrd1p in ER degradation . The Hrd1p RING-H2 motif is conserved in a group of ubiquitin ligases or E3 proteins, including Ubr1p , Hrt1p/Rbx1p , Apc11p , and c-Cbl . In fact, we have recently demonstrated that Hrd1p is a ubiquitin ligase, catalyzing the transfer of ubiquitin from Ubc7p to ERAD substrates by the activity of its RING-H2 motif, which is required for a direct in vivo physical interaction between Hrd1p and Ubc7p (our unpublished results). Unlike other family members, the Hrd1p RING-H2 domain is anchored to the ER membrane by an NH 2 -terminal, multimembrane spanning domain. Although the Hrd1p NH 2 -terminal transmembrane domain contains conserved sequences, no function has been suggested for this anchor. Section title: Introduction Educational score: 4.4331865310668945 Domain: biomedical Document type: Study Language: en In this paper, we have performed an extensive study of the functional interplay between Hrd1p and Hrd3p. From these studies, we have demonstrated for the first time a direct physical interaction between the two proteins, defined domain-specific functions for each protein, ascertained the correct topology of Hrd1p, demonstrated transmembrane communication between Hrd1p and Hrd3p, and evaluated the importance of the stoichiometry of the Hrd1p–Hrd3p complex. Importantly, we have clearly shown that the Hrd1p transmembrane domain alone mediates the interaction between Hrd1p and Hrd3p. This interaction regulates the activity of the Hrd1p cytosolic RING-H2 domain to allow ERAD by preventing Hrd1p degradation. Hrd3p-mediated regulation of the cytosolic Hrd1p RING-H2 domain occurs across the ER membrane, as a completely lumenal version of Hrd3p allowed ERAD through normal regulation of Hrd1p stability. These studies further demonstrated that the lumenal functions of Hrd3p include both transmembrane regulation of Hrd1p RING-H2 domain activity and as yet undefined aspects of ERAD separate from control of Hrd1p stability. Section title: Materials and Reagents Educational score: 1.7285951375961304 Domain: biomedical Document type: Other Language: en Rabbit anti-hemagglutinin (HA) polyclonal antiserum was obtained from Babco. Rabbit polyclonal anti–green fluorescent protein (GFP) antiserum was obtained from Dr. Suresh Subramani (University of California at San Diego). Rabbit polyclonal antiserum to the COOH-terminal residues 348–551 of Hrd1p or the NH 2 -terminal residues 220–417 of Hrd3p was made in collaboration with Scantibodies, Inc. All other materials and reagents were obtained from previously described sources . Section title: Plasmids and DNA Methods Educational score: 2.2827744483947754 Domain: biomedical Document type: Other Language: en All DNA manipulations were performed as described , using the reagents described therein. For PCR amplifications, primer sequences will be provided upon request. Section title: Plasmids and DNA Methods Educational score: 4.339113712310791 Domain: biomedical Document type: Study Language: en Plasmids expressing epitope-tagged versions of the HRD genes were constructed as follows. pRH1196 (YIp/ TRPI ) expressed Hrd1p with a triple HA epitope sequence fused in frame to the immediate COOH terminus. Integration at the HRD1 genomic locus resulted in expression of only 3HA-Hrd1p. The triple HA epitope sequence was fused to the HRD1 sequence through the splicing by overlap extension (SOEing) PCR technique . The 3HA-HRD1 gene complemented an hrd1 Δ mutation when integrated as a single copy behind the native HRD1 promoter. pRH1263 contained a triple HA epitope sequence inserted between codons 20 and 21 of the HRD3 sequence cloned from pYS14 . Integration of pRH1263 at the HRD3 genomic locus resulted in expression of only 3HA-Hrd3p, which complemented an hrd3 Δ mutation when expressed from the native promoter. pRH1048 allowed expression of 3HA-Hrd3p from the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) ( TDH3 ) promoter . The plasmid was constructed by subcloning a PCR-generated DNA fragment encoding the 5′ sequence of the 3HA-HRD3 allele between the XhoI and NheI sites in pRH632 (YIp/ LEU2 /P TDH3 - HRD3 ), thereby replacing the corresponding normal HRD3 sequence. Section title: Plasmids and DNA Methods Educational score: 4.2706217765808105 Domain: biomedical Document type: Study Language: en Plasmids expressing the Hrd1p NH 2 -terminal transmembrane domain, called hemi-Hrd1p, were constructed as follows. pRH936 (YIp/ TRP1 ) allowed hemi-Hrd1p expression from the GAPDH promoter. The hemi-Hrd1p coding region was prepared by PCR amplification of the 5′ end of HRD1 between the HRD1 start codon and the NcoI site just distal to the end of the transmembrane domain coding region. The PCR product was cloned into a TRP1 variant of pRH98-2 , placing hemi-HRD1 behind the GAPDH promoter, yielding pRH936. pRH986 (YIp/ TRP1 ), which allowed expression of 1myc-hemi-Hrd1p from the GAPDH promoter, was prepared from pRH936 by addition of a PCR-generated DNA fragment encoding a single myc epitope sequence (EQKLISEEDL) to the 3′ end of hemi-HRD1 . The DNA fragment replaced the sequence between the NsiI and KpnI sites in pRH936. pRH1297 (YIp/ TRP1 ) allowed expression of hemi-Hrd1p-GFP from the GAPDH promoter. hemi-Hrd1p-GFP contained a replacement of the Hrd1p COOH-terminal domain with the autofluorescent GFP protein. The GFP fusion sequence was created by SOEing PCR amplification of the GFP coding sequence from pRH469 , and replaced the sequence between the NsiI and KpnI sites in pRH936. Section title: Plasmids and DNA Methods Educational score: 4.203910827636719 Domain: biomedical Document type: Study Language: en Plasmids expressing the isolated COOH-terminal RING-H2 domain of Hrd1p, termed RING-Hrd1p, were constructed as follows. The plasmid pRH730 (YIp/ TRP1 ), which contained 3HA-HRD1 behind the GAPDH promoter, was digested with NcoI, the ends were filled in, and the resulting plasmid was religated to form pRH1227. The P TDH3 -RING-HRD1 allele was subcloned into pRH1153 (YIp/ LEU2 ), which contained the normal HRD1 gene, to yield pRH1303. The GFP coding sequence was fused to the 5′ end of HRD1 through the PCR SOEing method, and the resulting plasmid was named pRH684 (YIp/ TRP1 ). The GFP fusion portion of this construct was subcloned into the RING-HRD1 plasmid to yield pRH1231 (YIp/ TRP1/RING-HRD1-GFP ). Section title: Plasmids and DNA Methods Educational score: 4.032161712646484 Domain: biomedical Document type: Study Language: en The C399S Hrd1p point mutation was generated by PCR SOEing and introduced into the HRD1 coding region of the plasmids described above to yield pRH1245 (YIp/ TRPI/C399S-HRD1 ) and pRH1314 (YIp/ TRPI/C399S-RING-HRD1 ). Section title: Plasmids and DNA Methods Educational score: 4.254445552825928 Domain: biomedical Document type: Study Language: en The HRD3 truncation plasmids were constructed as follows. Deletion of codons 768–833 was accomplished by generating a DNA fragment containing a stop codon and XbaI site placed after codon 767 in the HRD3 coding region by PCR SOEing and introducing the DNA into pRH508. The subsequent plasmid was digested with XbaI to remove the DNA encoding the transmembrane domain and cytosolic extension, and the resulting plasmid was named pRH1244. Deletion of codons 393–833 was accomplished by digesting pRH508 with NdeI and religating the vector fragment to yield the plasmid pRH1219. Deletion of codons 24–356 was accomplished by generating a DNA fragment containing an SalI site placed after codon 24 in the HRD3 coding region by PCR SOEing and introducing the DNA into pRH508 (YIp/ LEU2 ). The subsequent plasmid was digested with SalI to remove the intervening DNA, and the resulting plasmid was named pRH1242. The various HRD3 truncation alleles, HRD3 1–767 , HRD3 1–390 , and HRD3 357–833 , were cloned by the TDH3 promoter in pRH632 to yield pRH1249, pRH1252, and pRH1248, respectively. Section title: Strains and Transformations Educational score: 2.781994581222534 Domain: biomedical Document type: Study Language: en Yeast and bacteria were cultured as described . Harvest, lysis, and transformation protocols were all standard and identical to those described . Section title: Strains and Transformations Educational score: 4.146451950073242 Domain: biomedical Document type: Study Language: en All yeast strains were derived from the same genetic background used in all our previous work . The parent strain from which every subsequent strain was derived had the following genotype: his3D200 lys2-801 ade2-101 ura3-52 met2 hmg1::LYS2 hmg2::HIS3 . The subsequent parent strains used for each appropriate experiment had the additional following genotypes and phenotypes: RHY853 ( ura3-52::HMG2cd:: hmg2GFP trp1::hisG leu2 Δ) expressed Hmg2p-GFP and the catalytic domain of Hmg2p as the sole source of Hmg2p; RHY1383 ( ura3-52::1MYCHMG2:: hmg2GFP trp1::hisG leu2 Δ) expressed normal, mevalonate pathway–regulated 1myc-Hmg2p and Hmg2p-GFP; RHY1886 ( HMG2 ) expressed native Hmg2p; and RHY1914 ( HMG2 ura3-52::1MYC-HMG2 ) expressed normal, mevalonate pathway–regulated 1myc-Hmg2p and native Hmg2p. Section title: Flow Cytometry Educational score: 3.837277889251709 Domain: biomedical Document type: Study Language: en Flow cytometric analyses were performed as described . Living cells were analyzed by flow microfluorimetry using a FACSCalibur™ (Becton Dickinson) flow microfluorimeter with settings for fluorescein-labeled antibody analysis. Histograms were produced from 10,000 individual, log-phase cells. Section title: Cycloheximide Chase Degradation Assay Educational score: 4.084873199462891 Domain: biomedical Document type: Study Language: en Degradation of expressed, epitope-tagged proteins was evaluated by treatment of log phase strains with cycloheximide as described . In brief, log phase cells were treated with 50 μg/ml cycloheximide to halt protein synthesis, and then lysed and immunoblotted after various incubation times to evaluate degradation as described . myc-tagged or HA-tagged proteins were detected using the 9E10 or 12CA5 mAbs as described . Section title: In Vivo Cross-Linking Educational score: 4.233076095581055 Domain: biomedical Document type: Study Language: en The in vivo cross-linking assay was modified from the in vitro assay described . Cells expressing the appropriate epitope-tagged proteins were grown to log phase (OD 600 = 1.0) in 30 ml minimal medium, harvested by centrifugation, and resuspended in XL buffer to a density of 6.0 OD 600 units. Zymolase was added (final concentration 50 μg/ml) and dithiobis(succinimidyl-propionate) (DSP) was added in varying concentrations up to 400 μg/ml. Cells were incubated for 30 min at 30°C. Spheroplasts were harvested by centrifugation and lysed by resuspension in 300 μl SUME , plus the detergents deoxycholate (0.5%) and Triton X-100 (1%), which were in addition to SDS (1%), and 20 μM hydroxylamine to quench the cross-linking reaction. Supernatants were clarified by centrifugation for 15 min. Supernatants were added to 1 ml immunoprecipitation buffer (IP buffer) , containing 20–30 μl of the appropriate antisera. Samples were incubated for 16 h at 4°C. Protein A–sepharose CL-4B beads (100 μl of a 10% wt/vol solution) were added, and the samples were incubated for 2 h. Beads were harvested by centrifugation and washed once with IP buffer and once with wash buffer . Proteins were removed from the beads by addition of 50 μl 2× USB and incubation at 55°C for 10 min. Section title: In Vivo Cross-Linking Educational score: 4.064631938934326 Domain: biomedical Document type: Study Language: en Immunoblotting of precipitated proteins was performed as described . In brief, separate 5- and 35-μl volumes of each precipitated sample were resolved on 8% SDS-PAGE gels and transferred to nitrocellulose. The 12CA5 anti-HA antibody was used to detect HA-tagged proteins, and a polyclonal anti-GFP antisera was used to detect GFP fusion proteins. Section title: Native Coimmunoprecipitation Educational score: 4.204095840454102 Domain: biomedical Document type: Study Language: en The native immunoprecipitation was adapted from Stack et al. 1993 . Log phase yeast cells (0.75 OD 600 units) were harvested by centrifugation and washed once with distilled water. Cells were resuspended in 0.5 ml zymolyase buffer . Zymolase (10 μg) was added to the cells, which were then incubated for 45 min at 30°C. Cells were washed once with 0.5 ml of ice-cold IP buffer minus detergent, then resuspended in 1 ml IP buffer. The resuspended cells were lysed by incubation on ice for 5 min. The supernatant was clarified by centrifugation for 5 min at 4°C, and again by centrifugation for 30 min at 4°C. For evaluation of total lysate protein, 20 μl of the final supernatant was removed and added to 20 μl of 2× USB. 7 μl rabbit anti-HA antisera was added to the remaining lysate to immunoprecipitate HA-tagged proteins, and the mixture was incubated overnight at 4°C. Protein A–sepharose CL-4B bead suspension (100 μl of a 10% wt/vol solution) was added to the mixture, which was then incubated for 2 h at 4°C. Beads were harvested by centrifugation and washed twice with IP buffer. Proteins were removed from the beads by addition of 40 μl 2× USB and incubation at 55°C for 10 min. Immunoblotting of HA-tagged proteins (5 μl of sample) or coprecipitated myc-tagged proteins (20 μl of sample) was performed with the 12CA5 or 9E10 mAbs, respectively. Section title: Protease Protection Assay Educational score: 4.076389789581299 Domain: biomedical Document type: Study Language: en Microsomes were prepared as described previously . Increasing amounts of protease were added to isolated microsomes, which were incubated on ice for 30 min. Reactions were stopped, and proteins were denatured by addition of an equal volume of 2× USB containing PMSF, followed by incubation at 65°C for 15 min. Samples were resolved by SDS-PAGE and immunoblotted with the appropriate antisera to assess levels of each protein. Section title: Hrd3p Interacted with the Hrd1p Transmembrane Domain Educational score: 3.900322437286377 Domain: biomedical Document type: Study Language: en Previous studies indicated that Hrd1p and Hrd3p functioned in the same ER-associated degradation pathway and showed genetic interactions . Therefore, we first examined if Hrd1p and Hrd3p physically interacted by chemical cross-linking and native immunoprecipitation assays. Section title: Hrd3p Interacted with the Hrd1p Transmembrane Domain Educational score: 4.170517921447754 Domain: biomedical Document type: Study Language: en Physical interactions between Hrd1p and Hrd3p were determined using an in vivo cross-linking assay modified from a procedure for cell lysates . The strains used for the cross-linking assays expressed fully functional, triple HA epitope–tagged versions of Hrd1p and/or Hrd3p from their native promoters. Each modified protein completely complemented the respective null alleles for ERAD when expressed in single copy from their native promoters (data not shown). When lysates were derived from cells treated with increasing amounts of cross-linker and Hrd1p was immunoprecipitated using Hrd1p-specific antisera, Hrd3p coimmunoprecipitated in a cross-linker concentration–dependent fashion . In the absence of cellular Hrd1p, no cross-linker–dependent Hrd3p coimmunoprecipitation was observed . The small amount of Hrd3p that coimmunoprecipitated with Hrd1p in the absence of cross-linker was roughly equivalent to that seen in the absence of Hrd1p, indicating that this amount was nonspecific. Section title: Hrd3p Interacted with the Hrd1p Transmembrane Domain Educational score: 4.186953544616699 Domain: biomedical Document type: Study Language: en We evaluated the importance of each Hrd1p domain in the Hrd3p interaction by testing Hrd1p truncation mutants and fusion proteins. We first tested if the Hrd1p NH 2 -terminal transmembrane domain, defined by residues 1–233, cross-linked with Hrd3p. In these and the following genetic studies, we refer to versions of Hrd1p that contained only the NH 2 -terminal transmembrane domain as hemi-Hrd1p because this domain is roughly one half the size of normal Hrd1p . The Hrd1p/Hrd3p cross-linking experiment was repeated in a strain coexpressing hemi-Hrd1p-GFP, a fusion protein consisting of the NH 2 -terminal domain of Hrd1p fused to GFP, and 3HA-Hrd3p. Immunoprecipitation of hemi-Hrd1p-GFP with anti-GFP antisera resulted in coimmunoprecipitation of Hrd3p in a cross-linker–dependent manner . When the same experiment was performed on cells that did not express hemi-Hrd1p-GFP, no cross-linker–dependent Hrd3p coimmunoprecipitation was observed . Section title: Hrd3p Interacted with the Hrd1p Transmembrane Domain Educational score: 4.135380744934082 Domain: biomedical Document type: Study Language: en We also tested if the Hrd1p COOH-terminal RING-H2 domain cross-linked with Hrd3p when expressed as an isolated protein encompassing residues 224–551 of Hrd1p. We refer to the 327-residue COOH-terminal domain as RING-Hrd1p because it contains the RING-H2 motif . When the same cross-linking assay was performed in strains coexpressing 3HA-Hrd3p and RING-Hrd1p-GFP, which contained GFP fused in frame to the extreme COOH terminus of RING-Hrd1p, no cross-linker–dependent Hrd3p coimmunoprecipitation was observed . Similarly, the isolated RING-Hrd1p without GFP also did not cross-link to Hrd3p (data not shown). Thus, the isolated COOH-terminal RING-H2 domain did not appear to interact with Hrd3p, despite it having a dominant negative ERAD phenotype and ER membrane localization (see below). Section title: Hrd3p Interacted with the Hrd1p Transmembrane Domain Educational score: 4.138566970825195 Domain: biomedical Document type: Study Language: en These mapping experiments indicated that the Hrd1p NH 2 -terminal transmembrane domain was necessary and sufficient for the Hrd1p–Hrd3p interaction. Accordingly, we tested if overexpression of hemi-Hrd1p could compete with native Hrd1p for Hrd3p cross-linking. When hemi-Hrd1p was coexpressed from the strong TDH3 promoter in the strain used to examine the Hrd1p–Hrd3p interaction, Hrd3p cross-linking to Hrd1p was eliminated . It is important to point out that hemi-Hrd1p expression resulted in decreased Hrd1p levels in the cell . To account for this, the same amount of total Hrd1p immunoprecipitated was loaded in each lane so that the amount of cross-linked Hrd3p could be directly compared. Section title: Hrd3p Interacted with the Hrd1p Transmembrane Domain Educational score: 4.239106178283691 Domain: biomedical Document type: Study Language: en The interaction between Hrd3p and hemi-Hrd1p was further tested with a native coimmunoprecipitation assay, in which membrane proteins were solubilized under nondenaturing conditions to preserve protein–protein associations . We used a myc epitope–tagged version of hemi-Hrd1p to test if the NH 2 -terminal domain could, by itself, interact with Hrd3p. 1myc-hemi-Hrd1p, containing a single, myc epitope sequence placed in frame after residue 231 of hemi-Hrd1p, was coexpressed in cells that also expressed 3HA-Hrd3p. When 3HA-Hrd3p was immunoprecipitated using anti-HA antibodies, 1myc-hemi-Hrd1p was coimmunoprecipitated . In contrast, hemi-Hrd1p coimmunoprecipitation was not observed when untagged Hrd3p was coexpressed in the cells . Coimmunoprecipitation was not the result of association between the two proteins through the action of the detergent lysis buffers, as 1myc-hemi-Hrd1p coimmunoprecipitation only occurred when coexpressed in the same cells as 3HA-Hrd3p . Section title: Hrd3p Interacted with the Hrd1p Transmembrane Domain Educational score: 4.082479476928711 Domain: biomedical Document type: Study Language: en Together, these experiments demonstrated that Hrd1p physically interacted with Hrd3p through the Hrd1p NH 2 -terminal transmembrane domain. The COOH-terminal, RING-H2 domain was dispensable for the interaction and did not appear to be able to interact with Hrd3p when expressed as an isolated protein. Section title: Phenotypes and Suppression of HRD1 Truncation Alleles Educational score: 4.008077621459961 Domain: biomedical Document type: Study Language: en It has been shown that expression of a full-length, mutant version of Hrd1p, C399S-Hrd1p, results in a dominant negative block in ERAD , which is suppressed by increased Hrd3p expression . We tested each half of Hrd1p for dominant negative ERAD phenotypes and suppression by Hrd3p overexpression. Section title: Phenotypes and Suppression of HRD1 Truncation Alleles Educational score: 4.1890997886657715 Domain: biomedical Document type: Study Language: en Overproduction of hemi-Hrd1p in wild-type strains strongly stabilized the ERAD substrate Hmg2p-GFP, resulting in increased cellular fluorescence in hemi-HRD1 cells compared with control cells as measured by flow cytometry . The hemi-HRD1 stabilizing effect was identical to the effect of a previously reported dominant negative stabilizing HRD1 allele encoding a protein with the RING-H2 motif deleted . Expression of hemi-Hrd1p also stabilized the regulated ERAD substrate 1myc-Hmg2p and the constitutive ERAD substrate 6myc-Hmg2p (data not shown), as measured by the direct assay of each protein's stability. Section title: Phenotypes and Suppression of HRD1 Truncation Alleles Educational score: 4.246617794036865 Domain: biomedical Document type: Study Language: en Surprisingly, overexpression of the isolated Hrd1p COOH-terminal domain, RING-Hrd1p, also had a dominant negative stabilizing phenotype on Hmg2p-GFP degradation . The stabilizing effect of RING-Hrd1p expression required an intact RING-H2 motif, as a point mutation in a critical cysteine within the motif, analogous to C399S in full-length Hrd1p, strongly suppressed the stabilizing effect of the RING-HRD1 allele . This was in direct contrast to the stabilizing effect of the C399S point mutation in full-length Hrd1p . The diminished stabilizing effect of C399S-RING-Hrd1p occurred despite its increased levels compared with RING-Hrd1p (see below). The dominant phenotype of the RING-HRD1 allele and its reversal by the C399S point mutation were also observed for degradation of 1myc-Hmg2p and 6myc-Hmg2p (data not shown). Section title: Phenotypes and Suppression of HRD1 Truncation Alleles Educational score: 4.161947727203369 Domain: biomedical Document type: Study Language: en The full-length C399S-Hrd1p dominant negative phenotype is suppressed by Hrd3p overexpression . Therefore, we evaluated the effect of Hrd3p overexpression on the dominant stabilizing phenotype of each Hrd1p truncation mutant. The dominant negative phenotype of the hemi-HRD1 allele was reversed by increased expression of Hrd3p from the TDH3 promoter, as measured by cycloheximide chase assay of 1myc-Hmg2p degradation , or Hmg2p-GFP steady state fluorescence . Suppression by HRD3 was complete, since the fluorescence histogram of the suppressed cells could be superimposed on that of the wild-type strain . Importantly, neither the stability nor the steady state levels of hemi-Hrd1p were affected by increased Hrd3p expression (data not shown). Increased Hrd1p expression from the TDH3 promoter also reversed the hemi-HRD1 phenotype as expected . Section title: Phenotypes and Suppression of HRD1 Truncation Alleles Educational score: 4.240133762359619 Domain: biomedical Document type: Study Language: en Conversely, the dominant negative stabilizing phenotype of RING-Hrd1p was not suppressed by increased Hrd3p expression. The same expression levels of Hrd3p had no effect on the RING-HRD1 –dependent stabilization of 1myc-Hmg2p or Hmg2p-GFP . The lack of suppression was not due to an inability of the RING-HRD1 effect to be suppressed, since increased Hrd1p expression did completely reverse the stabilizing effect of the RING-HRD1 allele . Thus, although RING-Hrd1p expression inhibited ERAD, the stabilizing effect was not through Hrd3p sequestration or inhibition, as would be expected by its lack of interaction with Hrd3p as shown above in the cross-linking studies. Section title: The Hrd1p Transmembrane Region Mediated Hrd3p-dependent Stabilization of Hrd1p Educational score: 4.25881814956665 Domain: biomedical Document type: Study Language: en One function of Hrd3p in ERAD is regulation of Hrd1p stability. In the absence of Hrd3p, the stability of Hrd1p is greatly reduced . We evaluated the role of the Hrd1p NH 2 -terminal transmembrane domain in this action of Hrd3p. Expression of hemi-Hrd1p in a wild-type HRD3 strain mimicked the phenotype of the hrd3Δ allele, resulting in a similar decrease in the half-life and stability of Hrd1p . Hrd1p was restabilized in this strain by increasing the expression of Hrd3p from the TDH3 promoter . Thus, in this molecular readout of Hrd3p function, the isolated Hrd1p transmembrane domain sequestered Hrd3p and removed its stabilizing influence on endogenous Hrd1p. Section title: The Hrd1p Transmembrane Region Mediated Hrd3p-dependent Stabilization of Hrd1p Educational score: 4.488162040710449 Domain: biomedical Document type: Study Language: en The two domains of intact Hrd1p had distinct roles in Hrd3p-dependent regulation of Hrd1p stability. Hrd1p degradation was mediated by the COOH-terminal RING-H2 domain, whereas Hrd3p-dependent stabilization was mediated by the NH 2 -terminal transmembrane domain. In an hrd3 Δ strain, Hrd1p degradation was dependent on a functional UBC7 gene and an intact RING-H2 motif. Loss of UBC7 function by introduction of a ubc7 Δ allele completely stabilized Hrd1p , as did introduction of the C399S mutation . This was consistent with the previous analysis of Hrd1p degradation in the absence of HRD3 . This was also consistent with the ubiquitin ligase activity of the Hrd1p RING-H2 domain (our unpublished results), and demonstrated the essential nature of the Hrd1p RING-H2 motif's activity in the degradation of Hrd1p. Degradation of isolated RING-Hrd1p, which lacks the NH 2 -terminal transmembrane region, was similarly dependent on UBC7 and its own functional RING-H2 motif . However, RING-Hrd1p degradation was completely independent of Hrd3p, occurring with identical kinetics in the absence or presence of HRD3 . This indicated that the transmembrane domain was critical for Hrd3p regulation of Hrd1p stability. In contrast, hemi-Hrd1p, which encompasses the isolated transmembrane domain, was exceedingly stable regardless of Hrd3p presence . Section title: The Hrd1p COOH-terminal RING-H2 Domain Was Located and Functioned in the Cytosol Educational score: 3.9199459552764893 Domain: biomedical Document type: Study Language: en It has been previously reported that the COOH-terminal RING-H2 domain is in the lumen of the ER . However, the homology of this domain with a class of ubiquitin ligases, and our concurrent studies of the Hrd1p biochemical function as a ubiquitin ligase (our unpublished results), led us to rigorously reevaluate the membrane orientation of the Hrd1p RING-H2 domain. Section title: The Hrd1p COOH-terminal RING-H2 Domain Was Located and Functioned in the Cytosol Educational score: 4.202271461486816 Domain: biomedical Document type: Study Language: en First, we tested the localization of intact Hrd1p and its truncation mutants by using the GFP fusions described in the interaction studies above. To aid visibility, all GFP fusions were expressed in strains with the ubc7 Δ allele, since both the Hrd1p-GFP and the RING-Hrd1p-GFP were subject to degradation in a wild-type background . When expressed from the TDH3 promoter, both hemi-Hrd1p-GFP and RING-Hrd1p-GFP demonstrated identical cellular localization of GFP fluorescence . The GFP fluorescence was primarily localized to a perinuclear band, as observed by 4,6-diamino-2-phenylindole (DAPI) staining of the nucleus (data not shown), with some GFP fluorescence localized to the periphery of the cell. This type of cellular localization is common for ER-localized membrane proteins . The cellular localization of either hemi-Hrd1p-GFP or RING-Hrd1p-GFP was identical to that observed for full-length Hrd1p-GFP , which has been previously determined to be ER localized . C399S-Hrd1p-GFP had identical ER localization as Hrd1p-GFP, as expected . Furthermore, the cellular localization of each GFP construct was similar to Hmg2p-GFP , a known ER-localized membrane protein . Section title: The Hrd1p COOH-terminal RING-H2 Domain Was Located and Functioned in the Cytosol Educational score: 4.299708366394043 Domain: biomedical Document type: Study Language: en ER membrane localization was expected for the fusions containing the NH 2 -terminal transmembrane domain. It was surprising that RING-Hrd1p-GFP behaved the same way since there are no predicted transmembrane spans or signal sequences within this protein, although such localization was consistent with its dominant inhibition of ERAD. From this, it seemed likely that RING-Hrd1p would be bound to the outside of the ER membrane. Therefore, we performed protease protection assays to test for cytosolic exposure of the RING-Hrd1p protein. When intact microsomes were isolated from cells expressing 3HA-RING-Hrd1p, nearly all of the immunoreactivity was associated with the pelleted microsomes . Brief trypsin treatment at several concentrations of protease completely removed the HA immunoreactivity without need for added detergent (.2 and .5). The loss of epitope immunoreactivity was identical to that of 1myc-Hmg2p , which has a myc epitope sequence placed in the large COOH-terminal, cytosolic region . In contrast, the immunoreactivity of the completely lumenal Kar2p was unaffected in the absence of detergent , whereas 3HA-Hrd3p showed a small shift in molecular weight , consistent with removal of its small, cytoplasmic COOH-terminal region . All protected proteins were rendered completely trypsin sensitive by pretreatment of the microsomes with detergent . Section title: The Hrd1p COOH-terminal RING-H2 Domain Was Located and Functioned in the Cytosol Educational score: 4.3750715255737305 Domain: biomedical Document type: Study Language: en The Hrd1p COOH-terminal RING-H2 domain was associated with the ER and accessible from the cytosolic face of the ER membrane when expressed as an isolated protein. However, it has been reported that this domain in full-length Hrd1p was exclusively on the lumenal side of the ER membrane . Therefore, we also evaluated the protease sensitivity of the same region when part of intact Hrd1p by using 3HA-Hrd1p, which contains the epitope tag at the extreme COOH terminus and completely complements an hrd1 Δ allele for ER degradation. In an identical protease protection experiment, the HA immunoreactivity for the COOH-terminal tagged, full-length 3HA-Hrd1p was also completely destroyed by trypsin addition in the absence of any added detergent , exactly like the cytosolic tag on Hmg2p and in striking contrast to Hrd3p or Kar2p. No accumulation of any protease-insensitive bands was seen for 3HA-Hrd1p , indicating that the HA epitope tag was completely destroyed by protease treatment. Thus, the COOH terminus of full-length Hrd1p was also present on the cytosolic face of the ER membrane. Section title: The Hrd1p COOH-terminal RING-H2 Domain Was Located and Functioned in the Cytosol Educational score: 4.384617805480957 Domain: biomedical Document type: Study Language: en To ensure that these results were not caused by the added epitope sequences, we repeated our experiment using untagged Hrd1p and polyclonal antisera specific for residues 348–551 of Hrd1p. Using the polyclonal antisera, we found that COOH-terminal RING-H2 domain of normal, untagged, full-length Hrd1p was clearly trypsin sensitive in the absence of any added detergent and was no different from the HA-tagged version of full-length Hrd1p . Control proteins Hrd3p, Kar2p and 1myc-Hmg2p showed identical responses to those determined in Fig. 4 b (data not shown). However, in contrast to the experiments using the HA mAb for detection, use of the polyclonal Hrd1p antisera did result in detection of a 32-kD trypsin-insensitive proteolytic fragment when microsomes containing either tagged or untagged full-length Hrd1p were subject to detergent-free trypsin digestion . The isolated, untagged COOH-terminal RING-Hrd1p protein was similarly trypsin sensitive , also yielding a trypsin-protected proteolytic fragment that was 16 instead of 32 kD (gray arrow). To resolve whether the protease insensitivity of this fragment from the RING-H2 domains was specific for trypsin or was instead indicative of a protected lumenal localization, we repeated these studies using proteinase K. In fact, the entire COOH-terminal region of either full-length Hrd1p or the isolated RING-Hrd1p was completely proteinase K sensitive (>95% digestion) in the absence of detergent , with no accumulation of a protected, immunoreactive fragment. In contrast, the mostly lumenal 3HA-Hrd3p was insensitive to proteinase K addition in the absence of detergent , indicating that the microsomes were intact. Thus, the partial trypsin insensitivity of the Hrd1p COOH-terminal domain appeared not to be a result of lumenal sequestration, but rather a structural feature of the COOH-terminal region. Section title: The Hrd1p COOH-terminal RING-H2 Domain Was Located and Functioned in the Cytosol Educational score: 4.408364295959473 Domain: biomedical Document type: Study Language: en Our protease protection data indicated that the Hrd1p COOH-terminal RING-H2 domain, whether expressed alone or as part of Hrd1p, was exposed to the cytosolic face of the ER membrane. As an independent and stringent test of this idea, we asked if the isolated Hrd1p COOH-terminal domain could function in ERAD when expressed with the isolated transmembrane domain. Indeed, coexpression of hemi-Hrd1p and RING-Hrd1p partially complemented the hrd1 Δ allele for Hmg2p-GFP degradation, whereas expression of either hemi-Hrd1p or RING-Hrd1p alone did not . Furthermore, the partially restored Hmg2p-GFP degradation was normally regulated by inhibitors of the mevalonate pathway, as seen by the expected effects on cellular Hmg2p-GFP fluorescence from addition of the degradation-stimulating zaragozic acid or degradation-slowing lovastatin . Coexpression of both hemi-Hrd1p and RING-Hrd1p also complemented the hrd1 Δ allele for the constitutive degradation of misfolded 6myc-Hmg2p as measured directly by cycloheximide chase . Section title: The Hrd1p COOH-terminal RING-H2 Domain Was Located and Functioned in the Cytosol Educational score: 3.787707805633545 Domain: biomedical Document type: Study Language: en Thus, the COOH-terminal RING-H2 domain of Hrd1p was localized to the cytosolic side of the ER membrane when part of full-length Hrd1p or when expressed in isolation as RING-Hrd1p. Section title: Hrd3p Lumenal Determinants Mediated Stabilization of Hrd1p and a Separate Function in ERAD Educational score: 3.972687244415283 Domain: biomedical Document type: Study Language: en The bulk of the Hrd3p sequence is found in the ER lumen with a small COOH-terminal region that is located in the cytosol . The RING-H2 domain of Hrd1p, which is required for both ERAD and Hrd1p degradation, is located in the cytosol . Since Hrd3p regulates the activity of RING-H2 domain, we examined if this was mediated by the Hrd3p lumenal domain. Section title: Hrd3p Lumenal Determinants Mediated Stabilization of Hrd1p and a Separate Function in ERAD Educational score: 4.475009918212891 Domain: biomedical Document type: Study Language: en We prepared several truncation mutants of HRD3 in order to examine the role of the Hrd3p lumenal regions in both Hrd1p stability and ERAD. The domain organization of Hrd3p is shown in Fig. 5 a. The Hrd3p sequence includes an NH 2 -terminal lumenal domain (residues 1–767) containing a cleavable NH 2 -terminal signal sequence and a region (residues 391–767) with high homology to C. elegans SEL-1 and murine sel-1l (35% identity and 58% similarity) , followed by a COOH-terminal transmembrane span and a poorly conserved COOH-terminal cytosolic domain. We engineered a truncated HRD3 allele that expressed only the lumenal domain of Hrd3p from the normal promoter ( HRD3 1–767 ). When the resulting lumenal Hrd3p 1–767 was expressed from single copy in hrd3Δ cells, both Hrd1p stability and ER degradation of Hmg2p were restored . Complementation of the hrd3Δ allele by the purely lumenal Hrd3p 1–767 was complete, as observed by the superimposable Hmg2p-GFP cellular fluorescence histograms from the HRD3 1–767 cells and normal HRD3 cells . Thus, the transmembrane and cytosolic regions of Hrd3p were dispensable for both Hrd3p-mediated stabilization of Hrd1p and ERAD. Since the critical Hrd1p RING-H2 domain is in the cytosol and Hrd3p 1–767 is entirely lumenal (data not shown), the stable transmembrane domain of Hrd1p must transmit regulatory information across the ER membrane from lumenal determinants of Hrd3p to the cytosolic Hrd1p RING-H2 domain. Section title: Hrd3p Lumenal Determinants Mediated Stabilization of Hrd1p and a Separate Function in ERAD Educational score: 4.474201202392578 Domain: biomedical Document type: Study Language: en Although Hrd3p is clearly important for ERAD and Hrd1p stabilization, the effect of the hrd3Δ allele can be suppressed by overexpression of Hrd1p . This could be interpreted to mean that the only function of Hrd3p is maintenance of Hrd1p steady state levels. To address this issue, we have looked for and discovered an allele of HRD3 that still allows Hrd1p stability, but does not allow normal ERAD. When codons 24–356 were excised from HRD3 (so that the signal sequence was still made), expression of the truncated Hrd3p, Hrd3p 357–833 , allowed stabilization of Hrd1p , but did not allow ERAD of 1myc-Hmg2p or Hmg2p-GFP . In fact, comparison of the cellular fluorescence histograms showed that cells expressing Hrd3p 357–833 were equally deficient for ERAD as cells with the hrd3Δ allele, despite the restored stability and steady state levels of Hrd1p. Thus, the NH 2 -terminal region of Hrd3p (residues 1–356) appeared to be involved in aspects of ERAD distinct from Hrd1p stability. Expression of only the first 390 residues of Hrd3p did not allow Hrd1p stability or ERAD. Thus, Hrd1p stabilization required determinants in the COOH-terminal portion of Hrd3p lumenal domain (residues 357–783), whereas the NH 2 -terminal portion was dispensable for Hrd1p stability. Both regions were essential for ERAD when Hrd1p was expressed from its native locus. Section title: Stoichiometry of the Hrd1p/Hrd3p Complex Educational score: 4.2595930099487305 Domain: biomedical Document type: Study Language: en Hrd3p determines Hrd1p stability and, thus, Hrd1p steady state levels. The simplest model is that Hrd3p and Hrd1p form a complex and only Hrd1p molecules so engaged are stable. We compared the natural steady state levels of Hrd1p and Hrd3p to estimate the in vivo stoichiometry. To do this, we used strains coexpressing identically tagged (triple HA) versions of each protein from single coding regions under the control of their native promoters. In this way, each protein could be immunoblotted for identical epitopes using identical conditions and reagents. When identically tagged versions of Hrd1p and Hrd3p were expressed from their genomic loci, the resulting steady state levels of 3HA-Hrd1p and 3HA-Hrd3p were nearly equivalent, as determined by intensity of signal and diminution by dilution . The similarity of Hrd1p and Hrd3p levels was consistent with the simple model that Hrd1p was stabilized by formation of a stoichiometric complex with Hrd3p. Section title: Stoichiometry of the Hrd1p/Hrd3p Complex Educational score: 4.254133224487305 Domain: biomedical Document type: Study Language: en We next examined if the amount of cellular Hrd3p determined the steady state level of Hrd1p through transstabilization. If Hrd1p were only stable when bound to Hrd3p, then increasing Hrd3p expression would be expected to result in increased Hrd1p levels by allowing increased stable complexes. Indeed, elevation of Hrd3p levels ∼20-fold by expression from the TDH3 promoter resulted in a 3-fold increase in Hrd1p steady state levels . Clearly Hrd1p was not increased to the same extent as Hrd3p. However, by a costabilization model, the maximal possible effect of elevating Hrd3p would occur when every molecule of Hrd1p was stable. To evaluate this maximum effect, we compared the levels of 3HA-Hrd1p to the levels of the identically expressed, completely stable 3HA-C399S-Hrd1p . Cellular levels of the stable C399S-Hrd1p were approximately fourfold higher than wild-type Hrd1p levels . Furthermore, elevation of Hrd3p levels had no effect on the steady state level of C399S-Hrd1p (data not shown). Thus, the effect of Hrd3p overexpression on Hrd1p levels was close to the maximal effect caused by the in cis stabilization of every Hrd1p molecule synthesized. Section title: Stoichiometry of the Hrd1p/Hrd3p Complex Educational score: 4.519208908081055 Domain: biomedical Document type: Study Language: en The cellular level of Hrd1p is demonstrably important in ERAD. Hrd1p is rate limiting for a variety of substrates, including Hmg2p and related proteins. Overexpression of Hrd1p resulted in enhanced degradation of Hmg2p-GFP . Furthermore, Hrd1p overexpression suppressed the stabilizing effect of the hrd3Δ allele for Hmg2p-GFP degradation , and CPY* degradation . From these observations alone, it would seem that the sole function of Hrd3p is to ensure sufficient amounts of Hrd1p. If so, then Hrd3p overexpression, with the concomitant increase in the rate-limiting Hrd1p, would be expected to increase the degradation of HRD pathway substrates. However, the effect of Hrd3p overexpression on HRD -dependent degradation was more complex. The degradation of two separate substrates, mevalonate pathway–regulated Hmg2p-GFP and constitutive 6myc-Hmg2p-GFP, was slightly decreased , despite the fact that Hrd1p levels were increased. These results further suggested that Hrd3p has separate functions in addition to simple maintenance of Hrd1p levels, and are consistent with our discovery of a Hrd3p allele that allowed Hrd1p stabilization, but did not allow ERAD in strains with normal levels of Hrd1p . Section title: Discussion Educational score: 4.314541339874268 Domain: biomedical Document type: Study Language: en Both Hrd1p and Hrd3p are broadly involved in ER degradation. In this work, we have demonstrated that these two proteins interact via the NH 2 -terminal transmembrane region of Hrd1p. By rigorously evaluating the orientation of the Hrd1p, we have discovered that the RING-H2 domain is present and functions in the cytosol. The regions of Hrd3p required for control of Hrd1p stability and other aspects of ERAD reside solely in the ER lumen. Thus, the HRD complex, involving at least Hrd1p and Hrd3p, functions on both sides of the ER membrane and communicates across this barrier through the Hrd1p transmembrane domain to coordinate the cytosolic Hrd1p RING-H2 domain activity with events that occur on separate sides of the ER membrane. Section title: Cytosolic Localization of the Hrd1p COOH-terminal RING-H2 Domain Educational score: 4.146331787109375 Domain: biomedical Document type: Study Language: en To interpret our experiments and understand Hrd1p function, we evaluated the cellular location and orientation of the RING-H2 domain in relation to the ER membrane, both when part of the full-length, native protein and when expressed as an isolated protein. We have shown by numerous direct biochemical studies that, in either case, the Hrd1p COOH-terminal domain was exposed to the cytosol. Furthermore, the isolated Hrd1p COOH-terminal domain functioned normally in ERAD when expressed in the cytosol in trans with the isolated NH 2 -terminal transmembrane domain. Section title: Cytosolic Localization of the Hrd1p COOH-terminal RING-H2 Domain Educational score: 4.194080829620361 Domain: biomedical Document type: Study Language: en Thus, despite the report of a lumenal localization for the Hrd1p RING-H2 domain , we have clearly shown that the COOH-terminal RING-H2 domain of full-length Hrd1p was exposed to the cytosol. The reason why our results directly opposed those reported is not clear. The previous conclusions were reached by observation of Hrd1p's complete resistance to trypsin . Although we did not observe this, we did find that the Hrd1p COOH-terminal domain, whether expressed alone or as part of Hrd1p, was partially resistant to this protease. Perhaps the intrinsic trypsin insensitivity of the Hrd1p COOH terminus varies between different strains, allowing for the observation of complete protection in some circumstances. However, the Hrd1p COOH-terminal region was completely proteinase K sensitive in the same protocol, indicating that the trypsin resistance is a feature of Hrd1p structure and not lumenal sequestration. Section title: A Functionally Important Stoichiometric Hrd1p–Hrd3p Complex Mediated by the Hrd1p Transmembrane Domain Educational score: 4.3325724601745605 Domain: biomedical Document type: Study Language: en Using different biochemical and genetic assays, we have shown that Hrd1p and Hrd3p physically interacted through the Hrd1p NH 2 -terminal transmembrane domain, which was both necessary and sufficient for complex formation. In the absence of Hrd3p, Hrd1p is subject to RING-H2 domain–dependent degradation mediated by the ER-localized Ubc7p , whereas in the presence of Hrd3p, it is stable and abundant. The isolated Hrd1p RING-H2 domain similarly programs its own degradation, but in a manner independent of Hrd3p. Thus, the Hrd1p transmembrane domain allowed Hrd3p-dependent stabilization of the Hrd1p RING-H2 domain. Since the transmembrane domain itself was quite stable regardless of Hrd3p presence, it appeared that the transmembrane domain served as a transducer for lumenal Hrd3p regulation to control the cytosolic Hrd1p RING-H2 domain activity. Section title: A Functionally Important Stoichiometric Hrd1p–Hrd3p Complex Mediated by the Hrd1p Transmembrane Domain Educational score: 4.499359130859375 Domain: biomedical Document type: Study Language: en A model for the Hrd1p–Hrd3p complex required knowledge of the relative levels of the two proteins. It has been suggested that levels of Hrd1p and Hrd3p were highly disparate with Hrd1p in excess , thus favoring a catalytic role for Hrd3p stabilization of Hrd1p. However, we directly evaluated the relative natural levels of Hrd1p and Hrd3p using identically tagged versions of each protein expressed in the same strain from the native promoters. By our analysis, the relative levels of Hrd1p and Hrd3p were actually quite similar. Thus, we propose that Hrd3p forms a stoichiometric complex with Hrd1p, via the Hrd1p transmembrane domain, allowing stabilization of Hrd1p and normal ERAD. By this model, the levels of Hrd1p are contingent on the levels of Hrd3p, with the maximal amount of Hrd1p occurring when all Hrd1p molecules are Hrd3p associated. Any residual, unbound Hrd1p would be degraded by the unbridled action of the RING-H2 domain. Indeed, the level of Hrd3p did influence the amount of Hrd1p in the cell, and this effect was similar to that observed when every Hrd1p molecule was stabilized by the in cis C399S mutation. Section title: The Lumenal Functions of Hrd3p Educational score: 4.293064594268799 Domain: biomedical Document type: Study Language: en Most of the Hrd3p sequence resides in the lumen of the ER , yet one of its critical functions is to limit the degradation of Hrd1p, which depends on the ubiquitin ligase activity of the cytosolic Hrd1p RING-H2 domain. This suggested that Hrd3p regulation of Hrd1p stability was through lumen to cytosol signaling mediated by the Hrd1p transmembrane domain. We directly tested this idea by engineering an allele of HRD3 that expressed a version of Hrd3p containing only the signal sequence and the lumenal domain. This version of Hrd3p completely complemented the stabilizing effect of the hrd3Δ allele by restoration of both Hrd1p stability and ERAD, indicating that purely lumenal determinants of Hrd3p functioned in this regard. Section title: The Lumenal Functions of Hrd3p Educational score: 4.372913837432861 Domain: biomedical Document type: Study Language: en This regulatory arrangement, in which the Hrd1p hydrophobic anchor functions in transmembrane communication between another molecule and the active RING-H2 domain, has interesting implications. Although the complete role of this signaling process in ERAD is not known, it could well be part of the communication that must occur in coupling lumenal substrate scanning to cytosolic substrate tagging and destruction. More generally, there are other examples of proteins with a RING-H2 domain connected to a transmembrane anchor . If these and other unknown membrane-associated RING-H2 domains are regulated in a similar manner as Hrd1p, then the interactions and location of the corresponding anchors will play a key role in their function and biology. Section title: The Lumenal Functions of Hrd3p Educational score: 4.608366012573242 Domain: biomedical Document type: Study Language: en In parallel studies, we have demonstrated that Hrd1p functions both in vivo and in vitro as a RING-H2 ubiquitin ligase (our unpublished results). Thus, Hrd1p and Hrd3p appear to be part of an ERAD-specific ubiquitin ligase complex that functions in the detection and targeting of ERAD substrates. One main function of Hrd3p is to effect the stabilization of Hrd1p, preventing the RING-H2 domain from programming Hrd1p degradation possibly through autoubiquitination. However, our studies with truncation alleles of Hrd3p and overexpression of Hrd3p indicated that Hrd1p stabilization, although clearly important for ERAD, was not the only function of Hrd3p. By our current model, Hrd3p acts to modulate Hrd1p RING-H2 ubiquitin ligase activity by communication through the Hrd1p NH 2 -terminal transmembrane domain. In the absence of substrate, Hrd3p stabilizes Hrd1p by binding the Hrd1p transmembrane domain and suppressing the cytosolic RING-H2 domain ubiquitin ligase activity. When substrate is present, Hrd3p senses the requirement for Hrd1p RING-H2 domain ubiquitin ligase activity, possibly by directly binding substrate, and signals through the Hrd1p transmembrane domain to activate Hrd1p function in a correct temporal and spatial manner, perhaps by coordinating retrotranslocation with Hrd1p RING-H2 ubiquitin ligase activity. From our truncation analysis, it may be that the sensing function is mediated by determinants in the NH 2 -terminal portion of the Hrd3p (1–390), whereas determinants that regulate the RING-H2 domain ubiquitin ligase activity and Hrd1p stability lie in the COOH-terminal portion of the lumenal domain, although clearly more analysis is required. It is important to note that Hrd1p overexpression did allow ERAD substrate degradation in the absence of Hrd3p, suggesting that Hrd3p was not required for ERAD. However, it is likely that the elevated levels of Hrd1p under these conditions, approximately eightfold higher than in wild-type cells (data not shown), are sufficient to allow lower efficiency interaction between Hrd1p and ERAD substrates, which may normally be aided by Hrd3p. Section title: Implications of the Hrd1p–Hrd3p Complex Educational score: 4.779397964477539 Domain: biomedical Document type: Study Language: en Hrd3p determines Hrd1p–Hrd3p stoichiometry by controlling Hrd1p degradation as a likely result of Hrd1p autoubiquitination. Stabilization as a tactic to ensure correct stoichiometry is a feature of other complexes such as T cell receptors, low density lipoproteins, and yeast transcription factors . This mechanism may also be involved in self-regulation of ubiquitination machinery. In several cases, isolated RING-H2 proteins from known E3 complexes, including Hrd1p, catalyze their own ubiquitination in vitro . Similarly, the F box subunit of the SCF E3 ligase is susceptible to complex-catalyzed ubiquitination . Furthermore, it has recently been demonstrated that the RING-H2 component of the SCF E3 complex, ROC1, is degraded in a proteasome-dependent manner, but is stabilized by association with cullins . Thus, it is likely that self-catalyzed ubiquitination and subsequent degradation of E3 components is a general mode of self-regulation to maintain complex stoichiometry. In any event, it is clear that the HRD gene–encoded ERAD E3 complex is a dynamic, self-regulating one, with a separate region of Hrd1p mediating ubiquitination of self and substrate and another region mediating Hrd3p-dependent communication across the ER membrane. | Study | biomedical | en | 0.999997 |
0006072 | Section title: Introduction Educational score: 4.626532554626465 Domain: biomedical Document type: Study Language: en It is generally believed that proteins are transported through a protein-conducting channel of the ER membrane in an unfolded conformation. In cotranslational translocation, an unfolded state is maintained simply by the fact that the nascent polypeptide chain is transported while being synthesized on a ribosome that is bound to the membrane channel. In contrast, for posttranslationally transported proteins there must be a mechanism that prevents their aggregation or premature folding into a stable structure before they are transported through the channel. Cytosolic chaperones are likely involved to keep a translocation substrate in an unfolded or loosely folded conformation before it enters the channel . However, the identity of the chaperones and the mechanism by which they function remain largely unknown. In addition, once bound to the substrate, chaperones and other interacting cytosolic proteins would seem to pose a problem for the subsequent translocation step. How are they released to allow the polypeptide chain to slide through the channel? One possibility is that there is an active mechanism by which they are released when the substrate binds to or translocates through the channel. Alternatively, release may occur spontaneously in the cytosol, and the membrane channel may only capture free polypeptide chains. Section title: Introduction Educational score: 4.620469570159912 Domain: biomedical Document type: Study Language: en Posttranslational translocation occurs both in yeast and in mammalian cells, although in the latter case only substrates smaller than 70 amino acids are translocated and the mechanism of transport is not well understood . Studies in yeast have shown that the posttranslational translocation channel is formed from a seven-component membrane protein complex, the Sec complex . The Sec complex binds the translocation substrate through its signal sequence. The latter intercalates into two transmembrane domains of the Sec61p subunit of the complex, resulting in the insertion of the polypeptide chain into the channel as a loop . The subsequent movement of the chain through the channel involves the function of lumenal Kar2p (also called BiP), a member of the 70-kD heat shock protein (Hsp70) family in the lumen of the ER . At least in the case of the translocation substrate prepro-α factor (ppαF, 165 amino acids), Kar2p can function as a molecular ratchet to move the polypeptide chain across the membrane . It binds to the incoming polypeptide chain and prevents its backsliding through the channel. Section title: Introduction Educational score: 4.478729248046875 Domain: biomedical Document type: Study Language: en Cytosolic chaperones implicated in posttranslational protein translocation include Hsp70 and its cofactor, the J protein Ydj1p . Experiments in Saccharomyces cerevisiae have shown that loss-of-function mutants in the corresponding genes affect the translocation of some proteins . In addition, biochemical experiments have provided evidence that Hsp70 can associate with the substrate ppαF and can stimulate its posttranslational translocation in vitro . It is unknown whether chaperones other than the Hsp70–Ydj1p system interact with posttranslational translocation substrates. Significantly, however, cytosolic proteins are not necessary for the translocation process per se. The requirement for the binding and release of cytosolic factors can be bypassed by denaturing the substrate in urea, and the translocation process can be reconstituted with purified components in the absence of any cytosolic proteins . In addition, the Hsp70–Ydj1p system is required for translocation into both the ER and mitochondria. Thus, it is possible that cytosolic chaperones may only keep polypeptides in a loosely folded conformation and may have no specific interactions with either targeting signals or the translocation machinery in the membrane. Section title: Introduction Educational score: 4.663543224334717 Domain: biomedical Document type: Study Language: en While it is conceivable that a posttranslational translocation substrate interacts with the same cytosolic chaperones as a polypeptide chain remaining in the cytosol, some differences may exist. For polypeptides lacking signal sequences, Hsp70 and Hsp40 (a mammalian cytosolic J protein) have been found to associate with ribosome-bound nascent chains . In addition, the chaperonin-containing tailless complex polypeptide 1 (TCP1) complex has been reported to interact with ribosome-bound nascent chains , unlike the bacterial homologue GroEL, which only binds completed polypeptides . Ribosome-bound, but not full-length polypeptide chains also interact with the nascent polypeptide–associated complex (NAC) . Although no systematic study on the interaction with cytosolic proteins has been performed for posttranslational translocation substrates, the situation may be quite different. During synthesis of the polypeptide chain, the signal sequence likely interacts with the signal recognition particle (SRP), although the interaction may be weaker than with the more hydrophobic signal sequences that direct substrates into the cotranslational translocation pathway . Thus, SRP may block the association of other cytosolic proteins with the polypeptide chain, particularly as long as it is still bound to the ribosome. In addition, it is possible that there may be cytosolic proteins that, although not essential for translocation, specifically recognize the signal sequence and at some point replace SRP. Regardless of whether the cytosolic interaction partners are the same for polypeptides with and without signal sequences, there is the specific issue of how cytosolic proteins are released during posttranslational protein translocation. Section title: Introduction Educational score: 4.32992696762085 Domain: biomedical Document type: Study Language: en Here, we have used a systematic photo-cross-linking approach to probe interactions of cytosolic proteins with translocation substrates during early steps of their posttranslational translocation in yeast, until their binding to the Sec complex. Our results indicate that a posttranslational substrate synthesized in the reticulocyte lysate system interacts with SRP and NAC as long as it is associated with the ribosome. After termination of translation, it interacts with several cytosolic chaperones, including Hsp70 and TRiC/CCT, and has thus likely the same interaction partners as a polypeptide lacking a signal sequence. In fact, no specific cytosolic receptor for signal sequences of posttranslational substrates could be detected. Release of the cytosolic proteins from the translocation substrate occurs spontaneously, and the Sec complex plays no active role. Once bound to the Sec complex, the polypeptide chain is not associated with any cytosolic protein, explaining how its subsequent translocation through the membrane channel can occur without interference of cytosolic proteins. Section title: Constructs Educational score: 4.2049713134765625 Domain: biomedical Document type: Study Language: en cDNA coding for wild-type (wt) ppαF was cloned into the vector pAlter (Promega). All lysine codons in wt ppαF were altered to arginine codons (76, 84, 96, 103, 117, 124, 138, 145, and 159), and single lysines were introduced using appropriate oligonucleotides . K5Δ ppαF and M2 ppαF are signal sequence mutants of K5 and wt ppαF, respectively, with a deletion of amino acids 10–14 or a substitution of alanine for proline at position 13. The signal sequence of ppαF (165 amino acids) comprises amino acids 1–22. Section title: Transcription, Translation, and Photo–Cross-linking Educational score: 4.246473789215088 Domain: biomedical Document type: Study Language: en mRNAs coding for the different full-length ppαF polypeptides and proOmpA were generated as described . Truncated mRNAs coding for 160 amino acids of the respective ppαF molecules were produced after linearization of the plasmid with NciI by in vitro transcription with T7 RNA polymerase. In vitro translation was carried out in a reticulocyte lysate system for 25 min at 30°C in the presence of [ 35 S]methionine and trifluoromethyl-diazirino-benzoic acid (TDBA)-lysyl tRNA . Translation was stopped by addition of 2 mM cycloheximide. In the case of full-length proteins, ribosomes were removed by centrifugation for 10 min at 100,000 rpm in a Beckman TLA-100 rotor, and cross-links were induced by irradiation with UV light for 15 min on ice. Ribosome–nascent chain complexes (RNCs) were diluted with 10 volumes of buffer A (50 mM Hepes-KOH, pH 7.5, 150 mM potassium acetate, 2 mM magnesium acetate, 5 mM β-mercaptoethanol), and subsequently isolated by centrifugation through a sucrose cushion (buffer A with 500 mM sucrose) for 1 h at 100,000 rpm in a Beckman TLA-100.3 rotor. Finally, RNCs were resuspended in buffer A containing 250 mM sucrose and irradiated with UV light. Truncated nascent chains of 160 residues were released from the ribosome by addition of 10 mM EDTA and ribosome-depleted reticulocyte lysate. Section title: Immunoprecipitations of Cytosolic Cross-linked Products Educational score: 4.2146806716918945 Domain: biomedical Document type: Study Language: en For immunoprecipitation (IP) after denaturation, SDS was added to the irradiated sample to a final concentration of 2%. The mixture was incubated for 15 min at 65°C, and the SDS concentration was adjusted to 0.1% by dilution with IP buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1% Triton X-100) containing 1% BSA. Antibodies to Hsp70 , to TCP1α (CTA123 from StressGen Biotechnologies; CTA-191, CTA-122, and CTA-184 gave similar results), to Hsp70/Hsp90 organizing protein (p60/Hop) , to SRP54, and to the α and β subunits of NAC were added for 5 h at 4°C and collected with protein G–Sepharose. The resin was washed three times with IP buffer, and the bound material was eluted with SDS sample buffer. For immunoprecipitations under native conditions, the irradiated samples were diluted 20-fold with BT buffer (20 mM NaCl, 20 mM Tris-HCl, pH 7.5, 5 mM MgCl 2 , 0.1% Triton X-100) containing 1% BSA, the antibodies were added for 5 h at 4°C, then collected with protein G–Sepharose, and the resin was washed three times with BT buffer. Section title: Sucrose Gradient Centrifugation and Translocation Assays Educational score: 4.1428728103637695 Domain: biomedical Document type: Study Language: en 30 μl of the irradiated translation mixture containing full-length ppαF or proOmpA was diluted twofold with buffer A, layered on top of a 540-μl sucrose gradient (0.3–1.0 M sucrose in buffer A), and centrifuged for 6 h at 48,000 rpm in a Beckman SW55 rotor. After centrifugation, 50-μl fractions were collected starting from the top. To analyze the translocation competence of different ppαF complexes, a translation mixture containing wt ppαF without photoreactive probes was separated in a sucrose gradient. The translocation assays with different fractions of the sucrose gradients were performed with reconstituted proteoliposomes containing purified yeast Sec complex in the membrane and recombinant Kar2p and ATP in the lumen, as described . Section title: Dissociation of Cytosolic Complexes Educational score: 4.138212203979492 Domain: biomedical Document type: Study Language: en After in vitro translation of full-length ppαF and sedimentation of ribosomes, 10 μl of the translation mixture was added to 100 μl of buffer A containing 2 mM ATP, 40 mM creatine phosphate, and 1 mg/ml creatine kinase. Where indicated, 1.5 μmol GroEL trap (D87K; provided by F.U. Hartl and his laboratory, Max-Planck-Institute for Biochemistry, Martinsried, Germany), or reconstituted proteoliposomes containing Sec complex were present. After mixing, 10 μl was immediately removed and placed on dry ice to stop the reaction (0.5-min time point). The remainder of the mixture was incubated at 30°C. 10-μl samples were removed at the indicated time points and immediately frozen on dry ice. At the end, all samples were irradiated with UV light for 15 min on dry ice. Section title: Dissociation of Cytosolic Complexes Educational score: 4.120866298675537 Domain: biomedical Document type: Study Language: en To test whether the dissociation of cytosolic complexes leads to a reduction of translocation competence, in vitro–synthesized full-length ppαF was incubated for 30 min at 30°C or 0°C. Subsequently, half of the sample was irradiated with UV light for 15 min on ice, and the other half was incubated with reconstituted proteoliposomes containing Sec complex for binding of ppαF to Sec complex. Section title: IP of ppαF Bound to Sec Complex Educational score: 4.113565444946289 Domain: biomedical Document type: Study Language: en Binding of ppαF to reconstituted proteoliposomes containing Sec complex was done essentially as described . In brief, 4 μl of ppαF translation mixture was either first irradiated for 15 min on ice and subsequently incubated with 30 μl of reconstituted proteoliposomes for 15 min at 30°C, or first incubated with proteoliposomes and then irradiated. The samples were subsequently solubilized in digitonin, and the Sec complex was immunoprecipitated with antibodies to Sec62p. Section title: Electrophoresis Educational score: 3.423570156097412 Domain: biomedical Document type: Study Language: en Analysis of all samples was performed by SDS-PAGE with 7.5–17.5% linear acrylamide gels, followed by autoradiography or analysis with a Fuji PhosphorImager BAS1000. Section title: Interactions of Ribosome-bound ppαF in the Cytosol Educational score: 4.554838180541992 Domain: biomedical Document type: Study Language: en We first used photo-cross-linking to investigate interactions of ribosome-bound nascent ppαF with cytosolic proteins. A truncated mRNA coding for a ppαF polypeptide chain missing only the last five amino acids was translated in vitro in a reticulocyte lysate system in the presence of [ 35 S]methionine. When the ribosome reaches the end of the mRNA, the radioactively labeled nascent chain remains bound to the ribosome as peptidyl tRNA because there is no stop codon to effect termination of translation. The translation system also contained a modified lysyl tRNA that carries a carbene-generating probe in the side chain of the amino acid . This results in the incorporation of photoreactive lysine derivatives at positions of the polypeptide chain where lysines would normally occur. After translation, RNCs together with bound cytosolic proteins originating from the reticulocyte lysate were isolated by sedimentation, and the sample was irradiated to induce cross-links of nascent ppαF. The cross-linked products were analyzed by SDS-PAGE and autoradiography. With wt ppαF, containing all of its nine lysines in the COOH-terminal half of the polypeptide chain, cross-links occurred mostly to the two subunits of NAC , as demonstrated by IP with antibodies to both NAC subunits after SDS denaturation (data not shown). To identify interaction partners of the signal sequence, we substituted all the lysines of wt ppαF with arginines and introduced a single lysine residue at position 5 within the signal sequence. As previously observed with cotranslational translocation substrates , the signal sequence could be cross-linked to SRP54 . Surprisingly, two bands of SRP54 cross-links were seen, which both could be immunoprecipitated with specific antibodies . Perhaps, ppαF is cross-linked to different regions of SRP54, resulting in cross-linked products with different mobilities in SDS gels, as observed with cross-linking of ppαF to Sec61p . Cross-links to the NAC subunits could also be detected , although they were weaker than those with the probes in the mature region of the chain. These results demonstrate that nascent ppαF interacts mainly with SRP and NAC. It should be noted that nascent ppαF interacts with SRP even when the chain is almost full length, in contrast to the cotranslational translocation substrate preprolactin, which only interacts with SRP when the nascent chain is short . Thus, the posttranslational substrate ppαF may have folding characteristics that keep the signal sequence exposed. Section title: Interactions of Ribosome-bound ppαF in the Cytosol Educational score: 4.178402900695801 Domain: biomedical Document type: Study Language: en Next, we tested whether the cross-linking pattern changes when ppαF carries a defective signal sequence (K5Δ and M2 ppαF mutants). M2 and wt ppαF, which contain the photoreactive probes at the same positions in the COOH-terminal domain, showed identical cross-linking patterns . However, the K5Δ mutant, containing a single probe at position 5 of its defective signal sequence, showed drastically reduced SRP54 cross-links but no other changes compared with K5 ppαF, containing an intact signal sequence . These data demonstrate that the interaction of ppαF with SRP, in contrast to that with NAC, requires a functional signal sequence, confirming previous results obtained with preprolactin . Section title: Release of SRP and NAC upon Termination of Translation Educational score: 4.349274635314941 Domain: biomedical Document type: Study Language: en Next, we studied whether the interaction of ppαF with cytosolic proteins changes upon termination of translation. To generate ribosome-released full-length ppαF, in vitro translation in a reticulocyte lysate system was performed with mRNA containing the natural stop codon, and the ribosomes were removed by centrifugation. In these experiments, we employed a ppαF mutant that carries a single photoreactive probe at position 10 of the signal sequence (K10 ppαF). Ribosome-associated, truncated K10 ppαF of 160 residues gave the same cross-linking pattern as nascent K5 ppαF studied before ; both SRP and NAC cross-links could be verified by IP with specific antibodies . In contrast, ribosome-released full-length K10 ppαF of 165 residues could not be cross-linked to either SRP or NAC , and several new cross-linked products appeared instead . Similar results were obtained when the truncated nascent chain of 160 residues was released from the ribosome with EDTA . The resulting cross-linking pattern was similar to that of full-length K10 ppαF . These results demonstrate that both SRP and NAC are released from the translocation substrate ppαF upon termination of translation. Section title: Interactions of Full-Length ppαF in the Cytosol Educational score: 4.113458633422852 Domain: biomedical Document type: Study Language: en To analyze interactions of full-length ppαF with cytosolic proteins in more detail, we generated 37 ppαF mutants that each contain a single lysine within either the signal sequence or the mature part of the protein. These mutants can be used to scan the environment of distinct regions of the translocation substrate in a systematic manner. All mutant proteins containing photoreactive probes were efficiently bound to the Sec complex and translocated across yeast ER membranes . Section title: Interactions of Full-Length ppαF in the Cytosol Educational score: 4.308408737182617 Domain: biomedical Document type: Study Language: en Each of the single lysine mutants gave cross-links to several cytosolic proteins present in reticulocyte lysate . In each case, the cross-links were dependent on the presence of photoreactive probes in the polypeptide chain (data not shown). The cross-linking pattern was remarkably similar for all positions probed throughout the polypeptide chain. The major cross-linking partners had molecular masses of ∼200, 180, 70, 62, 60, 50, and 20 kD (in each case, the molecular mass of ppαF [20 kD] was subtracted from the size of the cross-linked product). Some differences between regions were noted. Cross-links to the 70- and 50-kD proteins were more prominent with photoreactive probes at positions within the central part of the signal sequence (positions 5–15) than with probes at all following positions. At position 40, the cross-link to the 200-kD protein disappeared while the band containing the 180-kD protein became more prominent. Beyond position 95, a strong cross-link to a protein of ∼55 kD was observed. The fact that a single position of ppαF could be cross-linked to several proteins suggests that there are different populations of ppαF molecules that contact either different sets of proteins or the same set with different orientations. Section title: Interactions of Full-Length ppαF in the Cytosol Educational score: 4.168936729431152 Domain: biomedical Document type: Study Language: en With wt ppαF, which contains nine lysine residues in the COOH-terminal half, a complex cross-linking pattern was seen. Several cross-linked products corresponded to those seen with single-lysine mutants (e.g., the cross-links to proteins of ∼180, 62, 60, 55, and 20 kD). The many bands seen without irradiation are caused by extensive ubiquitination of ppαF at the lysine residues (data not shown). Taken together, these results show that both the signal sequence and the mature part of newly synthesized full-length ppαF interact with several proteins of the reticulocyte lysate, and that there are relatively small differences in the interaction patterns probed at various positions of the polypeptide chain. Section title: Interactions of Full-Length ppαF in the Cytosol Educational score: 4.140579700469971 Domain: biomedical Document type: Study Language: en Next, we tested whether the cross-linking pattern changes when ppαF carries a defective signal sequence. Regardless of whether the photoreactive probes were located in the signal sequence (K5Δ ppαF) or in the COOH-terminal region of the polypeptide chain (M2 ppαF), the cross-linking pattern of the signal sequence mutant was indistinguishable from that of the corresponding protein with a functional signal sequence . These data suggest that there is no cytosolic protein that specifically interacts with the signal sequence of the posttranslational translocation substrate. In addition, they suggest that completed polypeptides with and without signal sequence interact with the same set of cytosolic proteins. Section title: Interactions of Full-Length ppαF in the Cytosol Educational score: 4.121452331542969 Domain: biomedical Document type: Study Language: en We also observed a cross-linked product that migrates in SDS gels slightly faster than ppαF itself . This product is probably generated by internal cross-linking within the ppαF molecule, resulting in a more compact structure with higher mobility in SDS gels. Internal cross-links occurred with some variation in intensity throughout the entire polypeptide chain, suggesting that ppαF may be in a collapsed conformation. Section title: Full-Length ppαF Interacts with Cytosolic Chaperones Educational score: 4.14461612701416 Domain: biomedical Document type: Study Language: en To identify the cytosolic cross-linking partners of full-length ppαF, we performed IP experiments with antibodies directed against cytosolic chaperones and their cofactors. First, cross-linked products obtained with ppαF containing the photoreactive probe at position 10 of the signal sequence were analyzed . After denaturation of the irradiated sample in SDS, antibodies to Hsp70 and TCP1α, a subunit of TRiC/CCT, immunoprecipitated cross-linked products of the expected size . Minor cross-links to p60/Hop, a chaperone cofactor that is known to interact with TRiC/CCT, Hsp70, and Hsp90 , could also be identified by denaturing IP (data not shown). Section title: Full-Length ppαF Interacts with Cytosolic Chaperones Educational score: 4.202986717224121 Domain: biomedical Document type: Study Language: en To test whether Hsp70 and TCP1α interact with ppαF alone or in conjunction with other proteins, we performed IPs under native conditions. The efficiency of IP was significantly higher than under denaturing conditions. With both Hsp70 and TCP1α antibodies, the cross-linked product of the expected size corresponded to a major band among the total products , indicating that both Hsp70 and TCP1α are major interaction partners of the signal sequence of ppαF. Hsp70 antibodies also precipitated the prominent cross-linked products containing the proteins of ∼50 and 20 kD . TCP1α antibodies coprecipitated cross-links to a protein slightly smaller than TCP1α itself (p60) as well as products containing the 20-kD protein . The native IPs with Hsp70 and TCP1α antibodies together account for the majority of the bands seen among the total cross-linked products . These data suggest that ppαF is part of at least two distinct complexes, explaining why different cross-linked bands could be coprecipitated with Hsp70 and TCP1α antibodies under native conditions. Section title: Full-Length ppαF Interacts with Cytosolic Chaperones Educational score: 4.152683258056641 Domain: biomedical Document type: Study Language: en Hsp70 and TCP1α were also identified as cross-linking partners when the photoreactive probes were located in the COOH-terminal region of the ppαF molecule. With the probe at position 97, the pattern of immunoprecipitation with Hsp70 and TCP1α antibodies was very similar to that of ppαF with the probe in the signal sequence, both under denaturing and native conditions . p60/Hop was also identified as a cross-linking partner (data not shown). Similar results were obtained with wt ppαF containing probes in its nine lysine residues at the COOH terminus . As expected from the results described above , both Hsp70 and TCP1α were also identified as cross-linking partners with M2 ppαF containing a defective signal sequence . The fact that Hsp70 cross-links more strongly to the signal sequence than to the mature part of ppαF may be explained by its preference for hydrophobic sequences. Section title: Full-Length ppαF Interacts with Cytosolic Chaperones Educational score: 4.196821212768555 Domain: biomedical Document type: Study Language: en Significant cross-links to TCP1α were only observed with ppαF released from the ribosomes , similar to results obtained with GroEL, the bacterial homologue of TRiC/CCT . The results are also in agreement with data obtained with a heterogeneous mixture of nascent chains and with recent data obtained for the cotranslational translocation substrate preprolactin . For Hsp70, cross-linking of RNCs was observed, but the intensity of the cross-links became stronger upon termination of translation . Section title: Full-Length ppαF Interacts with Cytosolic Chaperones Educational score: 4.191483497619629 Domain: biomedical Document type: Study Language: en We also tested a second posttranslational translocation substrate, proOmpA. Wt proOmpA contains lysine residues both in the signal sequence and in the mature part (a total of 19 lysines). Like ppαF, it could be cross-linked to several cytosolic proteins in the reticulocyte lysate, although a high background was seen in the absence of irradiation, probably caused by ubiquitination . IP experiments after denaturation of cross-linked products with SDS demonstrated that proOmpA was cross-linked to Hsp70 and TCP1α . Native IP suggested again that the translocation substrate is contained in at least two distinct complexes, one with Hsp70 and the other with TCP1α . Section title: Distinct Translocation-competent Complexes Educational score: 4.330556869506836 Domain: biomedical Document type: Study Language: en To confirm that posttranslational translocation substrates are present in at least two distinct complexes with cytosolic proteins, we performed sucrose gradient centrifugation. Specifically, a ppαF mutant containing a single photoreactive probe at position 10 was synthesized in vitro, irradiated, and separated in a sucrose gradient. The cross-links to Hsp70 and the 50-kD protein were found in fractions of relatively low molecular weight . In fact, the pattern of cross-links in these fractions was remarkably similar to that seen in native IPs with Hsp70 antibodies . The cross-links to TCP1α and to the slightly smaller 60-kD protein were found in a high molecular weight peak , whose size is consistent with that of TRiC/CCT (970 kD). Again, the cross-linking pattern looked similar to that of native IPs with TCP1α antibodies . p60 is thus likely a subunit of TRiC/CCT that contains several polypeptides of almost the same size. The Hsp70 and TRiC/CCT peaks also contained the maximum amounts of non-cross-linked ppαF, although the latter was found in all fractions (quantitation not shown). These data support the existence of at least two distinct ppαF populations, one in a complex with Hsp70 and the other with TRiC/CCT. The other cross-linked products were also found in distinct fractions of the sucrose gradient, and only the cross-links to the 20-kD protein were distributed throughout the gradient. It should be noted that internal cross-links of ppαF were observed in all fractions of the sucrose gradient , indicating that despite association with different chaperone proteins, ppαF does not attain a fully extended conformation. Section title: Distinct Translocation-competent Complexes Educational score: 4.154979228973389 Domain: biomedical Document type: Study Language: en Similar results were obtained with wt ppαF and ppaF carrying a photoreactive probe at position 97 of the mature region, with a signal sequence mutant, or if the order of sucrose gradient centrifugation and cross-linking was changed (data not shown). Moreover, proOmpA behaved similarly to ppαF in sucrose gradient centrifugation , indicating that posttranslational translocation substrates are generally associated with different cytosolic chaperone complexes. ppαF synthesized in yeast lysate was also found to be contained in different complexes, since it showed a broad distribution in sucrose gradients (data not shown). Unfortunately, cross-linking experiments with ppαF synthesized in yeast lysates did not give distinct cross-linked bands, most likely because of the fast hydrolysis of the modified lysyl tRNA (data not shown). Section title: Distinct Translocation-competent Complexes Educational score: 4.148315906524658 Domain: biomedical Document type: Study Language: en Next, we tested whether non–cross-linked ppαF present in the different complexes could be translocated. Wt ppαF without photoreactive probes was separated in a sucrose gradient, and the various fractions of the gradient were incubated with proteoliposomes containing Sec complex in the membrane and Kar2p and ATP in the lumen . Translocation was assessed after treatment with protease. Regardless of whether equal volumes or equal amounts of substrate of the individual fractions were tested, all were clearly active in translocation . The fact that translocation competence was found for fractions between the TRiC and Hsp70 peaks may be explained either by significant overlap of the peaks or by the presence of a chaperone in all fractions . Taken together, these results indicate that different sets of cytosolic proteins keep ppαF in a translocation-competent conformation. Section title: Dissociation of ppαF from Cytosolic Proteins Educational score: 4.2840704917907715 Domain: biomedical Document type: Study Language: en We next investigated the fate of the ppαF–chaperone complexes during the next step in posttranslational protein translocation, the binding of ppαF to the Sec complex. Specifically, ppαF containing a photoreactive probe at a single position was synthesized in vitro in a reticulocyte lysate system, the ribosomes were removed by centrifugation, and proteoliposomes containing the purified yeast Sec complex were added. The vesicles lack Kar2p and thus allow binding of ppαF to the cytosolic face of the Sec complex but no translocation . After different incubation times at 30°C, the samples were irradiated and the cross-linked products were separated by SDS-PAGE and quantitated. For these experiments, we used ppαF with a photoreactive probe at position 11 because it gives only weak cross-links to the Sec61p subunit of the Sec complex , which could obscure the presence of some cross-links to cytosolic proteins in SDS gels. When the Sec complex was present, the cross-links to the 50-kD protein and to Hsp70 both diminished very rapidly with the same kinetics . Cross-links to the p60 subunit of TRiC/CCT also decreased, but more slowly . The kinetics of dissociation of cytosolic proteins from ppαF are consistent with those of binding of ppαF to the Sec complex, as determined by the appearance of cross-links to Sec62p and Sec71p, two other subunits of the Sec complex . The cross-links to the proteins of ∼20 kD also disappeared rapidly (those to the 180- and 200-kD proteins could not be well quantitated; data not shown). Section title: Dissociation of ppαF from Cytosolic Proteins Educational score: 4.146275043487549 Domain: biomedical Document type: Study Language: en When the proteoliposomes were omitted, cross-links to Hsp70, p60, and the 50-kD protein also diminished with time but remained significantly stronger than in the presence of Sec complex throughout the experiment . Liposomes lacking Sec complex gave the same result (data not shown). Incubation of ppαF–chaperone complexes on ice resulted in all cross-links remaining constant . These results show that, at elevated temperatures, spontaneous net dissociation of complexes between ppαF and cytosolic proteins occurs; net dissociation is significantly faster in the presence of Sec complex. Section title: Dissociation of ppαF from Cytosolic Proteins Educational score: 4.295121669769287 Domain: biomedical Document type: Study Language: en To test whether the Sec complex stimulates dissociation in an active manner or simply captures free ppαF molecules spontaneously released from their cytosolic partners, we used a mutant of the Escherichia coli chaperonin GroEL as a passive trap . This mutant (D87K, in which the aspartate at position 87 is replaced by a lysine) is able to bind unfolded proteins, but cannot release them and is not expected to actively stimulate the dissociation of ppαF from cytosolic proteins . In the presence of the GroEL trap, the cross-links to all cytosolic proteins disappeared with similar rapid kinetics as in the presence of the Sec complex . In addition, cross-links to GroEL appeared with the same kinetics as those to proteins of the Sec complex . We conclude that in both cases the binding partner serves as a simple trap for spontaneously released ppαF molecules. Dissociation of the complexes between ppαF and cytosolic proteins seems to be the rate-limiting step in the transfer of the substrate to the respective binding partner. Section title: Dissociation of ppαF from Cytosolic Proteins Educational score: 4.208501815795898 Domain: biomedical Document type: Study Language: en To test whether the spontaneous dissociation of chaperone–substrate complexes in the absence of Sec complex leads to a reduction of translocation competence, we preincubated in vitro–synthesized ppαF at 30°C or 0°C, and then tested in parallel cross-linking of ppαF to cytosolic proteins and binding of ppαF to the Sec complex . While after preincubation on ice both the cross-linking to cytosolic chaperones and the binding to the Sec complex remained unchanged compared with a sample without preincubation, at 30°C both were reduced to the same extent (∼50%). These data suggest that substrate molecules released from cytosolic chaperones aggregate and become incompetent for translocation if they cannot immediately interact with the Sec complex. Section title: Dissociation of Cytosolic Complexes Required for ppαF–Sec Complex Interaction Educational score: 4.446505546569824 Domain: biomedical Document type: Study Language: en To confirm the release of cytosolic proteins from ppαF during initiation of translocation, we analyzed interactions of the substrate bound to the Sec complex. ppαF molecules with probes in the signal sequence at positions 11 or 13 were first incubated with proteoliposomes containing the purified Sec complex and then irradiated . The samples were either analyzed directly by SDS-PAGE (total) or solubilized in digitonin, and were then subjected to IP with antibodies to Sec62p to isolate the Sec complex and the associated cross-linked and non-cross-linked ppαF. As reported previously , ppαF with a probe at position 11 cross-linked weakly to Sec61p and strongly to Sec62p and Sec71p . ppαF with a probe at position 13 gave strong cross-links to both Sec61p and Sec62p/71p . With both positions of the probe, ppαF bound to Sec complex did not give any cross-link to cytosolic proteins , indicating that the cytosolic binding partners were released from the signal sequence upon its binding to the Sec complex. To test whether cytosolic proteins are also released from the mature portion of ppαF, we repeated the experiments with mutants containing probes at positions 117, 138, and with the wild-type protein containing the probes at nine COOH-terminal positions . With all three proteins, strong cross-links to Sec62/71p and Sec72p were seen, while the single-lysine mutants showed additional very weak cross-links to Sec63p, Sec61p, and Sbh1p (Sec72p, Sec63p, and Sbh1p are also subunits of the Sec complex). Significantly, in all cases no cytosolic cross-links of ppαF bound to the Sec complex were discernible . Thus, during the binding of ppαF to the Sec complex, all cytosolic proteins must have been released, even from COOH-terminal parts of the polypeptide chain which are not inserted into the translocation channel. Section title: Dissociation of Cytosolic Complexes Required for ppαF–Sec Complex Interaction Educational score: 4.2719597816467285 Domain: biomedical Document type: Study Language: en Finally, we tested whether the release of cytosolic proteins is required for the binding of ppαF to the Sec complex. To this end, samples containing ppαF with photoreactive probes at different positions were first irradiated on ice, conditions that maximize the extent of cross-linking to cytosolic proteins, and then proteoliposomes containing the Sec complex were added to allow binding of ppαF to the Sec complex . Again, both the total products and those associated with the Sec complex were analyzed. Although a large number of cross-links to cytosolic proteins were visible among the total products, most of them either were not bound or were only inefficiently bound to the Sec complex . The only clear exception are the cross-links to the 55-kD protein (p55), seen with probes in wt ppαF, which were coprecipitated with the Sec complex. Thus, cross-linking of most cytosolic proteins appears to prevent the interaction of ppαF with the Sec complex, suggesting that their release is a prerequisite for initiation of translocation. Section title: Discussion Educational score: 4.348962783813477 Domain: biomedical Document type: Study Language: en We have used photo-cross-linking to follow the route of a posttranslational translocation substrate from its synthesis on a free ribosome to its binding to the Sec complex, the component in the yeast ER membrane that forms the translocation channel. Our data suggest the following sequence of events . First, while still bound to the ribosome, the nascent polypeptide chain interacts with SRP and NAC. Second, when the chain is completed and released from the ribosome, it no longer interacts with these proteins and instead associates with several different cytosolic proteins. At least two distinct complexes could be identified, one containing Hsp70 and the other TRiC/CCT. Third, the cytosolic complexes dissociate spontaneously, without active participation of the Sec complex. Finally, substrate bound to the Sec complex is no longer associated with any cytosolic proteins and can now begin translocation through the membrane channel without interference by cytosolic proteins. Section title: Discussion Educational score: 4.278379917144775 Domain: biomedical Document type: Study Language: en For technical reasons, we have used in our experiments a heterologous system comprised of cytosol from rabbit reticulocytes and Sec complex from S. cerevisiae . However, we believe that the results are also relevant to the homologous system in yeast, and even to in vivo conditions, for the following reasons. First, ppαF and proOmpA synthesized in a reticulocyte lysate system are posttranslationally transported into yeast ER vesicles with the same high efficiency as substrates synthesized in yeast lysate (data not shown). Although posttranslational translocation appears to be more prominent with yeast ER membranes than with mammalian ones, the difference is not due to cytosolic factors. Second, the cytosolic chaperone proteins are highly conserved among species, making it unlikely that entirely different pathways are employed in different eukaryotes. Furthermore, cytosolic chaperone proteins are equally abundant both in reticulocyte and yeast lysates . Third, in sucrose gradients, ppαF synthesized in yeast lysate runs as a heterogeneous population, similarly to ppαF made in reticulocyte lysate. Fourth, in yeast, Hsp70 is involved in the translocation of ppαF both in vivo and in vitro , similarly to its role in our heterologous system. Despite these arguments, we cannot strictly exclude that yeast chaperones, in contrast to mammalian chaperones, are actively dissociated from the substrate by the Sec complex, but the data demonstrate that active dissociation is not required for translocation and, if it occurred, would have little effect on the translocation efficiency. Section title: Discussion Educational score: 4.613731384277344 Domain: biomedical Document type: Study Language: en Our results show that a posttranslational translocation substrate differs from a polypeptide lacking a signal sequence, as long as it is associated with the ribosome, by being associated with SRP. However, once released from the ribosome, both classes of proteins seem to interact with the same cytosolic proteins. These include Hsp70 and TRiC/CCT, but also p60/Hop. Several other, unidentified binding partners of posttranslational translocation substrates may also be identical to those known to interact with cytosolic polypeptides. The 50-kD protein might be Hsp40, a mammalian cytosolic J protein, since it comigrated with Hsp70 in sucrose gradients and could be coimmunoprecipitated with it. The cross-linked proteins of ∼20 kD might be subunits of prefoldin, a cofactor of TRiC/CCT that has been shown to interact with some newly synthesized polypeptides . Unfortunately, no cross-links could be precipitated with Hsp40 or prefoldin antibodies (data not shown). No evidence was seen for cytosolic factors that interact specifically with the signal sequence of completed chains, suggesting that the signal sequence of a posttranslational substrate is only recognized by the Sec complex and not by a cytosolic signal sequence receptor. Thus, the only function of cytosolic binding partners may be to keep a polypeptide in a loosely folded conformation, a function not specific for translocation substrates. The essential role of cytosolic chaperones in translocation is illustrated by our observation that a decrease in cross-linking to cytosolic proteins after incubation at elevated temperature results in reduced substrate binding to the Sec complex . Our data also support the idea that there may be more than one way to keep a polypeptide unfolded and thus competent for translocation. Redundant pathways are also suggested by in vivo data in yeast, in which depletion of functional Hsp70 affects the posttranslational translocation of some substrates but not of others . Section title: Discussion Educational score: 4.196498870849609 Domain: biomedical Document type: Study Language: en Our data suggest that the cytosolic proteins continuously associate with and dissociate from a completed polypeptide chain. The addition of an unspecific GroEL trap accelerated the net dissociation of all chaperones, indicating that in its absence a portion of the substrate is able to reassociate with them. Reassociation of chaperones is apparently not very efficient in our in vitro system. In vivo, the chaperones may associate and dissociate as long as the translocation substrate remains in the cytosol , since the polypeptide cannot reach its native conformation in this compartment. Section title: Discussion Educational score: 4.252043724060059 Domain: biomedical Document type: Study Language: en Given that completed polypeptides with and without a signal sequence apparently interact with the same cytosolic proteins, and that there is continuous binding and dissociation, it may not be very surprising that we found that the Sec complex plays no active role in releasing these proteins from the translocation substrate. This conclusion is based on the result that the Sec complex and the unspecific GroEL trap accelerate net dissociation to the same extent. The binding of the substrate to these components appears to prevent the reassociation with cytosolic proteins. Surprisingly, Sec complex and the GroEL trap even stimulated the disappearance of all cross-links after depletion of ATP by addition of hexokinase and glucose (data not shown), suggesting that dissociation of the chaperones can occur in their ADP form. A similar observation has been made for the release of cytosolic Hsp70 from mitochondrial precursor proteins . Section title: Discussion Educational score: 4.364577770233154 Domain: biomedical Document type: Study Language: en Eventually, the polypeptide chain bound to the Sec complex is entirely stripped of all cytosolic proteins. It is possible that the polypeptide chain needs to be fully stripped before its binding to the Sec complex because, with the exception of a 55-kD protein, all cytosolic proteins need to be released from the substrate to allow its stable interaction with the Sec complex. Alternatively, only the signal sequence may have to be stripped initially, allowing substrate binding to the Sec complex. The chaperones would then dissociate from the mature part of the chain but reassociation would be prevented, possibly by the large cytosolic domains of the Sec62p and Sec63p subunits of the Sec complex. Section title: Discussion Educational score: 4.380777359008789 Domain: biomedical Document type: Study Language: en Remarkably, once bound to the Sec complex, the substrate is free of cytosolic proteins even in regions that are protruding into the cytosol or are only loosely associated with the cytosolic aspect of the Sec complex. As the bound polypeptide chain is fully stripped, it can move freely through the channel, uninhibited by any interaction with cytosolic proteins. In fact, thermal motion has been demonstrated to be sufficient for the movement of a chain through the channel . The existence of a “naked” polypeptide chain would allow a Brownian ratcheting mechanism to operate and would also facilitate a mechanism in which Kar2p would pull on the polypeptide chain and accelerate its forward movement through the channel. Section title: Discussion Educational score: 4.565007209777832 Domain: biomedical Document type: Study Language: en Posttranslational translocation appears to be quite different from the tightly regulated process of cotranslational translocation in which the signal sequence is recognized first by SRP in the cytosol and then by the Sec61p complex in the membrane. In the posttranslational pathway, the only essential recognition step seems to be the binding of the signal sequence to the Sec complex. Although we have found that the signal sequence of a ribosome-bound posttranslational substrate can bind to SRP, the interaction may be of low affinity in living yeast cells, explaining why the substrate escapes cotranslational membrane targeting. The recognition of the signal sequence by the Sec complex must be sufficient to distinguish proteins to be exported from those staying in the cytosol, despite the fact that both can associate with the same chaperones. The situation may be similar for at least some proteins imported into mitochondria. Although some targeting sequences may be recognized by specific cytosolic receptors, others associate with Hsp70 and are only recognized by receptors on the outer mitochondrial membrane . | Study | biomedical | en | 0.999996 |
0006125 | Section title: Introduction Educational score: 4.311325550079346 Domain: biomedical Document type: Study Language: en Mammalian cells internalize a large quantity of plasma membrane-associated and soluble molecules, and sort these molecules into recycling or lysosomal pathways. A great deal of this sorting occurs in early sorting endosomes, which segregate most of the membrane lipids and proteins, and a small fraction of the soluble contents, into tubular extensions that give rise to recycling endosomes . In nonpolarized cells, the contents of recycling endosomes then return to the cell surface. Most of the soluble contents of sorting endosomes, and a small fraction of the membrane, are collected into endosomal carrier vesicles , which are transported to, and fuse with, late endosomes . Section title: Introduction Educational score: 4.636396884918213 Domain: biomedical Document type: Study Language: en Virtually all of the soluble contents of late endosomes are delivered to lysosomes for degradation. However, a number of membrane proteins recycle to the TGN . This allows receptors that fail to recycle to the cell surface from early endosomes to recycle to the cell surface instead via late endosomes, the TGN, and the secretory pathway. This process increases the overall recycling efficiency for cell surface receptors significantly . Recycling from late endosomes to the TGN plays a particularly important role in the trafficking of mannose 6-phosphate receptors (MPR). The cation-independent (CI) and cation-dependent MPR carry nascent lysosomal enzymes from the TGN to late endosomes. The enzymes dissociate from the receptors at the low pH of late endosomes, and the receptors are then recycled efficiently to the TGN to mediate additional rounds of enzyme sorting . The high efficiency of MPR recycling from late endosomes is due at least in part to a cytoplasmic sorting determinant recognized by a sorting protein, TIP47 , in late endosomes. Section title: Introduction Educational score: 4.689414978027344 Domain: biomedical Document type: Study Language: en A different endosomal sorting phenotype was identified through studies of P-selectin, a member of a family of cell adhesion proteins expressed in the vascular system . Whereas expression of the cell adhesion protein E-cadherin at the cell surface is regulated by internalization of those molecules not bound to substrates , P-selectin is stored in secretory granules in platelets and endothelial cells. Stimulation of granule exocytosis in these cells transfers P-selectin rapidly to the plasma membrane, allowing binding of circulating neutrophils and monocytes expressing the P-selectin ligand . This interaction contributes to extravasation of the leukocytes, an early event in the inflammatory response. P-selectin is rapidly internalized at rates similar to other recycling receptors. However, in addition to cytoplasmic sorting determinants that mediate targeting to secretory granules and rapid internalization from the cell surface , P-selectin contains a sorting determinant that mediates sorting of internalized P-selectin away from typical recycling receptors, such as low density lipoprotein (LDL) receptor and transferrin receptor, leading to rapid delivery of P-selectin to lysosomes . Its half-life of 2.3–3 h in transfected cell lines is several times shorter than the half-lives of the LDL receptor or transferrin receptor. This sorting event affords temporal regulation of the adhesive activity of P-selectin after stimulation of granule exocytosis. Section title: Introduction Educational score: 4.704314231872559 Domain: biomedical Document type: Study Language: en Whereas it is clear that sorting of P-selectin from efficiently recycled receptors (e.g., LDL receptor) occurs in endosomes , it is not possible to determine from the existing data whether this sorting occurs in early endosomes, in late endosomes, or a combination of the two. If sorting of P-selectin occurs only in early endosomes, it would reach late endosomes faster than LDL receptor, and could then recycle from late endosomes to the TGN with the same efficiency as LDL receptor . This hypothesis predicts rapid transport of P-selectin from the cell surface through the TGN, which would enable the protein to recycle efficiently into nascent secretory granules. Indeed, internalized anti–P-selectin antibodies can reach Weibel-Palade bodies (secretory granules) in cultured endothelial cells , although with unknown efficiency. In this model, rapid turnover of P-selectin would be due to frequent trips through late endosomes, but with the same low probability as LDL receptor of entering lysosomes on each trip. Alternatively, if P-selectin recycles from sorting endosomes to the cell surface as efficiently as LDL receptor, the short half-life of P-selectin would require selective delivery of P-selectin from late endosomes to lysosomes, which would preclude extensive recycling to the TGN . Section title: Introduction Educational score: 4.228057861328125 Domain: biomedical Document type: Study Language: en To determine the location and consequences of endosomal sorting of P-selectin, we have measured transport of P-selectin in PC12 cells, where we previously measured transport of LDL receptor, cation-independent mannose 6-phosphate receptor (CI-MPR), the synaptic vesicle protein synaptophysin , and in CHO cells, where we have characterized endosomal sorting mutants of P-selectin . Our results strongly support the hypothesis that constitutive endosomal sorting of P-selectin from recycling receptors occurs only in early sorting endosomes, and not in late endosomes. The implications of these findings for sorting of membrane proteins in endosomes, and for trafficking of secretory granule membrane proteins, are discussed. Section title: Cells Educational score: 4.117212295532227 Domain: biomedical Document type: Study Language: en Ricin-resistant PC12 A1 cells and transfected clones derived from them were grown as originally described , and were plated for most experiments on dishes precoated with poly- d -lysine. Secretory granule exocytosis was stimulated by adding KCl to a final concentration of 50 mM to cells in complete growth medium. CHO cells expressing P-selectin or P-selectin −ΔC1 (a deletion of the 11-amino acid residue C1 region of the cytoplasmic domain, previously denoted P-selectin ΔD762-S772; Setiadi et al. 1995 ; or P-selectin-C2 were maintained in normal or LDL-depleted medium as described . Section title: Expression of Human P-Selectin in PC12 A1 Cells Educational score: 4.121556282043457 Domain: biomedical Document type: Study Language: en PC12 A1 cells in 6-cm plates were transfected with 6 μg of the plasmid pCDL-SRα containing the human P-selectin cDNA and 0.1 μg pSV2neo, using the lipofection protocol of Muller et al. 1990 . Cells were passaged into medium containing 0.4 mg/ml G418 (GIBCO-BRL) 2 d after transfection. Drug-resistant clones were picked, expanded, and screened for expression of P-selectin by immunofluorescence microscopy (see below) using mixed monoclonal anti–P-selectin antibodies . Section title: Radioisotopes Educational score: 1.1658028364181519 Domain: biomedical Document type: Other Language: en UDP[ 3 H]galactose was purchased from Amersham Pharmacia Biotech and from American Radiochemical Corporation. [ 125 I]sodium iodide and ExpreSS label [ 35 S]amino acid mixture were from New England Biolabs. [ 3 H]glucosamine was purchased from American Radiochemical Corporation. Na 35 SO 4 was from NEN Life Sciences. Section title: Enzymes Educational score: 1.7808672189712524 Domain: biomedical Document type: Other Language: en Recombinant Flavobacterium meningosepticum peptide:N glycosidase F produced in E . coli (N-Glycanase) was purchased from Roche or from Glyko, Inc. Streptococcus pneumoniae β-galactosidase was purchased from Roche or from Prozyme. Bovine milk galactosyltransferase was from Sigma-Aldrich. Section title: Antibodies Educational score: 3.794620990753174 Domain: biomedical Document type: Study Language: en mAbs S12, G1, G5, and 2B8 recognizing P-selectin, and goat polyclonal antiserum recognizing P-selectin, were generously supplied by Rodger McEver (University of Oklahoma, Oklahoma City, OK). Antipeptide antiserum recognizing the COOH terminus of P-selectin was affinity-purified as described . Purified antipeptide antibody was biotinylated by reaction with a 10-fold molar excess of sulfosuccinimidyl 6-(biotinamido) hexanoate (Pierce Chemical Co) for 30 min in PBS, followed by addition of a 20-fold molar excess of glycine to quench remaining reactive groups. S12 antibody was labeled with Alexa 488 (Molecular Probes) according to the manufacturer's protocol. Polyclonal rabbit antiserum was generated by a commercial service (Covance) against soluble P-selectin , and showed specificity in immunofluorescence and immunoprecipitation experiments identical to that obtained with the mAbs. Polyclonal rabbit antibodies against rat or bovine CI-MPR were from William Brown (Cornell University, Ithaca, NY). Rabbit antiserum recognizing synaptophysin was from Regis Kelly (University of California, San Francisco, CA). Rabbit antiserum recognizing chromogranin A was from John Hutton (University of Colorado, Denver, CO). mAb H68.4 recognizing transferrin receptor was provided by Ian Trowbridge (Scripps, La Jolla, CA). Goat anti–rabbit IgG, rabbit anti–mouse IgG, Texas red-conjugated goat anti–rabbit IgG and FITC-conjugated goat anti–mouse IgG were from Cappel. Oregon green- and Texas red-conjugated deglycosylated egg avidin (Neutralite) were from Molecular Probes. Section title: Immunofluorescence Labeling Educational score: 4.171009063720703 Domain: biomedical Document type: Study Language: en Immunofluorescence labeling was performed as previously described . For screening, cells were incubated for 1 h with a mixture of ascites fluid containing mAbs S12, G5, and 2B8. For double-labeling experiments, primary antibodies recognizing chromogranin A, synaptophysin, CI-MPR, and transferrin receptor were applied, followed by the appropriate secondary antibody. After labeling endogenous proteins, cells were incubated in preimmune rabbit serum (1:50) for 15 min, and were then labeled with biotinylated anti–P-selectin COOH-terminal peptide antibody diluted 1:200 in buffer containing rabbit preimmune serum 1:50. After washing, samples were labeled with Oregon green or Texas red avidin 1:300 and washed again, with 10 μg/ml free biotin included in the last IF buffer wash. Cells were then washed three times in PBS, rinsed in distilled water, and mounted in ProLong (Molecular Probes). As a positive control for colocalization, cells were labeled only with biotinylated anti–P-selectin COOH-terminal peptide antibody, followed by a mixture of Oregon green and Texas red avidin. Section title: Image Collection and Analysis Educational score: 4.1216020584106445 Domain: biomedical Document type: Study Language: en Immunofluorescence images were collected using a Zeiss Axioplan 2 microscope equipped with a 63× Apochromat objective lens, n.a. 1.4, a Hamamatsu C4742-95 CCD camera, in some cases fitted with a Zeiss 4× magnifying adapter and OpenLab (Improvision) software. For PC12 cells, 30 conventional images were collected at 0.2-μm intervals, in the Texas red and fluorescein channels sequentially, using an automation to drive the microscope controls. Digital deconvolution of one image near the middle of each series was performed using the OpenLab constrained iteration (confocal) algorithm, using 10–12 neighbors (20–24 images) to deconvolve each image. Grayscale matching and merging of deconvolved images was performed using Adobe Photoshop. Several cells were analyzed for each labeling condition, and representative results are presented. Section title: Uptake of LDL and S12 Antibody Educational score: 4.229440689086914 Domain: biomedical Document type: Study Language: en CHO cells expressing native P-selectin or P-selectin−ΔC1 were passaged onto coverslips and grown overnight. The medium was replaced with LDL-depleted medium and the cells were grown for an additional 16–18 h to increase surface expression of LDL receptor. After incubation for 10 min at 37°C in PBS containing 1 mg/ml glucose and 0.2% BSA (PBS/BSA), cells were incubated for 5 min at 37°C in PBS/BSA containing 10 μg/ml AlexaFluor 488-labeled S12 antibody and 5 μg/ml DiI-LDL (Molecular Probes). Cells were rapidly rinsed in PBS/BSA and recultured in PBS/BSA for 5–60 min. Cells were then rinsed in PBS and fixed in 3% formaldehyde; 100 mM NaPO 4 , pH 7.4, for 20 min at room temperature. Fixed cells were washed three times for a total of 5 min in PBS, rinsed once in distilled water, and were then mounted in FluorMount G (Fisher Scientific) containing 2.5 mg/ml n-propyl gallate (Sigma-Aldrich). Images were collected using FITC and Cy3 filter sets for Alexa 488 and DiI, respectively. Four or five fields, containing a total of 15–21 cells, were analyzed for each data point. In each field, 13 images, beginning at a focal plane 0.6–1.0 μm beneath the base of the cells, were collected at 0.2-μm intervals using a 63× objective lens . Six or seven sequential images, starting from the base of the cell and comprising 80–90% of the LDL-labeled structures in each cell, were deconvolved using 2 neighbors (above and below) in each channel. The deconvolved images from each channel were merged, and transferred to Photoshop to compress the grayscales and overlay the two channels. To quantitate the fraction of LDL-labeled vesicles that contained both labels, prints were made of the red channel (LDL) images and total LDL-positive vesicles were counted. The red channel image was then compared with the merged image to identify vesicles that also contained S12 antibody, and double-labeled structures were counted. An average of 926 LDL-positive vesicles were counted for each sample, and the data are expressed as the percentage of these vesicles that also contained S12 antibody. The entire experiment was performed twice, with similar results. Section title: Metabolic Labeling Educational score: 3.747929811477661 Domain: biomedical Document type: Study Language: en Metabolic labeling with [ 3 H]glucosamine or [ 35 S]amino acids was carried out for 1–16 h as previously described . Section title: Exogalactosylation Educational score: 4.171133995056152 Domain: biomedical Document type: Study Language: en Exogalactosylation of cells was carried out as described , with minor modifications. Cells on polylysine-coated 15- or 10-cm tissue culture plates were labeled at 80–90% confluence, usually 1–2 d after plating. Cells were cooled to 4°C and washed twice with cold exogalactosylation buffer . The labeling medium contained exogalactosylation buffer with the following additions: 1.7 U/ml galactosyltransferase, and 80–100 μCi/ml UDP[ 3 H]galactose. Cells were labeled with 3 ml/15-cm dish for 60 min. At the end of the labeling, cells were washed twice with PBS, 1 mg/ml glucose, and were then recultured in complete culture medium at 37°C. Cells were then washed twice with PBS lacking divalent cations before harvesting in PBS containing 5 mM EDTA by trituration. Section title: Lysis and Immunoprecipitation Educational score: 4.109190464019775 Domain: biomedical Document type: Study Language: en Cells were lysed, and proteins were immunoprecipitated as previously described . For biotinylated cells, 5 mg/ml iodoacetamide was included in the lysis buffer. Immune complexes were recovered using protein A–Sepharose or protein G–Sepharose (Amersham Pharmacia Biotech), and were eluted by heating in a boiling water bath for 3 min into either 25 μl N-glycanase buffer (1% octylglucopyranoside, 0.2% SDS, 40 mM Tris, pH 8.0, 5 mM EDTA, 1% β-mercaptoethanol) or 40 μl gel sample buffer (4% SDS, 125 mM Tris pH 6.95, 10 mM EDTA, 15% sucrose, 40 mM dithiothreitol). Immunoprecipitations with each of the antibodies and cell lines used were titrated to recover 90–95% of antigen, as assessed by second round precipitations. Section title: Analysis of N-linked Oligosaccharides after Exogalactosylation Educational score: 4.098984241485596 Domain: biomedical Document type: Study Language: en N-linked oligosaccharides were analyzed using a minor modification of the method of Duncan and Kornfeld 1988 , except that β-galactosidase digestion of oligosaccharides was carried out in 50 μl 50 mM MES, pH 6.0, or 50 mM NaH 2 PO 4 , pH 6. The digested samples were analyzed by gel filtration chromatography on Sephadex G25. 350-μl fractions were counted in 4 ml ReadySafe (Beckman) scintillation fluid. Section title: Electrophoresis, Fluorography, and Quantitation of Radioactivity in Gel Bands Educational score: 4.007892608642578 Domain: biomedical Document type: Study Language: en Proteins were analyzed on SDS polyacrylamide gels containing 7.5% acrylamide for P-selectin, or a gradient for analysis of multiple proteins. For fluorography, gels were fixed in 30% methanol/5% acetic acid, rinsed with water and impregnated with 0.5 M sodium salicylate. The dried gels were exposed to Fuji X ray film at −70°C. Section title: Supplemental Material Educational score: 3.825396776199341 Domain: biomedical Document type: Study Language: en Control experiments and descriptions of the relevant methods demonstrating that P-selectin resides in functional secretory granules, is rapidly internalized, and is rapidly degraded in PC12 A1-PS cells, and evidence that binding of mAb S12 to P-selectin is stable at low pH can be found at http//:www.jcb.org/cgi/content/full/151/1/107/DC1. Section title: Immunofluorescence Localization of Antigens in PC12 A1-PS Cells Educational score: 4.4079670906066895 Domain: biomedical Document type: Study Language: en Preliminary experiments indicated that the original clones used to study P-selectin traffic in PC12 cells did not have sufficiently high cell surface expression levels for these experiments. Therefore, a clone of PC12 A1 cells stably expressing higher levels of P-selectin was generated and designated PC12 A1-PS. Indirect immunofluorescence labeling of PC12 A1-PS cells was performed to determine the distribution of P-selectin with respect to other relevant organelle marker proteins. Epifluorescence and confocal microscopy show extensive apparent overlap of almost all markers in these cells, so digital deconvolution was used. As a control for spatial resolution and Z-axis registration, cells were labeled with biotinylated anti–P-selectin antibody and a combination of Oregon green- and Texas red-labeled avidin. After digital deconvolution, the Oregon green label and the Texas red label show nearly identical patterns, as demonstrated in the overlay . In double-label experiments, P-selectin and chromogranin A , a soluble content marker of regulated secretory granules in neuroendocrine cells, showed significant overlap . Many structures contained both labels, but with different relative intensities in the two channels . Synaptophysin was localized to punctate structures in the cell periphery . There was little colocalization between P-selectin and synaptophysin , and in areas where there was apparent overlap , the size and shape of the structures appeared different . Transferrin receptor was found in peripheral structures, and in a concentration of structures in the juxtanuclear region . P-selectin was rarely detected in structures enriched in transferrin receptor . CI-MPR antibodies labeled less numerous structures with a distinct juxtanuclear accumulation typical of late endosomes. There was little overlap in the distributions of P-selectin and CI-MPR . These experiments show that P-selectin is primarily concentrated in secretory granules in PC12 cells at steady state. Section title: Immunoprecipitation of P-Selectin, Synaptophysin, and CI-MPR Educational score: 4.114432334899902 Domain: biomedical Document type: Study Language: en The selectivity of each of the antibodies was assessed by immunoprecipitation of labeled proteins. PC12 A1-PS cells were labeled metabolically with [ 3 H]glucosamine or [ 35 S]amino acids . P-selectin, CI-MPR, and synaptophysin were sequentially immunoprecipitated from the cell lysates, and the immunoprecipitates analyzed by SDS-PAGE and fluorography. The bulk of the radioactivity in each immunoprecipitate migrated as a single band of the appropriate mobility. Section title: Sorting of P-Selectin in PC12 A1-PS Cells Educational score: 4.379478454589844 Domain: biomedical Document type: Study Language: en Control experiments were performed to insure that sorting of P-selectin in PC12 A1-PS cells occurred as described previously , and can be seen at http//:www.jcb.org/cgi/content/full/151/1/107/DC1. Stimulation of granule exocytosis for two minutes increased the fraction of P-selectin accessible to biotin labeling at the cell surface, whereas this fraction decreased after four minutes of stimulation . The transient increase in cell surface expression confirms sorting of P-selectin to functional secretory granules, and the subsequent rapid decrease suggests that internalization of granule membrane-derived P-selectin is rapid. Similar results have been obtained in primary cultures of endothelial cells . Since ∼25% of the P-selectin synthesized in PC12 cells is packaged into secretory granules , and these cells rapidly release only 15% of their granule pool upon stimulation , the amount of P-selectin delivered to the cell surface by granule exocytosis was expected to be ∼4% of the total. Although it was not possible to measure internalization of this small pool specifically, we measured internalization of the total steady state cell surface P-selectin pool, which includes protein delivered to the cell surface continuously via the constitutive secretory pathway and by recycling from early endosomes . The rate of internalization was found to be equal to the rate measured in transfected NRK fibroblasts . Degradation of metabolically labeled P-selectin occurred with a half-time of 3.8 h, similar to its turnover in other transfected cells . Section title: Rapid Transport of P-Selectin from the Cell Surface to the Golgi Apparatus Educational score: 4.344091415405273 Domain: biomedical Document type: Study Language: en The rate of P-selectin transport from the cell surface to the TGN was measured using an assay that is a minor modification of the method of Duncan and Kornfeld 1988 . PC12 A1-PS cells, which are deficient in the incorporation of galactose into glycoproteins , were exogalactosylated with UDP[ 3 H]galactose and galactosyltransferase at 4°C to label cell surface glycoproteins. After reculture at 37°C for varying times, cells were lysed, and specific glycoproteins were immunoprecipitated and digested exhaustively with N-glycosidase F to release N-linked oligosaccharide chains. Released oligosaccharides were isolated by acid precipitation of the proteins, followed by gel filtration chromatography of the supernatant, and were then digested exhaustively with β-galactosidase. The free galactose liberated by β-galactosidase was separated from intact oligosaccharides by gel filtration chromatography, and the radioactivity in the two peaks was quantitated. Only terminal galactose residues are sensitive to β-galactosidase, so additional glycosylation of these residues by any of several Golgi glycosyltransferases that act on terminal galactose renders the labeled galactose resistant to removal from the oligosaccharides. The readout of the assay is the percentage of galactose that has acquired resistance to β-galactosidase digestion, reflecting its passage through the TGN. Section title: Rapid Transport of P-Selectin from the Cell Surface to the Golgi Apparatus Educational score: 4.128389835357666 Domain: biomedical Document type: Study Language: en Previous studies of synaptophysin, CI-MPR, and LDL receptor showed that the fraction of galactose resistant to β-galactosidase reached a plateau by four hours of reculture for all three proteins . The plateau levels for P-selectin were determined by reculturing the cells for four or six hours, to provide a basis for subsequent measurement of the rate of transport . The fractions of galactose resistant to β-galactosidase at four hours of reculture were: P-selectin, 53 ± 11%; synaptophysin, 36 ± 3.7%; and CI-MPR, 35% , and were defined as the maximum signal for each protein for the purpose of determining rates of transport. Section title: Rapid Transport of P-Selectin from the Cell Surface to the Golgi Apparatus Educational score: 4.15234375 Domain: biomedical Document type: Study Language: en Preliminary experiments indicated that P-selectin acquired resistance to β-galactosidase much more rapidly than synaptophysin and CI-MPR, so the analysis focussed on short reculture periods. For all three proteins, the fraction of galactose resistance to β-galactosidase at 20 min of chase was similar to that measured in cells that were not recultured at all (t = 0), indicating a lag period preceding any significant transport of the labeled proteins to the TGN . By 40 and 60 min of chase, the signals for synaptophysin and CI-MPR had increased, but the signal for P-selectin had increased to a greater extent. The 40- and 60-min measurements were extrapolated back to estimate a lag period of ∼25–30 min before acquisition of resistance to β-galactosidase, similar to the lag period for transport from the cell surface to the TGN observed using a different assay . Section title: Rapid Transport of P-Selectin from the Cell Surface to the Golgi Apparatus Educational score: 4.274118423461914 Domain: biomedical Document type: Study Language: en After this lag, P-selectin oligosaccharides acquired resistance to β-galactosidase rapidly, reaching 60% of the plateau level by 60 min of chase. In contrast, synaptophysin and CI-MPR oligosaccharides had reached only 35 and 25% of maximum levels, respectively, by 60 min . Since the fraction of galactose resistant to β-galactosidase exceeds the half-maximal value for P-selectin by 60 min of chase, these data provide an estimate of the half-time for transport of the P-selectin pool labeled at the cell surface. We calculated this half-time to be ∼20–25 min for P-selectin, after correcting for the 25–30 min lag. Correcting for the lag period identified in the current study, we refine the value reported previously for the half-time for transport of synaptophysin, CI-MPR, and LDL receptor from the cell surface to the TGN to 2–2.5 h, which is slower than transport of P-selectin by a factor of six to seven. Section title: Internalized LDL and Anti–P-Selectin Antibodies Follow the Same Pathway Educational score: 4.45242977142334 Domain: biomedical Document type: Study Language: en To determine whether P-selectin was delivered to the TGN via late endosomes or directly from early endosomes, the transport pathway of internalized P-selectin was compared with that of internalized LDL, using mAb S12 to track internalized P-selectin. This antibody dissociated from P-selectin with a half-time of 156 min at 37°C and pH 5.5, conditions typical of late endosomes . CHO cells expressing P-selectin, or P-selectin-ΔC1, which is deficient in endosomal sorting , were incubated for 5 min with DiI-LDL and Alexa 488-labeled mAb S12. Cells were recultured for up to 60 min before fixation. Epifluorescence images were collected and analyzed after digital deconvolution (see Materials and Methods). In both cell lines, S12 antibody was observed in numerous punctate structures throughout the cytoplasm, and in a concentration of structures at one pole of the nucleus, visible in epifluorescence, but not in the deconvolved images (our unpublished observation). The juxtanuclear staining was not prominent after 5 min of chase, but the pattern of S12 labeling did not change or diminish significantly between 10 and 60 min of chase. At early chase times, LDL was observed in a smaller number of structures, many of which contained S12 antibody, in both native P-selectin and P-selectin-ΔC1 cells . After 20 min of reculture, few LDL-labeled structures in P-selectin-ΔC1 cells contained detectable levels of S12 antibody , whereas in cells expressing native P-selectin, double-labeled structures persisted . After 40 min of reculture, few double-labeled structures were seen in either cell line . Quantitation of double labeling on deconvolved images showed that LDL separated rapidly from S12 antibody in P-selectin-ΔC1 cells, whereas little separation occurred in cells expressing native P-selectin for 20 min . The clear difference between the two cell lines indicates that binding of S12 antibody does not divert P-selectin-ΔC1 to the lysosomal pathway, which can occur when using cross-linking antibodies . This result indicates that native P-selectin, unlike the efficiently recycled P-selectin-ΔC1 mutant, becomes concentrated in LDL-containing carrier vesicles (maturing endosomes) with relatively high efficiency soon after internalization, indicating selective sorting in early endosomes for efficient delivery to late endosomes en route to the TGN. Section title: P-Selectin Traffic Educational score: 4.413186073303223 Domain: biomedical Document type: Study Language: en We show here that P-selectin is sorted from long-lived recycling receptors in early sorting endosomes, and not in late endosomes: P-selectin, synaptophysin, CI-MPR, and LDL receptor are all rapidly internalized , reflecting rapid delivery to sorting endosomes. Sorting into cell surface recycling or late endocytic pathways at this stage is reflected in the rates of transport from the cell surface to the TGN, since access of these proteins to the TGN recycling pathway is most likely through late endosomes (see below). Transport of P-selectin from the cell surface to the TGN is six to seven times faster than the long-lived proteins (e.g., LDL receptor), showing that the P-selectin pool in sorting endosomes in PC12 cells must enter endosomal carrier vesicles (maturing endosomes) to reach late endosomes six to seven times more efficiently than LDL receptor . Direct evidence that P-selectin does enter carrier vesicles efficiently is provided by the observation that internalized anti–P-selectin antibodies colocalized with internalized LDL for 20 min after internalization. Section title: P-Selectin Traffic Educational score: 4.642868995666504 Domain: biomedical Document type: Study Language: en P-selectin is not sorted from LDL receptor in late endosomes. P-selectin is transported to the TGN approximately eight to nine times faster than it is delivered to lysosomes in PC12 cells (20–25 min vs. 3–3.8 h), showing that P-selectin molecules entering late endosomes are eight to nine times more likely to recycle to the TGN than to go directly to lysosomes. The ratio of these two transport rates is similar for LDL receptor . The simplest explanation for these observations is that both proteins have the same sorting phenotype in late endosomes . Therefore, the short half-life of P-selectin is most likely not due to its selective targeting to lysosomes, as has been suggested , but is due to its selective delivery from sorting endosomes to late endosomes. One significant consequence of this sorting event is rapid recycling of P-selectin through the TGN. Rapid turnover of P-selectin can be considered a secondary consequence of its frequent passage through late endosomes. Consistent with this conclusion, a recent report demonstrates transport of internalized anti–P-selectin antibodies from the cell surface to Weibel-Palade bodies (secretory granules) in endothelial cells by way of lysobisphosphatidic acid-enriched late endosomes . Section title: P-Selectin Traffic Educational score: 4.3757710456848145 Domain: biomedical Document type: Study Language: en P-selectin has cytoplasmic sorting determinants that mediate rapid internalization and delivery to regulated secretory granules , in addition to the endosomal sorting determinant . Disruption of any of the three sorting activities would be expected to result in a significant change in the steady state distribution of the protein. Recent mutagenesis studies found that alanine substitution of L768, which abrogates endosomal sorting , reduces the steady state concentration of P-selectin in secretory granules, and increases the fraction at the cell surface. Whereas L768 may well be required for sorting to secretory granules, the altered steady state distribution could be due to a block in endosomal sorting , combined with a partial impairment of internalization activity in the L768A mutant . A direct biosynthetic sorting assay, as opposed to a steady state measurement, would be required to distinguish between these possibilities. Section title: P-Selectin Traffic Educational score: 4.346808910369873 Domain: biomedical Document type: Study Language: en Based on cell fractionation studies in PC12 cells transiently expressing HRP/P-selectin chimeric proteins, it has been proposed that the cytoplasmic domain of P-selectin mediates sorting to synaptic-like vesicles, with sequence dependence similar to that required for sorting into dense core secretory granules . The HRP/P-selectin chimera, but not synaptophysin, is rapidly depleted from a small vesicle fraction in the presence of brefeldin A , which was interpreted as evidence that P-selectin and synaptophysin reach the same small vesicles via two or three different pathways. However, our finding that P-selectin did not colocalize with synaptophysin in PC12 cells demonstrates that there is little or no native P-selectin in synaptic-like vesicles, even at high expression levels. Indeed, colocalization of the HRP-chimeric protein with synaptophysin has not been addressed. An alternative interpretation of the fractionation data is that the small vesicles enriched in the HRP/P-selectin chimera represent constitutive-like vesicles forming from maturing secretory granules, a pathway that can be blocked by brefeldin A treatment , and not synaptic-like vesicles. Section title: P-Selectin Traffic Educational score: 4.868322372436523 Domain: biomedical Document type: Study Language: en P-selectin is a secretory granule membrane protein in endothelial cells . Selective sorting of P-selectin from early to late endosomes has a number of consequences that bear on the function of P-selectin in inflammation, and possibly more generally on the trafficking of secretory granule membrane proteins. It is likely that sorting of secretory granule proteins into granules in endothelial cells is not perfectly efficient, and/or that granule maturation may include removal of some membrane to a constitutive-like pathway . In both cases, some P-selectin would be delivered to the cell surface in the absence of a secretory stimulus . Rapid internalization followed by endosomal sorting would prevent any significant accumulation of P-selectin at the cell surface under these conditions . After stimulated granule exocytosis, cell surface P-selectin levels are transiently high, and mediate binding of leukocytes expressing the P-selectin ligand . If P-selectin recycled to the cell surface as efficiently as LDL receptor, it would be cleared from the cell surface with kinetics similar to turnover of the LDL receptor (0.5–1 d). Endosomal sorting provides a mechanism to reduce cell surface P-selectin levels significantly within a few hours, providing temporal regulation of its binding activity. Finally, endosomal sorting of P-selectin results in its rapid transport to the TGN, where it can be reincorporated into nascent secretory granules, both before and after regulated exocytosis . Thus, early endosomal sorting may represent a general mechanism mediating efficient recycling of granule membrane proteins to the TGN for repackaging into nascent granules. Section title: The TGN Recycling Pathway Educational score: 4.49148416519165 Domain: biomedical Document type: Study Language: en Recently, it has been shown that TGN38 and Shiga toxin B chain , are rapidly transported to the TGN from sorting and/or recycling endosomes, and not via late endosomes . In contrast, the TGN resident protein furin follows the LDL pathway shortly after internalization, indicating transport to maturing and/or late endosomes, then rapidly accumulates in the TGN . Our results show that P-selectin, like furin, recycles to the TGN via late endosomes and not from sorting or recycling endosomes. It should be noted that P-selectin, like furin , is not readily detected in CI-MPR–enriched late endosomes , despite the fact that P-selectin is rapidly delivered to lysosomes, presumably after passing through late endosomes. We interpret this to mean that exit of P-selectin from CI-MPR–enriched late endosomes is rapid, precluding accumulation of the protein to levels that are detectable by morphological means. As noted above, internalized anti–P-selectin antibodies pass through late endosomes en route to recycling to Weibel-Palade bodies (secretory granules) in endothelial cells . One important implication of our study of P-selectin is that selective transport of membrane proteins from early endosomes to late endosomes, resulting in rapid delivery to the TGN, is not specific to TGN resident proteins. Rather, this may represent a more general sorting pathway used by many different types of proteins. Section title: The TGN Recycling Pathway Educational score: 4.5860185623168945 Domain: biomedical Document type: Study Language: en Several lines of evidence indicate that recycling of CI-MPR through the TGN also occurs primarily (or exclusively) from late endosomes, and not directly from early endosomes . The available evidence strongly suggests that LDL and transferrin receptors, which undergo transport from the cell surface to the TGN at the same rate as CI-MPR , also follow the same pathway. These receptors reach the TGN after internalization at rates far slower than either TGN38 , which is targeted from sorting/recycling endosomes, or furin and P-selectin, which recycle via late endosomes . The slow rates of transport to the TGN for LDL and transferrin receptors are readily explained by inefficient, nonselective transport (mis-sorting) from sorting to late endosomes, followed by recycling from late endosomes to the TGN with the same efficiency observed for P-selectin . In contrast, direct transport of these molecules from sorting/recycling endosomes to the TGN, bypassing late endosomes, is difficult to reconcile with their relative rates of transport to the TGN and to lysosomes. Section title: The Function of Early Endosomal Sorting Educational score: 4.501516819000244 Domain: biomedical Document type: Study Language: en The observation that endosomal sorting of P-selectin occurs in several cell types that do not normally express it suggests that the endosomal sorting machinery recognizing P-selectin is ubiquitous, and serves more general functions in the endocytic pathway than recycling of granule membrane proteins. These may include endosomal sorting of resident lysosomal membrane proteins that pass through the cell surface. More generally, this sorting activity may function to limit exposure of certain membrane proteins at the cell surface, either to minimize activities that are not useful or are detrimental when expressed on the surface (e.g., furin), or to provide critical temporal regulation of the activity, as we propose for P-selectin. In addition, sorting of receptors that undergo ligand-dependent degradation, such as epidermal growth factor receptor, is controlled both at the level of internalization and by endosomal sorting . Downregulation of growth factor receptors may also be mediated in part by the same endosomal sorting mechanism that recognizes P-selectin. | Study | biomedical | en | 0.999997 |
0006158 | Section title: Introduction Educational score: 4.778325080871582 Domain: biomedical Document type: Review Language: en Agrin is a heparan sulfate proteoglycan that was purified from basal lamina (BL) based upon its ability to induce clustering of acetylcholine receptors on cultured myotubes . It has since been shown to be a critical motoneuron-derived organizer of synaptic differentiation at the neuromuscular junction in vivo . However, its broad expression pattern suggests that it may play additional roles. For example, agrin is synthesized by many neuronal types in addition to motoneurons, as well as by some glial cells . In neurons, it has been localized to both synapses and neurites and has been shown to affect synthesis and phosphorylation of transcriptional regulators when applied to cultured neurons . In addition, agrin is present in distinct subsets of BLs in numerous nonneural tissues. In kidney, it is a major proteoglycan of the glomerular BL . In view of evidence that heparan sulfate proteoglycans are critical determinants of renal permeability , agrin has been suggested to be an essential part of the glomerular filter . Likewise, agrin is prominent in the BLs of the cerebral microvasculature, and its levels in this BL increase during the period that the blood–brain barrier acquires its mature properties. This pattern of expression has led to the speculation that agrin contributes to the integrity of this barrier . Despite these intriguing data, little is known about roles of agrin at sites other than the neuromuscular junction, or about how its expression and subcellular localization are regulated in any tissue. Section title: Introduction Educational score: 4.72770881652832 Domain: biomedical Document type: Study Language: en Clues to the mechanism of agrin's action at the neuromuscular junction have come from analysis of its multiple isoforms. Alternative splicing near the 3′ end of the agrin gene generates isoforms that contain or lack short segments in the COOH-terminal third of the protein. Inclusion of a four amino acid (aa) insert at a site called A in chicks and Y in mammals is required for agrin to bind to heparin. Inclusion of 8, 11, or 19 (8 + 11) aa segments at a nearby site, called B in chicks and Z in mammals, is required for agrin to induce postsynaptic differentiation at the neuromuscular junction . Here we describe heterogeneity in the 5′ end of the agrin gene that contributes to the diversity of agrin's localization, tissue distribution, and function. We show that mice express two isoforms of agrin in which distinct NH 2 -terminal peptides of 49 or 150 aa precede ∼1,900 aa of common sequence. We refer to the isoforms as short NH 2 -terminal (SN) and long NH 2 -terminal (LN), respectively. The existence of these distinct isoforms explains the previously noted lack of homology between the NH 2 termini of agrins isolated from rats and chicks . SN- and LN-agrins are likely to be transcribed from distinct promoters, and they are expressed in different patterns throughout development. SN-agrin is largely confined to the nervous system, whereas LN-agrin is broadly distributed in neural and nonneural tissues. Moreover, analyses of native and recombinant protein indicate that SN- and LN-agrin exhibit distinct subcellular localizations, determined by their NH 2 termini: LN-agrin associates with BLs (and all BL-associated agrin is LN-agrin), whereas SN-agrin remains attached to cell surfaces. Finally we use mutant mice in which expression of only LN-agrin is abolished to show that this isoform is essential for synapse formation at the neuromuscular junction. Thus, analyses of gene expression, protein localization, and mutant phenotype all support the idea LN-agrin is a component of BLs and critical for signaling at the neuromuscular junction, whereas SN-agrin may play distinct roles in neuron–neuron interactions. Section title: Analysis of cDNA and Genomic Clones Educational score: 4.266862869262695 Domain: biomedical Document type: Study Language: en To identify cDNAs encoding 5′ ends of agrin, we performed anchored PCR from an embryonic day (E) 18 mouse library (CLONTECH Laboratories, Inc.). A primer in exon 1 of agrin and a second primer in the vector were used for amplification. Reaction products were separated on agarose gels, blotted to filters, and hybridized with 32 P-labeled agrin-specific oligonucleotides. A fragment identified in this way was sequenced, and found to encode SN-specific–translated and 5′-untranslated sequences. Its sequence is available from EMBL/Genbank/DDBJ . LN-agrin was identified by a search of public databases; an EST was obtained and resequenced. Bacterial artificial chromosomes containing the 5′ end of the agrin gene were identified by PCR screening of gridded clones from a commercial library (Genome Systems). Two positive clones were mapped by restriction digestion and Southern blotting. Sequencing to determine the intron–exon boundaries was performed directly from the bacterial artificial chromosomes DNA using 5 μg of DNA per reaction. Section title: Expression of Recombinant Agrin Educational score: 4.181008338928223 Domain: biomedical Document type: Study Language: en CHO cells were grown on glass coverslips coated with laminin (GIBCO BRL; 20 μg/ml). The cells were transfected with cDNAs encoding either full-length rat agrin or with a construct expressing the first 83 aa of rat SN-agrin fused to a COOH-terminal FLAG tag. This construct was generated by PCR and contained the same 5′ UTR and the first 249 base pair (bp) of coding sequence as the full-length rat agrin. At the 3′ end, an MfeI site was added and the PCR product was ligated into an expression vector upstream of a FLAG tag. An LN-agrin expression construct was made in the same vector; it began with the first 15 aa of the chick sequence, followed by mouse LN-agrin sequence. Section title: Expression of Recombinant Agrin Educational score: 4.12381649017334 Domain: biomedical Document type: Study Language: en After transfection, living cells were stained at 37°C for 30 min using either a polyclonal anti-agrin antibody generated against the COOH-terminal 50 kD of human agrin (a gift of David Glass, Regeneron Pharmaceuticals, Tarrytown, NY), or with anti-FLAG monoclonal antibody M2 (Sigma-Aldrich). The cells were then washed briefly, fixed in 4% paraformaldehyde, and incubated with fluorescein-conjugated anti–mouse or Alexa 488–conjugated anti–rabbit secondary antibodies. Section title: Transcript Analysis Educational score: 4.209197521209717 Domain: biomedical Document type: Study Language: en RNA was isolated for reverse transcriptase (RT)-PCR by homogenization in guanidinium isothiocyanate and phenol extraction. For reverse transcription, 10 μg of total RNA was incubated with Avian Myelosis Virus RT and a mixture of random hexamers and oligo-dT. For PCR, aliquots of the resulting cDNA were amplified 40 rounds using primers indicated in the figures. For Northern blotting, mRNA was isolated by passage over an oligo-dT cellulose column. Denaturing gels were run and blotted onto nylon membrane. In most cases, 5 μg of poly A + mRNA was loaded per lane. Probes were generated using 32 P incorporation by random priming of PCR products specific for each transcript. Hybridization was carried out at 50°C in 50% formamide buffer, and the final wash was 0.2× SSC, 0.1% SDS at 65°C. Equivalent loading of lanes was assessed by stripping blots and reprobing them with elongation factor 1α (EF1α). Section title: Gene Trapping Educational score: 4.195614337921143 Domain: biomedical Document type: Study Language: en A mutation in the agrin locus was generated by the insertion of a β-geo gene (neomycin phosphotransferase fused to Escherichia coli β-galactosidase [lacZ]) between the LN and SN exons. This insertion was identified from an insertional mutagenesis screen in embryonic stem (ES) cells that had been designed to identify mutations in genes encoding secreted and transmembrane proteins . Individual clones from this screen were analyzed by 5′ RACE (rapid amplification of cDNA ends) to identify the intercepted transcripts . ES cells were injected into mouse blastocysts to generate germ line chimeras. Section title: Histology Educational score: 4.043210983276367 Domain: biomedical Document type: Study Language: en For lacZ staining, tissues were fixed in 4% paraformaldehyde, with or without 0.25% glutaraldehyde, at room temperature. Tissue was then equilibrated with 15% and 30% sucrose in PBS, frozen, and sectioned in a cryostat at 10–20 μm. Slides were stained at 30°C for 6–18 h as described by Sanes et al. 1986 and then mounted in 80% glycerol for viewing. Section title: Histology Educational score: 4.120999336242676 Domain: biomedical Document type: Study Language: en Tissue sections or whole muscles were prepared for immunohistochemistry as described in Burgess et al. 1999 . Agrin staining was done using a rabbit polyclonal antibody against the COOH-terminal 50 kD of human agrin. Nerves were visualized with anti-NF200 (Sigma and Sternberger Monoclonals) and anti-SV2 or anti-synaptophysin (Zymed Laboratories). AChRs were stained with rhodamine α-bungarotoxin (Molecular Probes). Section title: Histology Educational score: 4.122543811798096 Domain: biomedical Document type: Study Language: en For in situ hybridization, tissue was fixed overnight in 4% paraformaldehyde, equilibrated with 15% and 30% sucrose, frozen and sectioned as above. The tissue was allowed to air dry for up to 2 h and then hybridized immediately. Hybridization was done using a solution of ∼250 ng/ml digoxygenin-labeled riboprobes in a 50% formamide buffer at 65°C overnight and washed and developed according to Schaeren-Wiemers and Gerfin-Moser 1993 . The SN probe included the entire unique 5′ untranslated (466 nucleotides) and unique coding sequence to the HinfI site. Section title: Clustering Activity Assay Educational score: 4.131294250488281 Domain: biomedical Document type: Study Language: en Brains and spinal cords from E18 mice were homogenized and used to induce AChR clustering on myotubes as described in Sanes et al. 1984 . In brief, tissue was dissected from the animals, homogenized in ground glass homogenizers at 25% wt/vol in DME with 10% horse serum. The homogenate was then spun at 4°C in a microfuge for several hours and the supernatant was sterilized by passage through a 0.2-μm syringe filter. The supernatant was diluted and applied to myotubes that had been cultured from neonatal mice as described by Gautam et al. 1996 . After 18 h of treatment, the myotubes were fixed in 2% paraformaldehyde, rinsed in PBS, and stained with rhodamine α-bungarotoxin for 2 h. The coverslips were then mounted and the number of AChR clusters was counted. Section title: Isolation of Chicklike and Ratlike Agrin Isoforms Educational score: 4.2081146240234375 Domain: biomedical Document type: Study Language: en cDNAs encoding agrin have been isolated from rat, Torpedo, chicken, human, and mouse . The deduced primary sequence is highly conserved among all of these species with one noteable exception: the NH 2 -terminal 50 residues of rat agrin are unrelated to sequences in other agrins or to any other sequences in public databases. In contrast, NH 2 termini of reported human and mouse agrins are homologous to the sequence initially isolated from chick (Torpedo sequence did not extend to the NH 2 terminus). Although this difference might reflect authentic interspecific differences, we considered an alternative possibility, that multiple agrin mRNAs with different 5′ ends exist in a single species . Because our previous studies of agrin function have been performed in mice , we began the present study by seeking an isoform of mouse agrin with a ratlike NH 2 terminus. Section title: Isolation of Chicklike and Ratlike Agrin Isoforms Educational score: 4.297022342681885 Domain: biomedical Document type: Study Language: en A cDNA encoding the NH 2 terminus of agrin was cloned by anchored PCR from an embryonic mouse cDNA library (see Materials and Methods). This 700-bp PCR product contained 466 bp of 5′-untranslated sequence (5′ UTR) followed by 148 bp of coding sequence homologous to rat but not chick agrin, and then 73 bp of sequence homologous to both chick and rat agrin . The 5′ UTR contained stop codons upstream of the proposed start ATG, confirming the NH 2 terminus of the agrin open reading frame. This mouse NH 2 terminus, like the rat sequence, lacks a canonical signal peptide. If the NH 2 terminus were to function as a leader sequence, its most likely cleavage site based on the parameters of von Heijne 1986 , is predicted to after residue 52, 3 aa into the common sequence. Section title: Isolation of Chicklike and Ratlike Agrin Isoforms Educational score: 4.175854206085205 Domain: biomedical Document type: Study Language: en We also confirmed the existence of murine cDNAs that encode a chicklike NH 2 terminus. In a search of public databases, several ESTs corresponding to the NH 2 terminus of agrin were identified and one was obtained and sequenced. The 5′ end of this cDNA encoded a peptide that was highly homologous to aa 16–211 of chick agrin but was dissimilar to rat agrin before aa 150 . In chick, alternative splicing leads to the inclusion or exclusion of a 7 aa stretch at approximately aa 150 . All of the ESTs we identified lacked this 7 aa stretch, and attempts to identify it by PCR and RT-PCR gave negative results (data not shown). Therefore, this exon may not be present in mice. Section title: Isolation of Chicklike and Ratlike Agrin Isoforms Educational score: 3.8762519359588623 Domain: biomedical Document type: Study Language: en These results, and others presented below, demonstrate that the agrin gene encodes two classes of patterns, in which unique sequences of 49 or 150 aa precede ∼1,900 aa of common sequences. Section title: Distinct Subcellular Localizations of LN- and SN-Agrin Educational score: 4.1814446449279785 Domain: biomedical Document type: Study Language: en Denzer et al. 1995 , Denzer et al. 1997 reported that recombinant chick agrin (LN-type) is secreted from transfected COS cells and binds to laminin in the extracellular matrix. In contrast, Campanelli et al. 1991 found that recombinant rat agrin (SN-type) is externalized by transfected CHO cells, but remains associated with the cell surface. Although these studies used different methods and analyzed agrin from different species, their results suggested that the localization of agrin is affected by its NH 2 terminus. To test this idea, we generated expression vectors in which the NH 2 termini of mouse LN- and SN-agrins were fused to the FLAG epitope tag. The SN–FLAG vector encoded the 49 aa of SN-specific sequence plus 34 aa of common sequences, whereas the LN–FLAG vector encoded 150 aa of LN-specific sequences plus the same 34 aa of common sequence. These vectors were transfected into CHO cells, which had been plated on dishes coated with laminin. Two days later, the cultures were stained either live or after fixation and permeabilization. Section title: Distinct Subcellular Localizations of LN- and SN-Agrin Educational score: 4.160770416259766 Domain: biomedical Document type: Study Language: en As shown in Fig. 2 A, the SN–FLAG fusion protein was efficiently externalized, and remained associated with the surface of transfected CHO cells. Staining extended to the tips of cellular processes. We did not observe any staining of presumably untransfected cells in the vicinity of SN–FLAG-rich (transfected) cells, suggesting that the fusion protein remained associated with the membrane after externalization, rather than being secreted and then retrieved from the medium. In all of these respects, the disposition of SN–FLAG was indistinguishable from that of full-length rat agrin . In contrast, cells transfected with the LN–FLAG vector were intensely stained after permeabilization, but not detectably stained when incubated with anti-FLAG before fixation . Section title: Selective Expression of SN-Agrin in Nervous System Educational score: 4.293184280395508 Domain: biomedical Document type: Study Language: en We used Northern blotting to compare expression patterns of SN- and LN-agrin mRNAs. Blots were probed sequentially with four probes: one comprising LN-specific sequences , a second comprising SN-specific sequences , a third comprising common sequences , and a fourth that recognized products of the ubiquitously expressed gene, EF1α . SN-specific, LN-specific, and common probes all hybridized to a band of 8.2 kb. In adults, LN-agrin RNA was present in all tissues tested, whereas SN-agrin was selectively expressed in brain. (A long exposure of the SN Northern blot revealed low levels of expression in lung; data not shown.) Both LN- and SN-agrin were expressed in embryos, at levels that declined with age. Likewise, SN- and LN-agrin RNAs were more abundant in P2 brain than in adult brain, and LN-agrin RNA was more abundant in P2 muscle than in adult muscle. In all tissues tested, RNAs detected with the common probes appeared to represent the sum of LN- and SN-specific signals, supporting the idea that LN and SN transcripts together account for all agrin transcripts. Section title: Selective Expression of SN-Agrin in Nervous System Educational score: 4.156867980957031 Domain: biomedical Document type: Study Language: en To date, agrin has been most intensively studied in the central nervous system and in muscle. To further examine the expression of SN and LN isoforms in these two tissues, we used RT-PCR. As shown in Fig. 3 E, a reverse primer in the second common exon was paired with either an SN- or a LN-specific primer. SN-agrin RNA was readily detected in E18 central nervous system (brain and spinal cord), but was barely detectable in E18 muscle. In contrast, LN-agrin RNA was detectable in both tissues. These results confirm that both LN- and SN-unique sequences are continuous with common sequence, and support the conclusion from Northern blotting that SN-agrin is selectively expressed in nervous tissue. Section title: Genomic Organization of the LN and SN Exons Educational score: 4.204012870788574 Domain: biomedical Document type: Study Language: en Knowing that LN- and SN-agrin RNAs are differentially expressed, we asked how LN-specific, SN-specific, and common sequences are arranged in the agrin gene. To this end, we isolated a >100-kb genomic clone that carried both SN- and LN-specific coding sequences. The clone was restriction mapped and partially sequenced to determine intron–exon boundaries. As shown in Fig. 4 , LN-specific sequences are encoded by at least three exons, all of which lie within a 5-kb stretch. These exons are 8-kb upstream of a single exon that encodes the entire SN-specific coding sequence as well as the SN-specific 5′ UTR. The SN exon, in turn, is separated from the first two common exons by introns of 0.4 and 4 kb. Section title: Isolation of a LN-specific Gene Trap Insertion Educational score: 4.1665167808532715 Domain: biomedical Document type: Study Language: en Skarnes et al. 1995 performed a “gene-trap” screen in which the mutagenic cassette contained a splice acceptor site followed by a β-geo fusion protein. Inclusion of a transmembrane domain in the cassette led to selection for proteins with signal sequences. 5′ RACE was then used to identify the transcripts that had been intercepted by the gene trap insertion . One RACE-derived sequence (Ex192) corresponded to the LN-specific NH 2 terminus of agrin, and ended precisely at the 3′ end of the last LN exon . This sequence suggested that the vector had integrated into the intron that separated the LN exons from the SN exon. Southern blot analysis of genomic DNA confirmed this location (not shown). We generated chimeric mice from these ES cells by blastocyst injection and then bred the chimeras to generate heterozygous and eventually homozygous mutants. Heterozygotes were phenotypically normal, and homozygotes exhibited defects described below. Section title: Isolation of a LN-specific Gene Trap Insertion Educational score: 4.177401542663574 Domain: biomedical Document type: Study Language: en Expression of agrin isoforms was examined in the mutant animals by Northern blotting, using LN- and SN-specific probes. The LN-specific probe recognized a transcript of 8.2 kb in wild-type mice and a transcript of 6.0 kb in agrin LN/LN mice . The decrease in size reflects the replacement of ∼7 kb of common agrin sequence by ∼5 kb of β-geo sequence. As expected, both 6.0- and 8.2-kb LN-reactive RNAs were present in agrin LN/+ heterozygotes. In contrast, the SN-specific probe recognized RNAs of identical size and abundance in mice of all three genotypes . Likewise, levels of SN-agrin were similarly low in nonneuronal tissues of both agrin LN/LN and control animals (not shown), indicating that SN-agrin is not upregulated in the absence of LN. Section title: Isolation of a LN-specific Gene Trap Insertion Educational score: 4.2152099609375 Domain: biomedical Document type: Study Language: en The complete absence of 8.2-kb RNA from the mutant suggested that the insert intercepted most if not all LN transcripts. To assess the completeness of the disruption, we used the more sensitive method of RT-PCR, using primers diagrammed in Fig. 5 D. LN transcripts were intercepted by the β-geo construct and wild type LN transcripts were undetectable in homozygous mutant . There was no evidence for splicing from the insert to the SN exons, consistent with the genomic analysis indicating that the insertion is upstream of the SN exon. Together, Southern, Northern, and RT-PCR analyses indicate that the gene trap insert has generated an effective null allele of LN-agrin, with no detectable effect on expression of SN-agrin. Section title: Distinct Patterns of LN- and SN-Agrin Expression in Central Nervous System Educational score: 4.373607635498047 Domain: biomedical Document type: Study Language: en In the agrin LN allele, genomic regulatory elements that normally direct expression of LN-agrin would be expected to direct expression of a LN–β-geo fusion protein, which is detectable with the histochemical stain for lacZ. We therefore used lacZ histochemistry to assess the cellular distribution of LN-agrin in the central nervous system of phenotypically normal agrin LN/+ heterozygotes at E14 and E18. Weak signals were present in numerous areas, including cerebellum, cortex, and hippocampus, but four sites of expression were especially prominent at both ages. First, small blood vessels were intensely stained throughout the nervous system . Second, neural progenitors were stained in the ventricular zones of the cerebral cortex , hippocampus , and spinal cord . Third, sensory neurons were lacZ positive in dorsal root ganglia and in the trigeminal ganglion (not shown). Fourth, motoneurons were intensely stained, indicating that these cells express high levels of LN-agrin. Selective staining of motoneurons was apparent both in the spinal cord and in the hindbrain . Staining was intense at both E14 and E18, and expression persisted into adulthood . Selective expression of LN-agrin by motoneurons is noteworthy in view of functional studies reported below. Section title: Distinct Patterns of LN- and SN-Agrin Expression in Central Nervous System Educational score: 4.189591407775879 Domain: biomedical Document type: Study Language: en In parallel we used in situ hybridization to confirm the localization of LN-agrin (not shown) and to map expression of SN-agrin . The pattern of SN-agrin expression was markedly different from that of LN-agrin. In forebrain, for example, levels of SN-agrin were highest in postmitotic neurons of the cortical plate, moderate in migrating neuroblasts of the intermediate zone, and lowest in progenitors of the ventricular zone. This pattern was the opposite of that seen for LN-agrin, which was expressed at highest levels in the ventricular zone and at lowest levels in the cortical plate . In spinal cord, SN-agrin was broadly distributed, in contrast to the motoneuron-selective expression of LN-agrin . SN-agrin RNA was also abundant in midbrain, hindbrain, retina, olfactory epithelium, trigeminal ganglion, and sympathetic ganglia (not shown). Section title: Distinct Patterns of LN- and SN-Agrin Expression in Central Nervous System Educational score: 4.10908317565918 Domain: biomedical Document type: Study Language: en The broad distribution of SN-agrin RNA in the CNS raised the possibility that this isoform was expressed by glial cells in addition to neurons. To test this possibility, we used isoform-specific primers to amplify agrin mRNAs from isolated, cultured cortical glia. LN-agrin was expressed by the glia (which include the astrocytes that contribute to the microvascular BL), but SN was not . Thus SN-agrin may be selectively expressed not only in the nervous system, but by neurons. Section title: BL-associated Agrin Is the LN Isoform Educational score: 4.195223808288574 Domain: biomedical Document type: Study Language: en Selective expression of SN-agrin in the nervous system suggested that BL-associated agrin in nonneural tissue is predominantly if not entirely the LN isoform. We tested this idea in two ways. First, we used lacZ histochemistry and in situ hybridization to map SN- and LN-agrin expression in cells that abut BLs. In skin, epidermal cells abutting the BL that separates the dermis from the epidermis were positive for LN-agrin based on lacZ staining . In the developing kidney, glomeruli and a subset of tubules were positive for LN-agrin . In the lung, LN-agrin was expressed primarily by pulmonary epithelial cells . In contrast, SN-agrin RNA was undetectable in all nonneural tissues tested, including lung, kidney, and skin, in which LN is abundant . Section title: BL-associated Agrin Is the LN Isoform Educational score: 4.1763224601745605 Domain: biomedical Document type: Study Language: en Second, we used immunohistochemistry to map the distribution of agrin protein in agrin LN/LN mutants. As reported previously and discussed above, agrin is present in numerous BLs of wild-type mice, including microvascular BLs in brain , glomerular BL in kidney , and meningeal sheaths , as well as skin, retina, and lung (not shown). No agrin immunoreactivity was detectable in any of these sites in agrin LN/LN embryos and neonates . The lack of staining did not reflect disappearance of the BL, as shown by staining with antibodies to broadly distributed BL components such as laminin . Together, these results show that all detectable BL-associated agrin is of the LN isoform, and that agrin is not essential for formation of BLs. Section title: Impaired Synapse Formation in the Absence of LN-Agrin Educational score: 3.784221887588501 Domain: biomedical Document type: Study Language: en Agrin LN/LN mice died at birth and exhibit no spontaneous movements, including respiratory movements. In this respect, the LN-specific mutants resembled mice lacking all forms of agrin or the bioactive Z-agrin isoform, both of which fail to form neuromuscular junctions . Section title: Impaired Synapse Formation in the Absence of LN-Agrin Educational score: 4.335109710693359 Domain: biomedical Document type: Study Language: en Histological analysis demonstrated that neuromuscular structure was drastically disrupted in agrin LN/LN muscles. In wild-type muscles, motor axons form a central nerve trunk; individual axons leave the trunk, branch, and terminate on myotubes . Each nerve terminal organizes a postsynaptic apparatus, rich in AChRs and acetylcholinesterase . In agrin LN/LN muscles, intramuscular nerve trunks form, but motor axons sprout abnormally and form few nerve terminals . Few AChR clusters and acetylcholinesterase deposits form . Those AChR clusters that do form are smaller and dimmer than those in control muscles , and fewer than half of them are apposed to nerve terminals. In all these respects, the synaptic defects observed in agrin LN/LN muscle are identical to those previously documented in detail for other agrin mutants . Section title: Impaired Synapse Formation in the Absence of LN-Agrin Educational score: 4.227499008178711 Domain: biomedical Document type: Study Language: en We also examined the distribution of agrin in muscles of control and agrin LN/LN neonates. In wild-type adult muscles, agrin is highly concentrated in the BL of the synaptic cleft . In embryos, however, agrin is present throughout the myotube BL ; a concentration of agrin immunoreactivity at synaptic sites becomes detectable around the time of birth . In agrin LN/LN mutants, no agrin was detectable in myotube BL, consistent with our conclusion that all BL-associated agrin is of the LN form . Importantly, agrin was also undetectable at sites of nerve–myotube contact in mutant muscles , indicating that most if not all synaptic agrin, presumably including motoneuron-derived agrin, is also of the LN form. Section title: SN-Agrin Has AChR Clustering Activity Educational score: 4.314950466156006 Domain: biomedical Document type: Study Language: en The similar neuromuscular defects of agrin LN/LN and agrin null mutants raised the possibility that SN-agrin is incapable of inducing postsynaptic differentiation. We tested this idea in two ways. First, we assessed the alternative splicing pattern of SN-agrin transcripts. Previous studies have shown that inclusion of either exon 32, 33, or both at a 3′ site called “Z” markedly enhances AChR clustering activity in vitro and is necessary for synaptic differentiation in vivo (see Introduction). In addition, inclusion of exon 28 at a nearby site called “Y” is required for agrin to bind to heparin, although the physiological significance of this binding is unknown . We therefore used RT-PCR to determine the Y and Z splice forms of agrin present in agrin LN/LN mice. As LN transcripts are undetectable in these animals , all residual agrin in these mutants is likely to be SN-agrin. The remaining transcripts included those that were Y + , Y − , Z + , and Z − . Moreover, the proportions of the various forms did not differ appreciably between mutants and littermate controls . Thus SN- as well as LN-agrin bears the Z exons required for synaptic organizing activity. Section title: SN-Agrin Has AChR Clustering Activity Educational score: 4.347326278686523 Domain: biomedical Document type: Study Language: en Second, we used myotube cultures to test whether LN-agrin accounts for all of the bioactive agrin in the central nervous system. Cultures were incubated for 18 h with extracts from wild-type brain and spinal cord and then stained with rhodamine–α-bungarotoxin to label AChRs. Extracts from wild-type brain induced a dose-dependent increase in the number of AChR clusters. Extracts from agrin null mutants were nearly inactive in this assay, indicating most of the AChR clustering activity in soluble extracts of the central nervous system is attributable to agrin . Extracts from agrin LN/LN tissue were nearly as effective in inducing AChR clusters as extracts from littermate controls . Together, these results provide evidence that SN-agrin has AChR-clustering activity in vitro. The result that levels of this bioactivity are similar in control and agrin LN/LN brains presumably reflects the fact that LN/Z + agrin has a limited distribution: there is more SN- than LN-agrin in neurons, most of the Z + agrin in brain is likely to be SN/Z + , and most of the LN agrin is likely to be LN/Z − . We therefore believe that the requirement for LN-agrin in postsynaptic differentiation reflects the appropriate cellular (motoneurons) and subcellular (BL) localizations of this isoform. Section title: Discussion Educational score: 4.689663887023926 Domain: biomedical Document type: Study Language: en Molecular cloning of agrin from rat and chick revealed that the protein sequences are highly conserved between species with the exception of their NH 2 termini, which display no similarity at all. We have now shown that this divergence reflects the existence of two agrin isoforms in which distinct NH 2 termini of 49 aa (ratlike or SN) or 150 aa (chicklike or LN) are fused to ∼1,900 aa of common sequence. The two isoforms are expressed in different patterns, with LN-agrin being broadly expressed and SN-agrin being selectively expressed in the nervous system. We believe these patterns are regulated transcriptionally by elements near the 5′ end of the gene. In addition, SN and LN-agrin are localized to different compartments, with LN being matrix associated and SN being attached to plasma membranes. These differences are determined by unique SN and LN NH 2 -terminal sequences. Interestingly, inclusion of alternatively spliced segments in the COOH-terminal quarter of agrin, which affect the bioactivity and binding properties of the protein, are regulated independently of the choice between SN and LN isoforms. We propose that transcriptional regulation, alternative use of NH 2 termini and alternative splicing of COOH termini act together to generate proteins with appropriate activities at appropriate sites. Section title: Gene Expression Educational score: 4.103096961975098 Domain: biomedical Document type: Study Language: en LN-agrin is broadly expressed in neural and nonneural tissues alike, whereas SN-agrin is selectively expressed in the nervous system. Within the nervous system, LN- and SN-agrins are also expressed in different patterns: SN is expressed by many postmitotic neurons whereas LN is expressed by neuroblasts, glia, cells of the microvasculature, and restricted neuronal subpopulations, most notably motoneurons. Section title: Gene Expression Educational score: 4.642635345458984 Domain: biomedical Document type: Study Language: en The distinct distributions of SN- and LN-agrin RNAs could result from any or all of three mechanisms: (a) tissue-specific alternative splicing; (b) alternative promoter usage; or (c) tissue-specific differences in mRNA stability. We have no data that bear directly on the third possibility, but view it as unlikely and note that it would likely to act in conjunction with one of the other two. In contrast, we have two reasons for believing that expression patterns reflect transcriptional regulation rather than regulated splicing of a common transcript. The essential point is that alternative splicing of a single transcript would require the existence of a common upstream exon encoding untranslated sequences, which would be spliced to either the LN or the SN exons. However, the size of the SN-agrin mRNA seen on Northern blots is accounted for by the size of the SN exon plus known common translated and untranslated sequences . Therefore, any common upstream exon would have to be quite small. Second, and more compelling, the gene trap insertion in the agrin LN allele would be expected to intercept all transcripts initiated from the hypothetical upstream exon, whether or not they also included LN exons. In fact, however, the trap intercepts LN transcripts virtually completely, yet has no detectable effect on the level of SN transcripts. This result suggests that the insertion downstream of the LN exons and upstream of the SN exon which is between the LN- and SN-agrin transcriptional start sites. This arrangement, in turn, implies that LN- and SN-agrin RNAs are transcribed from different promoters, each associated with tissue-specific regulatory elements. A testable prediction of our model is that an SN-agrin promoter and neuron-specific enhancer lie in the <4-kb interval between the agrin LN insert and the SN-specific exon. Section title: Protein Localization Educational score: 4.3559064865112305 Domain: biomedical Document type: Study Language: en Three lines of evidence indicate that LN-agrin is associated with BLs. First, recombinant full-length chick (LN) agrin binds to BL components, including laminin . Second, nonneural tissues, in which agrin is known to be associated with BLs, express LN-agrin but not SN-agrin . Third, no agrin is detectable in BLs of agrin LN/LN mice, which express SN-agrin but not LN-agrin . In contrast, SN-agrin appears to be associated with cell surfaces. Recombinant, full-length rat (SN) agrin remains attached to cell membranes when synthesized and secreted by transfected cells . Furthermore, agrin is associated with axonal and synaptic membranes of neurons, which express SN-agrin . Interestingly, motoneurons, which secrete agrin into BLs, are among the few neuronal types that express LN- as well as SN-agrin. Section title: Protein Localization Educational score: 4.415495872497559 Domain: biomedical Document type: Study Language: en The different subcellular localizations of LN- and SN-agrin are attributable to their unique NH 2 -terminal sequences. Denzer et al. 1997 generated a fusion protein consisting of the chick NH 2 terminus including LN-specific residues fused to an immunoglobulin tag. This protein bound to a BL extract (Matrigel) and to purified laminin. Conversely, a chick agrin protein lacking the NH 2 -terminal sequences was unable to bind Matrigel or laminin. In a conceptually similar experiment, we generated a fusion protein in which the NH 2 terminus of mouse SN-agrin is fused to a FLAG epitope tag, and showed that this fusion is externalized but remains associated with the surfaces of cells that express it . Thus, unique SN and LN sequences can endow heterologous proteins with the ability to associate with cell membranes and BLs, respectively. For LN-agrin, a direct binding of the LN sequences to domain I/II of the laminin γ1 chain has been demonstrated . For SN-agrin, the mode of membrane association remains to be determined. It is noteworthy that the unique SN sequences contain neither a canonical signal peptide nor a predicted transmembrane domain. It will be interesting to learn how SN sequences are externalized and how they associate with the membrane. Section title: Association of Y and Z Exons with LN and SN Educational score: 4.486196041107178 Domain: biomedical Document type: Study Language: en Previous studies of agrin isoforms have focussed on its COOH-terminal variants. As noted in the Introduction, inclusion of Z exons endows agrin with the ability to organize postsynaptic differentiation and inclusion of Y exons endows agrin with the ability to bind heparin. Our results , taken together with those of previous studies , provide several lines of evidence that both LN- and SN-agrin transcripts can either include or exclude the Y and Z exons. (a) Nonneural cells, for example in kidney or muscle, express LN-agrin but not SN-agrin and Z − but not Z + agrin; this agrin is therefore LN/Z − . (b) Likewise, the presence of both Y + and Y − agrin in nonneural tissues argues for the existence of LN/Y − and LN/Y + species. (c) Motoneurons express both LN and SN-agrin and both Z + and Z − agrin. However, LN and Z + agrin are required for synaptogenesis at the neuromuscular junction, whereas SN (either Z + or Z − ) and Z − (either SN or LN) agrin are insufficient. The simplest explanation is that the nerve-derived organizer of postsynaptic differentiation is LN/Z + agrin. (d) RT-PCR analysis of residual agrin in the agrin LN/LN mutant reveals both Z + and Z − populations. On the assumption that the residual agrin is SN-agrin, both SN/Z + and SN/Z − RNAs must exist. (e) Similar RT-PCR analysis provides evidence for SN/Y + and SN/Y − species. (f) The presence of abundant AChR-clustering activity in the brains of agrin LN/LN mutants provides additional evidence for the existence of SN/Z + agrin. Section title: Association of Y and Z Exons with LN and SN Educational score: 4.316781997680664 Domain: biomedical Document type: Study Language: en Proper agrin function requires that appropriate agrin isoforms be expressed by specific cells and localized to particular subcellular domains. We propose that combinatinatorial usage of putative transcriptional regulatory elements (that drive expression of SN and LN-agrin), NH 2 -terminal sequences (that promote association with membranes or BL) and alternatively spliced COOH-terminal sequences (that regulate bioactivity) account for many aspects of the proper matching of localization to function. Section title: Association of Y and Z Exons with LN and SN Educational score: 4.396087646484375 Domain: biomedical Document type: Study Language: en The only proven activity of agrin in vivo is to organize the neuromuscular junction. As discussed above, this activity is mediated by the LN/Z + form expressed by motor neurons. The LN/Z − splice form is a component of numerous BLs. Our work demonstrates that agrin is not required for the formation of these BLs, but it may have structural or signaling roles postnatally (see Introduction). We are presently unable to investigate such roles in vivo due to the neonatal lethality of currently available agrin mutants. Particularly intriguing is the possibility that SN-agrin plays roles in the development or function of central neurons. Several lines of evidence including the expression pattern and localization of agrin suggest it is involved in interneuronal synaptogenesis. Furthermore, signal-transducing receptors for agrin appear to be present on cultured neurons . Two studies of cultured neurons have failed to demonstrate roles for agrin in neuron–neuron synaptogenesis but a third study has reported that agrin antisense oligodigonucleotides inhibit synapse formation . Unfortunately, as for BL, subtle or postnatal roles of agrin in brain have not been testable in vivo, owning to neonatal lethality. Because most of the agrin in central neurons is SN-agrin, whereas motoneuronal agrin is largely of the LN isoform, selective elimination of the SN exon may provide a suitable strategy for studying roles of agrin in the brain. | Review | biomedical | en | 0.999998 |
10036122 | Section title: INTRODUCTION Educational score: 4.254120826721191 Domain: biomedical Document type: Review Language: en Changes induced by the currently used raw gas during insufflation to create a pneumoperitoneum induces laparoscopic hypothermia, causes postoperative pain and results in prolonged recovery room stay. 1 – 7 Because the gas must be bone dry, there is a stark difference between the characteristics of this regulated raw gas and the normal physiologic condition of the abdomen that causes this dramatic contrast. The difference between the temperature of standard raw gas of 21 degrees Centigrade (C) and 37.0 degrees C core temperature and no water vapor versus intra-abdominal steady state satu-ration results in alterations that upset normal abdominal homeostasis. These changes are iatrogenically induced due to these differences and the insufflation gas delivery system. The result is alterations that influence development of hypothermia, effect recovery room length of stay and postoperative pain perception. The annual cost of correcting for the iatrogenic intra- and postoperative con-sequences of laparoscopy due to prolonged recovery room stay and productive work loss in the United States is estimated to be between $2.26-1.56 billion per year. Changes that improve laparoscopic gas characteristics from its raw state to a more physiologic condition influences surgical outcome, reduces pain, has cost benefit and improves the level of safety. This recognition is compelling and establishes a new standard of care. Section title: INTRODUCTION Educational score: 3.6574695110321045 Domain: biomedical Document type: Other Language: en Principles of any surgery including laparoscopy include gentle tissue handling, reduction of foreign body contamination and prevention of tissue drying. The use of standard unconditioned gas is a contradiction to these long-standing principles. In the current raw state the gas and its delivery system contribute to foreign body contamination and tissue dessication. Maintaining or reproducing the normal physiologic intra-abdominal environment (contaminant free, warm and moist) is a standard that can be achieved and should be met. Section title: INTRODUCTION Educational score: 4.14013147354126 Domain: biomedical Document type: Study Language: en Laparoscopic gas filters were introduced in 1989. 1 It is long understood that the laparoscopic gas is a contributor to surgical hypothermia. 5 Attempts to correct laparoscopic hypothermia have been found inadequate by only heating the gas. It is known that without hydration only heating the gas has little effect on preventing laparoscopic hypothermia. 2 , 3 It is necessary to use heated gas containing water vapor to have maximal intraoperative heat preservation and be appropriate for tissue surfaces to result in minimizing tissue damage. The detrimental changes that occur due to the raw gas require modification by heating and hydrating. 2 , 3 A method to deliver pre-conditioned gas that accomplishes these necessary changes that modify standard raw gas to one more appropriate for laparoscopic procedures is advocated. 2 , 3 The use of heated gas compared to the current raw gas is found to decrease postoperative pain. 6 – 8 Methods that only heat laparoscopic gas in the insufflator or in warmed gas tubing are only marginal in their ability to heat, transmit and maintain the gas at a physiologic temperature when it enters the abdomen. 6 To the best of our knowledge, only one report describes the use of heated humidified gas which was shown to reduce the time to return to normal function and decrease postoperative pain. 7 A recently approved method and device is available that filters, heats and hydrates laparoscopic gas to a more physiologic condition as it enters the peritoneal cavity. The purpose of this study is to assess the efficacy of this device (Insuflow®), a method that pre-conditions by filtering heating and hydrating endoscopic gas, and to determine its effect in reducing laparoscopic-induced hypothermia, reducing pain and shortening recovery room length of stay. Section title: Study Design Educational score: 4.029717922210693 Domain: biomedical Document type: Study Language: en This prospective, randomized, blinded, controlled, multi-center study compared the efficacy and safety of the Insuflow device regarding the incidence, extent and severity of laparoscopic-induced hypothermia, postoperative pain perception and effect on the length of recovery room stay. Section title: Randomization Educational score: 2.4060723781585693 Domain: biomedical Document type: Study Language: en Before the procedure, patients at each center were randomly assigned to the Insuflow® (treatment group) or the raw gas (control group). Section title: Randomization Educational score: 4.103135108947754 Domain: biomedical Document type: Other Language: en The Insuflow system has three components: an AC/DC converter, a controller circuit and a disposable filter heater hydrator (Insuflow®) device. The converter delivers reliable safe electric current and is connected to a controller circuit box that adapts and retrofits to any insufflator. The Insuflow device attaches to the controller circuit box and insufflator. It provides filtration by a hydrophobic 0.2 micron CO 2 Guard® filter and is nine feet long. The heating and hydrating occurs within five centimeters of the intra-abdominal gas delivery point for delivery of optimally conditioned gas. The small heater hydrator section is filled with eight cubic centimeters of warm sterile water, normal saline or lactated ringers solution for each 150 liters of gas insufflation used during the procedure. The insufflator settings are independent of and not effected by the Insuflow® device. The gas is delivered to the patient at 36.2° C (97.2° F) and 95% relative humidity at constant low flow demand for 150 liters of gas (2 hours 30 minutes average). The intra-abdominal delivered gas characteristics can vary due to the demands placed on the device by the user and are dependent on gas flow rate, frequency and amount of gas evacuated. Section title: Test Conditions Educational score: 3.747272253036499 Domain: biomedical Document type: Study Language: en All centers used their own insufflators. All surgeons determined their own parameter settings for gas insufflation (flow rates and intra-abdominal pressure), followed their own institutional and personal standards for surgical procedures, set their own operating room temperatures, irrigation use and gas evacuation criteria. All anesthesiologists followed and determined anesthetic characteristics and individual methodologies for the surgical procedure. Individual institutional recovery room protocols were followed. No surface warming devices were used in any of the Insuflow® patients. Operating room ambient temperature and humidity were recorded and not modified during the procedures. Section title: Study Population Educational score: 2.700448513031006 Domain: biomedical Document type: Study Language: en Eight principal investigators at seven institutions enrolled 72 adult women in the Insuflow® and control groups from January 15, 1998 to May 29, 1998. The patients were adult women between 18 and 48 years of age. Patients' weights were between 97 and 252 pounds. All patients had laparoscopy. The protocol excluded pregnant or cancer patients. Section title: Surgery Educational score: 2.0919034481048584 Domain: biomedical Document type: Study Language: en The methodology and care of all patients was consistent with the investigators' standard surgical and medical practices. The methodology and care of patients in the two groups differed only in the use of the Insuflow® device for the study group. Data was kept of various characteristics of the procedure, insufflation and medication use. Section title: Evaluation Educational score: 2.5770018100738525 Domain: biomedical Document type: Study Language: en Preoperative evaluation included medical and surgical history, vital signs (temperature, blood pressure and heart rate), laboratory values (complete blood count and pregnancy test), eligibility criteria and informed consent documentation. Section title: Evaluation Educational score: 3.748938798904419 Domain: biomedical Document type: Study Language: en Intraoperative evaluation was done following each institution and physician's established practices. Evaluation of the following parameters was done every ten minutes during and after surgery including operating room temperature and humidity, patient core temperature monitoring by endotracheal temperature probe, insufflation gas volume, flow rate and pressure and irrigation volume consumed. Pain questionnaires were used with a visual analogue scale having scores from 0 (no pain) to 10 (unbearable pain) using established protocols. 9 Section title: Evaluation Educational score: 2.6379830837249756 Domain: clinical Document type: Other Language: en Post-anesthesia recovery room evaluation followed each institution's established protocols and included temperature, pain medication use, time in recovery room and pain perception. Section title: Evaluation of Safety Educational score: 1.9419184923171997 Domain: biomedical Document type: Study Language: en Throughout the study no adverse events occurred in the Insuflow® group. Physician and patient evaluations were solicited and evaluated. Section title: Statistical Analysis Educational score: 2.6198112964630127 Domain: biomedical Document type: Study Language: en All variables were summarized with descriptive statistics, including number, mean, median, standard deviation, range of continuous variables and number and percentage in each category. Section title: RESULTS Educational score: 2.1420109272003174 Domain: biomedical Document type: Study Language: en There were no statistically significant differences between the groups in demographic information. Surgical evaluations included diagnoses of uterine leiomyomata, infertility, pelvic adhesions and endometriosis. A total of 72 patients were in the study at seven different centers. Section title: RESULTS Educational score: 4.148981094360352 Domain: biomedical Document type: Study Language: en All patients started their procedures in a euthermic state. Operating room temperature ranged from 19.5-21.5° C (67.1-70.7° F). Relative humidity ranged from 42-59%. Operating time ranged from 38 to 262 minutes. The total intraoperative temperature drop in the Insuflow group ranged from 0.0-0.6° C (average total procedure loss 0.3° C, less than 0.1° C per hour) and 0.3-2.06° C (average total loss per procedure was 1.64° C, more than 0.6° C per hour) for the raw gas group. Carbon dioxide gas volume ranged from 82-680 liters. Irrigation volume ranged from 0.3-12 liters and at time of use was 26° C (78.8° F) or less. All cases utilized laser or electro-surgical devices. Figures 1 – 3 depict findings for three procedures in both groups. While procedures less than 35 minutes had little or no hypothermia, pain profiles for these short procedures were still statistically significantly improved in the Insuflow® group. Section title: RESULTS Educational score: 4.120991230010986 Domain: biomedical Document type: Study Language: en A total of 88.9% of questionnaires were completed. A significant difference in postoperative pain was found in the raw gas versus the Insuflow® warmed hydrated gas group. Consistently lower pain values were found in the Insuflow® group at all time intervals up to three days. Pain intensity was directly related to gas volume used and length of surgery in both groups but was statistically significant and reduced in the Insuflow® group. Shoulder and sub-phrenic pain were significantly reduced in the Insuflow® group at all time parameters compared to the standard raw gas group regardless of volume of gas consumed or length of surgery . Section title: Efficacy Educational score: 4.07867956161499 Domain: biomedical Document type: Study Language: en The Insuflow® group had a statistically significant reduction in intraoperative and postoperative hypothermia for procedures lasting over one hour, reduced pain perception postoperatively for all procedures regardless of length of surgery or gas consumed and shorter recovery room stay compared to the standard raw gas group. There was no reduction or enhancement effect of laser or electrosurgery with the Insuflow device. Section title: Safety Educational score: 4.1192426681518555 Domain: biomedical Document type: Study Language: en The safety profile of the Insuflow® device showed an improvement over the currently used raw gas group. No adverse effects were noted intraoperatively or postoperatively in the Insuflow group. Statistically significant differences were noted between the groups regarding hypothermia, pain perception and recovery room length of stay. These parameters showed that the Insuflow® group had significantly less intraoperative and postoperative hypothermia (8.3% versus 94.4%), postoperative pain improvement (56%) and shorter recovery room length of stay (88.9% versus 33.3% less than one hour). No Insuflow® patient had a recovery room stay longer than two hours. No Insuflow® patients became more than mildly hypothermic (36.0° C or 96.8° F). In the raw gas group, 44.4% were in the recovery room for two hours, 16.7% for three hours and 5.6% over four hours . For the raw gas group, 100% of patients were mildly hypothermic if the procedure was longer than 1 hour and 30 minutes, or if more than 110 liters of gas were used. Section title: DISCUSSION Educational score: 2.9019601345062256 Domain: biomedical Document type: Other Language: en Caeteris parabus , insufflation with standard current raw gas into the peritoneal cavity, results in physiologic changes directly attributable to the nature of the gas, ie, tissue dessication, hypothermia and postoperative pain. Section title: DISCUSSION Educational score: 4.1489458084106445 Domain: biomedical Document type: Other Language: en The gas standard as promulgated by the United States Pharmacopeia and National Formulary defines gas characteristics for purity and requires less than 200 parts per million of water vapor (bone dry). 10 Surgical principles and techniques that influence laparoscopic outcome include attention to detail, gentle minimal tissue handling, meticulous hemostasis, reduction of foreign body contamination, prevention of tissue drying or dessication and magnification when appropriate. 11 , 12 Therefore, it is important and beneficial to maintain or reproduce the normal physiologic environment of the abdomen during any surgery to ensure optimal outcome, including laparoscopy. This is not the case using the current raw gas. The gas delivery systems further compound the problem by throttling gas to extremely high flow rates. The normal abdominal environment is particle free, 37° C and has tissue surfaces moist with peritoneal fluid. The current standard raw gas (usually carbon dioxide) and delivery systems used to create a laparoscopic pneumoperitoneum contain contaminants (inorganic and organic debris from cylinders and insufflators), is 21° C (69.8° F) and is bone dry . 1 , 13 , 14 Section title: DISCUSSION Educational score: 4.161918640136719 Domain: biomedical Document type: Study Language: en The steady state of high water vapor content in the abdomen with the peritoneal fluid covering tissue sur-faces is severely altered by insufflation of the standard raw bone dry gas. Insufflation with CO 2 in rigorous experimental studies demonstrated significant fall in core temperature. 2 Experiments further demonstrate these differences by measuring and comparing the temperature and relative humidity differences for gas insufflation using cold dry gas versus warmed humidified gas to be 0.6° C per hour. 3 Prevention of water loss is the most important factor in preventing laparoscopic hypothermia. The difference between the warm moist tissue surfaces and the cool dry gas causes rapid evaporation resulting in tissue dessication, mesothelial damage and loss of peritoneal cells with exposure of the underlying connective tissue matrix. Immediately after peritoneal drying there is loss of mesothelial cells on the cecum. 15 Section title: DISCUSSION Educational score: 4.249856948852539 Domain: biomedical Document type: Study Language: en The laparoscopic gas delivery system throttles gas through small apertures at high flow rates resulting in a nozzle effect and jet streaming of the gas. The fluid dynamics of the gas delivered through constricted apertures at high flow rates on wet tissue surface leads to rapid evaporation, heat loss resulting in hypothermia, tissue dessication and peritoneal damage. This is the case when gas is delivered through a Veress needle, small diameter trocars or through ports that have a similar-sized object entering through a port (10 mm laparoscope through a 10 mm trocar). The result is extremely high flow jet streams of cold dry gas over wet tissue surfaces, severe local tissue hypothermia and generalized evaporative hypothermic effects. 16 This creates a condition akin to a wind chill effect from the nozzling of gas through small diameter openings onto the peritoneal sur-faces. Larger, unobstructed gas entry ports with low gas flow rates reduce deleterious effects on tissue from gas insufflation. Section title: DISCUSSION Educational score: 4.172555923461914 Domain: biomedical Document type: Review Language: en Factors that surgeons have control over that contribute to these problems are gas flow rate demanded, insufflator settings, amount of gas consumed, length of surgical exposure, gentleness of tissue handling, operating room temperature and temperature of irrigation solutions. Factors that contribute to operative hypothermia over which there is no control is patient age, size, pre-existing metabolic conditions and prior medication use. Awareness by anesthesiologists of operative hypothermia has long been noted. Anesthesiologists recognized the need for pre-conditioning gas delivered to the respiratory tract in the 1950's. It was determined that adding water vapor to gases prevented damage to the trachea and lung tissue. Heating and humidification devices that prevent tissue drying and hypothermia effects from respiratory tract gas delivery have been standards of care for over 35 years. The most recent effort to help control intraoperative laparoscopic hypothermia is the addition of surface convection warming devices. While any and all attempts to reduce hypothermia are desirable, this is an attempt to cure a problem after it has occurred. The prevention of a deleterious iatrogenic effect before it occurs is a more prudent approach. Pre-conditioning gas with heat and water vapor has been advocated 2 , 3 and is a practical technologic cost-effective reality with the Insuflow® device. The device is efficient, rapidly responds to use demands of insufflation and maintains a more normal intra-abdominal condition compared to using raw gas. Section title: DISCUSSION Educational score: 4.602269172668457 Domain: biomedical Document type: Review Language: en Intraoperative hypothermia requires extensive monitoring, care and time in the post-anesthesia recovery room. Postoperative hypothermia has both metabolic and economic costs. Normal core body temperature is 37° C. Clinical hypothermia is defined as a core temperature less than 36° C (96.8° F). 17 Changes resulting from mild and moderate hypothermia (32-35° C) are significant. Surgery stresses normal body temperature, normal regulatory mechanisms are modified and the balance of heat production and heat loss is altered. The result is a shift of temperature outside the normal physiologic range. The thermoregulatory system attempts to maintain the core temperature in a 0.2° C range. Intraoperative maintenance of the preoperative normothermic state is necessary to counteract the negative effects of hypothermia as they occur during surgery. Even mild operative hypothermia influences skin infection rate and perioperative wound infection by directly impairing immune function and vasoconstriction (by decreasing partial pres-sure of oxygen in tissues), induces hypokalemia, impairs myocardial function, depresses respiration, influences nitrogen balance, 18 depletes clotting factors, induces thrombocytopenia, 19 decreases activation of the coagulation cascade, 19 decreases collagen synthesis, 20 impairs chemotaxis and phagocytosis of neutrophils and decreases the production of antibodies. Mild hypothermia alters drug metabolism and pharmacokinetics, significantly prolongs recovery room time 21 , 22 and is associated with postoperative shivering, substantial adrenergic activation23 and patient discomfort. 24 A core temperature decrease of 1.5° C causes a three times increase in ventricular tachycardia, 25 increased postoperative ventilation, oxygen consumption and changes peripheral vascular tone. Section title: DISCUSSION Educational score: 4.245558261871338 Domain: biomedical Document type: Study Language: en The normal thermal steady state is one in which heat loss is equal to metabolic heat production. This is not uniformly distributed during surgery. Thermoregulatory mechanisms try to keep the core temperature nearly constant while the periphery is simultaneously at a lower temperature because of tonic vasoconstriction. 26 Due to the evaporative cooling effects caused by the dry laparoscopic gas, intra-abdominal conditions are altered, thus decreasing the heat available for redistribution. This upsets the normal balance of heat retention and loss. The heat normally sequestered in the abdomen is lost by vaporization of water from peritoneal surfaces, gas removal and as yet undetermined reflexes that contribute to thermal instability. This normal core heat sink is then unavailable for postoperative thermoregulation and redistribution and leads to a cool periphery and a cool core compounding the hypothermic effect. The use of heated laparoscopic gas containing water vapor allows for maintenance of the normal heat sink and reduction or prevention of the induced hypothermia. Section title: DISCUSSION Educational score: 4.1419830322265625 Domain: biomedical Document type: Study Language: en The alterations caused by peritoneal dessication result in intact mesothelial cells becoming absent from bowel sur-faces immediately after drying. 15 The effect of damaged peritoneum is denuded areas with release of chemically active kinins and prostaglandins that contribute to post-operative pain. The damaged peritoneum increases the susceptibility to adhesion formation from apposing defects and probably contributes to de-novo adhesion formation. The application of heated gas containing water vapor during insufflation allows for maintenance of the normal intra-abdominal condition to maximize peritoneal preservation at laparoscopy. Section title: DISCUSSION Educational score: 4.0345587730407715 Domain: biomedical Document type: Study Language: en Postoperative pain intensity has been shown to decrease when heated laparoscopic gas is utilized. 6 Heated humidified gas has been shown to improve return to normal activities by 54% in cholecystecomy procedures and significantly reduce pain extending to the third postoperative day. 7 The conclusions of these studies are that warmed CO 2 gas leads to significant reduction of pain, and humidified insufflation gas reduces postoperative pain and reduces postoperative recovery time. 6 , 7 This study reaches the same conclusions by using a device that combines the benefits of both heating and hydrating the gas. Exactly how the use of heated and hydrated gas reduces pain is not clear. It is postulated that the loss of peritoneal integrity due to dessication results in release of chemical agents responsible for pain perception. Temperature-sensitive transmitters may additionally account for the loss of integrity of temperature regulation and pain sensation. Further studies are necessary to elucidate the etiology of these effects and the benefits afforded by gas pre-conditioning. Section title: DISCUSSION Educational score: 4.025562286376953 Domain: biomedical Document type: Study Language: en Since the advisability of a procedure or intervention is a scientific judgement, clinical effectiveness is necessary. The effectiveness of heated hydrated gas is demonstrated in this and other studies. It is also important to evaluate economic utility as a factor in making medical judgements. Analysis of outcomes from previous studies show that the use of the conditioned gas at laparoscopy favorably influences outcomes (reducing pain and rapid recovery and return to work). 6 , 7 Use of the Insuflow® device was less expensive in this study and resulted in better outcomes than using the current standard raw gas. Section title: DISCUSSION Educational score: 4.056614875793457 Domain: biomedical Document type: Study Language: en Economic consequences (costs) of hypothermia have been previously reported. 27 These include increased time in the recovery area, use of alternative warming methods and increased need for analgesic medicines. 6 The financial penalty for not using pre-conditioned gas includes time, equipment, use of modifying techniques that only partially treat the effect and not the cause and slower return to full function. The economic savings per procedure using the Insuflow® device on every laparoscopy far exceeds the costs of the current methods it replaces, making it clearly a better strategy. There is decreased need to de-fog the laparoscope lens because the intra-abdominal dew point is not reached, eliminating the need for de-fogging agents. Time, operating room costs and frustration are saved with not having to de-fog. Hypothermia is reduced or eliminated, making surface warming methods less necessary. Recovery room time is decreased as a result of the patients' more euthermic postoperative state. The need for postoperative analgesic medicines is reduced because of significantly reduced pain perception. Return to full function activities is improved by over 50%. Section title: DISCUSSION Educational score: 3.9419262409210205 Domain: biomedical Document type: Review Language: en In summary, laparoscopic procedures that induce hypothermia are favorably influenced by heated hydrated gas and also reduce pain and shorten recovery room stay significantly. Laparoscopic procedures that minimally affect hypothermia reduce postoperative pain and shorten recovery room stay when heated hydrated gas is used. The Insuflow® device pre-conditions laparoscopic gas by heating and hydration, significantly reduces with regard to incidence, severity and extent of postoperative pain, shortens recovery room length of stay and reduces hypothermia by maintaining a more normal physiologic intra-abdominal state. The safety profile of heated hydrated gas exceeds that of current standard raw gas. The use of heated hydrated gas during laparoscopy is a significant breakthrough that is beneficial to the patient's outcome. | Review | biomedical | en | 0.999997 |
10036123 | Section title: INTRODUCTION Educational score: 4.09364128112793 Domain: biomedical Document type: Review Language: en The incidence of adenocarcinoma of the esophagus is rising at an alarming rate. 1 The prognosis is poor, with a five year survival of 5-10%. Randomized studies indicate that multimodality regimens have not been uniformly successful in improving survival compared to surgery alone. 2 One problem in the design of these trials is that pre- and post- treatment staging was performed by conventional imaging which has been shown to be inaccurate in up to 40% of patients with esophageal cancer when compared to minimally invasive surgical staging. 3 , 4 Even when surgical staging is performed, lymph node assessment may be inaccurate due to sampling errors at the time of nodal dissection. 3 , 4 In addition, micrometastases may escape detection by routine histopathologic methods in up to 20% of cases. 5 The application of recent advances in molecular biology may facilitate the detection of micrometases in lymph nodes, thereby limiting the requirement for extensive surgical sampling, and increasing the accuracy of staging. Section title: INTRODUCTION Educational score: 4.048832416534424 Domain: biomedical Document type: Study Language: en Carcinoembryonic antigen (CEA) is a recognized tumor marker that is expressed in a variety of malignancies. CEA has been most extensively studied as a serum marker of recurrence of colorectal cancer after surgical resection. 6 The complete gene for CEA has recently been cloned and complete sequence information is available. 7 Recently, a CEA-specific nested PCR assay to detect CEA producing cells from bone marrow aspirates has been described. 8 In a preliminary study, this assay was modified to detect micrometastases in lymph nodes in a small number of patients with esophageal, gastric, colorectal, and breast carcinoma. 9 Section title: INTRODUCTION Educational score: 4.108991622924805 Domain: biomedical Document type: Study Language: en The purpose of this investigation was to investigate the utility of RT-PCR for detecting micrometastases in histologically negative lymph nodes in patients with esophageal cancer. The ability to detect micrometastases by utilizing minimally invasive surgical techniques would provide concrete pathological evidence of lymph node status, without subjecting patients to unnecessary surgical intervention. Minimally invasive surgical techniques have been shown to be cost-effective as well as to improve patient outcomes, and in the application of lymph node sampling for staging purposes, this modality would ultimately serve as a “gold standard.” Section title: RNA Extraction Educational score: 4.063470840454102 Domain: biomedical Document type: Study Language: en Total cellular RNA was extracted from all samples by a modified guanidinium thiocyanate extraction method 10 using a RNA isolation kit (RNeasy, Qiagen, Chatsworth, CA). Tissue lysis and homogenization were performed with an automated tissue homogenizer (PowerGen 35 Homogenizer, Fisher Scientific), using disposable generator tips to prevent RNA cross contamination. Section title: Reverse Transcription Educational score: 3.6508848667144775 Domain: biomedical Document type: Study Language: en cDNA was synthesized from 1-5 μg of total RNA using a cDNA synthesis kit (Superscript preamplification system, GibcoBRL, Gaithersburg, MD). The oligo-dT priming method was utilized. Section title: Polymerase Chain Reaction Educational score: 4.174477577209473 Domain: biomedical Document type: Study Language: en Three CEA specific oligonucleotide primers for nested PCR were designed from previously published sequences. 7 – 9 The primer sequences for CEA were as follows: Primer A, 5′-TCTGGAACTTCTCCTGGTCTCTCAGCTGG-3′; Primer B, 5′-TGTAGCTGTTGCAAATGCTTTAAGGAAGAAGC-3′; and Primer C, 5′-GGGCCACTGTCGGCATCATGATTGG-3′. The nested PCR was performed in two successive steps. Step 1 was performed in a 20 μl reaction volume containing 1X PCR buffer (Boehringer Mannheim), 250 μM dNTPs, 1 μM each primers A and B, and 0.5 U Taq DNA polymerase (Boehringer Mannheim). Twenty rounds of amplification were performed in a thermocycler (DNA Thermocycler 480, Perkin Elmer) under the following cycling conditions: 95° C (1 min) denaturation, 72° C (1 min) annealing, 72° C (1 min) extension, followed by a final extension of 72° C (10 min). Two ul of the Step 1 reaction product was transferred to another tube containing primers B and C, PCR buffer, dNTPs, and Taq DNA polymerase in the same concentrations as in Step 1. Step 2 was also performed in a 20 μl reaction volume. The second PCR reaction was performed under the same conditions as Step 1. Step 1 yields a 160-bp PCR product; the second PCR reaction produces a 131-bp product. Section title: Polymerase Chain Reaction Educational score: 4.097797393798828 Domain: biomedical Document type: Study Language: en As a positive control for RNA quality, a PCR assay for aactin was performed for each sample. The primer sequences were as follows: 5′-CCTGGCACCCAGCACAATGA-3′, and 5′-ACGAAGGCTCATCATTCAAA-3′. The actin PCR was performed with 30 rounds of amplification under the following cycling conditions: 94° C (30 sec) denaturation, 68° C (45 sec) annealing, 72° C (1 min) extension, followed by a 7 minute final extension at 72° C. Section title: Polymerase Chain Reaction Educational score: 3.1503591537475586 Domain: biomedical Document type: Study Language: en To check for possible contamination with genomic DNA, RT-PCR reactions were also performed without a reverse transcription step. Section title: PCR Product Analysis Educational score: 3.880530595779419 Domain: biomedical Document type: Study Language: en PCR products were analyzed on a 1.5% agarose gel containing 0.5 μg/ml ethidium bromide. A 100-bp ladder (Boehringer Mannheim) served as a molecular weight marker. PCR products were separated by electrophoresis at 80 volts for 2-3 h. Section title: Lymph Node Samples Educational score: 4.06089973449707 Domain: biomedical Document type: Study Language: en Informed consent was obtained from all patients as specified in University of Pittsburgh Cancer Institute protocol 94-121. Sixty lymph node samples were obtained from 13 patients with carcinoma of the esophagus. Lymph nodes from patients with esophageal cancer were obtained during combined laparoscopic and videothoracoscopic staging or esophagectomy. As a control, 10 lymph nodes were obtained from 6 patients undergoing treatment of a benign esophageal disease, such as gastroesophageal reflux disease or achalasia. Five lymph nodes were evaluated from a patient with a history of long-standing reflux and Barrett's metaplasia. In addition to lymph nodes, 8 samples of esophageal carcinoma (7 adenocarcinoma, 1 squamous cell carcinoma), and 5 samples of normal esophagus were studied. Section title: Lymph Node Samples Educational score: 4.037657260894775 Domain: biomedical Document type: Study Language: en Each sample was carefully dissected from perinodal fat and soft tissue and then sectioned into two pieces. One piece was fixed and embedded in paraffin and used for histologic analysis. The remaining piece was immediately placed in liquid nitrogen and stored at -70°C until RNA extraction. Following the RT-PCR analysis the results of histologic examination and RT-PCR were compared. Section title: Normal Esophagus and Carcinoma Samples Educational score: 4.057319641113281 Domain: biomedical Document type: Study Language: en RT-PCR amplification of all 8 samples of esophageal carcinoma and 5 samples of normal esophagus demonstrated the 131-bp CEA band . No product was observed in control PCR reactions performed without the reverse transcription, demonstrating that there was no contamination with genomic DNA . Section title: Lymph Node Samples Educational score: 4.163614749908447 Domain: biomedical Document type: Study Language: en Histologic examination detected metastases in 29 of 60 (48%) lymph node specimens studied. All histologically positive lymph node samples demonstrated a positive CEA signal by RT-PCR. The number of lymph node metastases detected increased to 46 of 60 (77%) when RT-PCR was used. Seventeen of 31 (52%) histologically negative lymph nodes were found by RT-PCR analysis to express the mRNA for CEA by RT-PCR . In three patients, all regional lymph nodes were negative histologically. Of these patients, two demonstrated lymph node metastases by RT-PCR and both of these patients died of disease progression within 12 months. The remaining patient, negative histologically and by RTPCR, remains without disease recurrence 12 months after surgical resection. In nine patients, RT-PCR indicated an increased number of metastatic lymph nodes compared to histologic review. In three patients, histologic and RTPCR diagnostic methods detected the same number of lymph node metastases ( Table 1 ). Section title: Lymph Node Samples Educational score: 4.118662357330322 Domain: biomedical Document type: Study Language: en All lymph nodes with histologic evidence of metastases failed to demonstrate the characteristic 131-bp band when PCR without reverse transcription was performed, indicating that genomic DNA contamination did not occur. All lymph nodes from patients with benign esophageal diseases and Barrett's esophagus were negative for CEA by RT-PCR and negative by histologic examination. Section title: DISCUSSION AND CONCLUSIONS Educational score: 3.960397958755493 Domain: biomedical Document type: Study Language: en The identification of lymph node metastasis in patients with esophageal cancer is an important determinant of prognosis and impacts on the clinical decision making process. 11 – 13 Randomized trials of multimodality treatment regimens have been performed but have not uniformly improved survival compared to surgery alone. 2 A major drawback in these studies is that accurate pre-treatment staging prior to the initiation of therapy was not performed, thus limiting any meaningful evaluation of therapeutic response. This approach of grouping patients based only on the presence or absence of distant metastases without consideration of the extent of locoregional disease also limits evaluation of survival data on a stage by stage basis. Since a number of reports have shown that not only the presence of lymph node metastases but their number and location are important indicators of long-term survival, it is possible that a more accurate assessment of stage prior to treatment would allow a more thorough evaluation of efficacy. 11 – 13 Section title: DISCUSSION AND CONCLUSIONS Educational score: 3.9359800815582275 Domain: biomedical Document type: Review Language: en Conventional imaging modalities employed to assess lymph node and distant metastases are inaccurate in over 40% of patients when compared to minimally invasive surgical staging. 3 , 4 Positron emission tomography offers a significant improvement over computerized tomography in detecting distant metastatic disease but is only 45% sensitive in detecting metastases lymph nodes under 1 cm in diameter. 4 Endoscopic ultrasound is accurate in assessment of the depth of tumor invasion but its utility in detecting small lymph node metastases has been questioned. 14 – 17 Section title: DISCUSSION AND CONCLUSIONS Educational score: 3.980510950088501 Domain: biomedical Document type: Study Language: en Currently, the most accurate method to stage patients with esophageal cancer is by minimally invasive surgical biopsy. However, this approach is associated with a small but measurable morbidity, significant expense and requires an extensive surgical dissection to avoid sampling errors and under staging. Once lymph nodes are surgically biopsied, further errors may take place in the histologic evaluation since up to 20% of histologically negative lymph nodes are positive on re-examination. 5 Immunohistochemical staining of lymph nodes to detect tumor markers may result in a higher detection rate of micrometastases when compared to histological examination but this technique is time consuming and labor intensive, and is not in widespread clinical use. 18 – 21 Section title: DISCUSSION AND CONCLUSIONS Educational score: 4.014900207519531 Domain: biomedical Document type: Study Language: en Sensitive molecular methods, such as RT-PCR, have recently been employed to detect lymph node metastases in a variety of malignancies such as malignant melanoma, breast, colorectal, gastric, and esophageal carcinoma. 9 , 22 – 25 This method is designed to detect markers expressed in the carcinoma cells but not in normal lymph nodes. A CEA specific RT-PCR assay has been developed to detect carcinoma cells in bone marrow samples from patients with colorectal, pancreatic, or gastric carcinoma. 8 Recently, this method has been modified to detect lymph nodes metastases in patients with esophageal, gastric, colorectal, and breast cancer. 9 This preliminary report included a small number of patients with esophageal cancer. Section title: DISCUSSION AND CONCLUSIONS Educational score: 4.1623077392578125 Domain: biomedical Document type: Study Language: en Our preliminary experience with RT-PCR to detect the mRNA for CEA confirms that this technique has the potential to improve the accuracy of detecting lymph node metastases in esophageal cancer compared to histologic evaluation. As expected, CEA was present in all samples of esophageal carcinoma and normal esophagus and thus appears to be a marker specific for epithelial cells. Importantly, CEA was not present in any of the lymph nodes from patients without cancer. Thus in the absence of cancer there is no migration of epithelial cells from the esophagus to the lymph nodes. Histologic assessment of lymph nodes from patients with esophageal cancer detected 29 of 60 lymph nodes involved with metastatic disease. All histologically positive lymph nodes were positive by RT-PCR for CEA, indicating that there were no false negative results using this method. The number of positive lymph nodes increased to 49 of 60 when RT-PCR technology was applied. Two of three patients with histologically negative lymph nodes were determined to have nodal disease by RT-PCR staging. In nine of the 13 patients with esophageal cancer, the number of positive lymph nodes detected was increased by RT-PCR compared to histology. Section title: DISCUSSION AND CONCLUSIONS Educational score: 3.875462532043457 Domain: biomedical Document type: Study Language: en CEA-specific RT-PCR is an extremely sensitive technique that facilitates the detection of histologically occult lymph node metastases in patients with esophageal cancer. The technique has the potential to improve upon the accuracy of current staging, aid in the evaluation of response to treatment, and impact on clinical decision making in patients with esophageal cancer. Further studies with clinical follow-up will be required to evaluate the role of RT-PCR in the diagnosis and treatment of patients with esophageal cancer. | Review | biomedical | en | 0.999997 |
10036124 | Section title: INTRODUCTION Educational score: 3.831684112548828 Domain: biomedical Document type: Clinical case Language: en Double gallbladder is a rare anomaly of the biliary tract, occurring in about 1 per 3800 cases at autopsy. 1 Two cases of double gallbladders managed laparoscopically have been reported previously. 2 , 3 We report herein another patient in whom laparoscopic cholecystectomy was attempted. The case represents a very rare variety of a double gallbladder, only once previously reported in the literature. 4 It highlights possible anomaly of the accessory biliary system, emphasizing the need for an intraoperative cholangiography in order to prevent iatrogenic injuries to the bile ducts. Section title: CASE REPORT Educational score: 3.9209511280059814 Domain: clinical Document type: Clinical case Language: en A 69-year-old female presented with several months history of right upper abdominal and epigastric pain. Ultrasonography revealed a gallbladder containing multiple stones and a normal-size common bile duct. In addition, a cystic structure was noted lateral to the left hepatic duct, raising the possibility of an accessory gallbladder. Computed tomography (CT) and endoscopic retrograde cholangiopancreatogram (ERCP) confirmed the presence of an accessory, partially intrahepatic gallbladder, which also contained stones . No ductal stones were visualized, and liver function tests were normal. Since the accessory gallbladder did not have an identified cystic duct on ERCP, the laparoscopic procedure started with a double cholangiogram through the cystic duct of the normal gallbladder and the accessory gallbladder ( . No accessory cystic duct was, however, visualized, and the laparoscopic procedure was converted to an open procedure. Cholecystectomy of the primary gallbladder was completed, and a cholecystectomy of the accessory gallbladder was performed in a retrograde fashion. The accessory gallbladder was found to have no cystic duct and originated directly from the distal left hepatic duct. It was dissected off the lateral wall of the left hepatic duct, and the resulting 3 mm defect was closed with 5-0 polydioxanone. Completion cholangiogram revealed several small stones in the distal common bile duct, which was then explored . Recovery was uneventful, and the T-tube cholangiogram was normal. Pathology report described a 3 × 4 cm accessory gallbladder containing three stones. Histology revealed chronic cholecystitis with a mild dysplasia of the mucosa. Section title: DISCUSSION Educational score: 3.6605207920074463 Domain: biomedical Document type: Clinical case Language: en Double gallbladder is a biliary anomaly usually not diagnosed preoperatively. Instead, it often represents an intraoperative surprise 2 or is missed during the operation, only to be diagnosed at postoperative ERCP, performed for persistent biliary symptoms. 5 , 6 In our case, both gallstone-containing gallbladders were probably symptomatic. The presence of the accessory gallbladder was suggested by the sonogram and confirmed on pre-operative ERCP and CT scan. The operation was started laparoscopically, but was converted to an open procedure due to the absence of the accessory cystic duct. Section title: DISCUSSION Educational score: 4.1357855796813965 Domain: biomedical Document type: Clinical case Language: en This case represents a type VI in the large spectrum of accessory gallbladders as proposed by Mochizuki 7 ( Table 1 ), or type C as proposed by Harlafits et al. ( Table 2 ). This rare variety of the accessory gallbladder has been reported only once. 4 Among 207 cases reviewed by Harlafits et al., 4 the majority of the anomalies consisted of duplicated gallbladders sharing the same cystic duct (Type A), or accessory gallbladders, with two cystic ducts entering common bile duct separately (Type B). The large spectrum of ductal anomalies associated with a double gallbladder mandates an intra-operative cholangiogram prior to the resection of the accessory gallbladder. Such an approach would minimize the risks of inadvertent injury to the biliary ductal system. | Clinical case | biomedical | en | 0.999998 |
10036125 | Section title: INTRODUCTION Educational score: 4.058441162109375 Domain: biomedical Document type: Study Language: en In the 1960s, physicians of the Internal Clinic at the University of Erlangen, Germany, successfully performed endoscopic duodenoscopy, one of the earliest reports of that procedure. The first endoscopic retrograde cholangio-pancreaticography (ERCP) followed in 1970. 1 In 1973, two Erlangen internists, Classen and Demling, performed an endoscopic papillotomy using their own device (Erlanger Papillotom). In Erlangen, 159 patients underwent this endoscopic procedure by the summer of 1976. By 1977, more than 1,500 papillotomies had been performed in ten West German centers, with an overall success rate of 93 percent. Complications occurred much less frequently than with transduodenal papillotomy, and the mortality rate dropped to 1.1 percent. 2 Section title: INTRODUCTION Educational score: 1.6928648948669434 Domain: biomedical Document type: Other Language: en Many surgeons were sharply critical of internists' entrance into classical fields of surgery. At the University of Erlangen, on the other hand, the surgeons had a different reaction. According to Erich Mühe , then an assistant in the Surgical Clinic, the Erlangen surgeons “respected the internists' pioneering work” 3 on polypectomy in the upper and lower gastrointestinal tracts, loop biopsy, endoscopic tumor resection, and removal of foreign bodies. “We were very proud of ‘our’ internists,” he noted years after. 3 In 1982, Mühe left Erlangen and became the Head of the Surgical Clinic in the Böblingen county hospital. Section title: Laparoscopic Cholecystectomy Educational score: 1.8808974027633667 Domain: biomedical Document type: Other Language: en During this time, news of Semm's laparoscopic appendectomy was rippling through German medical circles. Although Semm's first report did not appear in the medical press until 1982, German surgeons were already aware of his path-breaking work. 4 Mühe, spurred by successes of the Erlangen endoscopists and fascinated by Semm's technique, came up with the idea of laparoscopic cholecystectomy (LC). He recalls his underlying motivation: I had the overwhelming feeling that we [general surgeons] had already lost traditional surgical fields like polypectomy, papillotomy, and now even endoscopic appendectomy was discussed. I was convinced that if we passed up this chance like endoscopic cholecystectomy, internists and gynecologists would again take away a piece of our competence. 5 Section title: Laparoscopic Cholecystectomy Educational score: 2.230562448501587 Domain: biomedical Document type: Other Language: en In late September 1984, four packages of gynecological endoscopic instruments were delivered to the Böblingen hospital. 6 Mühe realized that Semm's instruments were sufficient to remove a normal gallbladder, but he had concerns about the gallbladders containing numerous stones. Mühe gradually worked out the details of a new operative laparoscope, the “Galloscope.” It had side-view optics, instrumentation channel with valves, light conductor, and a duct for creating pneumoperitoneum. Section title: The First LC on September 12, 1985 Educational score: 2.8805315494537354 Domain: biomedical Document type: Other Language: en Almost five years to the date from Semm's first fully endoscopic appendectomy, Mühe adopted Semm's technique and employed his own “Galloscope.” He used a Veress needle to establish pneumoperitoneum and followed each step of the surgical laparoscopy established by Semm: pneumoperitoneum, trocars and insertion site for the “Galloscope.” Mühe then inserted a mandrin within a trocar sleeve and introduced his “Galloscope” through the umbilicus. 7 The operation took two hours and went exceptionally well . Section title: The First LC on September 12, 1985 Educational score: 1.3878451585769653 Domain: biomedical Document type: Other Language: en Like many laparoscopists, Mühe was astonished at his patients' rapid recoveries. “I couldn't believe it,” he recalled ten years later. “The patients had bowel movements almost immediately after the surgery and could eat supper the same evening. Over and over I asked myself what factors could be responsible.” 8 Mühe came to the conclusion that “the approach was like magic.” 9 That confirmed Semm's observations. Section title: Mühe's Modifications: Gasless Technique, LC through Trocar Sleeve, and “Open Laparoscope” Educational score: 2.1339123249053955 Domain: biomedical Document type: Other Language: en Based on this insight, Mühe concluded that he could do away with pneumoperitoneum, created only to open the path from umbilicus to gallbladder. After several laparoscopic cholecystectomies, Mühe slightly changed his technique to introduce the laparoscope under the right costal arch. “I soon noticed that the costal margin served as a firm bone roof above the gallbladder,” noted Mühe, explaining his modification. “I strongly believed that the gasless laparoscopic cholecystectomy would find more acceptance.” 10 Section title: Mühe's Modifications: Gasless Technique, LC through Trocar Sleeve, and “Open Laparoscope” Educational score: 1.8847814798355103 Domain: biomedical Document type: Other Language: en Mühe's creative simplification led him to other modifications. He was intent on adapting the technique to a wide range of surgeons: “With or without the Galloscope, the magic approach would still be the same.” 11 Mühe soon moved on to the next step and operated through the trocar sleeve, without the optical instrument. The “magic” endoscopic approach remained untouched. 12 Section title: Mühe's Modifications: Gasless Technique, LC through Trocar Sleeve, and “Open Laparoscope” Educational score: 1.8954287767410278 Domain: biomedical Document type: Other Language: en Since a surgeon had no light source inside the abdominal cavity without the Galloscope, Mühe affixed a procto-scope light cable to the proximal trocar end. He recalled, “I then asked myself if any simple ‘open laparoscope’ could replace the trocar. ... Of course it could. In fact, I intended to affix a proctoscope light cable to any ‘open tube’ available in our hospital workshop.” 13 Section title: Mühe at the Congress of the German Surgical Society (GSS) Educational score: 2.06823992729187 Domain: biomedical Document type: Other Language: en For a lecture on endoscopic cholecystectomy at the Congress of the German Surgical Society (GSS) in Munich, April 1986, Mühe prepared 42 slides. 14 He took the first two from Semm's publications on an endoscopic resection of an ovarian cyst and a laparoscopic appendectomy. The next four showed the “Galloscope.” Mühe then presented 23 slides of his “open tube,” much to the amusement of his colleagues. In conclusion, he summarized the advantages of cholecystectomy without laparotomy in five main points: 1) Abdominal muscles are not cut, 2) Little postoperative pain, 3) Short immobilization, 4) Short postoperative hospitalization (4-5 days), and 5) Rapid return to the workplace. Section title: Mühe at the Congress of the German Surgical Society (GSS) Educational score: 1.2973145246505737 Domain: biomedical Document type: Other Language: en The audience was skeptical of Mühe's claims. 15 Most surgeons thought that operating through a small incision was dangerous. Mühe later had to deal with derogatory remarks such as “Mickey Mouse surgery” and “small brain—small incision.” Section title: A Weak Response to Mühe's Lectures Educational score: 1.204666018486023 Domain: other Document type: Other Language: en Mühe did not achieve his hope for a breakthrough at the GSS congress. Only a handful of surgeons contacted him in the following months (among them, F. Hutterer of Cologne, 16 H. Troidl of Cologne, 17 and G. Wilkenau of Aachen 18 ). In October 1986, Mühe gave a lecture at the annual meeting of the Lower Rhine-Westphalian Surgical Society held in Cologne. 19 In the fall of 1987, he presented endoscopic cholecystectomy at a Symposium of CAES (Surgical Study Group on Endoscopy and Ultrasound) in Mainz. 20 The response to Mühe's lectures was disappointingly weak. 21 That same fall, Mühe was dealt another blow when the GSS failed to publish his lecture in its 1986 Proceedings. Only an abstract appeared, even though Mühe's talk had been one of the main features of the congress. 22 Section title: A Weak Response to Mühe's Lectures Educational score: 2.0761449337005615 Domain: biomedical Document type: Other Language: en By March 1987, Mühe had performed 97 endoscopic cholecystectomies. 23 Section title: 1) “Big Surgeon—Big Incision” Educational score: 1.9562668800354004 Domain: biomedical Document type: Other Language: en Seen from today's perspective, it is difficult to understand why the German surgical world closed its eyes to Mühe's achievement. Perhaps one of the reasons is that abdominal surgery at that time was one of the few areas still largely untouched by the concept of “minimally invasive therapy.” The introduction of ever more sophisticated drugs, the impressive results of intensive care medicine, and advances in anesthesia together had led to the development of “major surgery.” 24 Section title: 1) “Big Surgeon—Big Incision” Educational score: 2.8289520740509033 Domain: biomedical Document type: Other Language: en Many leading abdominal surgeons had focused their attention on organ transplantation and cancer treatment. Abdominal operations were radical and extensive. The battle for higher five-year survival rates was in full swing, and countless new surgical techniques were published every year. “General surgical thinking was oriented to the concept that big problems required big incisions, and that incisions healed side to side, not end to end,” observed Michael S. Kavic of McKees Rocks, Pennsylvania. 25 The idea that large problems require large incisions so deeply dominated surgical thinking in the 1980s that there was little room to appreciate the advances of “key-hole” techniques. Surgeons had little interest in modifying operations with low mortality rates, such as appendectomy and cholecystectomy. Section title: 2) Non-Surgical Treatment of Biliary Stones Educational score: 3.9016942977905273 Domain: biomedical Document type: Review Language: en In this context, it is important to emphasize another aspect of gallstone treatment, the non-surgical therapy of biliary calculi. Experiments and clinical alternatives for non-surgical treatment of biliary stones proliferated into the mid-1980s: clinical results with chenodeoxycholic acid (CDCA) , ursodeoxycholic acid (UDCA) , endoscopic papillotomy , and, finally, mechanical lithotripsy of bile and common duct stones . 26 Expectations were high. Many believed that the era of effective chemical dissolution or lithotripsy was only a question of time. 27 In light of such promising alternatives, little attention was paid to the development of surgical gallbladder removal. Section title: 3) The German Healthcare System Educational score: 1.787593126296997 Domain: other Document type: Other Language: en Perhaps more puzzling for the reader, especially in North America, is that German healthcare professionals were unmoved by the economic benefits of endoscopic cholecystectomies, such as rapid return to work. But the German healthcare system was not designed to accommodate such considerations; at that time, hospitals based their charges on the length of stay rather than the individual details of a patient's hospitalization! 28 In short, the longer a patient was hospitalized, the more money the hospital received from the insurance company. Consequently, hospitals tended to keep surgical patients longer to cover operating costs. Section title: 3) The German Healthcare System Educational score: 1.739163875579834 Domain: biomedical Document type: Other Language: en Why were private surgeons in Germany not interested in laparoscopic cholecystectomy? Here, even greater differences exist between Germany and the United States. Private surgeons in Germany generally do not operate in hospitals. They refer patients who require surgery to the hospital, where residents or professors carry out the operation—nor do they have much say as to the preparation for the operation, type of surgery, or postoperative treatment. Section title: 4) The German University System Educational score: 2.0423648357391357 Domain: biomedical Document type: Other Language: en The traditional structure of German medicine is of course a reflection of the centralized decision-making process in European politics and education. As noted by a European social scientist, “The centralization of academic decision-making that is characteristic of European countries, it is argued, contrasts so sharply with the decentralization of academic decision-making in the U.S. that comparisons are not useful.” 29 In this system, non-university centers command scanty respect among academic surgeons. In other words, most German surgeons distrust any invention developed outside the university. And, among surgeons, Mühe was not a recognized university expert in the field of endoscopy, not even a member of any endoscopic society. In 1995, Michael Jung, former senior physician in the Division of Endoscopy at the University of Heidelberg, gave a pithy account of Mühe's struggle: “Mühe was an outsider to the field of surgical endoscopy. And as an outsider, he was ignored. That's it.” 30 Section title: Mühe's Shortcomings Only a Few Publications in German Educational score: 1.551528811454773 Domain: biomedical Document type: Other Language: en During the 18 months of his clinical activity with the “Galloscope” and “open laparoscope,” Mühe held only three lectures, and demonstrated the method on an individual basis. This period stands in sharp contrast to his previous average of 30 publications a year. In recent statements, Mühe agrees that he was too intent on the German audience and underestimated the North American potential; in fact, he was a surgeon with deep German roots. Of his 342 publications between 1965 and 1983, only 7 percent appeared in English. 31 In contrast to Semm, Mühe did not recognize the potential audience in North America for innovations. Section title: Erich Mühe: A Surgeon Ahead of His Time Educational score: 1.9721146821975708 Domain: biomedical Document type: Other Language: en For Mühe, the technique of laparoscopic cholecystectomy was a logical step in the evolution of surgery, a further application of already-available technology. Today, Mühe's approach seems pragmatic and simple. In the heyday of “big surgeon—big incision” and pharmaceutical gallstone treatment, few people thought it would be necessary to alter gallbladder surgery. Mühe was an exception. While numerous surgeons were aware of the potential in gynecological laparoscopic surgery, only Mühe took the decisive step. Section title: Erich Mühe: A Surgeon Ahead of His Time Educational score: 1.6192160844802856 Domain: biomedical Document type: Other Language: en Perhaps the significance of Mühe's work is found in the fact that he could prepare the way for the introduction of laparoscopy into surgical practice. One wonders whether any individual would have been in a position to over-come the opposition in the German medical and social system of that time. It is not the first (nor the last) time in the history of medicine that the medical world has overlooked the work of a pioneering member . Section title: Erich Mühe: A Surgeon Ahead of His Time Educational score: 0.9860542416572571 Domain: other Document type: Other Language: en Laurence Olivier once listed three things necessary to a successful career: talent, luck, and stamina. He then said of luck: “Though this must vary, it must be good enough to believe in the truth of it yourself. You must see that it has provided you with the right opportunities at the right times.” 32 Erich Mühe was graced by neither time nor place. He was a surgeon ahead of his time . | Study | biomedical | en | 0.999997 |
10037783 | Section title: Cell Culture Educational score: 4.113435745239258 Domain: biomedical Document type: Study Language: en Mouse embryonic fibroblasts (MEFs), were prepared from day 13.5 embryos derived from wild-type (wt) and mTER −/− mice from different generations. The details are explained elsewhere . The first culture directly obtained from embryos was considered as population doubling 2 (PD 2). Serial cultures were done according to the 3T3 protocol in dishes of 10 cm diam, seeding 10 6 cells every 3 d. The doubling number of each passage was calculated using the formula PD = log (n f /n 0 )/log2; where n 0 is the initial number of cells (10 6 ) and n f is the final number of cells. The details on the establishment of the immortal cell lines are explained elsewhere . Section title: Fluorescence In Situ Hybridization with PNA-telomere Probe Educational score: 4.191098213195801 Domain: biomedical Document type: Study Language: en Plates containing primary MEFs or established cell lines were treated with colcemid (0.1 μg/ml) for 4–5 h and subsequently trypsinized and spun for 8 min at 120 g . After hypotonic swelling in sodium citrate (0.03 M) for 25 min at 37°C, cells were fixed in methanol/acetic acid (3:1). After 2–3 additional changes of fixative, cell suspensions were dropped on wet, clean slides and dried overnight. FISH with Cy-3 labeled (CCCTAA) 3 peptide-nucleic acid, and subsequent quantitative analysis of digital images were performed as described . The slide coordinates of the metaphase images captured were noted to relocate the same metaphase after FISH with minor satellite DNA (see below) and mouse chromosome probes (Oncor). Section title: Quantitative Image Analysis Educational score: 4.2036051750183105 Domain: biomedical Document type: Study Language: en Digital images were recorded with a MicroImager MI1400-12 camera (Xillix) on an Axioplan fluorescence microscope ( Zeiss ). Microscope control and image acquisition was performed with a dedicated software (SSM; Xillix). Separate DAPI and Cy-3 images were subjected to telomere fluorescence analysis by using ad dedicated computer program, TFLTELO . Chromosomes and telomeres were identified through segmentation of the DAPI image and the Cy-3 image, respectively. Both images were combined and corrected for pixel shifts. The integrated fluorescence intensity for each telomere was calculated after correction for image acquisition exposure time. Finally, the integrated fluorescence intensity of individual telomeres is expressed in a table for each chromosome, which can be subjected to editing. Each metaphase of 40 chromosomes (in the mouse) yields 160 telomere spots and a typical analysis of 15–20 metaphases produces several thousand telomere fluorescence values. Because of the large number of data points in Q-FISH analysis, the standard error of mean telomere fluorescence estimates is typically small (for example, less than a few percent of the average) despite considerable variation in individual telomere fluorescence values. Section title: Quantitative Image Analysis Educational score: 4.187521457672119 Domain: biomedical Document type: Study Language: en Details of the calibration procedures used to reliably measure telomere fluorescence intensity are explained elsewhere . In brief, two levels of calibration values were used. First, to correct for daily variations in lamp intensity and alignment, images of fluorescent beads (orange beads, size 0.2 μm; Molecular Probes) were acquired and similarly analyzed with the analysis software. Second, relative telomere fluorescence units (TFU) were extrapolated from the plasmid calibration. For this, we hybridized and analyzed plasmids with a defined (TTAGGG) n length of 0.15, 0.4, 0.8, and 1.6 kb. There was a linear correlation (r 2 = 0.99) for plasmid fluorescence intensity and (TTAGGG) n length with a slope of 48.7 . Therefore, the calibration corrected telomere fluorescence intensity (ccTFI) of each telomere was calculated according to the formula: ccTFI = (Bea1/Bea2) × (TFI/48.7), where Bea1 equals the fluorescence intensity of beads when plasmids were analyzed, Bea2 equals the fluorescence intensity of beads when sample x was analyzed, and TFI equals unmodified fluorescence intensity of a telomere in sample x. Section title: Quantitative Image Analysis Educational score: 3.8658230304718018 Domain: biomedical Document type: Study Language: en A restriction of this calibration method is that the actual telomeres are outside the range of (TTAGGG) n length of the plasmids. The assumption is made that the linear correlation obtained between fluorescence intensity and telomere insert size in plasmid is maintained in the higher range in chromosomal DNA. Section title: FISH with Minor Satellite DNA Educational score: 4.264042854309082 Domain: biomedical Document type: Study Language: en The murine minor satellite DNA probe was a gift from Drs. J.B. Rattner and Shu-Lin Liu (Calgary University, Calgary, Canada). The minor satellite probe labels all centromeres near the kinetochore except mouse chromosome Y . The probe was labeled with Spectrum-green dUTP by nick translation and then precipitated with ethanol, using salmon sperm DNA as carrier. The probe was dissolved in a hybridization buffer containing 30% deionized formamide, 2× SSC, 10% dextran sulphate and 50 mM phosphate buffer (pH 7.0) to a concentration of 20 ng/μl. The slides used for telomere FISH were washed and then hybridized with the minor satellite DNA probe. The hybridization was performed as described . Slides were incubated with pepsin (0.005%) in 10 mM HCl for 10 min at 37°C, washed with PBS containing 50 mM MgCl 2 , and then treated with 1% formaldehyde in PBS/MgCl 2 for 10 min at room temperature. After one more wash in PBS, slides were dehydrated in a 70, 90, and 100% ethanol series. The labeled probe was diluted with hybridization buffer to a final concentration of 4 ng/μl, and 20 μl were added on each slide. The probe and the target DNA were denatured simultaneously at 80°C, for 3.5–4.0 min. Hybridization was carried out overnight at 37°C in a moist chamber. After hybridization, the slides were washed three times with 30% formamide, 2× SSC buffer (pH 7.0) for 5 min at 37°C, followed by two washes in 2× SSC at room temperature. The slides were dehydrated and embedded with Vectashield mounting medium (Vector Labs) containing 1 μg/ml propidium iodide. About 15–25 metaphases per group that had already been used for telomere measurements were relocated and observed under the microscope for characterizing the end-to-end fusions. Section title: FISH with Mouse Chromosome Paint Probes Educational score: 4.02806282043457 Domain: biomedical Document type: Study Language: en Chromosome painting probes for chromosomes 2 and 11 were used to follow telomere length in these chromosomes. The same slides that were analyzed after FISH with telomere probe and minor satellite DNA were processed for the third FISH with chromosome painting probes. The chromosome painting probes were purchased from Oncor. FISH was performed according to the manufacturer's instructions. About 15–25 metaphases were relocated using the known coordinates in the microscope and analyzed. Section title: Telomere Dynamics of Individual Chromosomes in mTER − /− Telomerase-deficient Embryos from Different Generations Educational score: 4.11806583404541 Domain: biomedical Document type: Study Language: en Primary cells (passage 1 mouse embryonic fibroblasts, MEFs) derived from wt embryos and from mTER −/− embryos from the 1st (G1) to the 6th (G6) generation were obtained following the scheme previously described . The telomere length of individual chromosomes (chromosomes 2 and 11) was measured using Q-FISH and chromosome painting. Chromosome 2 was chosen because it consistently has relatively short telomeres in several mouse strains (Hande, M.P., and P. Lansdorp, unpublished results; this paper). Chromosome 11 is the mouse homologue of human chromosome 17, which was found to have relatively short telomeres in all individuals analyzed to date . Section title: Telomere Dynamics of Individual Chromosomes in mTER − /− Telomerase-deficient Embryos from Different Generations Educational score: 4.326051712036133 Domain: biomedical Document type: Study Language: en Fig. 1 shows the mean and standard error of telomere fluorescence intensity of all telomeres together (average of q- and p-arms), and also of q- and p-arms separately from primary MEFs of both wt and mTER −/− embryos of the 2nd (KO2-G2), 4th (KO7-G4), and 6th generation (littermate embryos KO9-G6 and KO11-G6; littermate embryos KO1-G6 to KO4-G6 and embryo KO5-G6). Despite considerable variation between individual telomere fluorescence values , the large number of data points (>1,000) resulted in insignificant standard error values in the telomere values of all chromosomes. The standard error was also small for individual telomeres on chromosomes 2 and 11, with smaller number of data points . The standard error rather than the standard deviation is shown for clarity and presentation purposes only. The average telomere fluorescence of all chromosomes decreased linearly during successive generations of mTER −/− mice. The average telomere shortening was 3.9 kb per generation . This shortening affected both q-telomeres (telomeres of the q-arms) that showed a shortening of 4.17 kb per generation, and p-telomeres (telomeres of the p-arms) that showed a shortening of 3.7 kb per generation. As a result, the difference in telomere length between p- and q-arm telomeres was maintained throughout the six mouse generations . The loss of telomere repeats in mTER −/− mice resulted in an average length of 14.5 and 22.4 kb for p- and q-telomeres, respectively, in cells derived from the 6th generation. When we measured the mean telomere fluorescence of chromosome 2, the estimated rate of telomere shortening per generation was 3.4 kb for both 2q- and 2p-telomeres . This telomere shortening resulted in 6th generation 2p- and 2q-telomeres of an average length of 7.6 kb and 16.2 kb, respectively (embryo KO9-G6 had an estimated 2p-telomere length of only 0.15 ± 0.1 kb), shorter than the average of all chromosomes. In the case of chromosome 11, the average telomere fluorescence of 11q and 11p-telomeres decreased at an average rate of 5.2 and 5.6 kb per generation, respectively, up to the 4th generation . Interestingly, from the 4th (embryo KO-G4) to the 6th generation (the average of seven different embryos) we did not detect the expected telomere shortening in any of chromosome 11 telomeres . In contrast, there was a 6-kb increase in the telomere length at the 6th generation . Altogether, these results suggest that in the absence of telomerase activity, telomere shortening occurs at a similar rate in all chromosome ends. However, it appears that mechanisms that prevent telomere shortening in the absence of telomerase act differentially on different telomeres. In our study, chromosome 11 telomeres did not show the predicted shortening with increasing generations in seven different embryos, while chromosome 2 telomeres continued to shorten throughout the six generations of mTER −/− mice (see Discussion). In this study, we cannot rule out that telomerase independent mechanisms of telomere maintenance are also operating in early generation mTER −/− or wt mice. Section title: Telomere Dynamics of Individual Chromosomes in mTER − /− Telomerase-deficient Embryos from Different Generations Educational score: 4.08284854888916 Domain: biomedical Document type: Study Language: en Interestingly, in the different mTER −/− cells derived from 6th generation embryos, we observed a marked heterogeneity in the mean telomere fluorescence. This heterogeneity affected both chromosome 2 and 11 telomeres and was also observed in cells derived from littermate embryos. This variation in telomere length could be the basis for the variable penetrance of the phenotypes described in 6th generation mTER −/− mice . Section title: Telomere Dynamics of Individual Chromosomes in Spontaneously Immortalized mTER − /− Cell Lines Educational score: 4.276100158691406 Domain: biomedical Document type: Study Language: en Serial passage of mouse embryonic fibroblasts allows the selection of oligoclonal populations with the capacity to stably proliferate in culture. We had previously described that serial passage of mTER −/− MEFs according to a 3T3 protocol resulted in the selection of immortal cell lines in a manner similar to that of mTER +/+ MEFs, indicating that telomerase activity is not essential for the immortalization of mouse cells . Although some differences were observed in the growth rate between wt and mTER −/− cell lines, all cultures have shown a continued growth that exceeded 500 PDs for G1 MEFs and 250 PDs for G6 MEFs (not shown). To understand the basis for the continuous growth of these telomerase negative cells we have analyzed their telomere dynamics. Fig. 2 A shows the mean telomere fluorescence of q- (black bars) and p-telomeres (gray bars), separately, during increasing PDs of wt and mTER −/− cells. We calculated that telomeres of wt cells, Wt14, underwent a modest shortening at an estimated rate of 24.8 bp per PD . The occurrence of telomere shortening in wt MEFs could indicate that the level of telomerase activity present in these cells is not sufficient to prevent telomere erosion as it has been proposed previously for other cell types or, alternatively, that telomere length in these cultured cells is not tightly regulated around a fixed length. Section title: Telomere Dynamics of Individual Chromosomes in Spontaneously Immortalized mTER − /− Cell Lines Educational score: 4.367621421813965 Domain: biomedical Document type: Study Language: en In contrast to wt cells, mTER −/− cell lines derived from 1st (KO16-G1 and KO19-G1), 2nd (KO2-G2), and 4th generation (KO7-G4) embryos, showed a marked decrease in the telomere fluorescence of both p- and q-telomeres . The estimated average telomere loss in the different cell lines ranged between 65 and 108 bp per PD, similar to the shortening rate described for human cells that do not express telomerase. This indicates that in these mTER −/− cells that had escaped senescence and are immortal, the mean telomere length continues to shorten with increasing passage number. Interestingly, the rate of telomere shortening at both p- and q-telomeres decreased at later passages (PDs 215 and 322) of the KO16-G1 cell line, suggesting the activation of telomere maintenance mechanisms when telomeres shorten to a critical length . In the case of two different mTER −/− cell lines, KO9-G6 and KO11-G6, derived from 6th generation embryos, the telomere fluorescence at both p- and q-telomeres was maintained or increased during the different PDs analyzed . In KO9-G6 cells, p- and q-telomeres were maintained at an average length of 10.8 and 24.8 kb, respectively, and in KO11-G6 cells at an average length of 16.3 and 25.7 kb. These observations point to telomerase-independent mechanisms for the maintenance of telomeres in the immortal cells derived from the 6th generation mTER −/− MEFs. Section title: Telomere Dynamics of Individual Chromosomes in Spontaneously Immortalized mTER − /− Cell Lines Educational score: 4.239595413208008 Domain: biomedical Document type: Study Language: en Representative FISH images of metaphase spreads from wt and mTER −/− cell lines at early and late passages are shown in Fig. 3 . As previously described for immortal MEF cultures , most of the cell lines studied here were aneuploid at late passages . Fig. 3 A shows two metaphases of Wt14 cells before, PD 2, and after immortalization, PD 243. At PD 243, all chromosome ends had TTAGGG repeats and the cells did not show an increase of end-to-end fusions except for a very long chromosome that was clonal . Metaphases of KO16-G1 and KO7-G4 mTER −/− cell lines show a decrease in telomere fluorescence when early and late PDs are compared. In contrast, KO9-G6 cells showed a similar telomere fluorescence signal at both early, PD 2, and late, PD 88, PDs, in agreement with the observation that the mean telomere length is maintained in these cells . Finally, all mTER −/− cell lines contain many chromosomes lacking detectable telomere signal at late PDs, as well as a significant increase of end-to-end fusions . Section title: Telomere Dynamics of Individual Chromosomes in Spontaneously Immortalized mTER − /− Cell Lines Educational score: 4.203685760498047 Domain: biomedical Document type: Study Language: en We have also studied the telomere fluorescence of chromosomes 2 and 11 as a function of the accumulated number of cell doublings . When telomere fluorescence of both chromosomes 2 and 11 was measured in the wt cell line Wt14, we observed a slight decrease with increasing PDs, . The calculated average rate of telomere shortening for chromosomes 2 and 11 in the Wt14 cells was 10 and 11 bp per PD, respectively. Interestingly, when we studied telomere dynamics of chromosome 2 and 11 in the different generation of mTER −/− cell lines, we could not detect the predicted pattern of telomere shortening with increasing PDs . The length of p- and q-telomeres at chromosomes 2 and 11 with increasing PDs suggests the activation of telomere maintenance mechanisms at different points during the growth of the cell lines. In this regard, it is interesting to note that in KO16-G1 cells, 11q-telomeres did not shorten from PD 19 to PD 81 or from PD 215 to PD 322. However, 11p-telomeres continued to shorten to an average length of only 5.6 kb at PD 215 and then were stabilized. The involvement of 11p telomere in the chromosomal instability of this cell line will be discussed later in this paper. The maintenance of telomere length was particularly clear in cumulative PDs of the two cell lines derived from the 6th generation mTER −/− embryos . Interestingly, in these cells telomeres from different chromosomes were maintained at different length and, in general, chromosome 2 telomeres were stabilized at shorter lengths than chromosome 11 telomeres . Section title: Telomere Dynamics of Individual Chromosomes in Spontaneously Immortalized mTER − /− Cell Lines Educational score: 4.168393135070801 Domain: biomedical Document type: Study Language: en Fig. 2 D shows the distribution of fluorescence intensity values for 2q, 2p, 11q, and 11p telomeres with increasing PDs in wt (Wt14) and in mTER −/− cell lines from the first (KO16-G1) and from the 6th (KO9-G6 and KO11-G6) generation. The telomere fluorescence in wt cells at 2p, 2q, 11p, and 11q telomeres with increasing PDs remained similar, in agreement with the fact that, overall, telomeres were maintained in this cell line. In contrast, the number of telomeres with low fluorescence values (0–10 TFU) increased with passage number in the mTER −/− cell lines. Interestingly, in the mTER −/− cell lines the heterogeneity in fluorescence intensity values increased with increasing PDs for some telomeres (i.e., 11q telomeres in KO9-G6 cell line), again suggesting the existence of alternative telomere maintenance mechanisms in these cells. Section title: Analysis of End-to-End Fusions Educational score: 4.220303535461426 Domain: biomedical Document type: Study Language: en To analyze the nature of the chromosomal fusions promoted by the absence of telomerase, we performed FISH on wt and mTER −/− metaphases using telomeric and centromeric probes, as well as chromosomes 2 and 11 painting probes (Materials and Methods). Fig. 4 shows the diagrams of the different fusions characterized in this study together with representative images. End-to-end fusions were classified into different types according to their structure as shown in Fig. 4 . Types I, II, and III involve p-to-p arms fusions. Type I fusions contain telomeric repeats at the fusion point and two copies of minor satellite centromere repeat sequences (b). Type II fusions do not contain detectable telomeric sequences at the fusion point and yield two centromere signals (b). Type III fusions lack telomeric signals at the fusion point and only have one centromere signal (b). Type IV and V fusions involve q-to-q arm fusion, and have or lack detectable telomeric signals at the fusion point, respectively. Finally, type VI involves p-to-q arm fusion. In some cases, we performed chromosome painting to determine whether the fusions were homologous (for example, chromosome 2-to-chromosome 2 in panel c of type II fusions) or nonhomologous (for example, chromosome 11 to an undetermined chromosome in panel c of type VI fusions). Section title: Chromosomal Instability in Primary mTER − /− Cells Educational score: 4.37747859954834 Domain: biomedical Document type: Study Language: en No fusions were detected in metaphases analyzed from early passage primary wt cells (Table I ). In the case of mTER −/− primary cells, the frequency of fusions increased significantly from 0.07 fusions per metaphase in mTER −/− cells from the 1st generation (KO19-G1) to an average of 1.04 fusions (range from 0.5 to 1.72) per metaphase in seven independently derived mTER −/− cells from 6th generation embryos (KO9-G6, KO11-G6, and KO1-G6 to KO5-G6; Table I ). Interestingly, cytogenetic analysis of the cells derived from seven independent 6th generation embryos revealed that an average of 41% of the fusions was type II fusions. Chromosome painting showed that 70% of these type II fusions were homologous fusions involving 2p and only 2% involved chromosome 11. The dramatic increase in chromosome 2 but not chromosome 11p-arm fusions in 6th generation mTER −/− cells, is probably the consequence of 2p-telomeres shortening from 26.0 ± 2.8 kb in the wt cells, to an average of 7 kb, shorter than the average of all telomeres in cells from the 6th generation. In this regard, in 6th generation cells derived from embryo KO9-G6, 2p-telomeres were only an estimated 0.15 kb long and 100% of type II fusions were 2p-to-2p fusions. The homologous nature of these fusions indicates that they are likely to be the result of a failure to separate sister chromatids during mitosis . Interestingly, fusions involving chromosome 2 were stably maintained in two different G6 cell lines studied, KO9-G6 and KO11-G6, at least for >80 PDs (Table II , see Discussion). Other fusions found in primary KO9-G6 cells included type I fusions (35%), and less frequently, type III (8%) and type V (18.4%) fusions . Taken together, chromosome 2 seems to be more frequently involved in chromosome fusions than other chromosomes in the mTER −/− MEFs, although we can not rule out that other chromosomes might occasionally be involved in fusions in mTER −/− cells . Section title: Chromosomal Instability in mTER − /− Cell Lines Educational score: 4.152315139770508 Domain: biomedical Document type: Study Language: en To study the consequences of continuous proliferation in the absence of telomerase activity on chromosomal stability, we also analyzed chromosomal aberrations in wt and mTER −/− cell lines as a function of the accumulated number of PDs (Table II ). The wt cell line, Wt14, did not show any end-to-end chromosome fusions in the first 20 PDs, except for a very long chromosome that was clonal. We determined that this long chromosome was the result of a terminal translocation between chromosome 11 and another chromosome , and was stably transmitted throughout all the PDs analyzed. In later passages of the Wt14 cell line (PD 350), a low percentage of p-arm fusions, 0.2 per metaphase, was also detected. These fusions could be a consequence of the moderate telomere shortening detected in these cells (see above). Section title: Chromosomal Instability in mTER − /− Cell Lines Educational score: 4.38979959487915 Domain: biomedical Document type: Study Language: en A dramatic increase in end-to-end fusions was observed with increasing PDs in all the mTER −/− cell lines studied . This chromosomal instability furthermore increased with the generation number (Table II ). The high frequency of p-arm fusions (89%) versus q-arm fusions (11%) in the cell lines could be due to the fact that mouse p-telomeres are shorter than q-telomeres. Whereas type I and type II fusions were the most common fusions in primary cells (Table I ), type III fusions (with only one pair of centromere signals) were the most abundant in the cell lines (65%; see also Table II ). Interestingly, 80% of the type II fusions detected in the two different G6 cell lines, KO9-G6 and KO11-G6 involved chromosome 2, in agreement with the observation that chromosome 2 telomeres were shorter than the average of all telomeres in the 6th generation MEFs and/or that there is an increased stability of fusions involving chromosome 2 relative to fusions involving other chromosomes. By chromosome painting, we determined that type III fusions present in mTER −/− cell lines, usually involve nonhomologous chromosomes. Interestingly, 20% of these fusions involved chromosome 11p fused to other chromosomes. In the KO16-G1 PD 81 cell line, >75% of all type III fusions involved chromosome 11, in agreement with the fact that 11p-telomeres were specially short in this particular cell line . Type VI fusions, visualized as chromosome rings, were also present in mTER −/− cell lines . Other chromosomal rearrangements appear at late passage in mTER −/− cells. For example, in the case of KO16-G1 (PD 215) cells, these rearrangements included reciprocal and terminal translocations involving chromosomes 2 or 11. The frequency of such chromosome exchanges was 0.06 and 0.1 per metaphase, respectively (not shown). Section title: Chromosomal Instability in mTER − /− Cell Lines Educational score: 4.293524742126465 Domain: biomedical Document type: Study Language: en Fusion between nonhomologous chromosomes could originate by the simultaneous existence of two different chromosomes with critically short telomeres. To estimate the minimal telomere length that triggers chromosome fusions, we have calculated the mean telomere length of intrachromosomal telomere repeats in all fusions involving the p-arm of one chromosome and q-arm of a different chromosome and in terminal translocations detected at PD 215 and at PD 322 of the KO16-G1 cell line (Type I fusions were excluded from the analysis). The average length of intrachromosomal telomere repeats (not considering the type I fusions) was 2.3 kb (ranging between 0.1 and 5.7 kb), indicating that this length is not sufficient to prevent chromosome fusions in mouse cells. Altogether, these results suggest that end-to-end fusions and other chromosomal aberrations detected in the mTER −/− cell lines are the result of telomere shortening to a critical length, and that chromosomes 2 and 11 are commonly involved in these fusions. Section title: Discussion Educational score: 4.301327705383301 Domain: biomedical Document type: Study Language: en To study the role of telomeres and telomerase, we have analyzed telomere length and chromosomal stability in mouse cells deficient for telomerase activity. Previously, we have shown that the average telomere length of mice genetically deficient in the RNA component of telomerase (mTER) decreases with each generation number . However, somatic cells derived from these mice have been able to grow in culture beyond 500 PDs and were shown to be transformed and form tumors in nude mice . These findings have raised the question of how telomeres are maintained in late generation mTER −/− cells in the absence of telomerase. Indeed, deletion of the mTER gene in embryonic stem (ES) cells was recently reported to result in a severe growth defect after >300 divisions, suggesting that the growth of ES cells is telomerase dependent . In our study we found no growth inhibition in primary somatic cells from mTER −/− mice, suggesting that the telomerase requirement for continued growth may vary between cell types. To address questions about the mechanism of telomere maintenance in primary somatic cells from mTER −/− mice, the length of individual telomeres was measured using quantitative FISH and chromosome painting. Chromosome painting with whole chromosome probes unequivocally identifies mouse chromosomes and has allowed us to follow telomere length of particular chromosomes and their involvement in chromosome fusions. Section title: Telomere Dynamics in Primary Cells Educational score: 4.335046768188477 Domain: biomedical Document type: Study Language: en We concentrated our attention on chromosomes 2 and 11. Initial banding analysis of mTER −/− 6th generation MEFs used in an earlier study suggested that chromosome 2 was frequently involved in fusions (unpublished results). Therefore, we sought to analyze the telomere length dynamics of this chromosome in mTER −/− cells. Chromosome 11 is the mouse homologue of human chromosome 17. Recently, it was found that chromosome 17p has relatively short telomeres in normal human cells . The average telomere length of all chromosomes decreased uniformly at a rate of 3.9 kb per mouse generation in a number of independent groups of telomeres such as p-telomeres and q-telomeres, as well as individual 2p- and 2q-telomeres. This uniform and constant rate of shortening indicates that probably all telomeres are subjected to the same erosive processes in vivo. In the case of chromosome 2p, telomeres shortened to an average length of 7 kb in the 6th generation; however, some 6th generation cells (KO9-G6) had an average 2p telomere length of only 150 bp. Heterogeneity in telomere length was also observed for 2q, 11p, and 11q telomeres in different KO-G6 mice. Such variation between littermates could be the basis for the fact that only a percentage of KO-G6 mice show defects associated with telomere loss . Interestingly, the average telomere length of chromosome 11 increased ∼6 kb from the 4th to the 6th generation. This telomere lengthening in the absence of telomerase activity is suggestive of alternative telomere maintenance mechanisms. Such mechanisms may act in a telomere chromosome-specific manner because telomere 11p and 11q but not chromosome 2 telomeres showed this type of elongation upon analysis of the average telomere length in seven different 6th generation mTER −/− embryos. Section title: Telomere Dynamics in Immortal Cells Educational score: 4.334665775299072 Domain: biomedical Document type: Study Language: en Additional evidence for the operation of telomerase-independent telomere maintenance mechanisms was obtained from the study of serially passaged cells. Serial passage of mouse embryonic fibroblasts allows the selection of oligoclonal populations with the capacity to stably proliferate in culture. This experimental system allows the analysis of telomere dynamics in a context less restrictive than the entire organism. The telomere length in wt embryonic fibroblasts was stabilized after 100 PDs after an initial modest decline. In contrast, the average telomere length of mTER −/− cultures derived from embryos of the 1st or 4th generation continued to decrease. As the cells spontaneously escaped senescence (around PD 10), this shortening occurred at a similar rate to the one estimated in primary cells during successive generations of mTER −/− mice . When individual telomeres were followed during successive passages, a variable telomere shortening was observed, and not all telomeres were affected to the same extent. These observations support the existence of telomerase-independent and chromosome-specific telomere maintenance mechanisms in these cells. Section title: Telomere Dynamics in Immortal Cells Educational score: 4.473415374755859 Domain: biomedical Document type: Study Language: en Previous studies in yeasts have suggested the existence of telomerase-independent mechanisms that are sufficient to ensure yeast viability . Moreover, there are a variety of human established cell lines that do not show detectable telomerase activity . Telomere maintenance in these cells has been proposed to occur through alternative mechanisms . The fact that human ALT cells were not engineered to be genetically deficient for telomerase genes left open the possibility that discrete bursts of telomerase activity could be compensating for telomere shortening in these cells. However, our studies in mTER −/− mouse cells unequivocally prove the existence of mechanisms in mammalian cells that are capable of maintaining telomeres in the absence of telomerase. The genes responsible for telomerase-independent telomere maintenance are currently not known. Several genes involved in DNA recombination, i.e., Rad 52 or in DNA repair, such as DNA-PK, ATM, Rad 3, tel1+, and TEL 1, have been proposed as candidates because their mutation results in accelerated loss of telomeres . Telomerase-deficient mTER −/− cells are a good system to identify genes involved in telomere maintenance that could be essential to sustain tumor growth in the absence of telomerase. The targeting of both telomerase and gene products involved in alternative telomere-maintenance mechanisms could be used to prevent cell proliferation beyond a threshold telomere length. Such telomere-directed strategies may offer a promising approach to cancer therapy. Section title: Telomere Shortening and Chromosomal Instability Educational score: 4.265567779541016 Domain: biomedical Document type: Review Language: en In 1941, Barbara McClintock made the observation that chromosomes that lack telomeres have a tendency to fuse and concluded that one of the functions of the telomeric complex is to prevent end-to-end fusions of chromosomes. It has been shown recently that telomere binding proteins are of crucial importance to prevent end-to-end fusions in human chromosomes . In addition, the study of Tetrahymena mutants with an altered telomerase RNA template suggests an important role of telomeres in chromatid separation during anaphase . Similarly, other studies have suggested a role of telomeres and telomere binding proteins in chromosome segregation and meiosis . Section title: Telomere Shortening and Chromosomal Instability Educational score: 4.419860363006592 Domain: biomedical Document type: Study Language: en We have performed a detailed cytogenetic analysis to examine the relationship between chromosomal fusions and telomere shortening in mammalian cells. We could not observe chromosomal fusions in primary cells derived from wt embryos, thus estimating that the frequency of fusions is lower than 0.04 per metaphase. In contrast, primary cells derived from mTER −/− embryos showed a dramatic increase in chromosomal fusions. The frequency of chromosomal fusions increased with the generation number from <0.04 fusions per metaphase in wt mice to 1.72 fusion per metaphase in some mTER −/− cells from the 6th generation (∼40 times the estimated maximum frequency in wt mice). We performed a similar study in serially cultured cells. The loss of control of the chromosomal number or aneuploidy is an alteration commonly observed in cultured cells . Indeed, most of the established cell populations that we have generated in this study were aneuploid, independently of the presence or absence of telomerase activity. Despite being aneuploid, established wt cells presented a very low level of chromosomal fusions (0.20 fusions per metaphase in cultures that have undergone 350 PDs). In comparison, mTER −/− cells showed a very high proportion of chromosomal fusions (for example, 8–9 fusions per metaphase in mTER −/− cultures of the 1st generation that have undergone 325 PDs), >40-fold the number of fusions found in wt cell lines. These observations indicate that telomere shortening promotes the accumulation of chromosomal fusions both in vivo and in vitro. Recently, it was shown that the junctions of human dicentric chromosomes are typically characterized by the absence or a low number of telomere repeats , providing further support for a critical role of telomere shortening in chromosomal instability. Section title: Telomere Shortening and Chromosomal Instability Educational score: 4.333632469177246 Domain: biomedical Document type: Study Language: en To investigate the mechanisms by which telomeres prevent end-to-end fusions, we performed a detailed FISH analysis of the chromosomal fusions observed in mTER −/− cells using whole chromosome and centromeric minor satellite probes. The results obtained from this study are different from the initial characterization of end-to-end fusions carried out in mTER −/− mice . In these studies, chromosome 3 was found to be frequently involved in fusions of chromosomes derived from splenocytes of one mTER −/− G6 mouse. Possible explanations for this discrepancy range from cell type specificity to technical artefacts. In our study with primary mouse embryonic fibroblasts, most of the end-to-end chromosome fusions detected in mTER −/− primary cells from the 6th generation, i.e., KO9-G6, unequivocally involved 2p-telomeres as determined by chromosome painting and in agreement with the fact that 2p-telomeres were shorter than average in these cells. Moreover, the emergence of 2p-to-2p fusions with increasing passage number in the KO11-G6 cell line also supports the involvement of chromosome 2 in the initial chromosomal instability detected in mTER −/− cells. It is tempting to speculate that the frequent occurrence of this particular abnormality is related to the relatively short length of telomeres on this particular chromosome arm in several mouse strains (Hande, M.P., M.A. Blasco, and P. Lansdorp, unpublished observations) as it seems unlikely that fusions involving 2p may have occurred as isolated clonal events in seven independently derived embryos. While the majority of the primary fusions seem to involve chromosome 2, we cannot rule out the involvement of other chromosomes, such as chromosome 3 or 14 . Section title: Telomere Shortening and Chromosomal Instability Educational score: 4.628020763397217 Domain: biomedical Document type: Study Language: en As illustrated in the model shown in Fig. 5 , telomere loss or telomere shortening to a critical length could trigger either end-to-end fusions between sister chromatids or a failure to separate them during mitosis. As a consequence of either situation, the sister chromatids could be drawn to the same spindle pole at anaphase resulting in chromosome nondisjunction or missegregation . Alternatively, the fusion product could break at fragile sites and trigger breakage-fusion-bridge cycles. In the first case, one of the daughter cells (daughter cell 1 in the model) will end up with a Robertsonian fusion-like chromosome (fusion types I or II) or a dicentric chromosome (fusion types IV or V), and the other cell (daughter cell 2) will lose the chromosome. Further rounds of cell division might result in the maintenance of the fusion provided that the sister chromatids segregate normally with two centromeres, or that one of the centromeres is inactivated (type III fusions). In this regard, in 6th generation mTER −/− cells 16% of the p-fusions are type II fusions and were stably transmitted for >88 PDs. Interestingly, q-arm fusions were very infrequent in mouse cells. Although the fact that q-arm telomeres are generally longer than p-arm telomeres may in part responsible for this observation, q-arm fusions may also be more likely to form an anaphase bridge and may thus be less stable than p-arm fusion products. Altogether, these results indicate that telomeres protect chromosomes from end-to-end fusion events in vivo by maintaining telomere length above a critical threshold. This observation supports the important role of telomeres in chromosome segregation as previously proposed . Section title: Telomere Shortening and Chromosomal Instability Educational score: 4.225902557373047 Domain: biomedical Document type: Study Language: en When chromosomal instability was studied in the spontaneously immortalized mTER −/− cell lines, we found a dramatic increase in nonhomologous type III end-to-end fusions, in p-to-q arms fusions and in various types of translocations. Again, 2p and 11p telomeres were involved in many of these fusions in the different cell lines studied (derived from different embryos), suggesting that the involvement of these chromosomes in fusions is a general phenomena for mouse fibroblasts. The existence of these chromosomal abnormalities in mTER −/− cell lines can be explained by breakage-fusion-bridge cycles triggered by different chromosomes with critically short telomeres . Section title: Telomere Shortening and Chromosomal Instability Educational score: 4.343197822570801 Domain: biomedical Document type: Study Language: en In general, the types of chromosomal aberrations detected in mTER −/− cell lines are similar to the ones described in tumor cells, supporting the notion that telomere loss is one of the main inducers of chromosomal instability in tumors . In agreement with this, tumors generally have short telomeres probably because telomere loss is not compensated at the earlier steps of tumorigenesis. Telomere shortening would lead to chromosomal instability associated with end-to-end fusions and translocations of the types described in this work, favoring the allelic loss of tumor suppressor genes, such as p53 in the case of mouse chromosome 11 or human chromosome 17 . Our data agrees with the idea that when telomeres are critically short and there is a high degree of chromosomal instability telomere-maintenance mechanisms are activated and selected . These mechanisms, which can be either telomerase dependent or independent , can prevent telomeres from further shortening, allowing the immortal growth of cells that have critically short telomeres and a high degree of genetic instability. In this regard, mTER −/− mice from late generations show an increased cancer incidence than the wt counterparts . | Study | biomedical | en | 0.999996 |
10037784 | Section title: Cloning of Xenopus ADAR1.1 and ADAR1.2 Educational score: 4.273192405700684 Domain: biomedical Document type: Study Language: en Part of a Xenopus ADAR1 cDNA was isolated from an expression screen of a lambda Zap cDNA library for RNA-binding proteins . Sequence analysis identified this cDNA as a homologue of human ADAR1 from which the 5′ end was missing. Rescreening of a cDNA library and 5′ RACE protocols resulted in the isolation of two variants of Xenopus ADAR1 that were identical to the previously published Xenopus ADAR1.1 and ADAR1.2 sequences. Whereas ADAR1.1 had a full-length open reading frame, ADAR1.2 had no AUG translational initiation codon. To allow expression of this clone, a self complementary oligonucleotide (5′-CTA GCG TGT AAT GCA TTA CAC G-3′) was inserted in frame upstream of the cDNA in the SpeI restriction site of the pBluescript polylinker. Section title: Antibody Production Educational score: 4.119091510772705 Domain: biomedical Document type: Study Language: en For antibody production a 564-bp long EcoRI fragment encoding amino acids 373–561 in ADAR1.1 was cloned into pGEX 1 vector ( Pharmacia ) from where the fragment was expressed as a glutathione S transferase (GST) fusion protein in E . coli BL21. The fusion protein was purified on glutathione Sepharose beads ( Pharmacia ) according to the manufacturer's protocol. Eluted fusion protein was at least 99% pure as judged on overloaded Coommassie stained gels. Fusion protein was dialyzed against 0.05% TFA in H 2 O and lyophilized. Two rabbits were immunized and boosted in 4-wk intervals. Test sera were taken 2 wk after each injection. After two booster injections antisera from both rabbits could recognize endogenous ADAR1 on Western blots at which time point animals were killed and all serum was collected. Antisera were produced by Eurogentec. Section title: Myc-tagging of ADAR1.1 and ADAR1.2 Educational score: 4.195824146270752 Domain: biomedical Document type: Study Language: en ADAR1.1 was tagged with six tandemly arranged myc epitopes at either the 5′ end the 3′ end or at both ends. To do this the ∼250 bp long region encoding the six myc tags was cloned in frame upstream, downstream or at both ends of the ADAR1.1 cDNA. ADAR1.2 cDNA was only tagged at its 3′ end. After insertion of the AUG start codon at the 5′ end of the cDNA the myc-encoding sequence was cloned in frame downstream of the ADAR1.2 cDNA sequence. Additionally, to stabilize in vitro synthesized RNAs when injected into oocytes, the 3′ UTR including a poly(A) + tail of the Xenopus NO38 cDNA was cloned at the 3′ end of all tagged ADAR variants . Section title: Construction of ADAR1.1 Deletions Educational score: 4.170749664306641 Domain: biomedical Document type: Study Language: en Construct ΔREP was made from a partial cDNA obtained from our original phage screen. This construct deletes the first 250 codons of the ADAR1.1 cDNA. ΔREP was myc tagged at its COOH terminus and the NO38 poly(A) + tail was added at its 3′ end . A self complementary oligonucleotide containing an AUG codon was introduced in-frame upstream of the cDNA in the SmaI site of the pBluescript polylinker (5′-GAT GCA TC-3′). Section title: Construction of ADAR1.1 Deletions Educational score: 4.120370388031006 Domain: biomedical Document type: Study Language: en Construct ΔZBD was made by digesting the ADAR1.1 clone containing a COOH-terminal myc-tag with NdeI and AvaI (partial). After polishing the ends the DNA was religated. This construct deletes 363 codons from the 5′ end of the ADAR1.1 cDNA. Translation starts at an internal Met at codon 364. Section title: Oocyte Injections Educational score: 4.126044273376465 Domain: biomedical Document type: Study Language: en Myc-tagged ADAR1 variants were linearized at a unique restriction site downstream of the NO38 poly(A) + tail. Capped run off transcripts were synthesized in vitro from the linearized templates using T3 RNA polymerase. Aliquots of all RNAs were checked for integrity on RNA gels by ethidium bromide staining. Oocytes were injected with 50 ng RNA per oocyte and incubated at 16°C for 48–72 h to allow protein synthesis to occur. Section title: Oocyte Injections Educational score: 4.100858211517334 Domain: biomedical Document type: Study Language: en 25 to 50 ng of oligonucleotides anti-U2b or unrelated oligonucleotides (M13-20 5′-GTAAAACGACGGCCAGT-3′, MJ 183 5′-ACGGAGGATCCAATGAGTGAAGAGGAGCA-3′) were injected into the cytoplasm of oocytes. Inhibition of transcription could be followed immediately by microscopic observation of lampbrush chromosome (LBC) preparations. Oocytes were subsequently incubated at 16°C for 24 h after which time transcription resumed. For double injection experiments with oligonucleotides and myc-tagged ADAR1.1 oocytes were injected with the oligonucleotide 6 h before injection of the RNA. Section title: Oocyte Injections Educational score: 3.4395313262939453 Domain: biomedical Document type: Study Language: en Destruction of U2 snRNA was monitored by Northern blotting as described . Section title: Inhibition of Transcription Educational score: 4.078975200653076 Domain: biomedical Document type: Study Language: en Transcription was inhibited either by oligonucleotide injection (see above) or by incubation of oocytes in AMD or alpha-amanitin at concentrations of 50 μg/ml or 400 μg/ml, respectively, in OR2 medium (82.5 mM NaCl, 2.5 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 1 mM Na 2 HPO 4 , 5 mM Hepes, pH 7.8). Oocytes were typically held for 12–24 h in the presence of those inhibitors before they were used for chromosome preparations and immunofluorescence staining. In some cases incubation was extended for up to 5 d. Also, to ensure uptake of the drugs, some oocytes were injected with AMD or alpha-amanitin stock solutions. In all cases transcription seemed completely inhibited. Section title: LBC Preparations and Immunofluorescence Stainings Educational score: 4.1804423332214355 Domain: biomedical Document type: Study Language: en LBC preparations and immunofluorescence stainings were performed as described in Wu et al. . For RNAse treatment, preparations were digested after centrifugation but before fixation in paraformaldehyde with a mixture of RNAse A (1 mg/ml) and RNAse T1 (10,000 U/ml) at 37°C for 30 min. Subsequently, slides were washed in PBS and fixed for an additional hour in 2% paraformaldehyde in PBS before antibody staining. Antibodies used were Sat3 and Sat4 preimmune and immune sera, directed against Xenopus ADAR1; mAb Y12, directed against the Sm core proteins found on most splicing snRNPs ; mAb K121, directed against the trimethyl guanosine cap present on most splicing snRNAs ; mAb H14 and mAb CC3, directed against PolII ; affinity-purified CBP20 antiserum ; and mAb 9E10 directed against the myc tag . For single immunofluorescence labeling primary antibodies were detected with a secondary FITC-labeled antibody. For double labeling experiments the rabbit polyclonal sera were detected with a rhodamine-labeled secondary antibody while mouse mAbs were detected with a FITC-labeled antibody. Pictures were taken on a Zeiss fluorescence microscope equipped with DIC on Kodak Tmax 100 film which was pushed during development to 400 ASA. Section title: Western Blots Educational score: 4.127357482910156 Domain: biomedical Document type: Study Language: en For Western blots, oocytes were hand enucleated and nuclei (GVs) and cytoplasms were collected separately. Typically five GVs and five cytoplasms were loaded per lane on a 7% SDS-PAGE. Gels were blotted to Immobilon P membranes ( Millipore , MA). Myc-tagged proteins were detected with mAb 9E10 and an alkaline phosphatase labeled goat anti– mouse antibody (Pierce) that was detected with the NBT-BCIP substrate. Endogenous proteins were detected with Sat3 or Sat4 antisera at 1:600 dilutions followed by detection with 125 I-labeled protein A ( Amersham ). Section title: Antibodies against ADAR1 Recognize both ADAR1.1 and ADAR1.2 Educational score: 4.106348037719727 Domain: biomedical Document type: Study Language: en To study the intracellular distribution of Xenopus laevis ADAR1 (xlADAR1), we generated antibodies against a 187–amino acid long fragment located in the NH 2 -terminal region of the protein. The region chosen showed only few amino acid exchanges between the conceptual translation products of both ADAR1.1 and ADAR1.2. It was thus assumed that both proteins would be recognized by antisera directed against the fusion protein. Section title: Antibodies against ADAR1 Recognize both ADAR1.1 and ADAR1.2 Educational score: 3.5942163467407227 Domain: biomedical Document type: Study Language: en Two antisera termed Sat3 and Sat4 recognized the GST fusion protein and were thus tested for their ability to detect the endogenous protein in oocyte extracts. Section title: Antibodies against ADAR1 Recognize both ADAR1.1 and ADAR1.2 Educational score: 4.314449310302734 Domain: biomedical Document type: Study Language: en Oocytes were hand enucleated and nuclei (GVs) and cytoplasms were probed by Western blotting with both Sat3 and Sat4 antisera. Both antisera showed virtually identical results and detected a single band of ∼125 kD in nuclei while no signal could be detected in cytoplasmic lanes. A single band of ∼125 kD was also detected in XlA6 cells, a Xenopus fibroblast tissue culture cell line . Both corresponding preimmune sera showed no signal (data not shown). The detected band of 125 kD is slightly smaller than the predicted molecular mass of 138 kD for xlADAR1.1. However, purified xlADAR1 also migrates with an apparent molecular mass of 120 kD . Similarly, ADAR1 proteins from other species also migrate faster than predicted from their amino acid composition . This anomalous migration has been attributed to proteolytic degradation at the NH 2 -terminal end of ADAR1 proteins, a phenomenon also observed for xlADAR1 . The presence of ADAR1 in oocyte nuclei is in good agreement with previous findings that showed that the enzyme is confined to the nucleus until GV breakdown occurs . Section title: Antibodies against ADAR1 Recognize both ADAR1.1 and ADAR1.2 Educational score: 4.159572124481201 Domain: biomedical Document type: Study Language: en To determine whether Sat3 and Sat4 antisera could detect both ADAR1.1 and ADAR1.2 proteins we performed immunoprecipitation experiments on oocytes that had been injected with mRNAs encoding either ADAR1.1 or ADAR1.2. Since ADAR1.2 lacks its own 5′ AUG codon an oligonucleotide containing a suitable translational initiation codon was cloned in frame at the 5′ end of the cDNA. Furthermore, to distinguish endogenous from injected ADAR1 proteins the cDNAs were fused in frame with a 6× myc-tag at both ends (ADAR1.1) or at the COOH terminus only (ADAR1.2). Capped mRNAs encoding the resulting fusion proteins were in vitro transcribed and injected into the cytoplasm of oocytes. After overnight incubation, nuclei were hand isolated and used for immunoprecipitation with Sat3 and Sat4 antibodies. The immunoprecipitated material was then tested for the presence of myc-tagged xlADAR1.1 or xlADAR1.2. Both antisera were capable of immunoprecipitating either protein, indicating that both proteins are recognized by both antisera . Section title: xlADAR1 Associates with the Nascent RNP Matrix and a Special Loop Educational score: 4.227620601654053 Domain: biomedical Document type: Study Language: en xlADAR1 is a nuclear protein. Furthermore, in some cases ADAR-mediated editing has been shown to require the presence of both exonic and intronic sequences indicating that this type of RNA editing might occur cotranscriptionally . ADAR acts exclusively on double-stranded RNAs. The interaction between ADAR and its double-stranded substrate RNAs is most likely mediated by the three double-stranded RNA-binding domains (dsRBDs) located in the central part of the protein. However, it is not known how substrate specificity is achieved as adenosines will be converted to inosines in virtually any synthetic double-stranded RNA when injected into oocytes . To test whether ADAR1 might indeed act cotranscriptionally and to determine whether ADAR1 might only associate with a specific subset of RNAs, we performed immunofluorescence stainings of spread GV contents with our ADAR1 antisera. Section title: xlADAR1 Associates with the Nascent RNP Matrix and a Special Loop Educational score: 4.321018218994141 Domain: biomedical Document type: Study Language: en Spread germinal vesicles or LBC preparations allow the detailed observation of several nuclear structures at high resolution in the conventional light microscope. First, LBCs can be well observed with a nascent RNP matrix emerging from the transcriptionally active loops which themselves protrude from the condensed chromosomal axes of the two paired, homologous chromosomes, termed bivalents. Second, ∼1,500 amplified nucleoli can be distinguished. Finally, two types of spherical structures can be distinguished in spread Xenopus GVs which, based on their association with snRNP components, have been termed B and C snurposomes . Section title: xlADAR1 Associates with the Nascent RNP Matrix and a Special Loop Educational score: 4.207254409790039 Domain: biomedical Document type: Study Language: en Staining of LBCs with both Sat3 or Sat4 antisera showed an extraordinarily prominent signal on a single set of loops located on bivalent no. 3. In addition, there was moderate staining of all other loops. Preimmune sera, in contrast, showed no chromosomal staining indicating that the signals were specific for xlADAR1 . Staining with Sat3 showed a weak background on C snurposomes that was also observed in the corresponding preimmune serum whereas staining with Sat4 showed a weak background on nucleoli also observable in the corresponding preimmune serum. Those weak background signals that were only observed at low dilutions of antisera were thus considered as nonspecific background signals. The staining of the brilliant loop was so intense that it could still be detected at antisera dilutions up to 1:3,000. In contrast, for the majority of all other loops staining was well visible at antisera dilutions of 1:500. Thus, it was hard to take photographic pictures with both the brilliant loop and the regular loops at good resolutions. Therefore, Fig. 2 shows images of bivalent no. 3 including the brilliantly labeling loop and images of other bivalents, showing the label observed on all other chromosome loops. Section title: myc-E–tagged ADAR1 Mimics the Distribution of Endogenous xlADAR1 Educational score: 4.229395389556885 Domain: biomedical Document type: Study Language: en To determine whether the observed antibody staining was indeed specific for ADAR1, we wanted to determine the localization of myc-tagged ADAR1.1. To do this, myc-tagged ADAR1.1 was expressed in oocytes by mRNA injection. The protein was well detectable on Western blots 24 h after injection of the mRNA. To obtain good in situ staining, however, it was usually necessary to incubate oocytes for at least 48 h at 16°C. As protein synthesis did not increase dramatically after 24 h (as judged by Western blots) we believe that the prolonged incubation was required to displace endogenous protein by the epitope-tagged version. Staining with the mAb 9E10 directed against the myc-tag revealed that the tagged protein colocalized with endogenous ADAR1. The majority of the regular loops were clearly stained. In addition, the brilliantly labeling loop was also intensely decorated by the myc-tagged protein, at least in most cases . This data indicates that ADAR1 indeed associates with the RNP matrix found on most loops and occurs in high concentrations on a loop on bivalent no. 3. Section title: myc-E–tagged ADAR1 Mimics the Distribution of Endogenous xlADAR1 Educational score: 4.026280403137207 Domain: biomedical Document type: Study Language: en As a further control, to show that the chromosomal staining with both Sat3 and Sat4 antisera was specific for ADAR1, we blocked both antisera with the fusion proteins used to generate antibodies. This blocking eliminated chromosomal staining almost completely, leaving only a faint signal on the intensely labeling loops on bivalent no. 3. Thus, the observed signals reflect the localization of endogenous ADAR1 . Section title: The Intensely Labeling Loop on Chromosome no. 3 Has Several Specific Features Educational score: 4.126330852508545 Domain: biomedical Document type: Study Language: en The brilliantly labeling loop on bivalent no. 3 was not always labeled by the myc-tagged ADAR1 protein, even when the majority of all other loops showed clear labeling of the RNP matrix. Labeling of this special loop not only varied with the animal used for oocyte injection but also required more time to accumulate high amounts of myc-tagged protein, indicating that ADAR1 turnover at this particular loop is somewhat slower than on other loops. Furthermore, this set of loops is also morphologically outstanding as it represents the rare occurrence of a “double loop bridge” where the chromosomal axis at the basis of the loop is interrupted, giving the impression of bridging two parts of a chromosomal axis by this loop. Therefore, we set out to analyze the brilliantly labeling loop and the regular loops showing ADAR1 staining in more detail. Section title: The Intensely Labeling Loop on Chromosome no. 3 Has Several Specific Features Educational score: 4.09246826171875 Domain: biomedical Document type: Study Language: en First, we tested whether the observed labeling of all loops was RNA dependent. Therefore, we treated LBC preparations with RNAse before staining with Sat3 or Sat4 antibodies. RNAse treatment clearly abolished all chromosomal staining with anti-ADAR antibodies, indicating that ADAR1 was indeed directly or indirectly associated with RNA . Next, we wanted to inhibit RNA synthesis with the transcriptional inhibitors actinomycin D (AMD) and α-amanitin. AMD is a DNA-binding drug that is a general inhibitor of transcription. α-Amanitin, in contrast, shows dose-dependent inhibition of transcription. At low concentrations α-amanitin is an efficient inhibitor of Pol-II while Pol-III is inhibited at higher concentrations. The two drugs were thus either added to the medium or injected into oocytes at concentrations high enough to inhibit all transcription. Oocytes treated this way were used to prepare LBCs which were subsequently stained with either Sat3 or Sat4 antibodies . Section title: The Intensely Labeling Loop on Chromosome no. 3 Has Several Specific Features Educational score: 4.223338603973389 Domain: biomedical Document type: Study Language: en An obvious sign for the inhibition of transcription is the lack of transcriptionally active chromosome loops. Staining of these preparations with DAPI shows the chromosomes to be condensed and shortened. Furthermore, as AMD also inhibits Pol-I the nucleoli show a changed morphology and appear swollen. Consistent with the lack of transcriptionally active loops, no staining of regular loops was observed with either antiserum directed against ADAR1. However, the brilliantly labeling loops on bivalent no. 3 were still intensely labeled by Sat3 and Sat4 antisera . Remarkably, this staining persisted even after 5 d of incubation in AMD or α-amanitin. Section title: The Intensely Labeling Loop on Chromosome no. 3 Has Several Specific Features Educational score: 4.1648759841918945 Domain: biomedical Document type: Study Language: en Another possibility to inhibit transcription on LBCs is the injection of oligonucleotides into the nucleus or cytoplasm of oocytes. Shortly after injection of the oligonucleotide transcription stops . 12 to 24 h after injection of the oligo transcription resumes producing large, transcriptionally active loops. Therefore, we tested several unrelated oligonucleotides for their effect on transcription and ADAR1 localization on regular loops and on the brilliantly labeling loops. The outcome of these experiments was similar to the ones obtained with chemical inhibitors of transcription. As all loops disappeared no ADAR1 staining was observed on the regular chromosomal loops. However, the brilliantly labeling loop was still clearly recognizable, both morphologically and by staining with Sat3 or Sat4 antisera, indicating that the injected oligonucleotide did not affect this special loop (data not shown). After transcription resumed, normal staining of most loops and the special loop on chromosome no. 3 could be seen . Taken together, these data suggest that whatever RNA is localized at this loop might not be transcribed by a conventional polymerase, or, alternatively, is not synthesized at this location. Section title: The Intensely Labeling Loop on Chromosome no. 3 Has Several Specific Features Educational score: 4.100386142730713 Domain: biomedical Document type: Study Language: en To test the former possibility we performed double immunofluorescence staining with mAbs H14 or CC3, both directed against Pol-II, and Sat4 antiserum . This data showed the presence of Pol-II on all regular loops, as a faint line of signal could be seen throughout the axis of loops. The brilliantly labeling loops, in contrast, showed no detectable signal with either mAb directed against Pol-II, suggesting that no Pol-II-dependent transcription occurs at this particular loop . Section title: The Intensely Labeling Loop on Chromosome no. 3 Has Several Specific Features Educational score: 4.266546249389648 Domain: biomedical Document type: Study Language: en Most transcriptionally active loops are decorated with a multitude of snRNP and hnRNP components. Interestingly, all splicing components are present on the majority of loops where they are distributed almost homogeneously . However, several loops, so-called giant loops, have been observed that lack those components found on most other transcripts. Therefore, we tested whether the brilliantly labeling loop on bivalent no. 3 might in fact represent such a loop by staining LBCs with several antibodies directed against splicing and hnRNP components. Among these was antibody K121, directed against the 3mG cap present on most splicing snRNAs, mAb Y12, directed against the Sm core proteins, the anti-SR protein antibody SC35 and an antibody directed against the cap binding protein CBP20 . All those antibodies clearly labeled all regular loops but also the brilliantly labeling loop on bivalent no. 3 which was identified by double staining the preparations with Sat3 antiserum . This indicates that, despite the lack of detectable Pol-II on the special loop on bivalent no. 3, the RNP matrix present there is associated with all components typical for Pol-II transcripts. Section title: Nuclear Splicing Is Not Required for ADAR1 Localization Educational score: 4.148200035095215 Domain: biomedical Document type: Study Language: en Editing sites in some mammalian substrate RNAs are defined by short basepaired regions formed between exonic and adjacent intronic sequences . This raises the possibility that RNA editing might be linked to splicing, at least in timing if not mechanistically. Thus, we tested whether inhibition of splicing influences the association of ADAR1 with the RNP matrix on chromosome loops. One would expect, for instance, if spliceosome formation is required for the association of ADAR with RNA that inhibition of splicing might lead to an increased or altered association of ADAR with the nascent RNP matrix. Section title: Nuclear Splicing Is Not Required for ADAR1 Localization Educational score: 4.108041763305664 Domain: biomedical Document type: Study Language: en Therefore, an antisense oligonucleotide directed against U2 snRNA was injected into oocytes which leads to destruction of U2 snRNA via RNAse H mediated cleavage . Oocytes depleted of U2 snRNA are defective in splicing. As mentioned, injection of any oligonucleotide temporarily inhibits transcription in a nonspecific manner. However, after several hours normal transcription resumes with the exception that U2 is missing from the loop matrix . Section title: Nuclear Splicing Is Not Required for ADAR1 Localization Educational score: 4.087128639221191 Domain: biomedical Document type: Study Language: en Destruction of U2 was monitored by Northern blotting of RNAs isolated from individual GVs of injected and control oocytes (data not shown). After transcription resumed, LBCs were tested for the localization of ADAR1 by staining with Sat3 antiserum . Interestingly, no difference in ADAR1 localization could be observed in U2 depleted oocytes, indicating that splicing and spliceosome formation is not required for the association of ADAR1 with the nascent RNP matrix. Section title: The NH 2 -terminal Peptide Repeats and a Putative Z-DNA Binding Domain Are Not Required for Chromosomal Localization Educational score: 4.230319976806641 Domain: biomedical Document type: Study Language: en Although ADAR1.1 and ADAR1.2 show a high degree of sequence identity in their central region and at their COOH termini, their NH 2 termini differ considerably . Part of this difference can be attributed to the presence of an 11–amino acids long repeat that is present in 14 almost perfect tandemly arranged copies in ADAR1.1 but only in a single copy in ADAR1.2. To test whether these peptide repeats are required for proper association of ADAR1 with the RNP matrix we have analyzed the nuclear distribution of myc-tagged ADAR1.2 and ADAR1.1 from which the NH 2 -terminal end including the peptide repeats had been removed . When compared for their in situ localization on Xenopus LBCs, both clones showed an identical distribution: Strong labeling of the special loop was observed whereas moderate labeling was detectable on all other loops . This indicates that the peptide repeats have no influence on the intranuclear association of ADAR1 with chromosome loops. Section title: The NH 2 -terminal Peptide Repeats and a Putative Z-DNA Binding Domain Are Not Required for Chromosomal Localization Educational score: 4.2600507736206055 Domain: biomedical Document type: Study Language: en The 11–amino acid long peptide repeats are followed by a 70–amino acid long region that is highly conserved among all ADAR1 isoforms from various species including Xenopus ADAR1.1 and ADAR1.2. This sequence motif isolated from human ADAR1 has been shown to bind Z-DNA in vitro and has thus been suggested to be required for the localization of ADAR1 to transcriptionally active DNA that would facilitate the association of the protein with nascent transcripts . To test this hypothesis, we constructed a myc-tagged version of Xenopus ADAR1 from which the entire NH 2 -terminal end including the putative Z-DNA binding region had been deleted . In situ, this deletion variant localized like wild-type ADAR1 indicating that the Z-DNA binding activity is not required for the recruitment of the protein to actively transcribing chromosome loops . Section title: The NH 2 -terminal Peptide Repeats and a Putative Z-DNA Binding Domain Are Not Required for Chromosomal Localization Educational score: 4.233489513397217 Domain: biomedical Document type: Study Language: en Interestingly, this deletion variant also removes one of two putative nuclear localization signals from the predicted protein sequence . However, Western blots of oocytes injected with this construct still showed clear nuclear accumulation of the protein . As this deletion still localizes to chromosomes and enters the nucleus we can conclude that this first NLS sequence located between amino acids 291 and 307 in ADAR1 is not required for nuclear localization of the protein. Section title: ADAR1 Undergoes NH 2 -terminal Proteolytic Cleavage Educational score: 4.179665565490723 Domain: biomedical Document type: Study Language: en ADAR1 protein purified from endogenous sources of various species is smaller than the calculated molecular mass predicted from the corresponding cloned cDNA sequences . This obvious discrepancy has been attributed to a proteolytic cleavage at the NH 2 -terminal end of the protein. It has to be noted, however, that the smaller purified protein is enzymatically active indicating that the proposed cleavage does not influence enzyme activity . Section title: ADAR1 Undergoes NH 2 -terminal Proteolytic Cleavage Educational score: 4.23732852935791 Domain: biomedical Document type: Study Language: en In the course of our experiments we have used ADAR1.1 variants that were epitope-tagged at their NH 2 terminus, COOH terminus or at both ends. Western blots of oocyte extracts that had been injected with any of those constructs confirmed the proposed cleavage of ADAR1.1 at its NH 2 terminus: oocytes injected with the NH 2 -terminally tagged construct showed a signal at 180 kD which was relatively faint. In contrast, oocytes injected with ADAR1.1 tagged at its COOH terminus or at both ends showed a much smaller signal of ∼130 kD that was much stronger than the signal obtained from the NH 2 -terminally tagged protein. Additionally, the latter constructs showed faint bands of 180 and 185 kD, respectively, representing the full-length protein . This finding is fully compatible with the proposed proteolytic cleavage in the NH 2 -terminal region of the protein. In the case of the NH 2 -terminally tagged construct we can only detect the full-length construct as the cleaved protein looses its tag. Since most of the protein is cleaved, the signal derived from this construct is relatively weak. The COOH-terminally tagged construct, in contrast, can be detected in its full-length and cleaved version, giving rise to the two bands detected in Western blots. As the majority of the protein is cleaved the larger band is much fainter than the lower band. Section title: ADAR1 Undergoes NH 2 -terminal Proteolytic Cleavage Educational score: 3.9560959339141846 Domain: biomedical Document type: Study Language: en To confirm these results we have also performed Western blots of oocytes injected with the mentioned tagged constructs on high percentage gels. As expected, the small NH 2 -terminal cleavage product of ∼25 kD can only be observed in oocytes injected with ADAR proteins tagged at their NH 2 -terminal end or at both ends (data not shown). Section title: ADAR1 Undergoes NH 2 -terminal Proteolytic Cleavage Educational score: 3.848771333694458 Domain: biomedical Document type: Study Language: en It should also be noted, that cleaved and uncleaved products could be detected both in the cytoplasm and the nucleus and that all tagged versions showed normal intranuclear distribution, indicating that the cleavage has no influence on the nuclear and intranuclear localization of ADAR1.1 . Section title: Discussion Educational score: 4.566025733947754 Domain: biomedical Document type: Study Language: en Editing by ADAR-like enzymes can be unspecific and affect many adenosine residues in a double-stranded region but can also be very specific as in the case of GluR-B mRNA where three adenosines are preferentially modified by ADAR1 or ADAR2 . Currently, it is not known how substrate specificity of ADAR1 is achieved. It could be, for instance, that the three dsRBDs of ADAR1 are solely responsible for substrate recognition. This view is supported by the finding that purified ADAR1 and ADAR2 can edit GluR-B pre-mRNA quite specifically without additional cofactors being required . Alternatively, substrate specificity could be mediated by the formation of a complex containing ADARs and other RNA-interacting proteins. However, since such complexes have not yet been reported this model is only speculative. Finally, the deaminase domain might discriminate among different substrates, a view supported by recent data on Tad1p a tRNA-specific adenosine deaminase lacking any dsRBDs . Section title: ADAR1 Localization on Transcriptionally Active Chromosome Loops Educational score: 4.3240966796875 Domain: biomedical Document type: Study Language: en We have shown that both endogenous and myc-tagged Xenopus ADAR1 are associated with the RNP matrix on LBC loops and specifically localizes to a special loop. Several conclusions can be drawn from this observation. First, ADAR1 mediated editing can, in principle, take place cotranscriptionally as the enzyme associates with the nascent RNP matrix before transcription is completed. This is particularly interesting when considering that intronic sequences define ADAR editing sites in GluR-B and serotonin 2C receptor mRNAs . Splicing is a cotranscriptional process as well . In fact, the entire splicing machinery can be found associated with most chromosome loops in Xenopus and Notophthalmus GVs showing a distribution similar to ADAR1 . Taken together, this leaves the possibility that editing substrates in Xenopus could also be defined by basepaired regions of intronic and exonic sequences. In fact, if editing takes place cotranscriptionally it could even be regulated by the rate of splicing. Section title: ADAR1 Localization on Transcriptionally Active Chromosome Loops Educational score: 4.268505096435547 Domain: biomedical Document type: Study Language: en However, oocytes depleted of their endogenous U2 snRNA showed a normal localization of endogenous and myc-tagged ADAR1, indicating that inhibition of splicing does not affect the enzyme's association with the RNP matrix. In this context, it is interesting to note that injection of anti-U2 oligonucleotides, just like injection of any other oligonucleotide, temporarily inhibits transcription leading to removal of the RNP matrix from the chromosome loops . After several hours, transcription resumes leading to well observable transcriptionally active chromosome loops. Therefore, the entire RNP matrix visible on those loops has to be assembled de novo, including ADAR1. We can thus conclude that ADAR1 assembly with hnRNAs does not require spliceosome formation. Section title: ADAR1 Localization on Transcriptionally Active Chromosome Loops Educational score: 4.388782978057861 Domain: biomedical Document type: Study Language: en The widespread distribution of ADAR1 on LBCs suggests that the enzyme's localization is not restricted to sites where substrate RNAs are being transcribed but indicates a substrate independent assembly of ADAR with the RNP matrix. No editing substrates for ADAR1 have been isolated from Xenopus . Nonetheless, as RNA editing is a rare process it is hard to imagine that all loops being associated with ADAR1 encode substrate RNAs. The general association of ADAR1 with chromosome loops therefore suggests that ADAR1-mediated editing is not regulated by the association of ADAR1 with the RNP matrix. Instead, other factors such as conformation or accessibility of the underlying RNA, or interaction with other protein components might regulate editing. However, in vitro editing reactions indicate that ADAR1 shows site-specific editing without the need for further cofactors . Section title: ADAR1 Localization on Transcriptionally Active Chromosome Loops Educational score: 4.334044933319092 Domain: biomedical Document type: Study Language: en The mechanism by which ADAR1 associates with the RNP matrix is not yet clear. Not all underlying hnRNAs might contain sufficient double-stranded structures to provide binding sites for the dsRBDs. Therefore, ADAR1 might associate with the RNP matrix as part of a multiprotein complex. Such a situation has been found for Xlrbpa, a protein exclusively consisting of three dsRBDs. Xlrbpa is associated with hnRNAs as part of an hnRNP complex . Similarly, hnRNP proteins might facilitate the interaction of ADAR1 with the RNP matrix. Nonetheless, at this point an interaction of ADAR1 with hnRNP proteins remains to be determined. It should be noted, however, that deletion of an individual dsRBD from ADAR1 prevents chromosomal localization of the protein, underscoring the importance of the RNA binding domains for proper targeting of the protein (C. Eckmann and M. Jantsch, manuscript in preparation). Section title: The Special Loop on Bivalent no. 3 Educational score: 4.214062690734863 Domain: biomedical Document type: Study Language: en ADAR1 was not only found on the majority of regular loops but was specifically enriched on a particular loop on bivalent no. 3. The concentration of ADAR1 on this loop was several-fold higher than on other loops. This finding can be interpreted in several ways. On the one hand more RNAs might be present on this special loop than on any other loop. Alternatively, the RNAs on the special loop could contain more binding sites for ADAR1. A third possibility would be that ADAR1 is localized to that loop as pure protein or a multiprotein complex without RNA. Protein storage loops have been demonstrated on the LBCs of Drosophila hydei . However, the sensitivity of the loops on bivalent no. 3 to RNAse digestion clearly demonstrates that RNAs are present and required for the localization of ADAR1 to the special loops. Section title: The Special Loop on Bivalent no. 3 Educational score: 4.301109313964844 Domain: biomedical Document type: Study Language: en However, several features of the special loop are unusual. First, the loop represents the rare case of a so-called double loop bridge where the condensed chromosomal axes is disrupted only held together by the two loops formed from each chromatid on each of the two homologous chromosomes . Second, the special loops differ morphologically when compared with regular loops. They appear somewhat smoother in texture and are more diffracting when observed by phase contrast microscopy. Finally, and most important the special loops do not stain with antibodies directed against RNA Pol-II and are insensitive to treatment with inhibitors of known RNA polymerases including injection of oligonucleotides. Section title: The Special Loop on Bivalent no. 3 Educational score: 3.6790502071380615 Domain: biomedical Document type: Study Language: en These data suggest that the RNAs present on the special loops might either be synthesized by a novel, unconventional RNA polymerase or, alternatively, might not be synthesized there but only stored or edited at that particular site. Section title: The Special Loop on Bivalent no. 3 Educational score: 4.595791339874268 Domain: biomedical Document type: Study Language: en While the first possibility appears rather unlikely, several arguments make us favor the latter hypothesis. First, our double staining experiments indicate that the composition of the RNP matrix on the special loops resembles that of other Pol-II derived hnRNPs as they contain Sm proteins, trimethyl guanosine caps, cap binding protein CBP20 and accessory splicing factors such as SC35. It is thus conceivable that a Pol-II transcript is made somewhere else and then transported to that particular loop where it is found associated with ADAR1, possibly to be edited. Such a scenario would also explain why ADAR1 localization to this loop is sensitive to RNAse digestion but not to inhibition of transcription. The hypothesis is also consistent with our finding that localization of ADAR1 to this loop is not always observed and typically requires more time than localization to the normal loops. Transport of a transcript made on a regular loop to the special loop on bivalent no. 3 would certainly take more time than association of a primary transcript with myc-tagged ADAR1 would require. In this respect, the ADAR1-positive loops resemble the so-called sequentially labeling loops (SLL) described in urodeles. The hallmark of SLLs is their failure to incorporate 3 H-labeled ribonucleosides at a fast rate and the nonhomogeneous distribution of label along the loop . Regular loops incorporate label homogeneously over their entire length, within an hour or so, at all sites where transcription is taking place. SLLs, instead, incorporate 3 H-labeled ribonucleosides very slowly beginning from the thin end of the loop axes. Over a period of several days incorporation of label continuously progresses along the loop axes, being pushed forward from the thin end always leaving a sharp margin between the labeled and unlabeled part of the loop . It has thus been suggested that SLLs do not represent sites of RNA synthesis but instead become labeled by associating with transcripts made at other sites . Therefore, it seems conceivable that the ADAR1 positive loop on bivalent no. 3 represents such a SLL which accumulates transcripts synthesized elsewhere, possibly for ADAR1 mediated editing. However, further time course experiments with both 3 H-labeled nucleosides and myc-tagged ADAR1 will be required to clarify this point. Section title: The Variable NH 2 Terminus of ADAR1 Educational score: 4.522383689880371 Domain: biomedical Document type: Study Language: en The NH 2 -terminal end of ADAR1.1 contains an 11–amino acid long peptide repeat followed by a 70–amino acid long region which, in human ADAR1, has been shown to bind Z-DNA in vitro . It has thus been suggested that Z-DNA binding might be required to target the enzyme to sites of transcription where Z-DNA conformation is frequently observed. Targeting the enzyme to sites of transcription by this means could then facilitate the enzyme's association with nascent RNAs and thus help to target newly transcribed RNAs. Our comparison of myc-tagged ADAR1.1 and ADAR1.2, and of a deletion variant lacking the putative Z-DNA binding motif clearly showed that neither the 11–amino acid long peptide motif nor the Z-DNA binding region is required for targeting of the enzyme to chromosome loops. All three constructs localized to both regular loops and to the brilliantly labeling loop on bivalent no. 3. It thus seems as if the two naturally occurring variants of ADAR1 have similar if not equal functions. The repeated, 11–amino acid long peptide motif has so far only been found in ADAR1.1 of Xenopus and might thus represent a peculiar gene only found in this pseudotetraploid species. Section title: The Variable NH 2 Terminus of ADAR1 Educational score: 3.899080514907837 Domain: biomedical Document type: Study Language: en However, while we could show that the Z-DNA binding domain is not required for ADAR1 association with the nascent RNP matrix we cannot exclude that this domain is required for enzyme function in vivo. Further studies to dissect the regions required for chromosomal localization and enzyme function of Xenopus ADAR1 are currently in progress. | Study | biomedical | en | 0.999998 |
10037785 | Section title: Cells and Cell Culture Educational score: 4.251619338989258 Domain: biomedical Document type: Study Language: en Two strains of normal human diploid myoblasts, both of satellite cell origin, were used in these studies. The strain designated 077 was a gift from R. Brown (Massachusetts General Hospital, Charlestown, MA) and was derived from skeletal muscle tissue from a 43-yr-old female with no known muscle pathologies. Myoblast strain 50Mb-1 denotes myoblast preparations flow-sorted to substantially remove contaminating fibroblasts . They were obtained from the vastus lateralis muscle of a 10-yr-old male. 50Mb-1 myoblasts were kindly provided by H. Blau (Stanford University, Palo Alto, CA). For both myoblast preparations, cells were seeded onto coverslips that had been treated by boiling in 0.1 N HCl, rinsed, and then autoclaved in 0.5% gelatin ( Sigma ). For propagation, cells were cultured at subconfluent density in serum-rich medium (see below), with medium changes every other day. To induce muscle differentiation, cultures were grown to near confluence and then maintained in low-serum medium (see below) without further medium changes until the appearance of myotubes. For 077 cells, serum-rich growth medium was low-glucose Dulbecco's modified Eagle's medium (DME-low) containing 20% fetal bovine serum (FBS; Hyclone). Low-serum differentiation medium was DME-low with 10% FBS or horse serum (HS; Hyclone Labs). Both contained 10 mg/ml gentamycin. For strain 50Mb-1, propagation medium was Ham's F-10 supplemented with 20% FCS and 1% vol/ vol chick embryo extract (60 Å ultrafiltrate, GIBCO-BRL ), whereas differentiation medium contained DME-low, 2% HS, 1 mM insulin, and 1 mM dexamethasone ( Sigma ). Both contained 100 U/ml penicillin and 100 μg/ml streptomycin. The human diploid fibroblasts WI-38 (American Type Culture Collection) were grown in high-glucose DME supplemented with 10% FBS and gentamycin. Section title: Cardiac Myosin Heavy Chain Probes. Educational score: 4.232203960418701 Domain: biomedical Document type: Study Language: en Detection of RNA from the β-cardiac myosin heavy chain gene (cMyHC), was performed using clone p8-1A, obtained from L. Leinwand, (University of Colorado, Boulder, CO) or HM-1, a ∼32 kb β-cMyHC specific genomic probe obtained from C.-C. Liew (University of Toronto, Ontario, Canada) . p8-1A contains a 12.3-kb insert corresponding to a region of the cluster beginning in the intergenic spacer and continuing into the 5′ region of the α-cMyHC gene . The linked α-cMyHC and β-cMyHC genes are highly homologous , but only the β-cMyHC gene is active in cultured skeletal muscle . Signals detected with the HM-1 and p8-1A probes completely colocalized in DNA and RNA detection protocols, indicating that both probes were detecting the β-cMyHC RNA. Section title: Dystrophin Probes. Educational score: 4.1852803230285645 Domain: biomedical Document type: Study Language: en The approximate positions of the dystrophin probes are presented in Fig. 5 A. The designations and probe descriptions are as follows: (a) mid-genomic, a probe specific for the portion of the dystrophin gene surrounding exon 44, (15lDMD), obtained from C.T. Caskey (Baylor College of Medicine, Houston, TX). It contains a 16-kb insert corresponding to a portions of intron 43, exon 44 (147 bp), and intron 44. This probe is >99% intron sequences. (b) 5′ Genomic, dystrophin clone 24A2, contains a 10-kb genomic sequence from the 5′ region of the gene. (c) 5′ cDNA, DMD13 (exons 1–11, ∼1.67 kb). (d) mid-cDNA, DMD10 (exons 17–27, ∼2.8 kb), the 5′ genomic and cDNA probes were provided by L. Kunkel (Harvard School of Medicine, Boston, MA). Section title: Dystrophin Probes. Educational score: 2.672640562057495 Domain: biomedical Document type: Study Language: en Genomic clones (16–20 kb) of lamin A/C (LA6), lamin B1 (LMB-B), and lamin B receptor (LBR) were provided by H. Worman (Columbia University, New York, NY). The genomic clone of E2F4 (λ10-1, 15 kb) was obtained from D. Livingston (Harvard School of Medicine, Boston, MA). Section title: Antibody. Educational score: 1.5183883905410767 Domain: biomedical Document type: Other Language: en A mouse monoclonal antibody reactive against the arg-ser spliceosome assembly protein SC-35 was generously supplied by X.-D. Fu (University of California, San Diego, CA). Section title: In Situ Hybridization Educational score: 4.119658470153809 Domain: biomedical Document type: Study Language: en To be successful in detecting nuclear sequences within the relatively dense structure of the myotube, it was necessary to develop a protocol that both reduced background resulting from the thick cytoplasm and increased penetrability of the nucleus. Hence, the methods for detecting nuclear RNA within the myotube are distinct from those optimized for the detection of cytoplasmic RNA. The methods used here, including procedures for nonisotopic probe preparation and fluorescence in situ hybridization, have been published in detail . Two-color detection of RNA and genes simultaneously was done by sequential hybridization and detection of RNA, followed by fixation, RNase incubation, and DNA hybridization . In some experiments, after hybridization samples were reacted with monoclonal antibodies raised against the spliceosome assembly factor SC-35 , as described previously . Section title: Microscopy and Analysis Educational score: 3.958561658859253 Domain: biomedical Document type: Study Language: en A Zeiss Axioplan microscope equipped with a multiband-pass epifluorescence filter (Chroma) was used for analysis. Images were analyzed by direct analysis of cells through the microscope and by representative images captured with a Photometrics series 200 charge-coupled device camera using a high-resolution, low depth of field objective (100×, NA = 1.4) ( Zeiss ). For two-color samples, image shifts were avoided by the use of a filter wheel in conjunction with multiband pass filters. All scoring was done by at least two investigators. Section title: Results Educational score: 4.278580188751221 Domain: biomedical Document type: Study Language: en As illustrated in Fig. 1 (top), MyHC and dystrophin nuclear RNAs were simultaneously visualized in postmitotic nuclei of cultured skeletal myofibers using fluorescence hybridization techniques optimized for detection of nuclear RNA. Initial experiments used genomic probes that targeted sequences of similar size, thereby facilitating comparison of the signals. Although in situ hybridization does not provide information on the rate of transcript production, it can provide reliable comparisons of the relative amounts of RNA present at a given time. Most often there were two striking accumulations of MyHC RNA and one for dystrophin, as expected for expression of both alleles of the autosomal MyHC gene and one allele of the X-linked dystrophin gene in female cells. Perhaps less anticipated was that both the dimensions and intensity of the dystrophin and MyHC nuclear RNA accumulations were comparable, as illustrated in Fig. 1 (right). Although dystrophin RNA is a less abundant cytoplasmic mRNA than MyHC , and the probes used detect <1% of the dystrophin sequence versus ∼80% of MyHC, dystrophin nuclear RNA signal was similar to the MyHC RNA signal. This was seen in multiple experiments irrespective of the detection method used and even when the MyHC probe targeted a somewhat larger sequence than the dystrophin probe (32 versus 16 kb). Hence, the apparent nuclear abundance of the dystrophin RNA signal does not result from the extremely large size of the full dystrophin RNA. Rather, these results indicate a roughly comparable number of transcripts associated with each MyHC and dystrophin allele. Section title: Results Educational score: 4.267879486083984 Domain: biomedical Document type: Study Language: en These results show that cytoplasmic abundance does not necessarily correlate with nuclear abundance, as many factors will influence the number of transcripts associated with a gene. Since the dystrophin gene is over 50-fold larger than the MyHC gene, ∼50-fold more dystrophin transcripts would have to be in production at any given moment to achieve a given rate of completion. Other factors that will affect the amount of RNA accumulated in the nucleus are the RNA stability, transcription initiation rate, and potentially other rate-limiting steps in processing and transport. For most genes, immature nuclear transcripts comprise a small, often undetectable fraction of total cellular levels. The conclusion of in situ results presented here, that the molar accumulation of dystrophin nuclear pre-mRNA is large relative to its cytoplasmic abundance, is supported by the work of Tennyson et al. , who reported that nascent nuclear dystrophin transcripts are more abundant than mature transcripts in the whole cell, in a molar ratio of ∼2.5:1. Section title: Results Educational score: 4.157360076904297 Domain: biomedical Document type: Study Language: en If MyHC and dystrophin nuclear RNA accumulations exclusively reflect nascent transcripts on their genes, then it would be difficult to explain why the enormous difference in gene length did not result in a significant difference in the dimensions of their nuclear RNA accumulations. As addressed further below, localized concentrations of a specific RNA may also reflect other rate-limiting steps in the progression of transcripts from the gene to the cytoplasm. Section title: Association of RNA Foci with SC-35–defined Domains Educational score: 4.340884685516357 Domain: biomedical Document type: Study Language: en To determine the distribution of MyHC and dystrophin nuclear RNA foci relative to SC-35 domains, cultures containing myotubes were probed for either MyHC or dystrophin nuclear transcripts and subsequently stained with antibodies against the spliceosome assembly factor SC-35 . Using a very narrow depth of field objective (NA = 1.4) and focusing through the RNA and SC-35 signals, it was apparent if these signals occupied the same space. In essentially all of the 50 cells analyzed, the MyHC RNA foci colocalized with a prominent SC-35 domain . Most commonly, the MyHC RNA accumulation detected with the genomic probe did not fill the entire ∼1–3-μ domain and had a different contour. Unlike nuclear accumulations of fibronectin and actin mRNAs which localized to the domain periphery , the MyHC RNA was also clearly detected within the inner region of the domain, as previously shown for collagen 1α1 RNA. The accumulation of MyHC RNA within SC-35 domains provides further evidence for the presence of pre-mRNA in these regions, contrary to the expectation that large factor-rich domains are storage sites, but consistent with the uniform presence of poly A RNA in them . These results also suggest that MyHC transcription and splicing is associated with the SC-35 domain, as directly demonstrated for fibronectin and collagen 1α1 RNA . In contrast to the similarity in size and appearance of their nuclear RNA accumulations, MyHC and dystrophin RNA showed completely different distributions relative to the SC-35 domains. The dystrophin RNA accumulations were never within the domains, whereas the MyHC RNA foci consistently were. In fact, as illustrated in Fig. 2 , D–F, with the exception of a small fraction of cells, there was no discernible increase in SC-35 concentration coincident with the large, bright dystrophin RNA accumulation. Since the linear dimensions of this enormous gene can extend 1–2 μ or more at interphase , this analysis was repeated with cDNA and genomic probes to different regions of the primary transcript. These results confirmed that no part of the dystrophin RNA focus was preferentially associated with SC-35 domains . At most, the edge of the dystrophin RNA accumulation occasionally appeared to touch an SC-35 domain, but the body of the RNA accumulation did not ever closely associate with domains . Section title: Association of RNA Foci with SC-35–defined Domains Educational score: 4.4088358879089355 Domain: biomedical Document type: Study Language: en The diametrically opposed relationship of MyHC and dystrophin RNAs to SC-35 demonstrates for the first time that the association between RNA polymerase II (pol II) gene transcripts and SC-35 domains is specific to some, but not all, active intron containing genes. Dystrophin now provides a precedent for RNA from an actively transcribed and spliced protein-coding gene that consistently is excluded from SC-35 domains. Association does not appear to correlate simply with the complexity or concentration of transcripts. Although dystrophin is an extremely complex gene associated with a relatively large accumulation of nuclear transcripts of similar molar abundance to MyHC, unlike MyHC dystrophin shows no association with discrete accumulations of SC-35. These results suggest the possibility that dystrophin and MyHC RNA accumulations reside in different structural compartments of the nucleus. Section title: Overall Spatial Relationship of Dystrophin and MyHC RNAs Educational score: 4.239512920379639 Domain: biomedical Document type: Study Language: en The demonstration of distinct gene distributions relative to SC-35 domains , coupled with the specific three-dimensional topography of the domains themselves , raises the question of whether patterns of overall nuclear position exist. In human fibroblasts and myoblasts, SC-35 domains are excluded from the nuclear periphery rich in heterochromatin and are further confined to a plane in the ventral half of the nuclear volume . If a given gene or chromosome is constrained to a subregion within nuclear space, as has been recently reported in Drosophila , the distribution relative to SC-35 domains could be a coincidental result of the gross overall positioning rather than a specific relationship to SC-35. Section title: Overall Spatial Relationship of Dystrophin and MyHC RNAs Educational score: 4.183887481689453 Domain: biomedical Document type: Study Language: en The relative positions of MyHC and dystrophin RNA foci were examined to determine whether any association or pattern of spatial arrangement was apparent for these coordinately expressed sequences within reiterated myofiber nuclei. Cultured myofibers most often do not have well-aligned nuclei, hence, for part of our analysis we attempted to focus on nuclei arrayed in single file and apparently aligned relative to the linear axis of the myotube. Cells were probed for MyHC and dystrophin RNA simultaneously, and the relative positions of signals in 51 nuclei from 15 different 077 myotubes were recorded on drawings as represented in Fig. 4 . The three nuclear RNA accumulations (two MyHC and one dystrophin) showed highly variable locations relative to one another. Since sometimes only one MyHC RNA focus was detected, we cannot rule out the possibility that the two MyHC alleles associate in a minority of cells. Within the limits of our analysis, neither nuclear RNA accumulation showed precise coordinates or had a clearly preferred pattern of distribution. Although the dystrophin RNA focus tended to be slightly more peripheral than MyHC (data not shown), neither RNA showed the marked peripheral location commonly seen for inactive neurotensin and albumin genes . Dystrophin RNA was clearly not confined to the peripheral region, from which SC-35 domains are excluded in these cells . Section title: Overall Spatial Relationship of Dystrophin and MyHC RNAs Educational score: 4.394851207733154 Domain: biomedical Document type: Study Language: en An interesting aspect of the spatial arrangement of dystrophin RNA is most apparent in considering its distribution relative to SC-35 domains . As viewed in two dimensions, the dystrophin RNA accumulation was positioned between the SC-35 domains, in the interdomain compartment, rather than in the upper half of nuclear volume largely devoid of SC-35 domains . Dystrophin RNA was not observed above or below any of the 20–40 prominent SC-35 domains, but rather appeared to distribute within the same Z-axis plane as the domains . Hence, dystrophin and MyHC are distributed in the same nuclear plane, and in a small fraction of nuclei (∼6%) could even be found adjacent to one another. However, even when they were abutting, the two RNA accumulations remained as separate entities and did not intermingle . In contrast, we have observed other RNAs which do intermingle (Shopland, L., and C. Johnson et al., unpublished data). Similarly, even when dystrophin RNA was adjacent to an SC-35 domain, the concentration of factors did not extend over the dystrophin RNA. These observations show that dystrophin and MyHC RNA accumulations behave as separate structural entities, consistent with their residing in distinct nuclear compartments. Section title: Overall Spatial Relationship of Dystrophin and MyHC RNAs Educational score: 4.096011161804199 Domain: biomedical Document type: Study Language: en Our results show that the difference in distribution relative to SC-35 domains is not a coincidental result of a fixed position within a narrow nuclear region. These results also indicate that these coordinately expressed genes are not specifically positioned relative to one another, but do not rule out positional differences at a more refined level. Finally, we note that cultured muscle fibers remain physiologically immature, thus it remains possible that within the more structured architecture of in vivo tissues a greater topographic organization of individual active genes exists. However, such organization is clearly not necessary for either viability or differentiation in vitro. Section title: Visualization of Cotranscriptional Splicing within the Dystrophin RNA Accumulation Educational score: 4.253802299499512 Domain: biomedical Document type: Study Language: en The finding that the large dystrophin RNA accumulation exists outside of SC-35 domains suggests that sufficient splicing factors are present in the interdomain space for processing of a substantial accumulation of transcripts from a gene containing 79 exons. To evaluate directly whether the RNA accumulation detected was indeed undergoing splicing, we used simultaneous hybridization of probes for different sequences within the gene. As previously indicated , for typical genes the size of actin (6 kb) or even fibronectin (70 kb) it has not been possible by fluorescence microscopy to demonstrate cotranscriptional splicing, since either co- or posttranscriptional events would generally occur below the limits of resolution (0.2 μ). However, the enormous size of the dystrophin gene allowed a level of resolution of splicing events not previously possible by light microscopy. Simultaneous hybridization with genomic and cDNA probes was performed to evaluate the relative distribution of transcripts with and without introns, as shown previously . Because of the size of the gene, the analysis included different combinations of probes from the 5′ and midgene regions. Section title: Visualization of Cotranscriptional Splicing within the Dystrophin RNA Accumulation Educational score: 4.1671905517578125 Domain: biomedical Document type: Study Language: en From the examination of different combinations of probes in numerous cells, key patterns emerged. Results from hybridization of cDNA probes suggested that the 5′and 3′ ends of the RNA accumulation could be discriminated. For example, using 5′ cDNA (exons 1–11) and mid-cDNA (exons 17–27) probes, reproducible structural features of the dystrophin RNA focus were visualized. The 5′ probe detected an elongated focus of RNA which was larger than and completely overlapped the mid cDNA focus . Signals from the more 3′ cDNA probe consistently overlapped one end of the RNA accumulation detected by the 5′ probe, consistent with the 5′ sequences being transcribed first and carried down the length of the gene to where the 3′ sequences were transcribed. This pattern was independent of the labeling method used. Section title: Visualization of Cotranscriptional Splicing within the Dystrophin RNA Accumulation Educational score: 4.441461563110352 Domain: biomedical Document type: Study Language: en Using a 5′ intron probe (genomic probe which detects 99% intron sequence encompassing exon 1) and a midgene intron probe (genomic probe encompassing introns 43 and 44) RNA foci were detected in a pattern consistently distinct from that revealed by the cDNA probes. Double-label experiments using both of these genomic probes produced nonoverlapping foci (at most only a trace of 5′ sequences was seen with the more 3′ foci) . This strongly suggests that these two introns, from distant parts of the gene, did not simultaneously exist within the same RNA molecules. The most plausible explanation of these results is that the more 5′ introns are spliced out of nascent RNA molecules before the 3′ end of the RNA is transcribed. This indicates cotranscriptional splicing of the primary transcript along this unusually large gene as represented in Fig. 5 H, consistent with results of previously reported PCR analyses . Hence, these results demonstrate that fluorescence hybridization is capable of resolving cotranscriptional splicing events along the dystrophin gene, and directly demonstrate that the large RNA accumulation localized in interdomain compartment is undergoing splicing. Section title: Tracks and Trees Educational score: 4.461703777313232 Domain: biomedical Document type: Study Language: en For several reasons it is of interest to understand whether nuclear RNA accumulations represent nascent transcripts or transcripts that have already dissociated from their respective genes and are in a subsequent step in the progression to nuclear export. Dystrophin and MyHC nuclear RNA accumulations may not represent the same structural and functional entities, i.e., they do not necessarily comprise transcripts at equivalent stages of maturation. Despite the enormous difference in gene lengths, the RNA accumulations associated with each gene are quite similar in size. As would be expected for a tree of nascent transcripts on the gene, the approximate length of the dystrophin RNA signal with the 5′ cDNA probe (∼2 μm) is very consistent with earlier measurements of the gene length in fibroblast interphase nuclei . However, the fact that the 35-kb MyHC gene sequence is much smaller than the 2,300-kb dystrophin gene suggests that the relatively large size of the MyHC RNA accumulation may constitute more than a tree of nascent transcripts on the gene. Hence, we postulate that MyHC and dystrophin RNA formations might be structurally distinct with respect to their genes. Section title: Tracks and Trees Educational score: 4.14091682434082 Domain: biomedical Document type: Study Language: en To address this directly, we investigated the spatial configuration of the MyHC and dystrophin genes relative to their respective RNA accumulations using a sequential RNA and DNA hybridization strategy . MyHC RNA and gene were visualized in two different colors using a 32-kb β-cMyHC–specific genomic probe . Dystrophin RNA was visualized by using the 5′ cDNA probe while different segments of the dystrophin gene (∼10–15 kb each) were delineated using the 5′ and midgenomic probes . Section title: Tracks and Trees Educational score: 4.262589454650879 Domain: biomedical Document type: Study Language: en The dystrophin RNA was examined in one color in the same nucleus with the two DNA sequences marking distant gene segments (1 Mb apart) in another color. The 50MB-1 cells used are male, so only 1 X-linked dystrophin gene is present. The pattern observed most often is shown in Fig. 6 C. Here we show that the dystrophin RNA accumulation generally surrounds the two point signals produced by DNA hybridization with the two genomic probes, and often the longer axis of the RNA signal lies parallel to the axis connecting the two gene segments. This overlap is in keeping with a tree of nascent transcripts around the gene locus, as represented in the model in Fig. 6 D. These and the above results collectively demonstrate that the dystrophin RNA accumulation represents a Christmas tree of nascent transcripts undergoing cotranscriptional splicing, with introns removed from the nascent pre-mRNA as transcription and splicing move in a 3′ direction across the gene . This process consistently occurs in a nuclear compartment that is comparatively low in splicing factors. Section title: Tracks and Trees Educational score: 4.292080402374268 Domain: biomedical Document type: Study Language: en Results of the DNA/RNA analysis were clearly different for MyHC. The DNA hybridization signal from a 32-kb MyHC probe was a small (0.1–0.2 μ) round spot, indistinguishable from that observed above for hybridization to either of the 10–15-kb dystrophin sequences . This is consistent with studies of the packaging of interphase chromatin which have shown sequences of this length to be at or below the resolution of light microscopy (0.2 μ) . When gene and RNA were examined simultaneously in two colors it was clear that the MyHC RNA signal was not only much larger than the DNA signal, but was consistently displaced to one side of it . Rather than being positioned amid the RNA accumulation, as were dystrophin DNA signals, the MyHC gene signals were usually at the edge of the MyHC RNA accumulation. The fact that this polar configuration of DNA and RNA was not typically seen for dystrophin supports the interpretation that the apparent track of MyHC RNA emanating to one side of the gene reflects a bona fide biological distribution and not an artifact of the visualization technique. Section title: Tracks and Trees Educational score: 4.255398750305176 Domain: biomedical Document type: Study Language: en These results indicate that nuclear accumulations of dystrophin and MyHC RNA do not constitute equivalent functional entities. The results with dystrophin are essentially as one would predict for nascent molecules amassed on the enormous gene. However, the MyHC results do not fit the tree model, but rather are indicative of a track comprised largely of a posttranscriptional RNA accumulation to one side of the gene. We interpret these results to suggest that transcription is likely the rate-limiting step in RNA export for the dystrophin locus, whereas for MyHC posttranscriptional events may not keep pace with the rapid transcription of MyHC, resulting in an accumulation of RNA at a step subsequent to transcription. Although we believe the track is comprised of intact transcripts that enter the SC-35 domain, we do not suggest that MyHC splicing is posttranscriptional (see Discussion). Section title: Association of Transcribed Genes with SC-35 Domains Is Locus Specific Educational score: 4.314931869506836 Domain: biomedical Document type: Study Language: en The above results conclusively demonstrate a differential localization of MyHC and dystrophin RNAs relative to large accumulations of splicing factors in muscle cell nuclei. If this reflects different structural relationships relative to a nuclear compartment rich in RNA metabolic factors, rather than merely different amounts of splicing factors bound to these two pre-mRNAs, then differences in the structural association of other active genes/pre-mRNAs would be expected. Although a variety of observations favor the former (see Discussion), the extreme difference in size and potential transcription rates of dystrophin and MyHC may be considered to complicate the interpretation. For this reason, we examined four other active genes/RNAs whose size and expression levels are not as disparate as MyHC and dystrophin. These included the genes for transcription factor E2F4 , the nuclear envelope protein lamin A/C (LMNA) , lamin B receptor (LBR) , and lamin B1 (LMNB1) . Although not muscle specific, these four commonly expressed genes are roughly similar in size and complexity. Furthermore, their expression levels are more moderate, in between the highly expressed MyHC gene and dystrophin. Thus, they are good candidates for addressing whether SC-35 domain association is contingent upon these factors. Section title: Association of Transcribed Genes with SC-35 Domains Is Locus Specific Educational score: 4.535346984863281 Domain: biomedical Document type: Study Language: en For all four of these genes, the nuclear RNA accumulations generally produced small spot-like signals, smaller than either the dystrophin or MyHC nuclear RNA signals. This suggests that the signals largely comprise nascent transcripts on the gene (although there was sometimes a suggestion of RNA tracks for LMNA). Analysis of these genes/RNAs relative to SC-35 in fibroblasts demonstrated striking differences in levels of association with the prominent SC-35 domains, as seen in Figs. 7 and 8 . In the vast majority of cells, LMNA and E2F4 were not visibly separate from the SC-35 domains, whereas the LMNB1 and LBR genes/RNAs remained spatially separate, even when they were in the vicinity of a domain. The low levels of apparent association of these two sequences are similar to that previously seen for inactive genes and are consistent with random expectations within the limits of microscopic resolution . As viewed by light microscopy, LMNA and E2F4 contacted SC-35 domains four- to fivefold more often than LMNB1 and LBR . This represents a significant level of association for genes that are not nearly as highly expressed as MyHC in muscle cells, and is higher than the level of association reported for fibronectin with SC-35 . It should be noted that these sequences were studied in a cycling fibroblast population, which may introduce some variability between cells; e.g., E2F4 expression varies during the cell cycle . The nonrandom association of a regulatory gene such as E2F4 establishes that the spatial association with domains is not restricted to genes for the most abundant structural proteins. Collectively, our results provide strong evidence that such organization is common and involves a substantial subset of protein coding genes. These results lead us to conclude that association with SC-35 domains is locus specific and not solely dependent upon gene size, complexity or expression level. They further indicate that within the constraints of nuclear structure, active loci have differential access to the immediate and copious supply of RNA metabolic factors in the interchromatin domains, visualized here by immunofluorescence to assembly factor SC-35. Further implications of these results are considered in the Discussion. Section title: Discussion Educational score: 4.491471290588379 Domain: biomedical Document type: Study Language: en This study demonstrates that endogenous genes/pre-mRNAs are preferentially expressed in a locus-specific manner in one of two distinct nuclear compartments, as defined by immediate proximity to regions highly enriched in splicing factors. In addition, results provide further evidence that newly synthesized RNAs are commonly associated with fluorescent SC-35 domains or prominent speckles. In the vast majority of cells, genes for MyHC, E2F4, and LMNA are expressed directly at the periphery of SC-35–defined domains, whereas dystrophin, LMNB1, and LBR are not. Results indicate that the difference is not a coincidence of fixed positions within overall nuclear topography or a result of the abundance of pre-mRNA transcripts present. Results support that splicing factor distribution does not simply reflect factors binding to pre-mRNAs from randomly distributed genes, consistent with earlier studies showing that interchromatin granule cluster and SC-35 domains are structurally and functionally specialized regions of the nucleoplasm. Although initially debated, there is increasing acceptance that transcription and splicing can occur in the periphery of fluorescent domains. Whether or not one views the interior region of the speckle as strictly a storage site devoid of pre-mRNA, results shown here demonstrate a previously unknown paradigm of nuclear architecture involving locus-specific differences in the immediate access of active genes to these supplies of splicing factors. As discussed below, whether or not a gene associates with these regions likely depends not only upon its expression and complexity, but also upon constraints of its chromosomal context. Section title: Discussion Educational score: 4.441763401031494 Domain: biomedical Document type: Study Language: en As suggested here for MyHC , other results directly demonstrate that, for genes that associate with domains, association is linked to cell type–specific expression (Moen et al., manuscript in preparation). For MyHC and dystrophin, the simplest interpretation might a priori be that the difference in SC-35 association merely reflects the amount of splicing factors bound to each of their pre-mRNAs. However, results show that it is not that simple, and that the domain associated with MyHC is not formed merely by splicing factors bound to unspliced MyHC transcripts. Although the RNA foci for MyHC and dystrophin are comparable in size and intensity when probes of similar size are used, suggesting similar numbers of transcripts, the difference in localization with SC-35 is striking. Clearly, the lower level factors throughout the nucleoplasm are sufficient to splice the large nuclear accumulation of dystrophin RNA with its 74 introns. The abundant accumulation of splicing factors associated with MyHC lies primarily adjacent to the gene, rather than on it, indicating the domain behaves more as a structure than as a diffuse accumulation of splicing factors on nascent or dispersed RNA. Further, the MyHC RNA track, as detected with a genomic probe, frequently does not occupy the whole SC-35 domain. The absence of overlap of dystrophin RNA and SC-35 foci gives the strong impression that this RNA is excluded from splicing factor domains; even when dystrophin and MyHC accumulations are extremely close they do not overlap, suggesting that structural constraints may separate them. Section title: Discussion Educational score: 4.218996047973633 Domain: biomedical Document type: Study Language: en The exclusion of dystrophin, LMB1, and LBR from SC-35 domains contrasts with other results on microinjected sequences or transfected sequences , which indicated that the presence of a single intron was sufficient to cause an association with snRNP or SC-35 speckles. Although the presence of intron sequences may be necessary for this association, results presented here clearly show it is not sufficient for endogenous genes/RNAs. However, the behavior of extrachromosomal sequences may not accurately reflect the structural relationships of endogenous genes. Unlike viruses, plasmids, or microinjected sequences (which have been more commonly examined), our results indicate that for endogenous genes organizational/structural differences in their chromosomal context can impact their proximity and access to factor-rich regions. Section title: Tracks Versus Trees: Different Rate-limiting Steps in RNA Metabolism for Different Genes Educational score: 4.298894882202148 Domain: biomedical Document type: Study Language: en These results provide further evidence for the existence of RNA tracks , and demonstrate that nuclear RNA accumulations from different genes do not necessarily represent the same structural or functional entities. Results show the dystrophin RNA accumulation represents primarily a tree of nascent transcripts on an unusually long gene, for which transcription is logically rate limiting. This contrasts with a track of transcripts extending vectorially beyond the much smaller, but also complex, MyHC gene. These results counter the argument that localized RNA accumulations logically represent nascent transcripts and not posttranscriptional accumulations or RNA tracks . Such interpretations may be based on two presumptions: (a) that the rate-limiting step in nuclear RNA metabolism and export for all pre-mRNAs is transcription, and (b) that Drosophila polytene chromosomes accurately reflect the structure of mammalian interphase nuclei. Section title: Tracks Versus Trees: Different Rate-limiting Steps in RNA Metabolism for Different Genes Educational score: 4.30278205871582 Domain: biomedical Document type: Study Language: en Despite our conclusion that the dystrophin accumulation is largely cotranscriptional and the MyHC RNA accumulation largely posttranscriptional, we do not interpret this to show that MyHC splicing itself is largely posttranscriptional, even though the RNA overlaps the SC-35 domain. As previously suggested and further addressed elsewhere for the similar case of collagen (col) 1α1 RNA, results favor that splicing is largely cotranscriptional and at the periphery of the SC-35 domain (Johnson et al., manuscript in preparation). If so, then the posttranscriptional track of MyHC RNA that accumulates in the domain is already largely spliced. This further supports that the domain may contain both specific mRNAs as well as an abundance of RNA metabolic factors, beyond those immediately engaged on transcripts of that gene. As discussed elsewhere , we hypothesize that at each SC-35 domain, multiple functions related to RNA metabolism occur including metabolism of some specific pre-mRNAs, preassembly and disassembly of splicing factors, and potential transport of mRNA. The possibility of a role in RNA export will be addressed more directly elsewhere (Johnson et al., manuscript in preparation). Section title: Categories of Spatial Relationships Educational score: 4.155168056488037 Domain: biomedical Document type: Study Language: en These results triple the number of endogenous active genes that have been examined relative to this subnuclear compartment, allowing us to begin to categorize the different spatial distributions observed, as shown in Fig. 9 A. Of genes that associate with SC-35 domains, all localize at the domain periphery, however there is a clear difference in the distribution of their nuclear RNAs. MyHC RNA extends from the domain periphery into the central region, similar to collagen RNA. As indicated in Fig. 9 A, we term this a Type I association, as distinct from the Type II associations exhibited by RNAs such as E2F4 and LMNA. Type II RNAs are visible only at the periphery of a domain, and are associated with high frequency (70–95%), but not as consistently as Type I RNAs which overlap SC-35 domains essentially 100% of the time . We speculate that the difference in whether the RNA is observed within the domain may be a function of different relative rates of transcription versus posttranscriptional steps, e.g., whether the RNA forms a track or a tree. Section title: Categories of Spatial Relationships Educational score: 4.618341445922852 Domain: biomedical Document type: Study Language: en The different categories of association observed contribute to a more detailed hypothesis for domain formation. Previously we showed that after mitosis, the clustering of poly A RNA in domains preceded the clustering of snRNPs, leading us to suggest that domain formation may be linked to gene expression in a manner roughly analogous to formation of the nucleolus . In this model, domains would be nucleated at sites of highest pre-mRNA transcription and splicing such as the highly expressed and complex Type I RNAs for MyHC and col 1α1. As a smaller accumulation of factors begins to associate with a site of one or more highly active Type I gene/ RNA(s), the tendency will be for that accumulation to grow into a large globular domain, reflecting an inherent tendency for splicing factors to cluster . Perhaps due to an affinity of their pre-mRNAs for those same factors, other active genes will, within the constraints of their interphase chromosome structure, tend to cluster at or around these regions where access to metabolic factors is great. This secondary clustering of other active genes/pre-mRNAs (Type II RNAs) will tend to draw more factors, contributing further to the growth of the domain in a dynamic process. But because protein-coding genes reside within chromosomal structure that imposes constraints, not all genes can have equal access to these regions. If Type I genes do have a dominant influence on where domains form, then a locus which resides in the neighborhood of such a gene may be in a favorable position to associate with the domain. In contrast, some active genes not residing in these neighborhoods would not associate with SC-35 domains at a frequency any different from that of inactive sequences . As previously suggested , these observations raise implications for locus-specific influences on levels of gene expression since association with domains could facilitate efficient gene expression. This might eventually even provide some rationale for the clustering distribution of genes in metaphase chromosome bands. Section title: Categories of Spatial Relationships Educational score: 4.11798095703125 Domain: biomedical Document type: Study Language: en Results presented here point to a nucleus that is functionally organized, but which resists attempts to reduce that organization to any single paradigm. We suggest that the spatial arrangement of endogenous genes or RNAs must be considered in terms of the combined effect of multiple structural and biochemical factors, including the expression and complexity of the sequence, the structural context within the chromosome, and the dynamics of different steps in RNA metabolism. As will be addressed elsewhere, disruptions of these structural relationships may be key to the cellular pathogenesis of specific diseases. | Study | biomedical | en | 0.999997 |
10037786 | Section title: Isolation of Primary Myoblasts, Cell Culture, and Immunohistochemistry Educational score: 4.147398948669434 Domain: biomedical Document type: Study Language: en Satellite cell–derived primary myoblasts were isolated from adult lower hindlimb muscle from 2–3-mo-old mice as described previously , with the exception that hepatocyte growth factor (10 ng/ml; R&D Systems Inc.) and heparin (5 ng/ml; Sigma Chemical Co. ) were included in the growth medium for the first 48 h after the plating of the final cell preparation, and supplemented with FGF2 (2.5 ng/ml FGF2) thereafter. The primary cultures were maintained on collagen-coated dishes in Ham's F10 ( GIBCO BRL ) supplemented with 20% FCS, 200 U/ml penicillin, 200 μg/ml streptomycin, and 0.002% Fungizone ( GIBCO BRL ). The medium was changed daily and cultures were routinely passaged 1:3 as they reached 60–70% confluence. To maintain the primary characteristics of the cells, all experiments were performed using cultures that had undergone between four and seven passages. Differentiation medium consisted of DME supplemented with 5% horse serum and antibiotics as described above ( GIBCO BRL ). Section title: Isolation of Primary Myoblasts, Cell Culture, and Immunohistochemistry Educational score: 4.197200775146484 Domain: biomedical Document type: Study Language: en The extent of culture purity and differentiation was determined by subjecting the purified myoblasts to c-met, desmin, and myosin heavy chain (MHC) immunostaining. Briefly, myoblasts in growth medium were fixed in 4% paraformaldehyde in PBS (PFA), stained with anti–c-met antibody SP260 ( Santa Cruz Biotechnology ), and antidesmin antibody DE-U-10 (DAKO). MHC expression was detected by fixing differentiated cultures with 90% methanol and staining with MF20 mAb . Immunostaining was similarly performed with anti–β-catenin antibody sc-1496 ( Santa Cruz Biotechnology ) and anti–M-cadherin antibody sc-6470 ( Santa Cruz Biotechnology ). Immunostaining with anti–c-met and anti– β-catenin antibodies were detected with fluorescein-conjugated anti–goat antibodies ( Sigma Chemical Co. ), and photographed on a Zeiss Axiophot microscope equipped with a UV source and FITC detection filters. Desmin, M-cadherin, and MHC staining was visualized using a HRP-coupled secondary antibody (Bio-Rad Laboratories) in PBS containing 0.6 mg/ml diaminobezidine ( Sigma Chemical Co. ). Section title: Differentiation Time Course and Growth Rate Measurements Educational score: 4.103877067565918 Domain: biomedical Document type: Study Language: en Growth rate analysis was determined by [ 3 H]thymidine incorporation of three independent isolates of wild-type and MyoD −/− cultures seeded in 24-well plates at 10 4 cells per well in growth medium (each in triplicate). For day 0, the cells were cultured 24 h in growth medium before addition of 2 μCi of [ 3 H]thymidine for 2 h. Incorporation of [ 3 H]thymidine was normalized to protein concentration as determined by Bradford assay. The remaining wells were exposed to differentiation medium and labeled with [ 3 H]thymidine for 2 h on sequential days. Section title: Differentiation Time Course and Growth Rate Measurements Educational score: 4.116315841674805 Domain: biomedical Document type: Study Language: en To assay the differentiation potential of wild-type and MyoD −/− cultures, 10 5 low passage cells were seeded into 35-mm dishes in growth medium and cultured for an additional 24 h (day 0) before addition of differentiation medium. On subsequent days (1–5), the cultures were fixed and immunostained for MHC with antibody MF20. To establish the differentiation potential of the cultures, at least 1,000 nuclei from MF20-positive cells were counted from several random fields. The percentage of differentiated cells was calculated as: ( nuclei within MF20-stained myocytes / total number of nuclei ) × 100; or the fusion index calculated as: ( MF20-stained myocytes containing ≥2 nuclei/total number of nuclei ) × 100. 5-bromo-2′-deoxyuridine (BrdU) incorporation assays on cultured cells were performed (cell proliferation kit; Amersham Pharmacia Biotech ). All experiments were performed in triplicate on three independent wild-type and MyoD −/− isolates. Section title: Northern and Western Analysis Educational score: 4.133951663970947 Domain: biomedical Document type: Study Language: en To analyze the expression of the MRFs and of differentiation-specific markers, total RNA from low passage cells in growth (day 0) or differentiation medium (days 1–5) was isolated and subjected to Northern analysis . Replicate filters were sequentially hybridized to MRF-specific cDNAs as well as α-cardiac and α-skeletal actin, and acetylcholine receptor δ subunit probes . Dr. Paul Hastings (McGill University) kindly provided the M-cadherin probe. The Musk cDNA probe was obtained by RT-PCR of C2C12 RNA. The β - catenin and PEA3 probes were kindly provided by Drs. Rolf Kemler (Max Planck Institute) and John A. Hassell (McMaster University), respectively. Western analysis with rabbit anti–Myf-5 antibody C-20 ( Santa Cruz Biotechnology ), mouse anti-MyoD antibody 5A8 ( PharMingen ), and mouse antimyogenin antibody F5D (Developmental Studies Hybridoma Bank) was performed on extracts prepared from cultures in growth medium as described previously . Section title: Mixing of LacZ-expressing MyoD−/− and Wild-Type Cultures Educational score: 4.1870036125183105 Domain: biomedical Document type: Study Language: en Early passage MyoD −/− cultures were lipofected with a 1:10 ratio of PGK-LacZ-MAR and PGK-Puro plasmids, and stable transformants were pooled after 10 d of selection in 2 μg/ml puromycin. Plasmid PGK-LacZ-MAR contains the phosphoglycerate kinase 1 promoter expressing nls-lacZ and a chicken lysozyme matrix attachment region to confer high level site-independent expression . Primary cells were plated with an initial density of 10 5 cells per 60-mm well, in ratios of 1:0, 1:4, 4:1, and 0:1 of MyoD −/− to wild-type cells. Duplicate cultures were grown overnight in growth medium before exposing to differentiation medium for 5 d. Wells were washed with PBS, fixed in 2% formaldehyde/0.4% glutaraldehyde followed by X-Gal staining. Cells were postfixed with 90% methanol for 7 min, rinsed with PBS and 0.3% Triton X, before immunostaining with antibody MF-20 as described above. Section title: Transfection with MyoD Plasmid and Generation of Stable MyoD+ Pools Educational score: 4.13389778137207 Domain: biomedical Document type: Study Language: en Low passage subconfluent cultures of MyoD −/− cells were transfected with pEMSV-MyoD/PGK-Puro or PGK-Puro alone by lipofectamine ( GIBCO BRL ), according to the manufacturer's instructions. The MyoD expression plasmid carries the murine MyoD cDNA driven by the EMSV promoter and enhancer, as well as a puromycin resistance cassette. The cultures were refed 24 h after transfection and daily with growth medium containing 2 μg/ml puromycin ( Sigma Chemical Co. ) for 10 d. The resulting colonies (>200) were pooled and expanded for further analysis. MyoD expression was evaluated by Western blot analysis using anti-MyoD mAb 5A8 ( PharMingen ). The differentiation and fusion potentials of MyoD-expressing pools were assayed as described above. Section title: Altered Cellular Phenotype of MyoD−/− Myogenic Cells Educational score: 4.1239728927612305 Domain: biomedical Document type: Study Language: en To gain insight into the role of MyoD in satellite cell activation, low passage primary cultures were isolated from 2–3-mo-old wild-type and MyoD −/− mice to facilitate the generation of highly purified satellite cell–derived cultures and preclude the inclusion of neonatal myoblasts. Cultures were grown for 48 h in the presence of hepatocyte growth factor and thereafter in medium supplemented with FGF2 to allow the rapid recovery of high numbers of low passage primary myoblasts as described previously . Section title: Altered Cellular Phenotype of MyoD−/− Myogenic Cells Educational score: 4.138269424438477 Domain: biomedical Document type: Study Language: en As suggested by our previous observations , MyoD −/− cultures displayed a stellate flattened morphology with an enlarged cytoplasm and extended cytosolic processes. By contrast, wild-type cells were highly refractile under phase-contrast microscopy and displayed the rounded morphology and small compact cytoplasm characteristic of primary myoblasts . Section title: Altered Cellular Phenotype of MyoD−/− Myogenic Cells Educational score: 4.276325225830078 Domain: biomedical Document type: Study Language: en Quiescent and activated satellite cells in vivo express the receptor tyrosine kinase c-met as do cultured myoblasts . Proliferating myogenic precursor cells in vivo and myoblasts in vitro express the intermediate filament desmin. However, satellite cells do not express desmin . Therefore, primary cultures were immunostained with antibody reactive with c-met and desmin to assess their developmental status. Virtually 100% of the primary cells derived from both wild-type and MyoD −/− animals expressed high levels of c-met as detected by indirect immunofluorescence. Fibroblast cell lines did not express c-met (not shown). Therefore, the isolation procedure generated highly purified cultures of myogenic cells . In contrast to the uniform expression of c-met, only 6.2 ± 5.4% of MyoD −/− myogenic cells expressed the myoblast marker desmin, whereas 89 ± 4.2% of wild-type cells expressed desmin . These data are consistent with the notion that MyoD −/− myogenic cells represent an intermediate developmental stage between a satellite cell and myogenic precursor cells. Section title: Reduced Differentiation Potential of MyoD−/− Myogenic Cultures Educational score: 4.109050273895264 Domain: biomedical Document type: Study Language: en To evaluate the differentiation potential of the MyoD −/− myogenic cultures the extent of myogenic differentiation was assessed at the cellular level by immunostaining cultures fixed at daily intervals after mitogen withdrawal with antibody MF20 reactive with MHC . Importantly, this analysis was performed on three independently isolated wild-type and MyoD −/− primary myogenic cultures. After MF20 immunostaining, the proportion of MHC-positive cells and the fusion index of both wild-type and MyoD −/− cultures were assessed . Section title: Reduced Differentiation Potential of MyoD−/− Myogenic Cultures Educational score: 4.2503557205200195 Domain: biomedical Document type: Study Language: en Under growth conditions, MyoD −/− myogenic cells exhibited about a 100-fold reduction in the rate of spontaneous differentiation (0.10 ± 0.14%) compared to wild-type cells (10 ± 1.4%) . Consistent with this observation, MyoD −/− cultures displayed a severe defect in their ability to differentiate and form multinucleated myotubes after mitogen withdrawal . Differentiated wild-type myocytes displayed the typical elongated and multinucleated morphology, whereas differentiated MyoD −/− myocytes were primarily mononuclear and retained a fibroblastic stellate cytomorphology . Section title: Reduced Differentiation Potential of MyoD−/− Myogenic Cultures Educational score: 4.204265594482422 Domain: biomedical Document type: Study Language: en About 50% of the cells in wild-type cultures after 24 h in differentiation medium had begun to undergo terminal differentiation as assessed by MHC immunostaining . The number of differentiated wild-type myocytes continued to accumulate in a linear manner reaching ∼94% 5 d after serum withdrawal . By contrast, 24 h after mitogen withdrawal, the number of differentiated myocytes in MyoD −/− cultures remained below the limit of detection . The number of differentiated MyoD −/− myocytes began to accumulate after 3 d and reached ∼70% 5 d after serum withdrawal . Section title: Reduced Differentiation Potential of MyoD−/− Myogenic Cultures Educational score: 4.219228744506836 Domain: biomedical Document type: Study Language: en Enumeration of differentiated myocytes containing two or more nuclei in these cultures (i.e., fusion index) revealed a marked reduction in fusion capacity in MyoD −/− cultures . The fusion index of differentiated wild-type cultures was ∼90% 5 d of differentiation with an average of 4.6 ± 0.3 nuclei per myocyte. By contrast, by day 5 only about 15% of MyoD −/− myocytes contained 2 nuclei, with an average of 1.2 ± 0.2 nuclei per myocyte . Section title: Reduced Differentiation Potential of MyoD−/− Myogenic Cultures Educational score: 4.326044082641602 Domain: biomedical Document type: Study Language: en The rate of cell-cycle withdrawal after induction of differentiation was assessed by measuring [ 3 H]thymidine incorporation at daily intervals after transfer into differentiation medium . In growth medium, [ 3 H]thymidine labeling experiments revealed that MyoD −/− cells exhibited an apparent twofold higher growth rate than wild-type cells . Direct cell count experiments in growth medium revealed that numbers of primary MyoD −/− cells accumulated ∼1.5-fold faster than wild-type cells (not shown). Moreover, MyoD −/− cells displayed a 1.6-fold increase in mitotic index as determined by BrdU incorporation . However, this increase in apparent growth can be partly accounted for by the reduced rate of spontaneous differentiation of mutant (0.10 ± 0.14%) versus wild-type cells (10.0 ± 1.4%) . In addition, interpretation of these results is also confounded by other potential variables: differences in the proportion of cells temporarily withdrawn from the cell-cycle, differences in rates of apoptosis, and differences in cell-cycle kinetics. Therefore, additional analyses are required to determine whether primary MyoD −/− myogenic cells exhibit altered cell-cycle kinetics relative to primary wild-type myoblasts. Section title: Reduced Differentiation Potential of MyoD−/− Myogenic Cultures Educational score: 4.252935409545898 Domain: biomedical Document type: Study Language: en Wild-type myoblasts and MyoD −/− myogenic cells both exhibited about a twofold increase in the rate of [ 3 H]thymidine incorporation 24 h after mitogen withdrawal . This apparent increase in DNA synthesis may reflect a characteristic of primary cells analogous to the transient increase in cell proliferation observed after the IGF-I treatment of myoblasts under culture conditions that induce differentiation . After the first day of mitogen withdrawal, wild-type myoblasts exhibit a rapid withdrawal from the cell cycle as evidenced by the low levels of [ 3 H]thymidine incorporation . By contrast, the rate that MyoD −/− myogenic cells incorporated [ 3 H]thymidine continued to increase during the 5 d after transfer into differentiation medium. Similar results were obtained using three independent preparations of low passage primary myogenic cultures. Section title: Reduced Differentiation Potential of MyoD−/− Myogenic Cultures Educational score: 4.196995735168457 Domain: biomedical Document type: Study Language: en Taken together, these experiments suggested that MyoD −/− myogenic cells displayed continued proliferation under conditions of low mitogens that normally induce cell-cycle withdrawal and terminal differentiation of wild-type myoblasts. To investigate cell proliferation under conditions of growth and differentiation, cultured cells were exposed to BrdU for 4 h followed by immunodetection of nuclear localized BrdU incorporated during DNA synthesis . Wild-type primary myoblasts in growth medium exhibited a 20% rate of BrdU incorporation, whereas MyoD −/− myogenic cells exhibited a 32% rate of labeling. After 5 d of differentiation, 93% of nuclei in wild-type cultures expressed MHC and 5.5% were labeled by BrdU. In contrast, after 5 d of differentiation, 49% of cells in MyoD −/− cultures expressed MHC and 17% were labeled by BrdU . Interestingly, subconfluent cultures of MyoD −/− cells in differentiation medium exhibited up to 20% rates of BrdU incorporation, whereas confluent cultures exhibited as low as 5% BrdU labeling (not shown). Section title: Reduced Differentiation Potential of MyoD−/− Myogenic Cultures Educational score: 4.275581359863281 Domain: biomedical Document type: Study Language: en Considered together, these data indicate that MyoD −/− myogenic cells continue to proliferate under low-mitogen conditions that normally induce terminal differentiation of wild-type myoblasts and suggest that MyoD −/− cells exhibit contact inhibition of growth during differentiation. The observation that MyoD −/− cells exhibit an enhanced proliferative potential under conditions that normally induce differentiation strongly supports the notion that MyoD −/− myogenic cells exhibit an increased propensity for self-renewal rather than progression through the differentiation program. Section title: Analysis of Muscle-specific Gene Expression Educational score: 4.066334247589111 Domain: biomedical Document type: Study Language: en To gain further insight into the differentiation defect in MyoD −/− primary cultures, total RNA was prepared from both wild-type and MyoD −/− cultures in growth medium and at daily intervals after induction of differentiation. Northern blot analysis was performed using a panel of muscle-specific probes. Section title: Analysis of Muscle-specific Gene Expression Educational score: 4.396024703979492 Domain: biomedical Document type: Study Language: en The expression of the MRFs was investigated to elucidate the regulatory relationships and the potential for functional compensation in the absence of MyoD. Northern analysis of the expression pattern of the four MRFs confirmed that MyoD was expressed at high levels in wild-type myoblasts and was absent in the MyoD -deficient myogenic cells. Furthermore, densitometric analysis and normalization to 18S rRNA revealed that MyoD was somewhat downregulated during the differentiation of wild-type cells . Previously, we observed a 3.5-fold increase in Myf-5 mRNA in newborn and adult muscles in vivo . We similarly observed a fourfold increase in Myf-5 mRNA in growing MyoD −/− myogenic cells relative to wild-type myoblasts, supporting the hypothesis that MyoD negatively regulates Myf-5 expression . In wild-type cells, Myf-5 mRNA levels decreased about twofold after differentiation, whereas in MyoD −/− cultures, Myf-5 mRNA levels were upregulated about twofold after mitogen withdrawal . In addition, consistent with the observed 100-fold reduction in the rate of spontaneous differentiation, MyoD −/− myogenic cells expressed fivefold lower levels of myogenin mRNA in growth medium relative to wild-type myoblasts . After the induction of differentiation, the relative levels of myogenin mRNA in MyoD −/− cells remained significantly reduced relative to wild-type cultures . In wild-type cells, the level of MRF4 mRNA remained unchanged until day 2 of differentiation and was thereafter upregulated about sevenfold. By contrast, MRF4 mRNA was upregulated in differentiating MyoD −/− cultures to levels approximately two- to threefold lower than that of wild-type cells . Section title: Analysis of Muscle-specific Gene Expression Educational score: 4.257161617279053 Domain: biomedical Document type: Study Language: en Western analysis of MRF expression in lysates of primary cultures in growth medium confirmed the absence of MyoD protein in MyoD −/− cells . Interestingly, Myf-5 protein was upregulated >10-fold in MyoD −/− cells, suggesting that posttranscriptional mechanisms may contribute to this increase . As suggested by the Northern analysis , myogenin protein was absent in MyoD −/− cells in growth medium, whereas low levels were detected in wild-type myoblasts . Section title: Analysis of Muscle-specific Gene Expression Educational score: 4.385512351989746 Domain: biomedical Document type: Study Language: en To assess the differentiation kinetics at the level of gene expression, Northern analysis was performed with a panel of muscle-specific markers. Analysis of mRNA levels for differentiation-specific markers revealed a pattern consistent with overall delayed kinetics of differentiation in MyoD −/− myogenic cells. For example, low levels of α-skeletal and α-cardiac actin mRNAs were detected in wild-type cells in growth medium and these increased rapidly after mitogen withdrawal. By contrast, MyoD −/− cells in growth medium expressed no detectable sarcomeric actin mRNA . After induction of differentiation α-skeletal actin mRNA increased about threefold in wild-type cells, whereas in MyoD −/− cells the levels increased ∼65% of wild-type levels by day 5 . Although lower than that of α-skeletal actin, the levels of α-cardiac actin were found to increase in a similar pattern . Cultured MyoD −/− myogenic cells displayed a twofold reduction in levels of acetylcholine receptor δ subunit (AchR δ) mRNA in growth medium. After the induction of differentiation, a 5-fold increase was observed in wild-type cultures compared to an ∼2-fold increase in MyoD −/− cells for a net 10-fold relative difference. Section title: Analysis of Muscle-specific Gene Expression Educational score: 4.512980937957764 Domain: biomedical Document type: Study Language: en The protein coding for M-cadherin, a muscle-specific adhesion molecule, has been suggested to be expressed in quiescent satellite cells as well as to play an important role during myoblast differentiation and fusion . However, RT-PCR analysis only detects M-cadherin expression in a small number of quiescent satellite cells suggesting that M-cadherin may not be useful as a marker for satellite cells . In wild-type cells, M-cadherin mRNA was detected at low levels in growth medium, M-cadherin increased fivefold by day 2 of differentiation, and was subsequently downregulated . By contrast, MyoD −/− myogenic cells in growth medium expressed no detectable M-cadherin mRNA. However, low levels were detectable after mitogen withdrawal. Immunohistochemical detection of M-cadherin on cells in growth medium confirmed the reduced level of protein detectable on MyoD −/− cells relative to wild-type myoblasts . The reduced levels of M-cadherin expression observed in MyoD −/− myogenic cells may account in part for the differentiation deficiency as M-cadherin appears to be required for efficient myogenic differentiation and fusion . Section title: Analysis of Muscle-specific Gene Expression Educational score: 4.419273376464844 Domain: biomedical Document type: Study Language: en The receptor tyrosine kinase Musk has been suggested to be expressed in activated satellite cells , and therefore may provide an additonal marker for early myogenic cells. Northern blot analysis with a Musk -specific probe revealed the expression of three distinct isoforms in differentiating myogenic cells . In wild-type cultures in growth medium, the large mRNA (isoform 1) was not expressed, the midsize mRNA (isoform 2) was expressed at intermediate levels, and the small mRNA (isoform 3) was expressed at higher levels. Induction of differentiation of wild-type cultures resulted in upregulation of isoforms 1 and 2, but little change in isoform 3. By contrast, MyoD −/− myogenic cells in growth medium expressed no detectable expression of Musk mRNA isoforms 1 and 2, and low levels of isoform 3. After induction of differentiation of MyoD −/− cultures, delayed upregulation of isoforms 1 and 2 was observed. Therefore, these data suggest that Musk is upregulated in a differentiation-dependent manner during muscle regeneration. Section title: Analysis of Muscle-specific Gene Expression Educational score: 4.305967807769775 Domain: biomedical Document type: Study Language: en Adhalin, a dystrophin-associated protein also known as α-sarcoglycan, is upregulated during myoblast differentiation and is required for fully functional myofibers . Human loss-of-function mutations in adhalin results in limb girdle childhood autosomal recessive muscular dystrophy (SCARMD) . The delayed differentiation in the limb girdles evident in MyoD −/− embryos raised the possibility that adhalin may represent a MyoD target gene. Northern analysis revealed that adhalin is completely absent in growing MyoD −/− cells . At the onset of differentiation, adhalin was slightly downregulated in wild-type cells, whereas it steadily increased in MyoD −/− cultures reaching ∼50% of wild-type levels . Therefore, these data substantiate the delayed differentiation of MyoD −/− myocytes, but do not elucidate whether adhalin is specifically upregulated in the MyoD -induced embryonic lineage that gives rise to hypaxial musculature. Section title: Analysis of Growth-associated Gene Products Educational score: 4.219220161437988 Domain: biomedical Document type: Study Language: en To investigate the continued proliferation of MyoD −/− cells in differentiation medium, we examined the mRNA expression levels of several growth-associated proteins that have been demonstrated to play important roles in the control of myoblast differentiation. The plakoglobin-related protein, β-catenin , is believed to play important roles in cellular growth and morphogenesis in response to cellular adhesion and Wnt signaling . Primary wild-type myoblasts expressed abundant β - catenin mRNA under growth conditions. These levels increased threefold after 2 d of differentiation but subsequently decreased . In MyoD −/− cultures, β - catenin mRNA levels were found to continuously increase and to stabilize at levels that were comparable to that of wild-type cells by day 5 of differentiation . Detection of β-catenin protein by immunofluorescence revealed a similar nuclear cytoplasmic distribution in wild-type and mutant myogenic cells . Therefore, these data do not support a role for β-catenin in the differentiation deficiency evident in MyoD −/− myogenic cells. Section title: Analysis of Growth-associated Gene Products Educational score: 4.245428085327148 Domain: biomedical Document type: Study Language: en The PEA3 gene product is upregulated in activated satellite cells in vivo, and has been suggested to be important for myoblast fusion in vitro . Wild-type primary myoblasts in growth medium expressed very low levels of PEA3 mRNA. However PEA3 levels increased about twofold by day 5 of differentiation . By contrast, MyoD −/− cells in growth medium displayed sixfold higher levels of PEA3 mRNA, which declined steadily to wild-type levels by day 4 of differentiation . However, this increased level of PEA3 was not associated with enhanced differentiation (see above). Nevertheless, because PEA3 is expressed in activated satellite cells , these data are consistent with the notion that primary MyoD −/− myogenic cells represent an intermediate stage between a satellite cell and a myogenic precursor cell. Section title: Analysis of Growth-associated Gene Products Educational score: 4.349076271057129 Domain: biomedical Document type: Study Language: en The p53-inducible cyclin-dependent kinase inhibitor p21/ WAF1 arrests proliferating cells when ectopically expressed and is upregulated by MyoD during the differentiation of C2C12 myocytes . Moreover, forced overexpression of p21 in myoblasts drives induction of differentiation-specific genes . Unexpectedly, we observed no significant differences in p21 mRNA levels between wild-type and MyoD −/− cells, in growth medium or during differentiation . Therefore, the normal induction of p21 in MyoD −/− myogenic cells after mitogen withdrawal suggests that p21 induction is not MyoD-dependent and that p21 requires MyoD to positively stimulate differentiation. Section title: Analysis of Growth-associated Gene Products Educational score: 4.318531036376953 Domain: biomedical Document type: Study Language: en The insulin-like growth factor, IGF-I, stimulates the proliferation and inhibits differentiation of cultured myoblasts . To assess whether IGF-I expression was altered in the absence of MyoD, we analyzed expression levels in primary myogenic cultures. Wild-type myoblasts in growth conditions expressed low levels of IGF-I mRNA and these levels were rapidly extinguished after mitogen withdrawal . By contrast, MyoD −/− myogenic cells expressed over threefold higher levels of the small IGF-I mRNA isoforms 1 and 2 and these remained constant after transfer of the cells to differentiation medium . Interestingly, the 7-kb pre- IGF-I mRNA (isoform 3) was not expressed under growth conditions but was rapidly upregulated after 3 d of differentiation. These observations strongly suggest that MyoD negatively regulates IGF-I expression and raises the possibility that MyoD is required for the repression of IGF-I expression during normal myogenic differentiation. Section title: The MyoD Mutant Cellular Phenotype Is a Cell Autonomous Deficit Educational score: 4.470505237579346 Domain: biomedical Document type: Study Language: en The reduced fusion and continued proliferation of MyoD −/− myogenic cells under conditions that normally promote cell-cycle withdrawal and terminal differentiation can be hypothesized to be a consequence of cell autonomous attributes. For example, the marked reduction in M-cadherin expression and the overexpression of IGF-I in MyoD −/− myogenic cells could both act to inhibit differentiation. Alternatively, MyoD −/− cells may have a unique developmental identity that precludes participation in the myogenic precursor cell differentiation program. To explore these possibilities, we mixed different proportions of wild-type myoblasts with lacZ-expressing MyoD −/− myogenic cells, and induced differentiation by culturing the cells in 5% horse serum for 5 d . Importantly, PGK-lacZ expression is unaffected by terminal differentiation in transfected wild-type myoblasts (not shown). Strikingly, lacZ-labeled nuclei were never detected within any myotubes containing more than two nuclei . Conversely, the differentiation of wild-type myocytes was completely unaffected by the presence of high numbers of MyoD −/− myogenic cells . Taken together, these data support the notion that Myf-5 expression may define a distinct cell identity in the satellite cell developmental program. Section title: The MyoD Mutant Cellular Phenotype Is a Cell Autonomous Deficit Educational score: 4.546805381774902 Domain: biomedical Document type: Study Language: en Our analysis suggested that in satellite cell–derived myogenic cell lineage, important aspects of cytomorphology, differentiation, and ultimately fusion of mononuclear cells into myotubes are highly dependent on MyoD activity and cannot be substituted for by other MRFs. To determine whether the observed phenotypic differences between MyoD −/− and wild-type primary myogenic cells were attributable to MyoD, a MyoD -expression plasmid was introduced into low passage cells and stable pools of transfectants were analyzed. Western blot analysis indicated that transfected MyoD −/− cells (termed MyoD+ ) expressed the exogenous MyoD protein . By densitometry, the levels were found to be approximately threefold lower than in wild-type cells but similar to MyoD levels in C2C12 myoblasts. As expected, untransfected or PGK-Puro transfected MyoD −/− cells did not express MyoD protein . In growth medium, pools of stable MyoD+ cells displayed an almost complete reversion of the fibroblastlike phenotype and exhibited a rounded compact cytomorphology similar to wild-type cells . Transfer of MyoD+ cultures into differentiation medium resulted in increased numbers of MF20-positive differentiated myocytes that displayed the elongated bipolar multinucleated myotube morphology typical of wild-type myocytes . After 3 d in low serum medium, the MyoD+ pools displayed fusion indices that were comparable to wild-type cultures and approximately fivefold higher than MyoD −/− cells . Differentiated MyoD+ myocytes contained 2.5 ± 0.4 nuclei on average, differentiated wild-type myocytes contained 2.7 ± 0.1 nuclei on average, and differentiated MyoD −/− myocytes contained only single nuclei . Lastly, transfection of MyoD −/− cells with a Myf-5 expression plasmid did not rescue cytomorphology or differentiation potential (not shown). Taken together, these observations suggest that expression of MyoD is necessary and sufficient to reestablish the progression of MyoD −/− myogenic cells through the differentiation program. Section title: Discussion Educational score: 4.233073711395264 Domain: biomedical Document type: Study Language: en Analysis of muscle regeneration in MyoD −/− mice led to the suggestion that MyoD is required for satellite cells to efficiently give rise to proliferative myogenic precursor cells . To characterize the phenotype of MyoD -deficient myoblasts and to gain insight into the role of MyoD in the activation and differentiation of satellite cells, an in-depth characterization of MyoD −/− primary myoblast cultures was undertaken. The establishment and propagation of stable cell lines can potentially result in aneuploidy as well as the introduction of additional mutations, which are necessary for immortalization and continuous proliferation in culture. To avoid such anomalies, all of our analyses were carried out using newly established low-passage primary myoblast cultures. Our characterization of early passage primary cultures reported here differs from the analysis of later passage MyoD −/− cultures (>15 doublings) that exhibited decreased growth, readily formed multinucleated myotubes, and grew in an FGF2-independent manner . Therefore, these results underscore the importance of characterizing early passage (<10 doublings) primary cultures before any adaptation to growth in tissue culture conditions. Section title: Discussion Educational score: 4.328600883483887 Domain: biomedical Document type: Study Language: en Primary MyoD −/− myogenic cells exhibited a fibroblastlike cytomorphology, and expressed c-met and high levels of PEA3 and IGF-I , but did not express desmin, M-cadherin , or Musk. Transfer of primary MyoD −/− cells into differentiation medium resulted in formation of reduced numbers of mononuclear myocytes and delayed induction of differentiation-specific markers. Mixing experiments revealed that MyoD −/− cells did not influence the differentiation of wild-type myocytes and did not fuse with differentiating myotubes. Forced expression of MyoD rescued both the cytomorphology and the differentiation deficit. Taken together, these data suggest that Myf-5 expressing cells represent an intermediate between a quiescent satellite cell and a proliferating myogenic precursor cell. Section title: Discussion Educational score: 4.68535041809082 Domain: biomedical Document type: Study Language: en Northern blot analysis revealed that MyoD −/− myogenic cells expressed fourfold higher levels of Myf-5 mRNA and Western analysis revealed an even greater increase in Myf-5 protein. Similar findings are observed for the in vivo expression of Myf-5 in the muscle of MyoD -deficient mice . Interestingly, delayed muscle differentiation has also been reported during limb development of MyoD −/− embryos in which Myf-5 expressing myogenic processors arrive in the limb but differentiate with markedly delayed kinetics . Embryos lacking MyoD display normal development of trunk musculature in the body proper, whereas muscle development in limb buds and branchial arches is delayed by ∼2.5 d. In contrast, embryos lacking Myf-5 display normal muscle development in limb buds and branchial arches, but exhibit a marked delay in development of trunk muscles. Although MyoD -mutant embryos exhibit delayed development of limb musculature, the migration of Pax-3–expressing cells into the limb buds and subsequent induction of Myf-5 in myogenic precursors occurs normally. These data, together with the observed regeneration deficit in MyoD −/− muscle, indicate that MyoD and Myf-5 cannot fully substitute for each other during embryogenesis and in satellite cells, and suggest that Myf-5 and MyoD activate discrete subsets of target genes that differentially define myogenic cell identity. Section title: Discussion Educational score: 4.5833563804626465 Domain: biomedical Document type: Study Language: en Forced expression of MyoD in a variety of cell lines induces growth arrest even in the absence of differentiation . Consistent with this, myogenic cells lacking MyoD displayed inefficient withdrawal from the cell cycle in response to low mitogens. However, the cell-cycle inhibitor p21 was upregulated to similar levels in wild-type and MyoD −/− cells in growth medium and following induction of differentiation. In C2C12 myoblasts, expression of p21 appears to be directly induced by MyoD upon cell-cycle arrest and terminal differentiation . Moreover, forced expression of p21 arrests proliferating cells , and induces the terminal differentiation of C2C12 myoblasts . Therefore, the relationship between cell-cycle control and differentiation appears to be uncoupled in primary MyoD −/− myogenic cells. Interestingly, depending on context, p21 expression can either arrest cells via inhibiting cdk activity, or promote cell division by acting as a cdk4/cyclin D1 assembly factor . Future characterization of cell cycle control in mutant cells should elucidate this aspect of the MyoD −/− myogenic cell phenotype. Section title: Discussion Educational score: 4.5097856521606445 Domain: biomedical Document type: Study Language: en The PEA3 transcription factor was observed to be expressed at about sixfold higher levels in MyoD -deficient myoblasts under growth conditions. The ETS-domain transcription factor PEA3 is rapidly induced after muscle damage and forced expression of PEA3 stimulates myogenesis in vitro when overexpressed in satellite cell–derived cultured myoblasts . These data led to the suggestion that PEA3 is an important regulator of activated satellite cell function . The lack of correlation between increased expression of PEA3 and differentiation potential in MyoD −/− myogenic cells supports the assertion that MyoD −/− myogenic cells have an identity distinct from wild-type myoblasts. Interestingly, overexpression of PEA3 is correlated with increased metastatic potential of mammary adenocarcinomas . Moreover, overexpression of PEA3 in cultured cells directly induces the upregulation of a subset of matrix metalloproteases (Hassell, J.A., personal communication). After muscle damage, activated satellite cells readily cross the basal lamina and are capable of migrating from surviving to damaged areas . By contrast, cultured primary myoblasts injected into muscle exhibit a very poor ability to migrate away from the injection site . Therefore, it will be of interest to determine whether MyoD −/− cells are more invasive than their wild-type counterpart. Section title: Discussion Educational score: 4.288805961608887 Domain: biomedical Document type: Study Language: en M-cadherin had been suggested to be expressed in satellite cells . However, recent RT-PCR analysis indicates that a small minority of satellite cells express M-cadherin mRNA . Consistent with this we observed markedly reduced expression of M-cadherin in MyoD −/− myogenic cells relative to wild-type cells . This observation raises the possibility that MyoD directly or indirectly regulates the expression of surface adhesion molecules involved in fusion and differentiation. Interestingly, incubation of antagonistic M-cadherin peptides or antisense RNA inhibits both myoblast fusion and cell-cycle withdrawal in conditions that normally promote differentiation . Therefore, these data suggest that cell-cycle withdrawal during terminal differentiation also involves cell–cell interactions. Section title: Discussion Educational score: 4.421032905578613 Domain: biomedical Document type: Study Language: en Cellular adhesion is clearly linked to regulation of proliferation, migration, and differentiation. For example, expression of dominant negative cadherin inhibits proliferation and stimulates terminal differentiation of human epidermal keratinocytes . Moreover, integrin and cadherin synergistically inhibit migration and promote the aggregation of myoblasts . Forced overexpression of an effector of adhesion mediated signaling integrin-linked kinase (ILK) in intestinal epithelial cells and mammary epithelial cells induces the activity of G1/S cyclin/Cdks, downregulates E-cadherin expression, induces nuclear translocation of β-catenin, and results in increased invasiveness . Therefore, examination of integrin and cadherin function in MyoD −/− myogenic cells should elucidate the role of adhesion in the control of proliferation and migration in early myogenic precursors. Section title: Discussion Educational score: 4.553182125091553 Domain: biomedical Document type: Study Language: en High level expression of IGF-I in L6 myoblasts stimulates proliferation and inhibits differentiation, whereas lower levels of IGF-I stimulate both proliferation and differentiation . A striking feature of MyoD −/− cells was the relatively high expression of IGF-I mRNA and its continued upregulation during differentiation. In contrast, wild-type myoblasts expressed lower levels of IGF-I in growth conditions and these levels decreased during differentiation. It is interesting to speculate that increased IGF-I expression in MyoD −/− cultures contributes to the observed differentiation delay. As IGF-I levels rapidly decrease in wild-type cultures induced to differentiate, an interesting hypothesis is that MyoD is required to downregulate IGF-I at the onset of differentiation. In MyoD −/− myogenic cells, continued IGF-I expression could result in an autocrine loop that acts to promote proliferation. However, the presence of MyoD −/− myogenic cells did not inhibit the differentiation of wild-type primary myoblasts raising the possibility that expression of other components of the IGF-I signaling pathway are downregulated in wild-type myoblasts. These data further underscore the assertion that MyoD −/− myogenic cells are distinct from wild-type myoblasts. Section title: Discussion Educational score: 4.680948257446289 Domain: biomedical Document type: Study Language: en Continuous myoblast cell lines lacking MyoD exhibit somewhat similar traits in comparison to primary MyoD −/− myogenic cells. For example, C2C12 cells expressing antisense MyoD RNA, display increased Myf-5 expression, decreased IGF-II expression, and are defective in differentiation . The brain tumor–derived BC3H1 myoblast cell line expresses Myf-5 but does not express MyoD, and exhibits a differentiation deficit with reduced ability to form multinucleated myotubes . However, unlike primary MyoD -deficient myogenic cells, BC3H1 myocytes in differentiation-inducing medium exhibit normal upregulation of myogenin together with normal induction of subsets of MHC isoforms and other differentiation-specific markers . Forced expression of myogenin in BC3H1 cells is unable to rescue the differentiation deficit, whereas forced expression of MyoD confers competency for myogenic differentiation . Forced expression of a functional MyoD protein in MyoD -deficient cells was sufficient to revert the MyoD −/− cytomorphology and rescue the differentiation defect as evidenced by a dramatic increase in fusion index of MyoD+ cells. Therefore, these data support the assertion that lack of MyoD results in a cell-autonomous deficit in the satellite cell differentiation program. Taken together, the parallels observed between primary MyoD −/− myocytes and continuous myoblast cell lines deficient in MyoD support a unique set of functions for MyoD that cannot be substituted for by Myf-5. Section title: Discussion Educational score: 4.549932956695557 Domain: biomedical Document type: Study Language: en The muscle regeneration deficit in MyoD −/− muscle suggests that expression of MyoD is required for satellite cells to efficiently form differentiation-competent myogenic precursor cells . RT-PCR analysis reveals that activated satellite cells first express either Myf-5 alone or MyoD alone, before coexpressing Myf-5 and MyoD , and subsequently progressing through the myogenic program . Our analysis of the phenotype of primary MyoD −/− myogenic cells can be interpreted to suggest that MyoD −/− myogenic cells represent an intermediate stage between a quiescent satellite cell and a myogenic precursor cell. Together, these data suggest the hypothesis that expression of Myf-5 alone may define an intermediate developmental stage that provides a mechanism for satellite cell self-renewal. In this model, activated satellite cells expressing only Myf-5 could undergo cell division and either return to quiescence by downregulating Myf-5 , or alternatively upregulating MyoD and progressing through the myogenic program . Clearly, further analysis of the developmental potential and phenotype of primary MyoD −/− myogenic cells may present a unique opportunity to investigate the early myogenic program of satellite cells. | Study | biomedical | en | 0.999995 |
10037787 | Section title: Production of mAbs Educational score: 4.1464524269104 Domain: biomedical Document type: Study Language: en mAbs were obtained essentially according to the procedure of Köhler and Milstein . 50 μg of denatured recombinant human Ran was initially intraperitoneally administered with Freund's complete adjuvant to a 16-wk-old BDF1 mouse (Japan SLC), followed by three subsequent injections at 3-wk intervals with the same dose in Freund's incomplete adjuvant. 1 mo after the fourth injection, the mouse was given a booster injection of the same dose. 4 d later, spleen cells isolated from the mouse were fused with the mouse myeloma cell line P3U1 using standard methods. Screening was performed by ELISA and immunoblotting using the recombinant human Ran. Ran-specific mAbs were typed by using mouse monoclonal antibody isotyping kit ( Amersham ). Ascites fluid was produced from a BDF1 × BALB/3T3 mouse (Japan SLC) which had been implanted with the hybridoma. The IgG fraction was obtained by precipitation with 50% saturated ammonium sulfate followed by chromatography on a protein A affinity column. Section title: Antibodies Educational score: 2.482729196548462 Domain: biomedical Document type: Study Language: en Rabbit anti-Ran polyclonal antibodies were produced as described previously . Chromatographically purified normal mouse IgG was purchased from Zymed. Rabbit anti–importin β polyclonal antibodies were prepared as described previously . Mouse anti–human CAS monoclonal antibody and mouse anti–human transportin monoclonal antibody were purchased from Transduction Laboratories. Section title: Expression and Purification of Recombinant Proteins Educational score: 4.225679397583008 Domain: biomedical Document type: Study Language: en Expression and purification of recombinant importin β were performed as described previously . The human CAS gene was amplified from a HeLa cDNA library via the polymerase chain reaction (PCR) using the synthetic oligonucleotide primers, 5′-TTTTTTGGATCCATGGAACTCAGCGATGCAAATCTGCAA-3′ and 5′-TTTTTTCTCGAGTTAAAGCAGTGTCACACTGGCTGCCTG-3′. The PCR product was inserted into BamHI and XhoI sites of pGEX-6P-2/hGFP vector, which was an expression vector of glutathione-S-transferase (GST)- fused green fluorescent protein (containing the S65A/Y145F mutation) (hGFP). This construct was transformed to express the recombinant GST–hGFP-CAS fusion protein in Escherichia coli strain BL21. The expressed GST–hGFP-CAS fusion protein was purified on glutathione– Sepharose ( Pharmacia Biotech ). After the digestion of GST portion with PreScission Protease ( Pharmacia Biotech ), hGFP–CAS protein was purified by ion-exchange chromatography on Hi-TrapQ ( Pharmacia Biotech, Inc. ), and then was dialyzed against 20 mM Hepes, pH 7.3, 110 mM potassium acetate, 2 mM DTT, and 1 μg/ml each of aprotinin, leupeptin, and pepstatin. Section title: Expression and Purification of Recombinant Proteins Educational score: 4.132197380065918 Domain: biomedical Document type: Study Language: en Human transportin/karyopherin β2 gene was amplified from HeLa cell cDNA library by the PCR using the synthetic oligonucleotide primers (5′-CTCAGCGGATCCATGGAGTATGAGTGGAAACCTGAC-3′ and 5′-CTCAGCGGTACCTTAAACACCATAAAAAGCTGCAAG-3′). The PCR product was inserted into the BamHI and KpnI sites of a modified pGEX-6P-3 ( Pharmacia ) and verified by DNA sequencing. Recombinant GST-transportin was expressed and purified as described for hGFP–CAS. Section title: Expression and Purification of Recombinant Proteins Educational score: 4.2933807373046875 Domain: biomedical Document type: Study Language: en E . coli strains expressing wild-type Ran were prepared as described previously . Mutation (Q69L) within Ran was created using QuikChange site-directed mutagenesis kit (Strategene). RanQ69L fragment was inserted into NcoI and BamHI sites of pET3d. Recombinant RanQ69L protein was expressed in E . coli and purified in the same manner as wild-type Ran. Recombinant wild-type and Q69L Ran were expressed, purified, and then charged with GTP or GDP according to the method of Bischoff and Ponstingl and Melchior et al. with slight modifications. In brief, expression was induced by addition of 1 mM isopropyl-β d -thiogalactopyranoside (IPTG) and incubation for 14 h at 20°C. The E . coli cells were lysed in buffer C (50 mM Tris-HCl, pH 8.0, 75 mM NaCl, 1 mM MgCl 2 , 0.1 mM PMSF, 1 mM DTT, 1 mg/ml each of aprotinin, leupeptin, and pepstatin) by freeze-thaw. The clarified lysates were applied to DEAE-Sepharose FF column ( Pharmacia ) and flow through fractions were collected. After 60% saturate ammonium sulfate precipitation, 2 mM GTP or GDP was added and incubated for at least 1 h in buffer D (50 mM phosphate buffer, pH 7.0, 1 mM 2-mercaptoethanol, 10% glycerol, 2 mM GTP or GDP) on ice. The samples were applied to HiPrep Sephacryl S-200 HR FPLC column ( Pharmacia ) equilibrated with buffer D, and peak fractions containing Ran proteins were pooled. GTP- and GDP-form of Ran were further separated on Fractogel EMDSO 3 − 650 (s) column (Merck) with linear gradient of buffer D containing 50 mM phosphate to 500 mM phosphate. GDP-bound form of Ran was eluted between 200 and 250 mM phosphate. GTP-bound form was eluted between 350 and 400 mM phosphate. The purified GTP- or GDP-form of Ran was then desalted with PD10 column ( Pharmacia ) equilibrated with transport buffer (see below), concentrated with centricon 30 (Amicon), and then frozen in small aliquots. Section title: Expression and Purification of Recombinant Proteins Educational score: 4.147407531738281 Domain: biomedical Document type: Study Language: en The guanine nucleotides bound to purified Ran were determined by FPLC ( Pharmacia ) after denaturation. The Ran preparations were heated at 95°C for 2 min. After filtration, the solutions were applied to a monoQ HR5/5 ( Pharmacia ) column and then the guanine nucleotides were eluted with a linear gradient from 10 mM potassium phosphate buffer, pH 8.0, to 50 mM potassium phosphate buffer, pH 8.0, containing 250 mM NaCl. Nucleotides were detected at 254 nm and quantified against a standard GDP/ GTP solution. The bound nucleotides were found to be virtually 100% GDP for the GDP-bound preparations and 95% GTP for the GTP-bound preparations. Section title: Expression and Purification of Recombinant Proteins Educational score: 4.181248664855957 Domain: biomedical Document type: Study Language: en All GST-fused deletion mutants of human Ran were prepared using PCR with appropriate oligonucleotides. The PCR product was inserted into the BamHI/EcoRI site of pGEX-2T vector. For the production of the GST-fused COOH-terminal domain of Ran, the synthesized oligonucleotide, corresponding to the COOH-terminal region of human Ran, TALPDEDDDL, was inserted into the BamHI/EcoRI site of pGEX-2T vector ( Pharmacia ). These resultant plasmids were sequenced to confirm the fidelity of the region, which had been amplified by PCR and in-frame ligation of the fused region. Recombinant GST-fused proteins were expressed by incubation with 1 mM IPTG for 6 h at 37°C in E . coli strain BL21(DE3). The purification of the GST-fused COOH-terminal domain of Ran with glutathione–Sepharose was performed as described for importin β. The protein was then dialyzed against 20 mM Hepes, pH 7.3, 110 mM potassium acetate, and 2 mM DTT. Section title: Expression and Purification of Recombinant Proteins Educational score: 4.248105049133301 Domain: biomedical Document type: Study Language: en Mouse RanBP1 was expressed as GST fusion protein. A fragment which is encoding the entire amino acid sequence of this protein was amplified from mouse Ehrlich ascites tumor cell cDNA by PCR using synthetic oligonucleotides (5′-CCTACGGATCCATGGCGGCCGCCAAGGACA-3′ and 5′-CCACTGAATTCTCATTGTTTCTCCTCAGAC-3′) and cloned into the BamH1-EcoRI sites of pGEX-2T, to produce an in-frame fusion with GST. Expression in BL21(DE3), lysis of bacteria, and purification of the fusion protein with glutathione–Sepharose were performed as described for importin β. The GST portion of chimeras was cleaved off by a 2-h incubation at room temperature with 1 NIH U of thrombin ( Sigma ) per 100 μg of chimeras. GST and thrombin were separated from recombinant protein on a MonoQ column ( Pharmacia Biotech ) at flow rate of 0.5 ml/min with a linear gradient from 0.025 to 1.0 M NaCl in 20 mM Hepes, pH 7.3, 2 mM DTT, and 1 μg/ml each of aprotinin, leupeptin, and pepstatin. Free RanBP1 protein was dialyzed against 20 mM Hepes, pH 7.3, 110 mM potassium acetate, 2 mM DTT, and 1 μg/ml each of aprotinin, leupeptin, and pepstatin. Section title: Preparation of Ehrlich Ascites Tumor Cells Cytosolic Extract Educational score: 3.4862425327301025 Domain: biomedical Document type: Study Language: en Cytosolic extract was prepared from Ehrlich ascites tumor cells as described previously . Section title: Gel Electrophoresis and Immunoblotting Educational score: 3.95865797996521 Domain: biomedical Document type: Study Language: en One-dimensional SDS-PAGE was based on the method of Laemmli and the gels were stained with Coomassie brilliant blue. For immunoblotting, after electrophoresis, proteins were electrophoretically transferred from gels to nitrocellulose sheets. After blocking with 3% skim milk in PBS, the sheets were then incubated with the first antibody followed by alkaline phosphatase-conjugated anti-mouse or anti-rabbit IgG (Bio-Rad). Section title: Immunofluorescence Educational score: 4.135522365570068 Domain: biomedical Document type: Study Language: en BHK cells, plated on coverslips, were washed twice in PBS and fixed with 3.7% formaldehyde in PBS (at room temperature for 20 min) followed by permeabilization with 0.5% Triton X-100 in PBS (at room temperature for 5 min). After blocking with 3% skim milk in PBS, the cells were incubated with ARAN1 (3 μg/ml) or affinity-purified rabbit anti-Ran polyclonal antibodies (3 μg/ml) overnight at 4°C and detected with Cy3-conjugated goat anti–mouse IgG ( Amersham ) or FITC-conjugated goat anti–rabbit IgG (Tago). The samples were examined using a Zeiss Axiophot fluorescent microscope ( Carl Zeiss ). Section title: Immunoprecipitation Using Recombinant Proteins Educational score: 4.136745452880859 Domain: biomedical Document type: Study Language: en 50 pmol of Ran (GTP-bound or GDP-bound) and/or 50 pmol of importin β were incubated with 50 pmol of ARAN1 in total 300 μl of transport buffer (TB: 20 mM Hepes, pH 7.3, 100 mM potassium acetate, 2 mM magnesium acetate, 5 mM sodium acetate, 1 mM glycoletherdiaminetetraacetic acid [EGTA], 2 mM DTT, and 1 μg/ml each of aprotinin, leupeptin, and pepstatin) for 1 h at 4°C. Protein A–bound agarose beads ( Calbiochem-Novabiochem ) were then added and the mixture was incubated for 1 h at 4°C. The beads were collected by centrifugation and washed five times with TB. The bound fraction and unbound fraction were analyzed by SDS-PAGE and immunoblotting. Section title: Immunoprecipitation Using Recombinant Proteins Educational score: 4.073174476623535 Domain: biomedical Document type: Study Language: en 150 pmol of transportin or hGFP-CAS and 150 pmol of Ran-GTP were incubated with 150 pmol of ARAN1 in the presence or absence of 150 pmol of importin α in TB for 1 h at 4°C. The proteins which bound to ARAN1 were then analyzed using the same procedure as above. Section title: Immunoprecipitation Using Cytosolic Extract Educational score: 4.107976913452148 Domain: biomedical Document type: Study Language: en To preclear the extract, anti-mouse IgG-conjugated agarose beads (American Qualex) were added to the Ehrlich ascetics tumor cell cytosolic extract and rotated for 1 h at 4°C. The beads were then removed by centrifugation. 300 pmol of ARAN1 and 300 pmol of Q69L Ran-GTP was added to 200 μl of the precleared cytosolic extract followed by 1 h of incubation at 4°C. 15 μl of protein A–agarose beads were then added to the mixture, which was then incubated for 1 h. After washing five times with TB, the proteins bound to the protein A–conjugated agarose beads were resuspended in SDS-PAGE sample buffer containing DTT and analyzed by immunoblotting using antibodies indicated in the figure legend. Section title: Solution Binding Assay Educational score: 4.124900817871094 Domain: biomedical Document type: Study Language: en Solution binding assays were performed in TB. 50 pmol of GST-importin β and 50 pmol of Ran-GTP was incubated with 10 μl of a packed volume of glutathione–Sepharose for 1 h at 4°C. After washing the beads twice with TB, 250 pmol or 2,500 pmol of ARAN1 was added to the mixture followed by a 30-min incubation, 50 pmol of RanBP1 was then added and the mixture incubated for an additional 30 min at 4°C. The beads were washed with TB and proteins bound to the beads were resuspended in SDS-PAGE sample buffer containing DTT and analyzed by SDS-PAGE. Section title: Solution Binding Assay Educational score: 4.111807346343994 Domain: biomedical Document type: Study Language: en 50 pmol of GST–importin β, 50 pmol of Ran-GDP, and 250 pmol of ARAN1 was incubated with 10 μl of a packed volume of glutathione– Sepharose for 1 h at 4°C. After washing the beads twice with TB, 50 pmol of RanBP1 was added to the beads and the mixture incubated for 1 h. For a control, 50 pmol of GST–importin β, Ran-GDP, and RanBP1 were incubated with glutathione–Sepharose for 1 h. The beads were then washed with TB and proteins bound to the beads were resuspended in SDS-PAGE sample buffer containing DTT, and then analyzed by SDS-PAGE. Section title: Preparation of Fluorescein-labeled NLS-containing Transport Substrates Educational score: 4.048437118530273 Domain: biomedical Document type: Study Language: en BSA was fluorescein-labeled and then chemically conjugated to the synthetic peptides which contained the amino acid sequence of SV-40 large T-antigen NLS (CYGGPKKKRKVEDP), for the transport substrates termed FITC–T-BSA, as described previously . Section title: Microinjection Educational score: 3.9771597385406494 Domain: biomedical Document type: Study Language: en BHK cells were grown on coverslips. Microinjection of proteins was performed as described previously . After incubation for the indicated time in each figure at 37°C, the cells were fixed with 3.7% formaldehyde in PBS. The injected fluorescently labeled proteins were detected by fluorescent microscopy (model Axiophot 2; Carl Zeiss ). Section title: A Monoclonal Antibody Specific for Ran Educational score: 4.215024948120117 Domain: biomedical Document type: Study Language: en To study the functional domain(s) of Ran, monoclonal antibodies were produced against recombinant human Ran. The screening of the monoclonal antibodies was based on two criteria: (a) that they react with only Ran protein in immunoblotting; and (b) that they show the same staining pattern with polyclonal anti-Ran antibodies in immunofluorescence microscopy. Through this screening procedure, we isolated only one clone, designated ARAN1, for further characterization. ARAN1 was typed using a mouse monoclonal antibody isotyping kit as an immunoglobulin kappa G2b. On an immunoblot, ARAN1 detected recombinant human Ran and specifically reacted with a single 25-kD band in cell extracts from mouse Ehrlich ascites tumor cells, BHK21 cells, human embryonic lung (HEL) cells , Madin-Darby bovine kidney-derived epithelial cells (MDBK), and Xenopus laevis oocytes (data not shown), which was also detected by affinity-purified rabbit anti-Ran polyclonal antibodies. Furthermore, ARAN1 recognized a single spot by immunoblotting analysis of HeLa total cell extracts after two-dimensional electrophoresis, which was also detected with the anti-Ran polyclonal antibodies (data not shown). Immunofluorescent image with ARAN1 in BHK cells showed the same typical staining pattern as that with the anti-Ran polyclonal antibodies . From these findings, it was concluded that the monoclonal antibody ARAN1 specifically binds to Ran. Section title: ARAN1 Recognizes an Epitope at the COOH-terminal Domain of Ran Educational score: 4.27833890914917 Domain: biomedical Document type: Study Language: en To determine the specific epitope recognized by ARAN1, we prepared a series of truncated forms of recombinant Ran fused with GST and analyzed the interaction of these with ARAN1 by immunoblotting. Although anti-GST polyclonal antibodies showed that all of the Ran mutants were appropriately expressed and transferred to nitrocellulose , ARAN1 recognized only the full-length of GST-Ran. Removal of the COOH-terminal seven amino acids (210–216) of Ran completely abolished the reactivity with ARAN1 in immunoblotting, suggesting that the COOH-terminal domain, which consists of seven amino acid residues in Ran, represents the epitope for ARAN1. To confirm this, we prepared the 10-mer peptide of the COOH-terminal domain, 207–216, of Ran fused to GST . As shown in Fig. 3 , B and C, ARAN1 clearly reacted with the residues between 207 and 216 of human Ran not only in immunoblotting but also in a solution binding assay. From these findings, it was concluded that the epitope of ARAN1 is located in the COOH-terminal portion of Ran, which is highly negatively charged (−DEDDDL) and conserved among species . Section title: ARAN1 Binds to the Ran–Importin β Complex But Not the Ran Molecule Alone in Solution Binding Assay Educational score: 4.375538349151611 Domain: biomedical Document type: Study Language: en It is known that the stable Ran-GTP–importin β complex causes the inhibition of GTP hydrolysis on Ran stimulated by RanGAP1. In contrast, Ran-GDP shows a much lower affinity to importin β than Ran-GTP. To determine which form of Ran is recognized by ARAN1 in solution, immunoprecipitation analysis of ARAN1 using recombinant proteins was performed. Purified recombinant human Ran-GTP or Ran-GDP was incubated with ARAN1 in the presence or absence of importin β followed by the precipitation with protein A–conjugated agarose beads. The results showed that, whereas ARAN1 precipitated neither Ran-GTP nor Ran-GDP alone, it reacted with both Ran-GTP and Ran-GDP only in the presence of importin β and precipitated both Ran and importin β. To clearly confirm that ARAN1 can't recognize Ran molecule alone, precipitated proteins were analyzed by immunoblotting with anti-Ran polyclonal antibodies . The molar ratio of the Ran and importin β precipitated with ARAN1 was estimated to be ∼1:1 (data not shown). Since ARAN1 did not bind to importin β directly in this assay, these results indicate that ARAN1 reacts only with Ran when bound to importin β, and suggest that Ran changes conformation when bound to importin β which, in turn, exposes its COOH-terminal portion. Section title: ARAN1 Also Binds to the Ran–Transportin Complex and the Ran–CAS–Importin α Complex Educational score: 4.353057384490967 Domain: biomedical Document type: Study Language: en It is well known that Ran interacts with some import and export receptors, e.g., transportin, CAS, and CRM1, which are members of the superfamily of Ran-GTP-binding protein/importin β . Tranportin forms a complex with Ran-GTP and CAS simultaneously binds to both Ran-GTP and importin α . Judging from the conservation of the Ran-binding domain in this family, it is likely that the COOH-terminal tail of Ran may also be exposed as the result of the interaction with these molecules. To confirm this, we performed the solution binding assay with different importin β–related transport factors, transportin, and CAS. The results clearly showed that ARAN1 binds to both Ran-GTP/transportin complex and Ran-GTP–CAS–importin α complex , suggesting that importin β–related transport factors might induce a general conformational change in Ran through their interaction. In the case of CAS, it should be noted that a slight recognition of a complex between CAS and Ran-GTP by ARAN1 can also be observed . This suggests that CAS directly binds to Ran-GTP in the absence of importin α, although the affinity is very low. Section title: ARAN1 Suppresses the Ternary Complex Formation of Ran, Importin β, and RanBP1 in a Solution Binding Assay Educational score: 4.339528560638428 Domain: biomedical Document type: Study Language: en Both Ran-GTP and Ran-GDP form a ternary complex with RanBP1 and importin β in a solution binding assay. Although it has been assumed that RanBP1 plays a role in the dissociation of the Ran-GTP–importin β complex, the biological significance of the ternary complex has not yet been established. The RanBP1–Ran-GDP–importin β complex has been proposed to play a role in nuclear protein import as a ternary complex , although it has not yet demonstrated how the complex functions in the nuclear protein import. We therefore examined the effects of ARAN1 on the interaction of RanBP1 with both Ran-GTP– importin β and Ran-GDP–importin β complexes in the solution binding assay. In the absence of ARAN1, GST-tagged importin β efficiently precipitated both Ran-GTP and Ran-GDP together with RanBP1 in a molar ratio of 1:1 . In contrast, in the presence of ARAN1, the amount of RanBP1 precipitated with GST-tagged importin β apparently decreased, although the Ran was efficiently precipitated. These results indicate that RanBP1 is not able to interact with the Ran-GTP–importin β and Ran-GDP– importin β complexes, when bound to ARAN1. That is, the binding of ARAN1 to the Ran–importin β complex suppresses the ternary complex formation of both RanBP1– Ran-GTP–importin β and RanBP1–Ran-GDP–importin β in solution. Section title: Ran–Importin β–related Transport Factor Complex Is Exported from the Nucleus to the Cytoplasm Educational score: 4.235657215118408 Domain: biomedical Document type: Study Language: en As described above, it has been proposed that the Ran-GTP– importin β complex and Ran-GTP–importin β–related transport factor (such as transportin and CAS) complex are formed in the nucleus and exported to the cytoplasm. To determine whether these complexes are actually formed in the nucleus and migrate to the cytoplasm as a single entity in living cells, ARAN1, which binds to only the Ran complexed with importin β and importin β–related transport factors in solution, was injected into the cytoplasm or nucleus of cultured BHK cells. It was found that nuclear-injected ARAN1 was efficiently exported to the cytoplasm within 30 min of incubation , whereas control mouse IgG was retained in the injected nucleus (data not shown). Since due to its size ( M r = 160,000), mouse IgG cannot passively traverse the nuclear envelope , these results indicate that ARAN1 migrates to the cytoplasm in a piggyback fashion. These findings suggest that it is most likely that ARAN1 binds to the Ran-GTP complexed with importin β–related transport factors in the nucleus, in which Ran would exist as the GTP-bound form, and is transported to the cytoplasm as a complex with them. That is, these results suggest that the Ran-GTP–importin β–related transport factor complex moves to the cytoplasm as a single entity without dissociation. On the other hand, cytoplasmically injected ARAN1 remained in the cytoplasm even after 3 h of incubation (data not shown). Section title: Ran–Importin β–related Transport Factor Complex Is Exported from the Nucleus to the Cytoplasm Educational score: 4.199590682983398 Domain: biomedical Document type: Study Language: en To determine whether ARAN1 actually recognizes the Ran-GTP–importin β–related transport factor complex in a crude cell extract, we performed immunoprecipitation analysis with mouse Ehrlich ascites tumor cell cytosolic extract in the presence of Q69L Ran-GTP, which should stabilize the complexes of importin β–related transport factors and Ran-GTP in the crude lysate. As shown in Fig. 6 B, importin β, CAS, and transportin were coprecipitated with the Ran from the cell lysate by ARAN1. This result supports the view that nuclear-injected ARAN1 binds to Ran-GTP–importin β–related factors and is exported from the nucleus to the cytoplasm in vivo. Section title: ARAN1 Affects the Distribution of Endogenous Ran Educational score: 4.128320693969727 Domain: biomedical Document type: Study Language: en Ran is localized predominantly in the nucleus. If Ran continuously shuttles between two compartments, the nucleus and cytoplasm, and the binding of RanBP1 to the Ran-GTP–importin β complex in the cytoplasm is required for the recycling of Ran to the nucleus, ARAN1 would be expected to affect the distribution of Ran. To examine this hypothesis, ARAN1 was introduced into the cell and the distribution of endogenous Ran was observed. As shown in Fig. 6 , 30 min after injection into either the nucleus or the cytoplasm, injected ARAN1 localized in the cytoplasm. 30 min after injection of ARAN1 into either the nucleus or the cytoplasm, the subcellular localization of endogenous Ran became apparently dominant in the cytoplasm of the ARAN1-injected cells, whereas control mouse IgG had no effect on this distribution . The relocalization of Ran was caused irrespective of the injection sites of ARAN1. Section title: ARAN1 Inhibits the Nuclear Import of Proteins Containing the Classical NLS Educational score: 4.210841178894043 Domain: biomedical Document type: Study Language: en Lastly, we examined the effect of ARAN1 on the nuclear protein import in living cells. For this, ARAN1 or control mouse IgG was injected into nucleus or cytoplasm before the cytoplasmic injection of FITC-labeled BSA conjugated with the NLS peptide of SV-40 T antigen (FITC–T-BSA). In both cases, in which ARAN1 was injected into the cytoplasm or the nucleus, the nuclear import of FITC–T-BSA, which was injected afterwards was clearly inhibited . In both cases, the injected ARAN1 was confirmed to be localized in the cytoplasm as expected from the results of Fig. 6 (data not shown). In contrast, the simultaneous cytoplasmic injection of ARAN1 with FITC– T-BSA failed to inhibit the nuclear import of the FITC– T-BSA (data not shown). These results suggest that the injected ARAN1 interrupts the Ran cycle in the cytoplasm probably by preventing RanBP1 from interacting with the Ran–importin β complex, which causes an abnormal distribution of Ran and further the suppression of nuclear protein import. Section title: The COOH-terminal Acidic Domain of Ran Is Exposed to the Molecular Surface as the Result of Its Interaction with Importin β or Importin β Families Educational score: 4.209053039550781 Domain: biomedical Document type: Study Language: en In this study we obtained a monoclonal anti-Ran antibody, ARAN1, which recognizes mono-specific Ran as confirmed by immunoblotting of total cell extract and immunofluorescent analysis of cultured cells. An epitope of ARAN1 was found to lie in the acidic COOH-terminal domain of Ran. A solution binding assay revealed that ARAN1 interacts only with Ran, when it is complexed with importin β, transportin, or CAS–importin α but not Ran alone. These results indicate that the COOH-terminal domain of Ran is exposed to the molecular surface probably as the result of a conformational change only when importin β–related transport factors bind to Ran. Section title: The COOH-terminal Acidic Domain of Ran Is Exposed to the Molecular Surface as the Result of Its Interaction with Importin β or Importin β Families Educational score: 4.176243782043457 Domain: biomedical Document type: Study Language: en Although ARAN1 recognizes only Ran complexed with importin β or its related transport factors in solution, it shows the same immunofluorescence pattern as a polyclonal anti-Ran antibodies . Since it was previously reported that importin β is located not only in the cytoplasm but also within the nucleus , it is likely that the apparent nucleoplasmic staining by ARAN1 means the complex formation of Ran with importin β and importin β–related transport factors in the nucleus. Alternatively, this can be explained by a similar degree of Ran denaturation during cell fixation as in immunoblotting. Section title: The COOH-terminal Acidic Domain of Ran Is Exposed to the Molecular Surface as the Result of Its Interaction with Importin β or Importin β Families Educational score: 4.3810954093933105 Domain: biomedical Document type: Study Language: en It was found that ARAN1 binds to Ran-GDP–importin β complex as well as Ran-GTP–importin β complex, whereas it is known that importin β binds to Ran-GDP with much lower affinity than to Ran-GTP. Chi et al. demonstrated that the addition of RanBP1 with Ran-GDP increased the affinity of Ran-GDP for importin β. By analogy, it is speculated that ARAN1 may stabilize the interaction of Ran-GDP with importin β like RanBP1. In both cases, whereas ARAN 1 cannot reach the COOH-terminal domain of Ran-GTP or Ran-GDP alone, the interaction of Ran with importin β appears to induce a conformational change of Ran so that ARAN1 is able to recognize the epitope domain. Richards et al. demonstrated that a polyclonal antibody against a peptide corresponding to residues 196–207 of Ran, which is adjacent to the acidic 211 DEDDDL-COOH 216 domain, preferentially recognizes the GTP-bound form of Ran. They therefore proposed that the COOH-terminal domain is structurally flexible and undergoes a nucleotide-dependent conformational change, which is not inconsistent with the results herein. Section title: The COOH-terminal Acidic Domain of Ran Is Exposed to the Molecular Surface as the Result of Its Interaction with Importin β or Importin β Families Educational score: 4.374432563781738 Domain: biomedical Document type: Study Language: en Due to the characteristic amino acid sequences and conformational flexibility, it is likely that the COOH-terminal domain has some functions. Richards et al. also proposed from their biochemical analysis using ΔDE-Ran that the DEDDDL sequence stabilizes the GDP form of Ran, possibly by folding into the guanine nucleotide binding pocket and mimicking the negative charge on the γ-phosphate of GTP. In addition, it was suggested that the COOH terminus of Ran is implicated in the regulation of interaction between Ran and RanBP1 . Consistent with their suggestion, we showed that the binding of ARAN1 to the COOH-terminal tail suppresses the interaction of RanBP1 with the Ran–importin β complex. Although ΔDE-Ran is able to bind to RanBP1, its affinity to RanBP1 is lower than wild-type Ran . Therefore, we speculate that exposure of the COOH-terminal tail may increase the accessibility of RanBP1 not only to the Ran–importin β complex but also to the Ran– importin β–related transport factors. Section title: The COOH-terminal Acidic Domain of Ran Is Exposed to the Molecular Surface as the Result of Its Interaction with Importin β or Importin β Families Educational score: 4.3720502853393555 Domain: biomedical Document type: Study Language: en A similar sequence in importin β ( 335 DENDDDW 342 ) with the COOH-terminal domain of Ran is immediately adjacent to sequences required for binding Ran-GDP/ RanBP1, but not Ran-GTP . They proposed that this sequence in importin β substitutes for the COOH-terminal Ran sequence in the nucleotide binding pocket of Ran when GDP is bound and that this rearrangement would expose the COOH terminus of Ran for binding to RanBP1, stabilizing the trimeric complex. Their proposal is consistent with our result that the COOH-terminal tail of Ran-GDP was also exposed by the binding of importin β. Section title: The Export of Ran–Importin β–related Transport Factor Complexes from the Nucleus Educational score: 4.505362510681152 Domain: biomedical Document type: Study Language: en Although it has been proposed that the Ran–importin β–related transport factor complexes form in the nucleus, are exported to the cytoplasm, and then dissociate in order to recycle , this proposal has not been experimentally verified in vivo. Recently, Izaurralde et al. showed that the depletion of nuclear Ran-GTP inhibits the export of importin α and β, transportin and NES-containing proteins in Xenopus laevis oocyte, suggesting that nuclear Ran-GTP is required for the export of importin α and β, transportin, and NES-containing proteins from the nucleus. However, their results do not necessarily mean that the Ran–importin β–related transport factor complexes are translocated from the nucleus to the cytoplasm as a complex. Thus, in regard to the dynamic behavior of Ran through the nuclear envelope, little concrete experimental data exist. In this study, we found that a monoclonal anti-Ran antibody ARAN1 failed to inhibit the export of Ran from the nucleus and inhibited reimport into the nucleus, and that nuclearly injected ARAN1 was efficiently exported from the nucleus. Our results indicate that the Ran–importin β–related transport factor complexes efficiently migrate from the nucleus to the cytoplasm as a complex in living cells, even when complexed with ARAN1. Although it can not be excluded that the nuclear export of ARAN1 injected into the nucleus could be due to complex formation with Ran-GDP–importin β–related factors rather than Ran-GTP–importin β–related factors, the current model of a steep Ran-GTP gradient across the nuclear membrane (see introduction) and the immunoprecipitation experiments with ARAN1 in the presence of Q69L Ran-GTP support that ARAN1 is exported from the nucleus as a complex with Ran-GTP and importin β–related factors. Section title: The Role of RanBP1 in the Recycling of Ran Educational score: 4.574319839477539 Domain: biomedical Document type: Study Language: en After the translocation of the Ran–importin β complex into the cytoplasm, it is predicted that the complex is disassembled for the next round of nuclear import and the recycling of Ran. The Ran-GTP–importin β complex is kinetically very stable (the dissociation constant is ∼1 nM, when nucleotide exchange and GTP hydrolysis are blocked) . Injected ARAN1 induced the relocalization of endogenous Ran in living cells. That is, endogenous Ran was found predominantly in the cytoplasm within 30 min after injection . Since the injected ARAN1 was also localized in the cytoplasm, irrespective of the injection site , it is concluded that ARAN1 prevented the event(s) from occurring in the cytoplasm but not in the nucleus. Our in vitro biochemical experiments strongly suggest that the injected ARAN1 efficiently interacts with the Ran–importin β complex in the nucleus or the cytoplasm and prevents the binding of RanBP1 to the Ran–importin β complex in the cytoplasm. Furthermore, it was found that the COOH-terminal tail of Ran is also exposed via interaction with other importin β family molecules. Therefore, we conceive that Ran probably remains in the cytoplasm complexed with importin β or its related transport factors due to the inhibition by ARAN1 on the binding of RanBP1 to Ran–importin β or its related transport factor complexes in the cytoplasm, although we did not examine in this study that ARAN1 actually inhibits the binding of RanBP1 to the complex of Ran with importin β–related transport factors other than importin β. At least part of our conclusion is consistent with the recent biochemical analysis by Bischoff and Görlich . They showed that the importin α and RanBP1 cooperatively relieved the GAP resistance of the Ran-GTP–importin β complex, suggesting that RanBP1 is critical for the release of Ran-GTP from importin β. Moreover, in our study, it was found that ARAN1 competes much better for RanBP1 complexed to Ran-GDP–importin β than to Ran-GTP–importin β, which means that RanBP1 has much higher affinity for Ran-GTP– importin β complex than for Ran-GDP–importin β complex. This is consistent with the idea that RanBP1 efficiently binds to Ran-GTP–importin β complex in the cytoplasm to promote the dissociation of the complex. Section title: The Role of RanBP1 in the Recycling of Ran Educational score: 4.232220649719238 Domain: biomedical Document type: Study Language: en RanBP2 contains four Ran-binding domains which are functionally equivalent to RanBP1 . It is located at the cytoplasmic filaments of the NPC and a fraction of RanGAP which is modified by the addition of a small ubiquitin-like peptide binds to RanBP2 . Therefore, RanBP2 may play the same role as RanBP1 in the recycling of Ran, although further experiments will be required to better understand the role(s) of RanBP2 on the nucleocytoplasmic transport of macromolecules as well as to understand the effect of ARAN1 on RanBP2. Section title: Nuclear Protein Import and Recycling of Ran and Importin β Educational score: 4.462125301361084 Domain: biomedical Document type: Study Language: en This study showed that ARAN1, when injected into cultured cells inhibits nuclear protein import mediated by basic-type classical NLS irrespective of the injection site, i.e., nucleoplasm or cytoplasm. It has been recently reported that RanBP1 is required for nuclear protein import and RNA export in vivo and stimulates the protein import in a permeabilized cell-free assay . Furthermore, it has been shown that the COOH terminus deleted Ran (ΔDE-Ran) represents the dominant-negative effect on the nuclear protein import. In this study, we found that ARAN1 inhibits the interaction of RanBP1 (and probably RanBP2) with the Ran–importin β complex. There are two possible explanations for the inhibitory effect of ARAN1 on the nuclear import of the classical NLS substrates. One possibility is that ARAN1 may inhibit the interaction of RanBP1 to Ran-GTP–importin β complex, which leads to the suppression of the disassembly of the complex. Alternatively, consistent with the suggestion of Chi et al. that RanBP1 promotes nuclear protein import by stabilizing the interaction of Ran-GDP with importin β, ARAN1 may block the function of RanBP1 on the interaction of Ran-GDP with importin β by binding to the Ran-GDP–importin β complex stably and, as a result, affect nuclear protein import. Section title: Nuclear Protein Import and Recycling of Ran and Importin β Educational score: 4.420722484588623 Domain: biomedical Document type: Study Language: en Thus, it seems most likely that the inhibition of the function of RanBP1 (and probably RanBP2) blocks the disassembly of Ran–importin β complex in the cytoplasm, which causes the depletion of nuclear Ran, an inhibition in the recruitment of importin β, and further, the decline of the recycling of importin α/β to the cytoplasm by the suppression of the dissociation of the nuclear pore-targeting complex (importin α/β bound to a karyophile) in the nucleus following the depletion of nuclear Ran. It appears that such an imbalance in the import factors totally inhibits the nuclear protein import. Further studies will be necessary in order to understand whether ARAN1 also affects import and export pathways other than the classical NLS import pathway. | Study | biomedical | en | 0.999998 |
10037788 | Section title: Cells, Viruses, Antibodies, and Chemicals Educational score: 4.11646842956543 Domain: biomedical Document type: Study Language: en HeLa cells (human cervical epitheloid carcinoma) were grown as described earlier . TC7 cells (African green monkey kidney) and the TC7 clone TC7/MAP4/MTB–GFP stably transfected with a cDNA encoding the MT-binding domain of the MT-associated protein 4 (MAP4) linked to enhanced green fluorescent protein (eGFP) were kindly provided by J. Bulinski (Columbia University, New York, NY) . Wt Ad2 and temperature-sensitive (ts)1 mutant Ad2 were grown and isolated as described . Labeling with Texas red (TR) was exactly as published . γ-Tubulin was visualized in 3% pFA-fixed (20 min at room temperature) and methanol-extracted (5 min at −20°C) HeLa cells using the mouse monoclonal antibody TU-30 (obtained from P. Draber, [Institute of Molecular Genetics, Prague, Czech Republic]) . Mouse monoclonal anti-tyrosinated α-tubulin antibody (mAb 1A2) was obtained from T. Kreis (University of Geneva, Geneva, Switzerland) . Anti–β-tubulin antibody (N357; Amersham ) was used on methanol-fixed cells. Rabbit anti-nuclear lamins A, -B, and -C peptide antibody 8188 was supplied by L. Gerace (Scripps Research Institute, La Jolla, CA) and used as described . Mouse monoclonal anti-Polyoma virus middle T tag was obtained from J.C. Perriard (Eidgenössische Technische Hochschule, Zürich, Switzerland) . Nocodazole was purchased from Sigma , taxol from Calbiochem-Novabiochem , and fluorescent dyes from Molecular Probes. Drugs were dissolved as 1,000× stocks in DMSO and used as indicated. No drug treatment included appropriately diluted DMSO. Section title: Time-lapse Fluorescence Microscopy Educational score: 4.281050682067871 Domain: biomedical Document type: Study Language: en HeLa cells, TC7, or TC7/MAP4/MTB–GFP cells were grown on glass coverslips (Assistent, Winigor) in DME-10% FBS medium to ∼20–50% confluency. Geneticin (100 μg/ml; Alexis Biochemicals) was included to maintain the TC7/MAP4/MTB–GFP cells. TR-labeled virus (1–2 μg/ml) was added in warm RPMI-0.2% BSA-20 mM Hepes medium, pH 7.4, for 5 min. Cells were washed several times in RPMI medium. The coverslip was mounted to a homemade temperature-regulated chamber, covered with a glass slide, sealed with silicon grease, and then perfused with warm RPMI medium using a plastic syringe. The system was mounted to an electronically controlled inverted fluorescence microscope (model DM IRBE B; Leica) equipped with a Leica 100× oil immersion objective (NA 1.3), a differential interference contrast (DIC) polarizer and fluorescein (490/7.3 nm), TR (600/10.1 nm), and Cy5 (649/ 11.4 nm) excitation filters with bandpass emission filter cubes of 510/20 nm (Dichroic 495), 615/30 nm (Dichroic 595) and 690/40 nm (Dichroic 660), respectively (Omega Optical). Fluorescence images were recorded with a digital, back-illuminated charge coupled device camera onto a 1,000 × 800 pixel chip (15 × 15 μm/pixel) at 12 bit/500 kHz/−40°C using a PCI interface card and the MetaMorph software package ( Universal Imaging ). Exposure times in the TR channel were between 0.2 and 0.4 s, yielding less than 1% saturation of individual pixels. Intervals between subsequent exposures were between 1.2 and 2.6 s depending on the size of the region of interest selected in a given experiment. Observations typically lasted 1–3 min and were made between 20 and 40 min postinternalization, unless otherwise indicated. At the beginning or end of the experiment, the GFP fluorescence and/or DIC was recorded to visualize either the MT network (MAP4/MTB–GFP) or GFP alone to identify transfected cells. Individual virus particles showing linear translocations were tracked and velocities (distance between subsequent frames per time interval) determined using the MetaMorph tracking module. Velocities <0.1 μm/s were within the experimental error of our setup. The uncertainty in the tracing analysis was ±1 pixel (15 μm at 100× magnification) yielding a mean error of 1.45 pixels/time interval. Section title: Statistical Analysis Educational score: 4.161375999450684 Domain: biomedical Document type: Study Language: en Elementary speeds (ES, elementary motion steps per time interval) were determined for motile TR-labeled particles by recording x,y coordinates as described above. Motile particles were operationally defined as those particles that could be unequivocally traced in at least 20 subsequent frames and translocated in more than five subsequent frames over at least 0.5 μm in either plus or minus end direction. Depending on the cell type and the length of the observation period, 20–80% of the particles showed continuous plus- or minus end–directed translocation in five or more subsequent frames over several micrometers. Plus end–directed motility was defined as d i − d i+1 < 0 and minus end–directed motility as d i − d i+1 > 0, ( d i is the particle distance from the presumptive MTOC in frame i, and d i+1 the distance from the MTOC in frame i+1). Calculated ES < 0.1 μm/s were counted as no motility events. An apparent net population velocity was determined by the difference between all minus- and plus end–directed distances divided by the total sampling time. A one-sided t test was applied at the 95% confidence level to compare the MTOC- and the periphery-directed mean population speeds of wt and ts1 virus in a given cell type under different conditions. Frequencies of MTOC- and periphery-directed speeds and no movements were compared in a chi square test and evaluated at 95% confidence level. Section title: Indirect Immunofluorescence and Laser Scanning Confocal Microscopy Educational score: 4.191740989685059 Domain: biomedical Document type: Study Language: en Cells were grown on 12-mm glass coverslips in DME-7% FBS, transferred to cold RPMI medium containing 0.2% BSA, buffered with Hepes-NaOH to pH 7.4 (binding medium) and incubated with TR-labeled Ad2 (0.2–0.5 μg per 0.2 ml) for 90 min, followed by an incubation in warm DME-BSA at 37°C for indicated times as described . Samples were fixed in pFA, mounted in paraphenylene diamine (0.5%)-glycerol (80%)-Tris (20 mM), pH 8.8, and analyzed for single or double epifluorescence on a Reichert-Jung Polyvar microscope (Merck) equipped with Nomarski DIC, a fluorescein filter set (485/10, BP 540/20), and a TR filter set (557.5/27.5, LP 615) linked to an eight-bit CCD video camera . Images were collected with the Argus-20 imaging acquisition software (Hamamatsu Photonics) and batch-processed with Adobe Photoshop (Adobe Systems) on an Apple Macintosh computer (Quadra 650). Triple fluorescence experiments were analyzed on a L eica DM IRBE inverted microscope equipped with a 63× lens and FITC, TR, and Cy5 filter sets as specified above. Confocal microscopy was conducted on a L eica DM IRBE inverted microscope equipped with a 100× lens and a L eica TCS 4D confocal laser scanning device . Typical section thickness was estimated to be 300–400 nm based on the pinhole settings. Section title: Plasmids and DNA Experiments Educational score: 4.195141792297363 Domain: biomedical Document type: Study Language: en GFP-encoding plasmid DNA (gift of S. Zimmermann; Invitrogen, Zürich, Switzerland) was transfected into TC7 or HeLa cells alone or together with dynamitin-encoding DNA (gift of R. Vallee, University of Massachusetts, Worcester, MA) at a molar ratio of 2:1 using Lipofectamine™ (5 μl/μg DNA; GIBCO BRL ) or TransFast™ (7 μl/μg DNA; Promega ). Under these conditions more than 90% of the transfected cells expressed both GFP and dynamitin at 24 h posttransfection. In situ hybridizations to detect incoming viral DNA were carried out as described , except that the 42° and 60°C wash steps were omitted. A control plasmid encoding hexon loop 1 (amino acids 160–336) was constructed by PCR using the synthetic oligonucleotide AGCTACAAGCTTGGATCCCATATGGAAGAGCAAAACGCTCGAGAT as an NH 2 -terminal primer and C T CGAT GCTGAGCCCCGGGTT-ATTCCATT GGCAT GTATT CTCGGGT CT GTTT GGCATAGATTG as a COOH-terminal primer, respectively. The latter also encoded the polyoma middle T tag (EEYMPME, single amino acid letter code) in front of the multiple cloning site. PCR products were ligated into the polylinker of pSCT2+ using the HindIII and SmaI restriction sites and amplified in Escherichia coli . The loop 1 insert of the clone used here was confirmed by dideoxy sequencing. Section title: Electron Microscopy and HRP Detection Educational score: 4.229337692260742 Domain: biomedical Document type: Study Language: en Alcian blue–coated glass coverslips were prepared as follows. Glass coverslips (12 mm; Assistent) were washed in a glass beaker with 1 N HCl for 10 min and rinsed with distilled water, 90% methanol, 50% methanol, and then again with distilled water. Alcian blue (1% wt/vol Alcian blue-tetrakis[methyl-pyridinium]chloride in water [ Sigma ]) was added and the liquid brought to boiling for 30 s in a microwave oven. Coverslips were rinsed several times in water, placed between filter paper and sterilized by autoclaving and drying. HeLa or TC7/MAP4/MTB-GFP cells were seeded onto glass coverslips coated with Alcian blue. Wt Ad2 (50 μg/ml) was bound to the cell surface in the cold for 90 min and internalized at 37°C in the presence of HRP (20 mg/ml, Type II, 150–200 U/mg; Sigma ) in DME containing 0.2% BSA for 15 min. HRP was washed out with warm DME containing 0.2% BSA and cells were further incubated at 37°C for 15 min. Samples were quickly washed in PBS several times and fixed in PBS containing 2% glutaraldehyde (Electron Microscopy Sciences, Merck) for 30 min at room temperature, washed in PBS, and then stained for HRP activity in 0.1 M Tris-HCl, pH 7.4, 1 mM MgCl 2 containing 0.5 mg/ml diaminobenzidine ( Sigma ) and 0.03% hydrogen peroxide (Fluka). Samples were then processed for Epon embedding and analyzed by EM as described . Section title: Results Educational score: 3.9787895679473877 Domain: biomedical Document type: Study Language: en We have generated two types of fluorescent adenoviruses, wt and an endosomal mutant virus, ts1, to visualize virus trafficking between the cell periphery and the nucleus in vivo and to determine the mechanism of cytoplasmic motility, which allows virus targeting to the nuclear membrane. Section title: Physiological Temperature Is Needed for Nuclear Targeting of Ad2 Educational score: 4.290977954864502 Domain: biomedical Document type: Study Language: en The structural organization of the cytoplasm typically restricts the diffusional mobility of artificial particles of 50 nm in diameter to a few square micrometers per second . Particles larger than 50 nm, such as Ad2, are expected to have essentially no diffusion mobility under physiological conditions. To test if cytosolic virus was localized to the nucleus in the absence of energy metabolism at a reduced temperature, we bound TR-labeled Ad2 to the surface of HeLa cells in the cold and internalized for 29 min at 37°C. During this time virus is taken up into the cytoplasm but is not yet targeted to the nucleus . Cells were subsequently shifted to 4°C and incubated in the cold for up to 60 min postinternalization, whereas control cells were kept at 37°C. Since exposure of cells to reduced temperatures causes depolymerization of MTs, targeting was also analyzed in cells that had been pretreated with the MT-stabilizing drug taxol. In control cells the large majority of virus particles was directed to the nucleus during the 60-min internalization, in agreement with previous results . In contrast, at reduced temperatures, with or without taxol treatment, virus particles were found randomly distributed in the cytoplasm at 60 min. The same result was obtained if the cold incubation was extended up to 90 min (data not shown). Virus particles also remained cytoplasmic if cellular ATP levels were reduced after 30 min of internalization by shifting cells into a glucose-free medium containing 10 mM sodium azide and 20 mM 2-deoxy- d -glucose (data not shown). These results confirmed that incoming Ad2 does not move through the cytoplasm by diffusion, but uses some form of mediated transport. Section title: Microtubule-dependent Minus End–directed Transport of Cytosolic Virus Educational score: 4.147075653076172 Domain: biomedical Document type: Study Language: en Previous EM studies have detected Ads in close association with MTs both in vivo and in vitro (data not shown) . To ask whether MTs are actually needed for directional transport of virus to the nucleus, we first tested if virus could be detected at the MTOC in cultured epitheloid HeLa cells. In these cells the MTOC predominantly contains the minus ends of MTs and is located in the perinuclear area as confirmed with an anti–γ-tubulin immunostaining and confocal microscopy . As expected , γ-tubulin was found all over the cytoplasm but was concentrated at a perinuclear location . Of a total of 101 examined cells, 75% had a perinuclear enrichment of incoming virus at 60 min postinfection (p.i.). At 15 min postinternalization no enrichment was observed. Section title: Microtubule-dependent Minus End–directed Transport of Cytosolic Virus Educational score: 4.475582122802734 Domain: biomedical Document type: Study Language: en We next analyzed if intact MTs were required for virus targeting to the nucleus. HeLa cells were incubated with 20 μM nocodazole for 30 min and virus was bound to the cell surface for 60 min in the cold to synchronize infection and to further depolymerize MTs. Cells were then shifted to 37°C in drug-containing medium for 75 min. Under these conditions, MTs were completely depolymerized as shown with mAb 1A2 and anti–β-tubulin immunostainings (data not shown). Nocodazole strongly, but not completely inhibited localization of TR-labeled virus to the nuclear envelope as indicated by confocal microscopy . Similar results were obtained if the internalization time was extended to 135 min (data not shown), thus suggesting that the low level of nuclear targeting was most likely due to particles entering from a plasma membrane area near the nucleus. If MTs were restored by washing out nocodazole for 75 min, significant virus targeting to the nucleus was observed, similar to cells not treated with the drug . These results were confirmed by analyzing viral DNA import into the nucleus in nocodazole-treated cells using fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy at 180 min p.i. Control cells (no drug) had a strong signal of viral DNA inside the nucleus as concluded from an anti-lamin immunostaining of the nuclear envelope . Noninfected cells had no viral DNA signal, indicating that the FISH conditions were specifically detecting viral genomes . In nocodazole-treated cells most of the DNA signal was present in the cytoplasm, sometimes in an aggregated form . Note that the combined cytoplasmic signal was somewhat lower than the nuclear signal in the control (no drug)-treated cells, partly because some cytoplasmic virus had been artificially released during the FISH treatments. The absence of viral DNA in the nucleus was not due to a lack of detection or rapid nuclear export, since viral genomes were readily found in the nucleus of cells that were treated with nocodazole beginning at 90 min p.i., when the majority of virus particles had already arrived at the nuclear envelope . The cytoplasmic DNA signals in the nocodazole-treated cells decreased after washing out the drug and a strong nuclear DNA signal appeared . Taken together, these results suggested that intact MTs are needed for targeting of incoming virus particles to and for import of viral DNA into the nucleus. Section title: Microtubule-dependent Minus End–directed Transport of Cytosolic Virus Educational score: 4.159969329833984 Domain: biomedical Document type: Study Language: en It was possible that nocodazole affected endocytosis and/or virus escape from the endosome. We therefore measured the internalization of [ 35 S]methionine virus into drug-treated cells using a cell surface trypsinization assay, which measures virus uptake by the emergence of a trypsin-resistant form of the major capsid protein hexon . The results indicate that in the presence of nocodazole half of the virus particles were endocytosed at 15 min postwarming, whereas in control cells the half-time of internalization was ∼9 min . The efficiency of entry at 30 min postwarming was close to 75% under both conditions. The remaining viruses were most likely shed from the cells into the medium . The data demonstrated that virus uptake into cells lacking intact MTs was slightly slower, but no less efficient than into control cells, in agreement with earlier observations showing that nocodazole had no effect on the formation of endocytic vesicles . Section title: Microtubule-dependent Minus End–directed Transport of Cytosolic Virus Educational score: 4.345195293426514 Domain: biomedical Document type: Study Language: en We next asked if virus was able to escape from endosomes in cells lacking MTs. To visualize both endosomes and virus particles at high resolution we used a fluid-phase endosomal marker, HRP, in combination with EM . HeLa cells were pretreated with nocodazole for 15 min. Wt or a mutant virus, ts1, which is unable to escape from endosomes , were bound to the cells at 4°C. Ts1 has a point mutation P137L in the protease reading frame and, when grown at the nonpermissive temperature, produces particles, which fail to escape from endosomes in a new round of infection . Virus internalization then occurred in the presence of HRP and drug for 15 min at 37°C, followed by a 15-min chase without HRP in the presence of nocodazole. HRP activity was visualized by incubating the fixed cells with diaminobenzidine, which is converted to an electron dense product by HRP . Control cells (no drug) contained numerous cytosolic wt particles, also near nuclear pore complexes, but only few viruses were detected within vesicular structures . Interestingly, these cells contained essentially no vesicles with HRP reaction product, in contrast to noninfected cells, suggesting that contents of endocytic vesicles were released . Nocodazole-treated cells contained numerous virus particles in the cytosol and only few HRP-positive endosomes, similar to control cells . These cytosolic particles were clearly distinguishable entities and often occurred in clusters. Ts1 viruses, on the other hand, were exclusively detected within HRP-containing vesicles, as expected . We concluded that MTs were not needed for Ad2 uptake into cells and delivery to the cytosol, but were required for targeting of cytosolic virus to the nucleus. Section title: Microtubule-dependent Transport of Cytosolic and Endosomal Viruses at Micrometers Per Second Educational score: 3.933645009994507 Domain: biomedical Document type: Study Language: en A recent report stated that fluorophore-labeled Ad5 particles were able to move in the periphery of human A549 cells over short distances with apparent velocities of 0.5– 0.6 μm/s . We aimed at analyzing the mechanisms underlying this motility and wanted to test a possible involvement of MTs. Section title: Microtubule-dependent Transport of Cytosolic and Endosomal Viruses at Micrometers Per Second Educational score: 4.290478229522705 Domain: biomedical Document type: Study Language: en We conducted extensive particle tracking analysis in TC7 cells (an African green monkey kidney cell line) and in HeLa cells. The TC7 cells have a particularly large and flat cytoplasm and are well suited for studies of cytoplasmic movement of Ad since they efficiently internalize and target virus to the nucleus (data not shown). To determine the approximate position of the MTOC we used in some experiments a TC7 clone, which was stably transfected with a cDNA encoding a GFP hybrid of the MAP4 MT-binding domain . This cell line was viable in our hands for more than 20 passages . TR-labeled Ad2 was internalized at 37°C for 30 min and the coverslip was mounted to a temperature-controlled heating chamber placed on an inverted fluorescence microscope. Fluorescence images were recorded in the TR channel at 1.2– 2.6-s frame intervals over several min as described in Materials and Methods. We use the term ES to define directional movement of particles between two subsequent frames, negative ES indicating movement to the MTOC (minus end) and positive ES to the periphery (plus end). At the end of the recordings the MAP4/MTB–GFP fluorescence was captured. A representative experiment is shown in Fig. 5 where we traced three different particles. One peripheral particle (arrow) was followed throughout the experiment and two MTOC-neighboring particles were traced at the end of the experiment in frames 92–98 . The peripheral particle moved ∼30 μm in a quasi-straight direction towards the MTOC. In Fig. 5 , C and D we plotted the distances of the particle from the MTOC and the ES for each frame. The ES defined two distinct types of activity periods. The first type of activity occurred between frames 39–44 and 68–79 and contained exclusively MTOC-directed motions. This activity resulted in efficient particle advancement towards the MTOC and typically lasted for several seconds reaching elementary speeds of 0.17–2.63 μm/s. The other type of activity was characterized by rapidly alternating minus- and plus end– directed ES up to 0.5 μm/s at nearly equal extents and frequencies . Such activities resulted either in a net minus end–directed speed of 2–4 μm/min (frames 44–68), or yielded no net movement (frames 1–38 and 80–104). Note that from frames 80–104, two additional MTOC-near particles still showed net minus- and plus end–directed movements, indicating that cells and viruses were still motion competent. Section title: Microtubule-dependent Transport of Cytosolic and Endosomal Viruses at Micrometers Per Second Educational score: 4.321529388427734 Domain: biomedical Document type: Study Language: en A population analysis of 958 ES from 15 different particles in TC7/MAP4/MTB–GFP cells and 729 ES from 19 different particles in the parental TC7 cells indicated a tailed distribution of both the MTOC- and the periphery-directed speeds, with maximal ES occurrence between 0.1 and 0.2 μm/s (Table I ). In MAP4/MTB– GFP cells, all the MTOC-directed speeds were more prominent than the periphery-directed speeds, in contrast to the parental TC7 cells, in which less minus- and more plus end–directed ES occurred. However, even in these cells, the minus end–directed motion was dominant over the plus end–directed motion as indicated by the mean population speed of −0.06 μm/s. The MAP4/MTB–GFP-expressing cells contained significantly more active particles than the parental TC7 or HeLa cells. The number of immobile particles (ES smaller than 0.1 μm/s) amounted to 12.8, 28.4, and 41.4% for TC7/MAP4/MTB–GFP, parental TC7 and HeLa cells, respectively (Tables I and II ). The mean minus end–directed speed was 0.50 μm/s in both TC7 cell types, but the mean and also the peak plus end– directed speeds were significantly smaller in the MAP4/ MTB–GFP expressing cells than in the parental cells (Tables I and II ). Accordingly, the population mean speed (comprising all the traced particles) was significantly shifted to the MTOC direction in the TC7/MAP4/MTB– GFP cells (−0.21 μm/s) compared with TC7 cells (−0.06 μm/s) or HeLa cells (−0.03 μm/s). Although the mean minus- and plus end–directed speeds in HeLa cells were significantly smaller than in TC7 cells (perhaps relating to the fact that the TC7 cells allowed longer tracking analysis than the HeLa cells), the calculated population average speeds for wt virus were in good agreement with the observed enrichment of virus near the MTOC starting at ∼25 min to 40 min p.i. This reflects the half-time of virus penetration of endosomes which is ∼15 min . Section title: Microtubule-dependent Transport of Cytosolic and Endosomal Viruses at Micrometers Per Second Educational score: 4.2488579750061035 Domain: biomedical Document type: Study Language: en To confirm that the above measured speeds were truly derived from cytosolic viruses, we analyzed the trafficking of an endosomal mutant Ad2, ts1, in TC7/MAP4/MTB– GFP cells and in parental TC7 cells. Unlike wt virus, ts1 had a population mean speed of −0.02 μm/s in TC7/ MAP4/MTB–GFP cells and +0.04 μm/s in TC7 cells indicating slow minus- and plus end–directed net movements (Table I ). Accordingly, we have not observed ts1 virus accumulation near the MTOC up to 60 min p.i. Like wt virus, ts1 showed a tailed distribution of ES, with the majority in the range of 0.1–0.2 μm/s . In both cell types, about 22.5% of the ES were less than 0.1 μm/s. A typical ts1 trace in a MAP4 overexpressing cell shows how a virus particle moved from a location proximal to the MTOC towards the periphery with ES of up to 1.7 μm/s and from there returned to an MTOC-near location with ES of up to 2.3 μm/s . Taken together, the data illustrate a distinct dynamic behavior of wt and endosomal viruses further supporting the notion that wt Ad2 travels as a naked particle along MTs. Section title: Switching between Minus- and Plus End–directed Motions Educational score: 4.057024955749512 Domain: biomedical Document type: Study Language: en The observation that periods of minus end–directed Ad2 transport were often followed by plus end–directed motions along an apparently linear track could in principle be explained by the dynamic instability of MTs . In fact, growing and shrinking MTs were readily observed in TC7/MAP4/ MTB–GFP cells, preferentially in the cell periphery (data not shown). Section title: Switching between Minus- and Plus End–directed Motions Educational score: 4.3252973556518555 Domain: biomedical Document type: Study Language: en Dynamic turnover of MTs can be experimentally eliminated by incubating cells in low concentrations of taxol or nocodazole . To first test if dynamic MTs were needed for nuclear targeting of Ad2 we pretreated HeLa cells with 25 nM taxol for 30 min. Such treatment was sufficient to stabilize MTs against cold-induced depolymerization . Ad2-TR was bound to the surface and then internalized in the presence of the drug for 75 min. Similar to control cells, the majority of virus particles were localized to the nuclear envelope . Viral DNA was readily detected inside the nucleus at 180 min p.i. using the FISH assay described above . Likewise, treatment of TC7/MAP4/MTB–GFP cells with low concentrations of nocodazole (1–10 μM for 15 min) in the absence of cold incubations neither affected the MT network nor virus targeting to the nucleus (data not shown). We next analyzed Ad2 motility in MAP4/MTB–GFP-expressing TC7 cells in the presence of taxol or low nocodazole. As expected from the static entry analysis , the population mean speeds towards the MTOC and the periphery were not significantly different from the control cells (Table I ). In cells treated with low concentrations of nocodazole, we also observed peak minus- and plus end– directed speeds of up to 1.7 μm/s (data not shown). Although the frequencies of either minus- or plus end– directed motions were significantly decreased in the presence of taxol and the no movement frequencies were increased from 12.8 to 40.8%, rapid switching between plus- and minus end–directed ES was readily observed, similar to control cells . Within 2.6 s, single virus particles were frequently switching from +0.4 μm/s to −0.4 μm/s and vice versa, suggesting that stable MTs supported rapid bidirectional transport. Section title: Switching between Minus- and Plus End–directed Motions Educational score: 4.246150016784668 Domain: biomedical Document type: Study Language: en In a number of cultured cells, including polarized cells, MTs can be detached from the MTOC and may occur as migrating antiparallel structures with free minus and plus ends at steady-state conditions . We therefore tested if the rapid switching between minus- and plus end–directed motions of wt Ad2 occurred near the MTOC in TC7/MAP4/MTB–GFP cells that were reestablishing MTs after nocodazole wash out. Such cells are expected to have a minimal amount of MTs that are not anchored to the MTOC. As indicated in Fig. 7 C, a perinuclear MT aster formed after 1–2 min of nocodazole wash out. MTOC-near viruses were observed to switch between plus- and minus end–directed motions at ES of 0.3–0.4 μm/s as indicated by a typical particle trace . Although these amplitudes were somewhat smaller than those in the control or the taxol-treated cells, they were significantly larger than the randomly directed mean ES in nocodazole-treated HeLa (0.15 and 0.17 μm/s, Table I ) or TC7 cells (data not shown). We thus concluded that single cytosolic Ad2 particles were able to engage with both plus- and minus end–directed motors and thus move bidirectionally along not highly dynamic MTs. Section title: Dynein/Dynactin Significantly Contributes to Minus End–directed Transport of Cytosolic Ad2 Educational score: 4.17268180847168 Domain: biomedical Document type: Study Language: en The major minus end–directed motor complex of interphase cells is cytoplasmic dynein . The motor activity can be experimentally dislodged from cargo by overexpression of p50/dynamitin, a component of the dynein-associated dynactin complex . We therefore tested if transient overexpression of human dynamitin in HeLa cells affected nuclear targeting of Ad2. Control cells transfected with eGFP-encoding plasmid DNA showed efficient nuclear targeting of TR-labeled Ad2 at 60 min p.i. . HeLa cells cotransfected with GFP and dynamitin plasmids showed reduced nuclear targeting of Ad2, particularly at high levels of overexpression . At moderate dynamitin overexpressions, variable levels of nuclear targeting occurred, perhaps due to incomplete inactivation of the dynactin complex. Section title: Dynein/Dynactin Significantly Contributes to Minus End–directed Transport of Cytosolic Ad2 Educational score: 4.21389102935791 Domain: biomedical Document type: Study Language: en To test if dynamitin overexpression interfered with Ad2 escape from endosomes, we assayed the release of cointernalized fluid-phase dextran-FITC from endosomes to the cytosol upon Ad2 infection in a qualitative assay, similar to the HRP assay described in Fig. 4 B. To reliably determine the endpoint distribution of the dextran we used a lysine-fixable 10-kD dextran, which readily diffuses from the cytosol to the nucleus. Wt Ad2 and, as a control, the mutant ts1 were bound to control cells or cells overexpressing dynamitin and internalized in the presence of dextran for 15 min, followed by a 15-min chase in the absence of dextran. About 80% of the cells infected with wt virus showed a prominent diffuse dextran pattern in the cytosol and the nucleus, independent of dynamitin overexpression . Similar results were obtained with a control plasmid overexpressing the loop 1 region of the hexon protein (see Materials and Methods) (data not shown). In contrast, none of the more than 200 analyzed cells infected with ts1 virus showed any diffuse cytoplasmic or nuclear dextran staining, but contained numerous punctuate FITC signals in the cytoplasm indicative of endosomes or lysosomes . These results were in full agreement with the HRP fluid-phase uptake experiments and suggested that dynamitin overexpression did not appreciably affect Ad2 release from endosomes. Section title: Dynein/Dynactin Significantly Contributes to Minus End–directed Transport of Cytosolic Ad2 Educational score: 4.337908744812012 Domain: biomedical Document type: Study Language: en To quantitate the effects of dynamitin overexpression on Ad2 trafficking, we compared the extents and frequencies of the minus and plus end–directed ES in dynamitin overexpressing HeLa cells with control and nocodazole-treated cells. Virus motility at the cell surface was, in addition, determined to account for cytosol-independent particle motion. As shown by an ES population analysis and exemplified by representative particle traces , dynamitin overexpression significantly reduced the extents and also the frequencies of the minus end–directed ES. The mean minus end–directed speed of 0.22 μm/s was significantly smaller than the plus end–directed mean speed of 0.30 μm/s (Tables I and II ), resulting in a plus end–directed population speed of 0.06 μm/s compared with a minus end–directed speed of 0.03 μm/s in control cells (Table I ). As pointed out earlier, not all minus end–directed transport of Ad2 was blocked by dynamitin overexpression. This was illustrated also by the maximal minus end–directed ES (∼2 μm/s) and a mean minus end speed, which was significantly higher than in nocodazole-treated cells (Table II ). This result may either reflect the heterogenous expression levels of dynamitin or, alternatively, indicate some form of dynein/dynactin-independent minus end transport of Ad2. In contrast to the effect of dynamitin on the minus end migration of Ad2, dynamitin overexpression did not significantly affect the mean plus end–directed velocity, as illustrated by the predominantly peripheral virus localization in these cells (Tables I and II ). However, dynamitin overexpression significantly increased the frequency of plus end motions to 31.5 from 25.5% in control cells or 25.8% in GFP-expressing cells (legend of Table I , chi square test at 95% confidence). Interestingly, dynamitin overexpression also significantly increased the fraction of immobile particles to 50.9 from 41.4% in control cells. The validity of our chi square analysis and of the sample volumes were confirmed by the fact that there were no significant differencies between the plus, the minus, and the no movement frequencies in control HeLa cells compared with GFP-expressing HeLa cells. That the expression of GFP had no significant effects on Ad2 trafficking was further supported by the observation that the mean minus- and plus end–directed speeds were identical in GFP-expressing cells and control cells (Tables I and II , one-sided t test, 95% confidence). Section title: Dynein/Dynactin Significantly Contributes to Minus End–directed Transport of Cytosolic Ad2 Educational score: 4.210168838500977 Domain: biomedical Document type: Study Language: en Taken together, the data show that dynamitin overexpression strongly affected but not completely blocked minus end–directed transport of cytosolic Ad2. It increased the frequency of plus end movements and supported long-range movements over several micrometers towards the periphery. In contrast, no long-range movements, either MTOC- or periphery-directed, were observed in nocodazole-treated cells. In these cells, short-range random movements (covering areas of 5–10 μm 2 ) with mean speeds of 0.15 μm/s were observed (Table I ). The motilities in nocodazole-treated cells were apparently randomly directed without any significant difference between plus- and minus end–directed mean speeds (Table II ). These motilities were, however, not an artefact of our experimental set up, since they were significantly larger than the motilities of surface-bound virus (Table I ). Possibly, in nocodazole-treated cells viruses were either moving on short MT fragments resistant to the drug, or they were using some MT-independent form of intracellular motility. Section title: Discussion Educational score: 4.42275333404541 Domain: biomedical Document type: Study Language: en In this study we have analyzed the dynamic properties of incoming Ad2 at various stages of entry. Our results identify the major minus end–directed motor complex, dynein/ dynactin, as a key mediator of nuclear targeting of Ad2. The data support the notion that the viral DNA remains wrapped by the capsid until arrival at the nuclear membrane. Capsids therefore are expected to contain all the necessary information for nuclear targeting. Virus particles on the cell surface remain apparently immobile, probably bound to a primary receptor . They are then captured into endosomes, penetrate the endosomal membrane, and then move as naked cytosolic particles along MTs to the MTOC. Our dynamic analysis at a resolution of up to one frame per second shows that minus end migration occurs stepwise with maximal ES of 2.6 μm/s, occasionally interrupted by periods of almost equally fast plus end–directed motions or periods of stalling. Consequently, the population speeds towards the MTOC are in the order of micrometers per minute, in agreement with the observation that wt virus starts to accumulate at the MTOC and the nuclear envelope at 30–40 min p.i. In contrast to wt virus, the endosomal escape-defective mutant virus ts1 does not end up near the perinuclear MTOC, but remains randomly distributed in the cytoplasm, despite the fact that it undergoes fast minus- and plus end–directed elementary motion steps. Section title: Cytosolic, but Not Endosomal Virus, Is Transported to the MTOC Educational score: 4.2891459465026855 Domain: biomedical Document type: Study Language: en Several lines of evidence indicate that cytosolic rather than endosomal viruses are transported to the MTOC. First, MT depolymerization with nocodazole effectively inhibited wt virus transport to the nuclear envelope and left capsids in the cytosol. These cytosolic capsids contain genomic DNA and are competent to translocate to the nuclear envelope and release their DNA into the nucleus if MTs are restored. Interestingly, microinjected viral DNA core structures lacking the remainder capsid components were not imported into the nucleus, but instead remained aggregated in the cytoplasm (data not shown). These results show that cytosolic capsid structures and intact MTs are of functional importance to nuclear import of viral DNA. Section title: Cytosolic, but Not Endosomal Virus, Is Transported to the MTOC Educational score: 4.414431571960449 Domain: biomedical Document type: Study Language: en The second evidence favoring transport of naked rather than endosomal viruses to the MTOC is based on the different dynamic properties of wt virus compared with the endosomal ts1 mutant. Whereas wt Ad2 moves predominantly to the MTOC with population mean speeds between 0.03 and 0.21 μm/s (depending on the cell type), plus- and minus end–directed movements occur with about equal frequencies and extents in the case of the mutant ts1. Ts1-positive vesicles contain fluid-phase endocytic markers, such as HRP or fluorescent dextran indicating that they were of endocytic origin. Unlike transferrin-containing endosomes ts1 did not accumulate near the MTOC within 60 min p.i., suggesting that the ts1 vesicles are perhaps routed towards lysosomes rather than a perinuclear recycling compartment. In accordance with this possibility, ts1 was degraded in a lysosomal protease-dependent manner starting at ∼60 min p.i. . MTs are, however, involved in ts1 trafficking, since nocodazole largely inhibited long-range ts1 movements (data not shown). Like in the case of wt virus, dynamic MTs are not required for ts1 motility. Elimination of the most dynamic MTs by taxol not only increased the mean minus- and plus end–directed speeds of ts1, but also increased the frequency of no movements, thus supporting the notion that dynamic MTs may facilitate establishing MT contact with cargo . Section title: The Nature of Ad2 Movements through the Cytosol Educational score: 4.35818338394165 Domain: biomedical Document type: Study Language: en The analysis of intracellular wt virus dynamics in nondrug-treated cells shows that between 12.8 and 41.4% of the elementary motion steps were less than 0.1 μm/s depending on the cell line. This was below the resolution of our set up and was therefore scored as no movement. Almost all the particles were, however, closely associated with MTs as suggested by confocal microscopy and earlier EM studies (data not shown) . It is therefore possible that cytosolic virus first attaches to MTs and remains immobile until some motor-dependent transport happens. Transport to the MT minus ends (or to a lesser extent also the plus ends) occurred by two types of motion, a slow transport mode working at micrometers per minute and a fast mode working at micrometers per second. Both modes are made up of numerous ES with peak speeds in the order of 0.5– 2.6 μm/s. The fast mode is reminiscent of vesicular trafficking and typically lasted for several seconds resulting in efficient virus advancement to the MTOC. It is not interrupted by stalling or plus end– directed motions. Unlike the fast mode, the slow mode can last for minutes and is composed of alternating minus- and plus end–directed ES, which are typically less than 0.5 μm/s in magnitude. Since these motions often follow a quasi-linear track and are completely eliminated in the presence of nocodazole, they are most likely mediated by MT-dependent motors. Section title: The Nature of Ad2 Movements through the Cytosol Educational score: 4.316468238830566 Domain: biomedical Document type: Study Language: en Rapid switching between plus- and minus end–directed motions is not only detected in nondrug-treated cells, but also in taxol-incubated cells and, importantly, in cells recovering from nocodazole treatment immediately after drug wash out. In the latter case, most of the MTs that were not anchored to the MTOC, including antiparallel MTs, are expected to be eliminated . We therefore concluded that a single Ad2 particle engages with both plus- and minus end–directed motor activities. The ability to use both types of motors might be important for Ad entry into polarized cells or, possibly, for virus egress from infected polarized or nonpolarized cells. Whether, and if so, how the motor activities are coordinated is subject of future studies. Besides motor regulation, MTs and associated proteins could have a potential role in determinating the directionality of transport. Interestingly, both the mean and also the peak periphery-directed speeds of wt Ad2 and endosomal mutant virus were significantly decreased in cells overexpressing the MT-binding domain of MAP4 compared with the parental cells. Whether this effect is related to the observed increase in MT stability in the MAP4/MTB–GFP–expressing cells is not known. Section title: The Nature of Ad2 Movements through the Cytosol Educational score: 4.1865010261535645 Domain: biomedical Document type: Study Language: en MT-dependent transport of cargo can, in principle, be driven by the energetics of plus end MT depolymerization and dynamic attachment of the motor to the shortening filament . Our data with taxol-stabilized MTs show that highly dynamic MTs are not required for long-range Ad2 transport. Dynamic MTs may, however, increase the probability of virus contact with MTs, since taxol strongly increased the fraction of nonmotile viruses. Section title: Dynein/Dynactin Is a Major Minus End–directed Motor for Ad2 Educational score: 4.571390151977539 Domain: biomedical Document type: Study Language: en Like other MT-based motors, the major minus end–directed motor dynein produces directed movement by a conformational change upon ATP-hydrolysis . Dynein is a protein complex of three different subunits of various stoichiometries translocating along MTs at speeds faster than 0.5 μm/s . Dynein attaches to cargo, such as cellular vesicles and mitotic chromosomes via the dynactin complex. Overexpression of the dynactin component dynamitin has been shown to disrupt the dynactin complex on the kinetochore of mitotic chromosomes and also affect vesicular trafficking in interphase cells . Our results show that overexpression of human dynamitin significantly inhibits nuclear targeting of Ad2 in HeLa cells and, to a lesser extent, also in monkey TC7 cells (data not shown). It is unlikely that dynamitin overexpression inhibited Ad2 penetration, since a fluid-phase endocytic marker is delivered to the cytosol upon Ad2 infection, independent of dynamitin overexpression. Accordingly, general receptor-mediated endocytic uptake is known to occur without intact MTs . Although not all minus end–directed transport of wt virus was blocked in the presence of excess dynamitin, our data imply that some component of the dynactin complex directly or indirectly interacts with cytosolic Ad2 capsid. Owing to the linearity of the remaining minus end–directed movements, this motility is unlikely caused by Brownian motion . It rather reflects either some dynein/dynactin-independent, but MT-dependent motor activity or, possibly, the heterogenous expression levels of dynamitin . Interestingly, dynamitin overexpression significantly increased the frequencies of no movements and of plus end–directed movements, which could suggest that a plus end–directed motor attaches to a site on the virus or even on a dynactin component, which is normally occupied by dynein/dynactin . Section title: Dynein/Dynactin Is a Major Minus End–directed Motor for Ad2 Educational score: 4.3034563064575195 Domain: biomedical Document type: Study Language: en With the advent of increasingly detailed models of motor interactions with MTs for kinesin and dynein and increasing structural understanding of kinesin's directionality , a more detailed knowledge of the mechanism of dynein motor directionality and interactions with simple cargo is desired. We already know much about the cell biology of motors involved in vesicular transport, such as dynein's role in trafficking of the intermediate compartment , Golgi vesicles , mature phagosomes , and endosomes , but we know very little about MT-dependent trafficking of nonmembranous organelles or of viral structures. Owing to its relative simplicity and high entry efficiency, Ad offers yet another opportunity to get additional insights into the complex regulation of polarized cytoplasmic trafficking. In addition, any successful application of gene transfer protocols requires overcoming of numerous subcellular barriers including the cytoplasm. Our present study, together with a recent report on Herpes virus , indicates that DNA viruses can overcome the cytoplasmic barrier by packaging their genomes into a capsid structure, which is capable of using the minus end–directed MT-dependent dynein motor. Section title: Dynein/Dynactin Is a Major Minus End–directed Motor for Ad2 Educational score: 4.241367340087891 Domain: biomedical Document type: Study Language: en Our present study is the first dynamic and functional analysis of an incoming viral structure. It demonstrates that individual Ad2 particles engage not only with a minus end–directed motor for DNA delivery into the nucleus, but also interact with a fast plus end–directed activity working at micrometer per second speeds. In HeLa and TC7 cells, the plus end–directed motions are of lower efficiency than the minus end motions, allowing nuclear targeting of virus at population speeds in the order of micrometers per minute. It is likely that long-range cytoplasmic motility of nucleic acids generally requires some form of active transport as demonstrated also for mRNAs in oocytes . For optimal efficiency of future synthetic or semisynthetic nucleic acid delivery systems viral and/or cellular elements may therefore be used to facilitate directional movement through the cytoplasm. | Study | biomedical | en | 0.999996 |
10037789 | Section title: Materials Educational score: 1.7201051712036133 Domain: biomedical Document type: Other Language: en N -(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza- s -indacene-3-pentanoyl)- d - erythro -sphingosine (C 5 -DMB-Cer) and 6-[ N -(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino]caproyl- d - erythro- sphingosine (C 6 -NBD-Cer) were purchased from Molecular Probes. Brefeldin A, fatty acid–free BSA, and endoglycosidase H (Endo H) were from Sigma Chemical Co. l -[U- 14 C]serine (5.85 GBq/mmol) was from Amersham Pharmacia Biotech . [ Methyl - 14 C]choline chloride (2.04 GBq/mmol) and [3- 3 H] d - erythro -sphingosine (0.74 GBq/mmol) were from American Radiolabeled Chemicals Inc. Section title: Cells and Cell Cultures Educational score: 4.145559787750244 Domain: biomedical Document type: Study Language: en The CHO-K1 cell line was obtained from the American Type Culture Collection (ATCC CCL 61), and LY-A strain, a CHO-K1–derived mutant cell line, has been established previously by us . Cells were routinely maintained in Ham's F-12 medium supplemented with 10% NBS, penicillin G (100 U/ml), streptomycin sulfate (100 μg/ml), and sodium hydrogen carbonate (1.18 g/liter) at 33°C . Nutridoma medium (Ham's F-12 medium containing 1% Nutridoma-SP [ Boehringer Mannheim ] and gentamicin [25 μg/ml]) was used as serum-free medium. Cells constitutively expressing a glycosylphosphatidylinositol (GPI)-anchored human placental alkaline phosphatase (PLAP) or a membrane-spanning PLAP (PLAP-HA), which is a chimera of PLAP with influenza hemagglutinin , were established as described . Section title: Metabolic Labeling of Lipids with [ 14 C]Serine and [ 14 C]Choline in Cells Educational score: 4.12387228012085 Domain: biomedical Document type: Study Language: en Subconfluent cell monolayers in 6-cm dishes were incubated in 2 ml of Nutridoma medium containing 37 kBq of [ 14 C]serine or [ 14 C]choline at 33°C for various times. After washing twice with 2 ml of cold PBS, cells were lysed in 1 ml of 0.1% SDS at 4°C, and 0.1 and 0.8 ml of the cell lysate were used for protein determination and lipid extraction, respectively. Lipids extracted from the cells were separated on TLC plates with solvent of methyl acetate/ n -propanol/chloroform/methanol/0.25% potassium chloride (25:25:25:10:9, vol/vol) in the [ 14 C]serine-labeling experiments, or chloroform/methanol/acetic acid/water (25:15:4:2, vol/vol) in the [ 14 C]choline-labeling experiments. After collecting gels from the plates by scraping, radioactivity in each lipid was determined by liquid scintillation counting. The values were normalized to cell protein. Section title: Metabolic Labeling of Lipids with [ 14 C]Serine and [ 14 C]Choline in Cells Educational score: 4.126335144042969 Domain: biomedical Document type: Study Language: en Since DCer and Cer were not separated by the TLC systems used, composition of metabolically labeled ceramides was determined after acid hydrolysis of them. Cells were labeled with [ 14 C]serine for 2 h at 33°C and labeled lipids were separated by the TLC as described above. Lipids containing both [ 14 C]DCer and [ 14 C]Cer were reextracted from the TLC fractions, hydrolyzed in 0.5 ml of 10 N hydrochloric acid/water/methanol (8.6:9.4:100, vol/vol) for 18 h at 70°C , and then the liberated sphingoid bases were separated on TLC plates with a solvent of chloroform/methanol/2 N ammonia solution (80:20:1, vol/vol) . Radioactivity in separated dihydrosphingosine or sphingosine was determined by liquid scintillation counting. Section title: Metabolic Labeling of Lipids with [ 14 C]Serine and [ 14 C]Choline in Cells Educational score: 4.11014461517334 Domain: biomedical Document type: Study Language: en For determination of distribution of SM to the external surface of cells, cells were labeled with [ 14 C]serine for 48 h at 33°C, fixed with 0.125% glutaraldehyde in PBS for 30 min at room temperature, and treated with or without 250 mU/ml of recombinant Bacillus cereus sphingomyelinase (Higeta Shoyu) for 30 min at 37°C. Radioactive SM extracted from the cells was analyzed as described above. Section title: Metabolic Labeling of Lipids with [ 3 H]Sphingosine in Cells Educational score: 4.155120849609375 Domain: biomedical Document type: Study Language: en For preparation of a complex of 100 μM [ 3 H]sphingosine (1.85 MBq/ml) with 200 μM fatty acid–free BSA in PBS, a stock solution of [ 3 H]sphingosine (370 KBq, 20 nmol) in ethanol was dried under a stream of nitrogen gas. The dried lipid was dispersed in 200 μl of PBS containing 200 μM fatty acid–free BSA by vortex mixing and sonication. Subconfluent cell monolayers were incubated in Nutridoma medium containing 1 μM [ 3 H]sphingosine complexed with BSA for various times. Cellular lipids were extracted under mild alkaline conditions for efficient recovery of the sphingoid bases or mild acidic conditions for efficient recovery of N -acetylneuramyl lactosylceramide (G M3 ), and were separated on TLC plates with a solvent of chloroform/methanol/water (65:25:4, vol/vol). Radioactivity in each lipid was determined as described above. Section title: In Vitro Enzyme Assays Educational score: 4.073003768920898 Domain: biomedical Document type: Study Language: en Cell lysates were used as the enzyme source for assays. Subconfluent cells were washed twice with PBS, harvested by scraping, and precipitated by centrifugation (400 g , 5 min). The cells were washed with buffer A (10 mM Hepes-Na, pH 7.5, containing 0.25 M sucrose and 5 mM EDTA), suspended in buffer A, and lysed with a probe-type sonicator. After removal of unbroken cells by centrifugation (1,000 g , 5 min), cell lysates were stored at −80°C until use. Section title: In Vitro Enzyme Assays Educational score: 4.101589202880859 Domain: biomedical Document type: Study Language: en An assay for SM synthase activity using C 5 -DMB-Cer was performed as described , except that C 5 -DMB-Cer was used instead of C 6 -NBD-Cer. GlcCer synthase activity was determined as described , except that 500 μM UDP-glucose was included in the assay mixture. Dihydrosphingosine N -acyltransferase (DCer synthase) activity was determined as described except for using [ 3 H]sphingosine instead of [ 14 C]acyl-CoA. Acid and neutral sphingomyelinase activities were determined as described . Section title: Metabolism of Fluorescent Cer Analogues in Cells Educational score: 4.144528388977051 Domain: biomedical Document type: Study Language: en Metabolism of C 5 -DMB-Cer and C 6 -NBD-Cer in cells was examined under the pulse–chase conditions as described with some modifications. In brief, subconfluent cell monolayers were incubated in Ham's F-12 containing 1.25 μM C 5 -DMB-Cer complexed with 1.25 μM BSA, or 5 μM C 6 -NBD-Cer complexed with 5 μM BSA at 4°C for 30 min. Monolayers were washed three times with Ham's F-12 and subsequently chased in Nutridoma medium containing 0.34 mg/ml fatty acid–free BSA at 33°C for various times. Lipids extracted from the cells were separated on TLC plates with a solvent of chloroform/ methanol/15 mM calcium chloride (60:35:8, vol/vol). After collecting the fluorescent spots by scraping, lipids were extracted with 0.5 ml of chloroform/methanol/0.1 M potassium chloride (1:2:0.8, vol/vol). NBD (excitation at 470 nm; emission at 530 nm) or DMB (excitation at 480 nm; emission at 515 nm) fluorescence of each lipid was measured with a spectrofluorometer. Values are presented as percentages of total fluorescence recovered (the sum of C 5 -DMB-Cer and C 5 -DMB-SM, or the sum of C 6 -NBD-Cer, C 6 -NBD-SM, and C 6 -NBD-GlcCer). Section title: Fluorescence Microscopy Educational score: 4.101076602935791 Domain: biomedical Document type: Study Language: en Cells grown on glass coverslips (22-mm diameter) were prelabeled with fluorescent lipids at 4°C and chased at 33°C under essentially the same conditions for the labeling of cells grown on culture dishes. After washing with PBS, the cells were fixed with 0.125% glutaraldehyde solution in PBS for 5 min at 4°C. The fixed specimens were observed and photographed under a fluorescence microscope. Note that the specimens were photographed soon after fixation, because redistribution of fluorescent lipid analogues might occur even in glutaraldehyde-fixed cells . Section title: Radiolabeling, Immunoprecipitation, and Endo H Treatment Educational score: 4.165632247924805 Domain: biomedical Document type: Study Language: en CHO transfectants expressing PLAP or PLAP-HA were pulsed with 9.25 MBq/ml of EXPRESS 35 S-protein labeling mix ( DuPont-New England Nuclear ) for 15 min at 33°C and chased for various times in Nutridoma medium containing 10 mM methionine. Radiolabeled cells were washed and lysed in 0.9 ml of lysis buffer, 50 mM Tris-HCl (pH 8.0) containing 150 mM NaCl, 0.1% SDS, and 1% Triton X-100. After addition of 100 μl of 10% BSA, the cell lysates were boiled for 5 min and clarified by centrifugation twice at 10,000 g for 10 min. The clarified extracts were incubated with 2 μl of rabbit antisera against human PLAP (Biomeda Corp.) at 4°C for 2 h. Resultant immunocomplexes were incubated with 50 μl of a 50% slurry of protein A–Sepharose CL-4B ( Amersham Pharmacia Biotech ) at 4°C for 2 h, pelleted, and washed once with lysis buffer containing 1% BSA and three times with lysis buffer at room temperature. Immunoprecipitates were resuspended in 50 mM sodium citrate (pH 5.5) containing 0.5% SDS, 1% 2-mercaptoethanol, and 1 mM 4-amidinophenylmethanesulfonyl fluoride, and boiled for 5 min. Immunoprecipitated proteins released from resin were incubated in the presence or absence of 6 mU of Endo H at 37°C for 16 h and separated by SDS-PAGE. Section title: Protein Determination Educational score: 3.5096206665039062 Domain: biomedical Document type: Study Language: en Protein was determined according to Lowry et al. or with a BCA protein assay reagent kit ( Pierce Chemical Co. ), using BSA as standard. Section title: Metabolic Labeling of Lipids with [ 14 C]Serine in LY-A Cells Educational score: 4.0734333992004395 Domain: biomedical Document type: Study Language: en We examined the effects of cell culture temperature on the activity of de novo SM synthesis in LY-A and wild-type cells by metabolic labeling of lipids with [ 14 C]serine for 2 h, and found that the relative levels of SM synthesis in LY-A cells to that in wild-type cells at 33, 37, and 40°C were 9.5, 10, and 16%, respectively. Thus, further characterizations of LY-A cells were performed with cells cultured at 33°C, unless otherwise described. Section title: Metabolic Labeling of Lipids with [ 14 C]Serine in LY-A Cells Educational score: 4.19072151184082 Domain: biomedical Document type: Study Language: en Time courses of metabolic labeling of lipids with [ 14 C] serine up to 12 h were compared between LY-A and wild-type cells. As shown in Fig. 1 , the SM labeling in LY-A cells was <10% of the wild-type level throughout the incubation. In contrast, time courses for Cer and GlcCer labeling in LY-A cells were comparable to those in wild-type cells, although the early labeling rates of these lipids in LY-A cells were slower than wild-type cells. LY-A and wild-type cells were virtually identical in the labeling of phosphatidylserine or phosphatidylethanolamine, both of which incorporate serine via metabolic pathways distinct from the pathway for sphingolipids. These results confirmed that LY-A cells are defective in SM synthesis, but not in glycosphingolipid synthesis. Section title: Metabolic Labeling of Lipids with [ 14 C]Serine in LY-A Cells Educational score: 4.158768177032471 Domain: biomedical Document type: Study Language: en Although DCer comigrated with Cer in the TLC system used, analysis of sphingoid base composition of metabolically labeled lipids in the TLC fraction containing both DCer and Cer showed that >80% of sphingoid bases derived from the fraction was sphingosine in both LY-A and wild-type cells, indicating that conversion of DCer to Cer is normal in LY-A cells. Section title: Metabolic Labeling of Lipids with [ 14 C]Serine in LY-A Cells Educational score: 4.171205520629883 Domain: biomedical Document type: Study Language: en For determination of the distribution of SM at the cell surface, cells were labeled with [ 14 C]serine for 48 h, fixed with glutaraldehyde, and treated with or without B . cereus sphingomyelinase. Analysis of radioactive lipids extracted from the cells showed that the pool size of sphingomyelinase-sensitive SM among the total pool of cellular SM was 56% in wild-type cells and 50% in LY-A cells. Thus, SM appeared to be preferentially distributed in the outer leaflet of the plasma membrane bilayer in LY-A cells, as in wild-type cells. Section title: Metabolic Labeling of Lipids with [ 14 C]Sphingosine and [ 14 C]Choline in LY-A Cells Educational score: 4.200754165649414 Domain: biomedical Document type: Study Language: en A defect in SM formation might result in some repression of de novo sphingoid base formation. Thus, to prevent such possible feedback repression from affecting Cer-to-SM conversion by metabolic labeling, we used [ 3 H]sphingosine as an alternative precursor for monitoring SM synthesis, since it has been shown that CHO cells are able to utilize exogenously supplied sphingosine for synthesis of Cer and complex sphingolipids . When cells were labeled with 1 μM [ 3 H]sphingosine at 33°C for various times up to 2 h, wild-type and LY-A cells similarly accumulated free [ 3 H]sphingosine, with a plateau level at 15–30 min after incubation, and showed almost equal levels of the total radioactivity of cell-associated lipids , indicating that the two cell types had an equal efficiency of sphingosine uptake. Under these conditions, formation of radioactive SM in LY-A cells was <30% of the wild-type level throughout the incubation, whereas formations of Cer, GlcCer, and G M3 were slightly higher in LY-A cells than in wild-type cells . When [ 3 H]dihydrosphingosine was used as another precursor, we obtained results similar to those obtained with [ 3 H]sphingosine (data not shown). Section title: Metabolic Labeling of Lipids with [ 14 C]Sphingosine and [ 14 C]Choline in LY-A Cells Educational score: 4.129042625427246 Domain: biomedical Document type: Study Language: en Synthesis of SM was also examined by metabolic labeling with [ 14 C]choline. Incorporation of [ 14 C]choline into SM in LY-A cells was ∼20% or less of the wild-type level, while incorporation of [ 14 C]choline into phosphatidylcholine (the phosphorylcholine donor for conversion of Cer to SM) was slightly higher in LY-A cells than in wild-type cells , eliminating the possibility that LY-A cells had a defect in phosphatidylcholine synthesis. Section title: Cell-free Assays for Activities of Enzymes Involved in SM Metabolism Educational score: 4.150418758392334 Domain: biomedical Document type: Study Language: en To identify the mechanism underlying the defect in conversion of Cer to SM in LY-A cells, we examined SM synthase activity in cell lysates by using C 5 -DMB-Cer, a fluorescent Cer analogue, as the substrate. LY-A cells showed essentially the same level of SM synthase activity as wild-type cells, as there was no difference in the dependence of C 5 -DMB-SM formation on C 5 -DMB-Cer concentration or in the time course of C 5 -DMB-SM formation between wild-type and LY-A cells . Similarly, when C 6 -NBD-Cer or N -[ 14 C]palmitoyl-sphingosine was used as the substrate, no difference in activity of SM synthase between the two cell types was observed (data not shown). Section title: Cell-free Assays for Activities of Enzymes Involved in SM Metabolism Educational score: 4.112358093261719 Domain: biomedical Document type: Study Language: en We also measured in vitro activities of various other enzymes related to SM metabolism including serine palmitoyltransferase, dihydroceramide synthase, GlcCer synthase, and acid and neutral sphingomyelinases. However, significant differences in these activities between wild-type and LY-A cells were not detected . Collectively, the results of these in vivo and in vitro experiments suggested that the specific defect of SM formation in LY-A cells was not due to alterations in enzymatic activities responsible for SM synthesis or degradation. Section title: Effect of Brefeldin A on De Novo SM Synthesis in LY-A Cells Educational score: 4.192744731903076 Domain: biomedical Document type: Study Language: en We next explored the possibility that LY-A cells had a defect in translocation of Cer from the ER to the Golgi apparatus where SM synthase exists. Treatment of CHO cells with brefeldin A, leading to fusion of the Golgi membrane with the ER , enhances the rate of conversion of Cer to SM, suggesting that in brefeldin A–treated cells Cer is accessible to the normally Golgi apparatus–localized SM synthase without the ER-to-Golgi apparatus trafficking process . Thus, if LY-A cells have a defect in ER-to-Golgi apparatus trafficking of Cer, brefeldin A treatment should restore SM synthesis in LY-A cells to the wild-type level. Section title: Effect of Brefeldin A on De Novo SM Synthesis in LY-A Cells Educational score: 4.269247055053711 Domain: biomedical Document type: Study Language: en After pretreatment with 1 μg/ml brefeldin A for 30 min at 33°C, cells were labeled with [ 14 C]serine in the presence of brefeldin A, and the labeling rates of lipids were compared between wild-type and LY-A cells. Under conditions without brefeldin A, the rate of SM synthesis in LY-A cells was only 10% of the wild-type level, while the labeling rates of other lipids in LY-A cells were comparable to the wild-type levels . In contrast, under brefeldin A–treated conditions, the rate of SM synthesis in LY-A cells was quite similar to the wild-type level, which was about threefold higher than the rate in brefeldin A–untreated wild-type cells, while labeling rates of Cer, GlcCer, phosphatidylserine, and phosphatidylethanolamine were also similar between wild-type and LY-A cells . In addition, we used [ 3 H]sphingosine and [ 3 H]dihydrosphingosine for monitoring the rate of Cer-to-SM conversion and observed restoration of SM synthesis in LY-A cells to the wild-type levels under brefeldin A–treated conditions (data not shown). These results demonstrated that LY-A cells had the Golgi apparatus–localized SM synthase normally and suggested that LY-A cells were defective in translocation of Cer from the ER to the Golgi apparatus. Section title: Metabolism of a Fluorescent Cer Analogue, C 5 -DMB-Cer, in LY-A Cells Educational score: 4.222161293029785 Domain: biomedical Document type: Study Language: en To examine further whether LY-A cells were defective in ER-to-Golgi apparatus trafficking of Cer, we used C 5 -DMB-Cer, a fluorescent Cer analogue, as a probe. Since the critical micellar concentration of C 5 -DMB-Cer is higher than that of natural Cer , externally supplied C 5 -DMB-Cer can be readily transferred to the plasma membrane and, after movement across the plasma membrane, this probe fluorescently labels various intracellular membranes including the ER membrane in living cells at 4°C. In addition, when cells prelabeled with C 5 -DMB-Cer are warmed up to physiological temperatures, C 5 -DMB-Cer is efficiently accumulated into the Golgi region and metabolized to C 5 -DMB-SM , indicating the possibility that the ER-to-Golgi apparatus trafficking behavior of C 5 -DMB-Cer mimics that of natural Cer produced at the ER. Section title: Metabolism of a Fluorescent Cer Analogue, C 5 -DMB-Cer, in LY-A Cells Educational score: 4.2206525802612305 Domain: biomedical Document type: Study Language: en For testing whether the behavior of C 5 -DMB-Cer in cells reflected the defect of SM synthesis in LY-A cells, the metabolic rate of C 5 -DMB-Cer to C 5 -DMB-SM was compared between wild-type and LY-A cells. Cells were preincubated with C 5 -DMB-Cer for 30 min at 4°C for transferring the probe to intracellular membranes and, after washing the extracellular C 5 -DMB-Cer, cells were incubated at 33°C for various times to start conversion of intracellular C 5 -DMB-Cer to C 5 -DMB-SM. During the chase, C 5 -DMB-Cer was metabolized to C 5 -DMB-SM in a time-dependent manner, resulting in conversion of 50% of the total lipidic fluorescence to C 5 -DMB-SM in wild-type cells 30 min after the chase, while the metabolic rate of C 5 -DMB-Cer to C 5 -DMB-SM in LY-A cells was about half of the wild-type level . In addition, the metabolic rate of C 5 -DMB-Cer to C 5 -DMB-SM in LY-A cells was restored to the wild-type level by brefeldin A treatment . Note that the level of total lipidic DMB fluorescence associated with cells was identical in the two cell types during the pulse and chase (data not shown). These results indicated that the behavior of C 5 -DMB-Cer at least partly reflects that of natural Cer in cells. Section title: Intracellular Behavior of Pulse-labeled C 5 -DMB-Cer in LY-A Cells Educational score: 4.219457626342773 Domain: biomedical Document type: Study Language: en Distribution patterns of intracellular DMB fluorescence were compared between LY-A and wild-type cells under the pulse–chase conditions. After prelabeling of cells with C 5 -DMB-Cer at 4°C, intracellular fluorescence was distributed throughout intracellular membranes including the ER in both wild-type and LY-A cells . After the chase at 33°C for 15–30 min, most of the intracellular fluorescence in wild-type cells accumulated in the perinuclear region corresponding to the Golgi complex , in agreement with previous studies . Interestingly, accumulation of intracellular fluorescence in the Golgi region was lower in LY-A cells than wild-type cells . The lower level of the Golgi fluorescence in LY-A cells was not due to an enhanced secretion of intracellular DMB fluorescence to the medium, because there was no significant difference in the total amount of DMB lipids associated with cells between the two cell types during the pulse and chase periods (data not shown). These observations also provided evidence that LY-A cells are defective in ER-to-Golgi apparatus trafficking of Cer. Section title: Energy Dependence of Redistribution of Intracellular C 5 -DMB-Cer to the Golgi Apparatus Educational score: 4.297706604003906 Domain: biomedical Document type: Study Language: en For examination of the energy dependence of intracellular redistribution of C 5 -DMB-Cer, cells were pretreated with or without energy inhibitors (50 mM 2-deoxy- d -glucose and 5 mM NaN 3 ) for 15 min at 33°C, incubated with C 5 -DMB-Cer for 30 min at 4°C, washed, and chased for 15 min at 33°C in the presence or absence of the energy inhibitors. Measurements of intracellular ATP showed that wild-type and LY-A cells had the same ATP levels under the inhibitor-minus control conditions, and ATP levels in both cell types were reduced by >95% in the presence of the inhibitors. ATP depletion did not affect the level and distribution of C 5 -DMB-Cer during the prelabeling of cells at 4°C; DMB fluorescence was distributed to various intracellular membranes (data not shown), as seen under the normal conditions . However, after the chase at 33°C, the level of DMB fluorescence accumulated into the Golgi region in wild-type cells was strikingly lower under the ATP-depleted conditions than under the control conditions, while in LY-A cells ATP depletion did not appreciably affect the Golgi level of DMB fluorescence . Consequently, ATP depletion reduced the DMB fluorescence level associated with the Golgi apparatus in wild-type cells to the level of LY-A cells. Section title: Energy Dependence of Redistribution of Intracellular C 5 -DMB-Cer to the Golgi Apparatus Educational score: 4.356832981109619 Domain: biomedical Document type: Study Language: en Biochemical analysis showed that the ATP-depleted conditions caused an ∼50% reduction of the conversion rate of C 5 -DMB-Cer to C 5 -DMB-SM in wild-type cells , indicating that the ATP-dependent redistribution of C 5 -DMB-Cer is responsible for synthesis of C 5 -DMB-SM in wild-type cells. The reduced rate in wild-type cells was almost identical to the rate in LY-A cells, which was not affected by ATP depletion , being consistent with the similar level of DMB fluorescence in Golgi apparatus between ATP-depleted wild-type and normally cultured LY-A cells . There was no difference in the total amount of lipidic DMB fluorescence associated with cells between the normal and ATP-depleted cells even after the 33°C chase (data not shown). Collectively, these results indicated that, in wild-type cells, redistribution of intracellular C 5 -DMB-Cer to the Golgi apparatus for C 5 -DMB-SM synthesis consists of an ATP-dependent pathway(s) and an ATP-independent (or less ATP-dependent) pathway(s), and that the ATP-dependent trafficking pathway of C 5 -DMB-Cer is almost completely impaired in LY-A cells. Section title: Metabolism and Intracellular Behavior of C 6 -NBD-Cer in Cells Educational score: 4.259522438049316 Domain: biomedical Document type: Study Language: en While C 5 -DMB-Cer is a poor substrate for GlcCer synthase , C 6 -NBD-Cer, another fluorescent analogue of Cer, serves as an efficient substrate for both GlcCer and SM syntheses . C 6 -NBD-Cer has an ∼10-fold faster rate at spontaneous transfer between artificial membranes than C 5 -DMB-Cer , and C 6 -NBD-Cer rapidly targets to the Golgi membrane even in glutaraldehyde-fixed cells, suggesting that no active process is required for ER-to-Golgi apparatus transfer of C 6 -NBD-Cer . Section title: Metabolism and Intracellular Behavior of C 6 -NBD-Cer in Cells Educational score: 4.331154823303223 Domain: biomedical Document type: Study Language: en Under normal culture or ATP-depleted conditions, wild- type and LY-A cells were preincubated with C 6 -NBD-Cer at 4°C to label intracellular membranes and, after washing extracellular C 6 -NBD-Cer, cells were incubated at 33°C for the chase. During the chase, C 6 -NBD-Cer was converted to C 6 -NBD-SM or C 6 -NBD-GlcCer in a time-dependent manner with redistribution of NBD fluorescence to the Golgi apparatus, in agreement with a previous study . In contrast to the cases with C 5 -DMB-Cer , redistribution of C 6 -NBD-Cer to the Golgi region or conversion of C 6 -NBD-Cer to C 6 -NBD-SM was not affected by ATP depletion even in wild-type cells, and C 6 -NBD-Cer behaved almost identically between LY-A and wild-type cells . The conversion rate of C 6 -NBD-Cer to C 6 -NBD-GlcCer under the ATP-depletion conditions was ∼70–75% of the normal level, probably due to a decrease in the intracellular UDP-glucose level by ATP depletion. These results indicated that C 6 -NBD-Cer is efficiently redistributed to the Golgi apparatus not via the ATP-dependent pathway, which is involved in the redistribution of C 5 -DMB-Cer to the Golgi apparatus. However, this nature of C 6 -NBD-Cer allowed us to demonstrate that the ATP-depleted conditions used did not cause an inhibition of SM synthase activity itself or extensive destruction of the Golgi apparatus. Section title: Effects of ATP Depletion on SM and GlcCer Formation from [ 3 H]Sphingosine Educational score: 4.429821968078613 Domain: biomedical Document type: Study Language: en We next examined the effect of ATP depletion on conversion of natural Cer to SM by metabolic labeling with [ 3 H]sphingosine. ATP depletion of wild-type cells resulted in the reduction of the [ 3 H]SM formation to the level of LY-A cells, which was not affected by ATP depletion , being consistent with the results of the pulse–chase experiments using C 5 -DMB-Cer . Although the [ 3 H]SM formation in wild-type cells under the ATP-depleted conditions was only 30% of the normal level, the [ 3 H]GlcCer formation under the ATP-depleted conditions was ∼65% of the normal level both in wild-type and LY-A cells . Considering that GlcCer formation appeared to be partially inhibited by a reduction of the UDP-glucose level under the ATP-depletion conditions, effects of ATP depletion on accessibility of [ 3 H]Cer to SM and GlcCer synthases could be estimated by the corrected values of [ 3 H]SM/C 6 -NBD-SM and [ 3 H]GlcCer/C 6 -NBD-GlcCer, respectively, although we did not eliminate the possibility that access of C 6 -NBD-Cer to SM and GlcCer synthases was not completely ATP independent. The corrected values suggested that the influence of ATP depletion on [ 3 H]Cer trafficking caused ∼70% and ∼20% inhibition of [ 3 H]SM and [ 3 H]GlcCer formation, respectively . ATP depletion did not reduce the level of [ 3 H]sphingosine associated with cells and did cause an increase in the level of [ 3 H]Cer along with the striking decrease in [ 3 H]SM, ruling out the possibility that the decreased formation of [ 3 H]SM under the ATP-depleted conditions was due to a decrease in cellular uptake of [ 3 H]sphingosine or formation of [ 3 H]Cer from [ 3 H]sphingosine. These results demonstrated evidence that trafficking of natural Cer from the ER to the site for SM synthesis is mainly ATP dependent and that this ATP-dependent trafficking of Cer is defective in LY-A cells. Section title: ER-to-Golgi Apparatus Trafficking of GPI-anchored and Membrane-spanning Glycoproteins in LY-A Cells Educational score: 4.229991912841797 Domain: biomedical Document type: Study Language: en To address the question of whether ER-to-Golgi apparatus trafficking of proteins was affected in LY-A cells, we determined the acquisition rate of Endo H resistance, a well-accepted determinant for arrival in the medial Golgi apparatus . For this, we used wild-type and LY-A cells constitutively expressing GPI-anchored alkaline phosphatase (PLAP) or chimeric alkaline phosphatase (PLAP-HA) having a membrane-spanning region in place of the GPI anchor. PLAP and PLAP-HA in these cells were pulse-labeled with [ 35 S]methionine/cysteine for 15 min and chased for various time at 33°C. Then, extracts from the cells were immunoprecipitated with anti-PLAP antibody, treated with Endo H, and separated by SDS-PAGE for detection of the Endo H–sensitive and –resistant forms. From the data shown in Fig. 11 , the acquisition rates of Endo H resistance of not only PLAP but also PLAP-HA were quite similar ( t 1/2 = ∼30 min) between wild-type and LY-A cells. Thus, it was likely that the machinery for ER-to-Golgi apparatus trafficking of glycoproteins operated normally in LY-A cells. Section title: LY-A Cells Have a Specific Defect in an ATP-dependent Trafficking Pathway of Cer from the ER to the Golgi Compartment for De Novo SM Synthesis Educational score: 4.0572028160095215 Domain: biomedical Document type: Study Language: en Previously, we isolated the LY-A strain, a CHO cell mutant resistant to an SM-directed cytolysin, and showed that LY-A cells have only a 30% level of SM content, but nearly a 100% level of glycosphingolipid content compared with wild-type cells . In this study, we attempted to identify a defective step causing the phenotype of LY-A cells. Section title: LY-A Cells Have a Specific Defect in an ATP-dependent Trafficking Pathway of Cer from the ER to the Golgi Compartment for De Novo SM Synthesis Educational score: 4.46002197265625 Domain: biomedical Document type: Study Language: en Metabolic labeling experiments with [ 14 C]serine, [ 3 H] sphingosine, and [ 14 C]choline indicated that conversion of Cer to SM was impaired in LY-A cells, but that the formations of Cer and phosphatidylcholine, both of which are immediate precursors for SM formation, are normal . However, enzyme assays in cell lysates showed no difference in the activities of SM synthase, neutral sphingomyelinase, or acid sphingomyelinase between LY-A and wild-type cells. Although reduced SM synthesis along with Cer accumulation was observed during the mitotic phase of the cell cycle , LY-A and wild-type cells grown in Nutridoma medium displayed virtually identical patterns of the phase distribution of the cell cycle; 30–32% of the total population of each cell type was in the G1 phase, 50–54% in the S phase, and 15–17% in the G2 plus M phases (data not shown). These in vivo and in vitro experiments suggested that the defect of SM formation in LY-A cells was not due to alterations in enzymatic activities responsible for SM metabolism nor to a secondary effect of cell-cycle phase on the lipid metabolism, leading us to the possibility that LY-A cells had a defect in translocation of Cer from the ER to the Golgi compartment where SM synthase exists. Section title: LY-A Cells Have a Specific Defect in an ATP-dependent Trafficking Pathway of Cer from the ER to the Golgi Compartment for De Novo SM Synthesis Educational score: 4.684699058532715 Domain: biomedical Document type: Study Language: en Several lines of evidence support this conclusion. First, when cells were treated with brefeldin A, SM synthesis in LY-A cells was restored to the wild-type level , in agreement with the expectation that in brefeldin A–treated cells Cer is accessible to the normally Golgi apparatus–localized SM synthase without the ER-to-Golgi apparatus trafficking process. Second, pulse–chase experiments with C 5 -DMB-Cer in intact cells demonstrated that redistribution of intracellular DMB fluorescence from intracellular membranes to the Golgi apparatus is strikingly slower in LY-A cells than in wild-type cells , along with the defect of conversion of C 5 -DMB-Cer to C 5 -DMB-SM in intact LY-A cells . Furthermore, ATP depletion in wild-type cells inhibited the conversion of C 5 -DMB-Cer to C 5 -DMB-SM by 50% and also inhibited redistribution of C 5 -DMB-Cer to the Golgi apparatus, whereas ATP depletion did not affect the behavior of C 5 -DMB-Cer in LY-A cells . Although conversion of C 6 -NBD-Cer to the SM metabolite may be necessary for trapping NBD fluorescence in the Golgi apparatus , at least a portion of the Golgi apparatus–accumulated DMB fluorescence in wild-type cells appears to be attributable to unconverted C 5 -DMB-Cer, because the amount of C 5 -DMB-SM formed in LY-A cells after a 30-min chase was equivalent to that of C 5 -DMB-SM formed in wild-type cells after a 15-min chase , whereas DMB fluorescence in the Golgi region was much denser in the latter cells than in the former . By contrast, intracellular labeling with C 6 -NBD-Cer, which rapidly transfers between membranes, displayed no difference in the Golgi redistribution between LY-A and wild-type cells, or between normally cultured and ATP-depleted cells . Thus, the impairment of the Golgi accumulation of C 5 -DMB-Cer in LY-A cells reflects a defect in the energy-dependent transport of this probe from intracellular membranes to the Golgi apparatus. In addition, the findings that intracellular DMB fluorescence is initially distributed throughout intracellular membranes after prelabeling with C 5 -DMB-Cer at 4°C and that >50% of the surface area of intracellular membranes is attributed to the ER membrane indicate that the energy-dependent redistribution of DMB fluorescence most likely represents an ER-to-Golgi apparatus transport of C 5 -DMB-Cer. Importantly, this energy-dependent trafficking of C 5 -DMB-Cer is relevant to physiological trafficking of natural Cer for SM synthesis, because ATP depletion in wild-type cells inhibited [ 3 H]SM formation from metabolically labeled [ 3 H]Cer, whereas [ 3 H]SM formation in LY-A cells was not affected by ATP depletion . Collectively, we conclude that LY-A cells are defective in ATP-dependent trafficking of Cer from the ER to the Golgi compartment where SM synthase exists. Section title: LY-A Cells Have a Specific Defect in an ATP-dependent Trafficking Pathway of Cer from the ER to the Golgi Compartment for De Novo SM Synthesis Educational score: 4.373275279998779 Domain: biomedical Document type: Study Language: en The defect in LY-A cells appears to be specific to Cer transport. When cellular lipids were metabolically labeled with [ 14 C]serine, the formation rates of [ 14 C]phosphatidylserine and [ 14 C]phosphatidylethanolamine were identical between LY-A and wild-type cells , indicating that transport of phosphatidylserine from the ER to the mitochondria proceeds normally in LY-A cells. Cholesterol metabolism in LY-A cells is also suggested to be normal, since the amount of free cholesterol in LY-A cells is the wild-type level . There was no appreciable difference in the acquisition rate of Endo H resistance of a membrane-spanning or a GPI-anchored protein between LY-A and wild-type cells , indicating that ER-to-Golgi apparatus trafficking of proteins is not defective in LY-A cells. Both wild-type and LY-A cells display a similar preferential distribution of SM at the plasma membrane (data not shown), suggesting that, once SM is produced in the Golgi apparatus, SM is subsequently sorted to the plasma membrane in LY-A cells similar to wild-type cells. Section title: LY-A Cells Have a Specific Defect in an ATP-dependent Trafficking Pathway of Cer from the ER to the Golgi Compartment for De Novo SM Synthesis Educational score: 4.54926872253418 Domain: biomedical Document type: Study Language: en Our findings that in wild-type CHO cells ATP depletion inhibits conversion of C 5 -DMB-Cer to C 5 -DMB-SM by 50% concomitantly with inhibition of redistribution of intracellular DMB fluorescence to the Golgi apparatus suggest that a ∼50% portion of ER-to-Golgi apparatus transfer of C 5 -DMB-Cer is mediated via the ATP-dependent pathway and that another 50% of the C 5 -DMB-Cer transfer is mediated by ATP-independent (or less ATP-dependent) mechanisms. The ATP-independent redistribution of C 5 -DMB-Cer to the Golgi apparatus may be at least partly attributable to a nonphysiological spontaneous transfer of C 5 -DMB-Cer between membranes due to less hydrophobicity of C 5 -DMB-Cer than natural Cer. For natural Cer, ATP-dependent and -independent (or less ATP-dependent) trafficking mechanisms were estimated to be responsible for 70–80% and 20–30%, respectively, of newly synthesized SM, since ATP depletion caused a 70–80% inhibition of formation of [ 3 H]sphingosine-derived SM without reduction of [ 3 H]Cer formation . In contrast, redistribution of intracellular C 6 -NBD-Cer to the Golgi apparatus or conversion of C 6 -NBD-Cer to C 6 -NBD-SM was not, or only slightly, affected by ATP depletion. The different trafficking behavior between C 5 -DMB-Cer and C 6 -NBD-Cer most likely reflects the fact that C 6 -NBD-Cer has a much faster rate of spontaneous transfer between artificial membranes than C 5 -DMB-Cer , implying that C 5 -DMB-Cer is embedded in membranes more firmly and thus mimics natural Cer more accurately, compared with C 6 -NBD-Cer. Section title: Different Pathways of Cer Transport for SM versus GlcCer Synthesis Educational score: 4.486083984375 Domain: biomedical Document type: Study Language: en It has been postulated that a yet-undefined pathway(s) mediates Cer trafficking from the ER to the Golgi apparatus and that, after arrival at the cytoplasmic surface of the Golgi apparatus, Cer molecules are used for GlcCer synthesis and the residual Cer molecules are subsequently used for SM synthesis after translocation to the lumenal side of the Golgi apparatus . Collins and Warren have shown that newly synthesized Cer can be delivered to the sites of SM and GlcCer synthases by a different pathway from the major vesicular-mediated trafficking pathway for newly synthesized proteins during the mitotic phase in HeLa cells. Slomiany et al. have demonstrated that Cer-containing vesicles derived from the ER fuse with the Golgi membrane in an N -ethylmaleimide– sensitive manner under cell-free conditions. On the other hand, studies with another cell-free system and with a semi-intact cell system to assay for the transfer of Cer between the ER and the Golgi membranes have suggested that Cer could be transferred from the ER to the Golgi apparatus through a nonvesicular pathway in ATP-independent and N -ethylmaleimide–insensitive manners . Section title: Different Pathways of Cer Transport for SM versus GlcCer Synthesis Educational score: 4.726733207702637 Domain: biomedical Document type: Study Language: en If ER-to-Golgi apparatus trafficking of Cer is mediated by only one pathway, it would be a paradox that LY-A cells synthesize GlcCer to the wild-type level or more, in spite of the defect of the ATP-dependent ER-to-Golgi apparatus trafficking of Cer. A possible explanation for the paradoxical phenotype of LY-A cells is that the putative sole pathway for ER-to-Golgi apparatus Cer trafficking might be narrowed in LY-A cells, but that the narrowed pathway might still supply enough Cer to produce a normal level of GlcCer in the Golgi apparatus, but not of SM. However, this explanation is not consistent with the observation that formations of [ 3 H]GlcCer and [ 3 H]G M3 from [ 3 H]sphingosine were somewhat higher in LY-A cells than in wild-type cells . It is unlikely that the reduced synthesis of SM results from an overconsumption of newly synthesized Cer at the GlcCer synthesis step, since the level of [ 3 H]sphingosine-derived Cer was higher in LY-A cells than in wild-type cells and there was no difference in GlcCer synthase activity between the two cell types (data not shown). Another possibility is that a transbilayer transport of Cer across the Golgi membranes is impaired in LY-A cells. However, the observation that SM synthesis in LY-A cells was restored to the wild-type level after brefeldin A treatment favors the interpretation that the phenotype of LY-A cells is primarily due to a defect in the interorganelle transport of Cer, although we cannot completely exclude the possibility that brefeldin A treatment facilitates a transbilayer transport of Cer across the membrane where SM synthase exists, thereby restoring SM synthesis to LY-A cells. Considering that SM synthase as well as ganglioside synthases are suggested to exist more distally in the Golgi complex than GlcCer synthase , an intra–Golgi apparatus transport of Cer might be specifically blocked in LY-A cells. If so, one could expect accumulation of Cer in a pre- or early Golgi region. However, there was no sign of such specific redistribution of C 5 -DMB-Cer in LY-A cells , although this observation does not conflict with the alternative expectation that a block of the intra–Golgi apparatus transport of Cer causes recycling of C 5 -DMB-Cer to the ER instead of its accumulation in the early Golgi apparatus. Note that LY-A cells normally produce G M3 , eliminating the possibility that intra–Golgi apparatus transport pathways of various sphingolipid types are nonspecifically blocked in LY-A cells. Section title: Different Pathways of Cer Transport for SM versus GlcCer Synthesis Educational score: 4.45012092590332 Domain: biomedical Document type: Study Language: en Therefore, we considered another possible mechanism that Cer, produced at the ER, is accessible to GlcCer synthase by different pathways from the ATP-dependent trafficking pathway which is impaired in LY-A cells. This mechanism appears to be consistent with the characteristics of LY-A cells; for example, the overflow of Cer to glycosphingolipids in LY-A cells could be mediated by the putative pathway responsible for GlcCer synthesis. The observation that an ATP depletion of wild-type cells caused ∼70% inhibition of [ 3 H]sphingosine-derived [ 3 H]SM formation, but only ∼20% inhibition of [ 3 H]GlcCer formation , may also be accounted for by the notion that Cer is efficiently accessible to GlcCer synthase by ATP-independent (or less ATP-dependent) trafficking, although there could be the alternative explanation that a minor population of GlcCer synthase is distributed to the ER in CHO cells, so that Cer produced at the ER is accessible to the ER-distributed GlcCer synthase without an interorganelle transport. Collectively, we propose that there are at least two pathways for Cer trafficking from the ER to the site of de novo SM synthesis: the ATP-dependent major pathway, which is impaired in LY-A cells, and the ATP-independent (or less ATP-dependent) minor pathway(s). This scenario appears to occur in various types of mammalian cells since depletion of intracellular ATP in HeLa cells and human skin fibroblasts affected redistribution of C 5 -DMB-Cer to the Golgi apparatus and conversion of C 5 -DMB-Cer to C 5 -DMB-SM (data not shown), as observed in wild-type CHO cells . Section title: Different Pathways of Cer Transport for SM versus GlcCer Synthesis Educational score: 4.615932941436768 Domain: biomedical Document type: Study Language: en Two hypothetical models may be conceivable to explain the preferential delivery of Cer to SM synthase by the ATP-dependent mechanism. In one model , a portion of newly synthesized Cer molecules is rapidly internalized to the lumenal side of the ER, and the lumenal Cer is delivered to the Golgi apparatus via an ATP-dependent and vesicle-mediated pathway for SM synthesis, while Cer remaining at the cytosolic side of the ER is delivered to the cytosolic side of the Golgi apparatus via an ATP-independent (or less ATP-dependent) pathway for GlcCer synthesis. In another model , regardless of the Cer topology in the ER membrane, a portion of the Cer molecules enters an ATP-dependent pathway, which is specifically directed to the Golgi subcompartment where SM synthase is localized, while Cer molecules associated with an ATP-independent (or less ATP-dependent) pathway have a broader specificity for their destination and are eventually accessible to both GlcCer and SM synthases. If LY-A cells have a deficiency in an intra–Golgi apparatus transport of Cer, it may be conceivable that the ATP-dependent step for specific delivery to the SM synthase– localizing compartment is located at the intra–Golgi apparatus transport of Cer. Section title: Different Pathways of Cer Transport for SM versus GlcCer Synthesis Educational score: 4.251087188720703 Domain: biomedical Document type: Study Language: en Membrane proteins are delivered from the ER to the Golgi apparatus by vesicular transport mechanisms , and the same mechanisms for protein trafficking may be involved in the ATP-dependent trafficking of Cer. However, since LY-A cells exhibit no appreciable defect in ER-to-Golgi apparatus trafficking of proteins, we currently speculate that the machine defective in LY-A cells could be a specific device for Cer transport. There might be multiple, independent vesicular transport pathways from the ER to the Golgi apparatus, one of which is exclusive for Cer. For further elucidation of the molecular mechanism underlying the intracellular trafficking of Cer, additional studies, including identification of the genes related to this function, will be necessary, and the LY-A strain will be a useful tool for this purpose as well. | Other | biomedical | en | 0.714285 |
10037790 | Section title: Antibodies Educational score: 2.0734283924102783 Domain: biomedical Document type: Other Language: en Monoclonal antibody against rat GP2 has been described previously and was a gift from Dr. Anson Lowe (Stanford University). Monoclonal antibody KT3 against SV-40 large T antigen was a gift from Dr. Gernot Walter (University of California, San Diego, CA). Polyclonal antibody against the cytoplasmic domain of cadherin has been described previously . Monoclonal antibody against human CD7 (T3.3A1) was obtained from American Type Culture Collection. Section title: Recombinant cDNA Construction Educational score: 2.4643890857696533 Domain: biomedical Document type: Study Language: en Due to the numerous recombinant cDNA constructs used in this study and that some constructs are derivatives in nature, construction details are not described for all constructs but are available upon request (E-mail: etchen@cmgm.stanford.edu ). Individual constructs were confirmed by restriction mapping and DNA sequencing (PAN facility, Stanford University). Section title: Plasmid for Expression of E-cad sol . Educational score: 4.433541774749756 Domain: biomedical Document type: Study Language: en U-GFP, a previously unpublished plasmid based on CDM8 , containing a fusion protein with the extracellular domain of canine E-cadherin jointed with green fluorescent protein (GFP), was the starting material. The chimeric coding region is flanked by Hind3 and Not1 sites at the 5′ and 3′ end, respectively, with a Xho1 site as a linker (reading frame CTC-GAG, Leu-Glu) between the E-cadherin extracellular domain and GFP. The majority of the cadherin region (Hind3-Xho1, 2.1 kb) is derived from a cDNA clone (Hind3-Bgl2, 1.9 kb). The small remaining portion of the cadherin extracellular domain (Bgl2-Xho1, 0.2 kb) was from a PCR product. The GFP region was replaced with a pair of complimentary oligonucleotides encoding for the epitope tag of KT3 (amino acid sequence KPPTPPPEPET) and a stop codon, while retaining the Xho1 linker and the 3′-end Not1 site. The resulting plasmid is named U-SVLT1. The encoded soluble, KT3-tagged extracellular domain of canine E-cadherin is named E-cad sol . A PCR reaction was carried out to amplify the coding region of E-cadherin from the internal endogenous Bgl2 site to the end of the coding region (the stop codon was replaced with a Xho1 site), using canine E-cadherin cDNA (a gift from Dr. Lee Rubin, University College London, UK) as the template. Note that the PCR product contains a small part of the extracellular domain and the complete transmembrane and cytoplasmic domains of E-cadherin and, after restricting with Bgl2 and Xho1, was used to replace the Bgl2 and Xho1 portion of U-SVLT1, resulting in the plasmid U-SVLT2. Thus, U-SVLT2 encodes a complete canine E-cadherin fused with KT3 epitope tag through a Xho1 (Leu-Glu) linker. Further mutagenesis of the putative basal-lateral sorting signals was carried out by PCR reactions to amplify the region of the Bgl2/Xho1 of U-SVLT2, as detailed below. Section title: Plasmids for Expression of E-cad BL1, BL2, and BL12. Educational score: 4.300364017486572 Domain: biomedical Document type: Study Language: en Plasmid U-SVLT3, which encodes the mutant protein E-cadherin BL2 (Y→ A mutations in BL2), was generated by PCR using the pair of oligonucleotides Cad BL-P6 (5′-CGC GGG CC A GAT CT T CCC CCC AAC ACA TCT-3′; Bgl2 site is underlined) and Cad BL-P3 (5′-CGC GGG CTC GAG GTC GTC CTC GCC ACC GCC GGC CAT GTC CGC CAG CTT-3′; Nae1 site is underlined). Note that the mutations introduced were accompanied by the introduction of a Nae1 site. The PCR product was used to replace the Bgl2 and Xho1 portion of U-SVLT2. Plasmid U-SVLT5, which encodes the mutant protein E-cadherin BL1 (Y→ A mutations in BL1), was generated by PCR from three reactions. A complimentary pair of oligonucleotides, Cad BL-P4 (5′-GAC ACC CGG GAC AAT GTT GCA G CT GC A G AT GAA GAA GGA GGT GG-3′) and Cad BL-P5 (5′-CCA CCT CCT TCT TCA T CT GCA G CT GC A ACA TTG TCC CGG GTG TC-3′), encode the (Y→ A) mutations (italics in the sequences given above) and were synthesized. Note that the introduction of a Pst1 site (underlined, CTGCAG) accompanied the introduction of the mutations. Two PCR reactions were carried out with pairs of oligonucleotides Cad BL-P6 (sense) and Cad BL-P5 (anti-sense), Cad BL-P4 (sense) and Cad BL-P2 (antisense, 5′-GCG CCC CTC GAG GTC GTC CTC GCC ACC TCC-3′; Xho1 site is underlined). The reaction products were mixed and served as the template for the third reaction with a pair of oligonucleotides, Cad BL-P6 (sense), and Cad BL-P2 (antisense). The PCR product was subcloned and replaced the Bgl2-Xho1 portion of the U-SVLT2 to give rise to U-SVLT5. Section title: Plasmids for Expression of E-cad BL1, BL2, and BL12. Educational score: 4.087886810302734 Domain: biomedical Document type: Study Language: en The plasmid U-SVLT4 which encodes E-cadherin BL12 (Y→ A mutation in BL1 and BL2) was constructed in an identical manner as that used to construct U-SVLT5, except oligonucleotide Cad BL-PL3 was used in place of Cad BL-P2. Note that in the series of plasmids U-SVLT3, 4, and 5, the identity of the introduced mutations were checked by the presence of the Pst1 and/or Nae1 restriction sites. Section title: GP2S. Educational score: 4.163477897644043 Domain: biomedical Document type: Study Language: en Amino acids (aa) 1–504 of the coding region of rat GP2 cDNA (a gift from Dr. Anson Lowe, Stanford University) was amplified by PCR with a stop codon introduced downstream to amino acid 504, and with Hind3 and Not1 sites added to the 5′ and 3′ ends, respectively. The PCR product was subcloned into CDM8 through Hind3 and Not1 sites and resulted in the plasmid, GP2S. Section title: GP2CAD1. Educational score: 4.34576416015625 Domain: biomedical Document type: Study Language: en GP2CAD1 is a chimeric protein of rat GP2 in which the GPI modification signal has been deleted (named GP2ΔGPI), and the transmembrane domain and cytoplasmic domain of canine E-cadherin added. To create GP2CAD1, the following oligonucleotides were used as PCR primers: a pair of complimentary oligonucleotides with the COOH terminus sequence of rat GP2 (without the GPI modification signal) joined in-frame with canine E-cadherin transmembrane sequence (italic), GP2CAD1-1S (5′-CAG AAT CCT GAT ACC TCC GCG CCT TAC GCC GAA GCA -3′) and GP2CAD1-3A (5′- TGC TTC GGC GTA AGG CGC GGA GGT ATC AGG ATT CTG-3′); an oligonucleotide hybridizing with the COOH terminus of canine E-cadherin, including the stop codon, GP2CAD1-2A (5′-CGC GGG GCG GCC GCT TTA GTC GTC CTC GCC ACC TCC); an oligonucleotide hybridizing with the NH 2 terminus of GP2, including the start codon, GP2-1S (5′-CGC GGG AAG CTT AGG ATG GTG GCT TGT GAC-3′). Primers GP2-1S and GP2CAD1-3A were used to amplify GP2 using a cDNA clone of rat GP2. Primer GP2CAD1-2A was used with primer GP2CAD1-1S to amplify the transmembrane and cytoplasmic domains of canine E-cadherin using a cDNA clone of canine E-cadherin as the template. The PCR products from the two reactions were mixed and used as template for amplification using oligonucleotides GP2-1S and GP2CAD1-2A as primers. The final PCR product was then subcloned into the Hind3-Not1 sites of CDM8, and the plasmid was named GP2CAD1. GP2CAD1 was then used as the template to create chimeric proteins in the following section. Section title: GP2CAD1 Truncation Mutants. Educational score: 4.196357727050781 Domain: biomedical Document type: Study Language: en GP2CAD2, 4, 6, 7, 8, and 10 are simple truncation mutants of GP2CAD1. GP2CAD1 was used as the PCR template. Individual antisense oligonucleotides hybridizing with the 6 aa upstream to the desired truncation, with a stop codon and Not1 site, were used with the oligonucleotide primer GP2-3S (5′-CGC GGG CAA GTC GAC TTC GCA GTA GTG AAC C-3′), which hybridizes to the region of the endogenous Sal1 site (underlined in the sequence) close to the COOH terminus of GP2 coding region, for individual PCR reactions to amplify parts of the cytoplasmic domain of E-cadherin. The resulting PCR products were cloned into GP2CAD1 through Sal1-Not1 sites. The cytoplasmic domain carried by each chimeric protein is shown in the diagram in Fig. 4 , and described in detail in the Results section. Section title: GP2CAD1 Truncation Mutants. Educational score: 4.221066474914551 Domain: biomedical Document type: Study Language: en GP2CAD3 is a loop-out mutant constructed using a method similar to that used in the construction of GP2CAD1. A pair of complimentary oligonucleotides with the sequence corresponding to aa 77–82 and 130–135 of the cytoplasmic domain of E-cadherin were joined. The primer pair was used with oligonucleotides GP2-3S and GP2CAD1-2A to create GP2CAD3 using recombinant PCR reactions identical to those used to construct U-SVLT4 and GP2CAD1. Section title: GP2CAD1 Truncation Mutants. Educational score: 4.242004871368408 Domain: biomedical Document type: Study Language: en The other loop-out mutants, GP2CAD5 and GP2CAD9, were constructed in a similar manner as follows: aa 83–129 (BB1) of the cytoplasmic domain of canine E-cadherin were PCR amplified using the canine cDNA clone as template with a Xho1 site added to the 5′ end and a Not1 site added to the 5′ end downstream to a stop codon. The PCR product was subcloned into CDM8FluTag through Xho1 and Not1 sites and resulted in an in-frame fusion with the influenza virus hemagglutinin tag. The resulting plasmid was named HA-BB1. A PCR reaction were carried out to amplify the GP2 and the transmembrane domain of E-cadherin from GP2CAD1 to replace the HA portion in HA-BB1. The resulting plasmid encodes the chimeric protein GP2CAD5, which comprises GP2 and the transmembrane domain of E-cadherin fused with 83–129 of the cytoplasmic domain through a Xho1 linker (Leu-Glu). GP2CAD9 was constructed similarly. Section title: CD7BB1. Educational score: 4.151435375213623 Domain: biomedical Document type: Study Language: en A human CD7 cDNA clone, CD7-31 (a gift from Dr. Brian Seed, Harvard University Medical School), was in a CDM8-based expression vector and was used directly for transfection. CD7-31 was used as the template for PCR to amplify the complete coding region of CD7 with Hind3 and Xho1 sites added to the 5′ and 3′ ends, respectively. The amplified CD7 was subcloned into HA-BB1 through Hind3 and Xho1 site which generated an in-frame fusion of CD7 and BB1. The resulting plasmid was named CD7BB1. Section title: E-cad8. Educational score: 4.231781005859375 Domain: biomedical Document type: Study Language: en E-cad8 is a canine E-cadherin deletion mutant with 116–151 (the most COOH-terminal 36 aa) of the cytoplasmic domain deleted. The deletion in cytoplasmic domain is equivalent to that in GP2CAD8. Oligonucleotides Cad BL-P6 (see above) and E-CAD8-A (5′-CGC GGG CTC GAG GCT CAG ACT AGC AGC TTC AGA-3′, the Xho1 site is underlined) were used to perform PCR using canine E-cadherin cDNA as the template. The PCR product was restrited with Xho1 and Bgl2, and then was subcloned into the plasmid U-SVLT1 after the Bgl2 and Xho1 digestion. The ligation resulted in an in-frame fusion with KT3 epitope tag through the Xho1 site with Leu-Glu as a linker at the COOH terminus of the truncated E-cadherin. Section title: Cells and Transfection Educational score: 4.123364448547363 Domain: biomedical Document type: Study Language: en MDCK clone IIG cells , maintained in DME supplemented with 10% fetal bovine serum, were transfected using the calcium phosphate method with pSV2neo as the selection marker. G418 resistant (500 μg/ml) colonies were isolated using cloning rings, and individual clones were expanded and screened by immunofluorescence staining or Western blotting using appropriate antibodies. For each construct, multiple clones were isolated and characterized. No difference in immunofluorescence staining or Western blot profile was found among clones transfected with the same construct. All of the transfected MDCK cells used in this study developed transepithelial electric resistance (TER) similar to that of parental MDCK cells. Two independent clones of each construct were used for experiments. Section title: Metabolic Labeling, Immunoprecipitation, and Related Methods Educational score: 4.228906631469727 Domain: biomedical Document type: Study Language: en To monitor intracellular trafficking of newly synthesized protein, cells were grown on 24-mm Transwell™ filters for 7 d, incubated in DMEM without Met and Cys for 1 h, and then labeled with 250 μCi 35 S-Met/Cys (Pro-Mix; Amersham ) for the time specified in individual experiments (20 min or 1 h). To metabolically label proteins to steady state, cells were labeled with 250 μCi 35 S-Met/Cys supplemented with 1.5 μg/ml of methionine and 3.0 μg/ml of cysteine-HCl for 24 h before extraction. When required, membrane domain-specific biotinylation was performed using NHS-SS-Biotin (Pierce) as described previously . Labeled cells were extracted with Triton X-100 Lysis buffer (10 mM Tris-HCl, pH 7.5, 120 mM NaCl, 25 mM KCl, 2 mM EDTA, 2 mM EGTA, and 0.5% Triton X-100) or with 1× NDET extraction buffer (1% NP-40, 2% sodium deoxycholate, 66 mM Na-EDTA, and 10 mM Tris-HCl, pH 7.4). After treating with specific antibody bound on protein A–Sepharose ( Pharmacia ), immunoprecipitates were washed with High Stringency Buffer (HS-B): 20 mM Tris-HCl, pH 7.5, and 120 mM NaCl, 25 mM KCl; 5 mM EDTA; 5 mM EGTA; 0.1% SDS; 1% Na-deoxycholate; 0.5% Triton X-100, and High Salt Wash Buffer (Hs-B containing l M NaCl). If cells were biotinylated before extraction, biotinylated immunoprecipitates were collected with immobilized avidin (Pierce) as described previously . Samples were then resolved by SDS-PAGE and radioactive signals were collected by fluorography or a PhosphorImager (Molecular Dynamics). Signals were quantified with a PhosphorImager or a laser densitometer (Hoefer, San Francisco, CA). Section title: Metabolic Labeling, Immunoprecipitation, and Related Methods Educational score: 4.145789623260498 Domain: biomedical Document type: Study Language: en To perform digestion of GP2CAD10 with endoglycosidase H, GP2CAD10 MDCK cells were grown on 24-mm Transwell™ filters for 7 d, labeled with 250 μCi of 35 S-Met/Cys for 24 h, then extracted with 1× NDET and immunoprecipitated with the monoclonal antibody against rat GP2. Immunoprecipitates were released from protein A–Sepharose ( Pharmacia ) and antibodies by boiling in 50 μl of 1% SDS, 200 mM dithiothreitol for 5 min. An equal volume of 100 mM citric acid buffer (pH 5.5) was added to the immunoprecipitates, and 1 U of endoglycosidase H ( Boehringer Mannheim ) was added and the sample was incubated at 37°C for 16 h. The processed samples were resolved by 10% SDS-PAGE and detected by fluorography. Section title: Metabolic Labeling, Immunoprecipitation, and Related Methods Educational score: 4.007957458496094 Domain: biomedical Document type: Study Language: en Radioactive signals collected with a PhosphorImager were transferred directly to Adobe Photoshop, and radioactive signals collected with fluorography were scanned from autoradiography film ( Kodak ) into Adobe Photoshop with a Hewlett-Packard Scan-Jet IIc scanner. The contrast of images was adjusted in Photoshop, the images were arranged and labeled for individual figures using Canvas 5 (Deneba Software). In some cases, samples containing apical or basal-lateral membrane biotinylated proteins were separated in gels with an empty lane between the sample lanes in order to avoid any cross contamination of protein samples. The empty, intervening lane was removed in Canvas 5 and the two sample lanes are shown side-by-side. The rearranged images were printed using a Tektronix dye sublimation printer. The printed images are representative of the original data. Section title: Size Exclusion Chromatography Educational score: 4.1174116134643555 Domain: biomedical Document type: Study Language: en Preparation of samples and elution of extracted proteins of MDCK cells from Superose 6 column was preformed using identical procedures as previously published . The collected fractions were resolved by 7.5% SDS-PAGE and proteins were transferred onto nitrocellulose membranes. The membranes were then probed with a monoclonal antibody against the extracellular domain of E-cadherin (Transduction Laboratories) or a monoclonal antibody against β-catenin (Transduction Laboratories). After incubation with horseradish peroxidase conjugated secondary antibody ( Amersham ), the signals were recorded using ECL reagent ( Amersham ) and autoradiography film ( Kodak ). Section title: Immunofluorescence Staining and Other Staining Methods Educational score: 4.139986515045166 Domain: biomedical Document type: Study Language: en For immunofluorescent staining of GP2 chimeric proteins, cells on collagen coated coverslips were fixed with 3.7% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) at room temperature, permeabilized with 0.5% Triton X-100 in PBS, and processed for immunofluorescence staining with monoclonal antibody against rat GP2. NBD-ceramide (Molecular Probes) staining of the Golgi apparatus performed on fixed MDCK cells as published previously . Acridine orange staining of lysosomes in live MDCK cells on coverslips was performed by adding acridine orange (1 μg/ml; Sigma Chemical Co. ) to growth medium for 30 min at 37°C, followed by incubation in normal growth medium without acridine orange for 15 min. Then the coverslip was then mounted on a microscope slide with spacers to form a chamber for direct observation. Section title: Immunofluorescence Staining and Other Staining Methods Educational score: 4.076481819152832 Domain: biomedical Document type: Study Language: en Fluorescent antibody-stained cells, NBD-ceramide-stained fixed cells, or acridine orange stained living cells were observed using a Zeiss Axioplan fluorescence microscope with a 63× objective and the appropriate filter sets. Note that for observation of acridine orange staining, the normal filter set for fluorescein isothiocyanate fluorescence was reconfigured by replacing the barrier filter with a long pass band filter (590 nm). Fluorescent images of stained cells were recorded using Kodak Tri-X film or Kodak Ektachrome Elite II (ASA 400) film . Photographic images were then digitized with a slide scanner ( Nikon ) and resized using Adobe Photoshop. Images in Fig. 5 and Fig. 12 were arranged and labeled using Canvas 5 and printed from the computer file using a dye sublimation printer. Section title: LDL-R-like Basal-lateral Sorting Motifs Are Not Responsible for Basal-lateral Sorting of E-Cadherin Educational score: 4.311277866363525 Domain: biomedical Document type: Study Language: en The primary structure of the cytoplasmic domain of E-cadherin contains two regions with amino acid sequences and spacing that are similar to previously identified basal-lateral sorting signals in the cytoplasmic domain of the LDL-R . We assessed whether these two motifs function as basal-lateral sorting determinants for E-cadherin. All tyrosine residues in each motif of canine E-cadherin were mutated into alanine in region 1 (BL1), or region 2 (BL2), or both (BL12); similar mutations in both motifs of LDL-R resulted in random sorting to the cell surface in MDCK cells . Each mutant was epitope-tagged with the SV-40 large T antigen epitope and stably transfected into MDCK cells . Confluent monolayers of cells were grown on Transwell™ filters for 7 d, and then labeled with 35 S-Met/Cys for 1 h. At the end of the 1 h labeling period, the first wave of newly synthesized E-cadherin that arrived at the apical or basal-lateral membrane was detected by cell surface biotinylation. Mutant E-cadherin was distinguished from endogenous E-cadherin with the KT3 monoclonal antibody to the epitope tag. Section title: LDL-R-like Basal-lateral Sorting Motifs Are Not Responsible for Basal-lateral Sorting of E-Cadherin Educational score: 4.167697906494141 Domain: biomedical Document type: Study Language: en Greater than 95% of newly synthesized mutant E-cadherin, lacking tyrosine residues in either region 1 (BL1), or region 2 (BL2), or both (BL12) was delivered to the basal-lateral domain of MDCK cells with an efficiency and fidelity similar to those of endogenous E-cadherin . This result indicates that the basal-lateral sorting determinant(s) of E-cadherin are different from those of LDL-R. To further dissect basal-lateral sorting determinants in E-cadherin, we next examined the overall localization of the basal-lateral sorting determinant in the whole molecule. Section title: LDL-R-like Basal-lateral Sorting Motifs Are Not Responsible for Basal-lateral Sorting of E-Cadherin Educational score: 4.18473482131958 Domain: biomedical Document type: Study Language: en A secreted form of E-cadherin (E-cad sol ), in which the transmembrane (TM) and cytoplasmic (CYT) domains had been deleted, was expressed stably in MDCK cells . Delivery of newly synthesized E-cad sol to different membrane domains was detected by sampling apical and basal-lateral medium for 35 S-Met/Cys labeled E-cad sol . Note that E-cad sol was efficiently secreted into the culture medium, did not associate with the cell surface of MDCK cells, and did not disrupt TER (Y.-T. Chen, unpublished result). As shown in Fig. 2 , newly synthesized E-cad sol was secreted equally into the medium of both apical and basal-lateral compartments of the Transwell™ filter. We conclude that the extracellular domain of E-cadherin does not carry signals that specify E-cadherin sorting or delivery to the basal-lateral membrane. Section title: Basal-lateral Sorting Activity Resides in the Cytoplasmic Domain of E-Cadherin Educational score: 4.220677852630615 Domain: biomedical Document type: Study Language: en To locate the basal-lateral sorting signal(s) in E-cadherin, we constructed a chimeric protein comprising the transmembrane and specific regions of the cytoplasmic domain of E-cadherin fused to the extracellular domain of GP2, a highly glycosylated protein of zymogen granules in pancreatic exocrine cells . Normally, GP2 is attached to the membrane via a GPI anchor, and is delivered exclusively to the apical domain of MDCK clone II/G cells . A deletion mutant of GP2 that lacked the GPI modification signal was expressed in MDCK cells. As shown in Fig. 3 A, newly synthesized GP2ΔGPI was secreted equally into both the apical and basal-lateral medium. This result confirms that GP2 sorting to the apical membrane requires the GPI-anchor , and reveals that the extracellular domain of GP2 does not contain specific sorting signals for the basal-lateral membrane. Section title: Basal-lateral Sorting Activity Resides in the Cytoplasmic Domain of E-Cadherin Educational score: 4.2590742111206055 Domain: biomedical Document type: Study Language: en GP2ΔGPI was fused to the TM and CYT of E-cadherin, producing the chimeric protein GP2CAD1 . Significantly, >95% of newly synthesized GP2CAD1 was preferentially delivered to the basal-lateral domain, which represents an efficiency and fidelity of delivery similar to that of endogenous E-cadherin. We conclude the transmembrane and cytoplasmic domains of E-cadherin contain a basal-lateral sorting signal(s). Section title: Systematic Deletions of E-Cadherin Cytoplasmic Domain Result in Retarded Exit from ER and Random Delivery of GP2/Cadherin Chimera to the Apical and Basal-lateral Cell Surface Educational score: 4.180172920227051 Domain: biomedical Document type: Study Language: en To map in detail the basal-lateral sorting domain of E-cadherin, a series of chimeric proteins were created in which GP2ΔGPI was used as the extracellular domain (reporter) fused to the transmembrane domain and specific regions of the cytoplasmic domain of E-cadherin . Each mutant was designed to explore whether distinct features/ activities of the cytoplasmic domain, such as binding to β-catenin and individual putative sorting signals, could be separated from each other. Section title: Systematic Deletions of E-Cadherin Cytoplasmic Domain Result in Retarded Exit from ER and Random Delivery of GP2/Cadherin Chimera to the Apical and Basal-lateral Cell Surface Educational score: 4.25 Domain: biomedical Document type: Study Language: en In general, two types of deletion mutants were constructed. The first type involved the introduction of simple truncations in the cytoplasmic domain: GP2CAD2 contained amino acids (aa) 1–33 of the cytoplasmic domain, and ended at the COOH terminus of the first putative basal-lateral sorting signal (BL1); GP2CAD4 only contained the first 4 aa of the cytoplasmic domain; GP2CAD6 contained aa 1–99 of the cytoplasmic domain, and ended just before the start of the previously defined minimal β-catenin binding domain (see Introduction); GP2CAD7 contained aa 1–129 of the cytoplasmic domain, and ended at the COOH terminus of the minimal β-catenin binding domain (aa 100–129); GP2CAD8 contained aa 1–115 of the cytoplasmic domain, and ended in the middle of the minimal β-catenin binding domain; GP2CAD10 contained almost all of the cytoplasmic domain except the COOH-terminal three acidic amino acids (EDD). The second type of mutants were loop-out mutants in which two halves of the cytoplasmic domain were joined after an intervening sequence had been deleted: GP2CAD3 contained a deletion of aa 83–129 of the cytoplasmic domain (minimal β-catenin binding domain is aa 100–129); GP2CAD5 contained aa 83–129 of the cytoplasmic domain; GP2CAD9 contained aa 1–33 and aa 83–129 of the cytoplasmic domain. Rather unexpectedly, none of the mutants (GP2- CAD1–GP2CAD10) gave distinct sorting patterns compared with endogenous E-cadherin or GP2CAD1 . In all cases, mutant proteins were delivered equally to both apical and basal-lateral membranes. Note that the relative amounts of each chimera delivered to either the apical or basal-lateral membrane domain are directly comparable, but a comparison cannot be made between the amount of cell surface delivery of different chimeras from these data. However, as described above, with the exception of E-cadherin and GP2CAD1, the amount of each chimera that reached the cell surface was <10% of that synthesized. Section title: Systematic Deletions of E-Cadherin Cytoplasmic Domain Result in Retarded Exit from ER and Random Delivery of GP2/Cadherin Chimera to the Apical and Basal-lateral Cell Surface Educational score: 4.243572235107422 Domain: biomedical Document type: Study Language: en To examine the subcellular location of each chimeric protein, cells were grown on glass coverslips and processed for immunofluorescence. An anti-GP2 antibody was used to examine the distributions of the GP2CAD chimeras, and to distinguish them from endogenous E-cadherin. As shown in Fig. 5 A, GP2CAD1 was localized mainly at the cell surface, including cell–cell contact sites, similar to endogenous E-cadherin. In contrast, staining of all other chimeric proteins (GP2CAD2–GP2CAD10) revealed an intracellular, reticular staining pattern; only staining for GP2CAD10 is shown in Fig. 5 B, but the staining of other chimeric proteins (GP2CAD2–GP2CAD9) was indistinguishable from that of GP2CAD10 (Y.-T. Chen, unpublished results). In addition, larger, punctate structures were stained for GP2CAD10. The reticular pattern of GP2CAD10 staining , and other GP2CAD mutants was very similar to that of the ER resident protein BiP in MDCK cells . It was distinct from the Golgi revealed by NBD-ceramide staining of fixed cells , and lysosomes revealed by labeling live cells with acridine orange . Thus, the majority of GP2CAD10 is most likely localized to the ER. Section title: Systematic Deletions of E-Cadherin Cytoplasmic Domain Result in Retarded Exit from ER and Random Delivery of GP2/Cadherin Chimera to the Apical and Basal-lateral Cell Surface Educational score: 4.324110507965088 Domain: biomedical Document type: Study Language: en To confirm biochemically that GP2CAD1 deletion mutants were retained in the ER, we examined the sensitivity of GP2CAD10 to digestion by endoglycosidase H (endo H). GP2CAD10 MDCK cells were incubated with 35 S- Met/Cys for 24 h to label proteins to steady state. Total cell lysates were prepared, and GP2CAD10 was immunoprecipitated with anti-GP2 antibody and then subjected to endo H digestion. As shown in Fig. 6 , endo H digestion resulted in a significant increase in electrophoretic mobility of the major GP2CAD10 protein band. Note that the electrophoretic mobility of a minor protein band was not affected by incubation with endo H. This upper band appears to be specific to the GP2 antibody as a minor protein with a slower electrophoretic mobility was also present in other GP2 chimeric protein immunoprecipitates. It is likely that this protein band represents a small portion (∼10%) of the chimeric protein that exited the ER and had undergone processing in the Golgi to remove the high mannose carbohydrate chain. From the data on the sensitivity of GP2CAD10 to endo H digestion, we conclude that at steady state >85% of GP2CAD10 retained high mannose carbohydrate chains and had not undergone further processing consistent with its location in the ER. Section title: Truncations of E-Cadherin Cytoplasmic Domain Result in both Reduced Cell Surface Delivery and Random Sorting to Plasma Membrane Domains Educational score: 4.15383243560791 Domain: biomedical Document type: Study Language: en The difference in staining patterns of GP2CAD1 and GP2CAD10 might reflect differences in the efficiency of protein passage through the secretory pathway. Therefore, we compared the kinetics of cell surface delivery of newly synthesized GP2CAD1, GP2CAD10, and endogenous E-cadherin . MDCK cells stably expressing GP2CAD1 or GP2CAD10 were grown on Transwell™ filters for 7 d, pulse labeled with 35 S-Met/Cys for 20 min, and chased in DMEM containing an excess of nonradioactive Met/Cys for 40 min. At the end of the chase period, either whole cell lysates were collected, or cells were treated with the plasma membrane-impermeant biotinylating agent NHS-SS-biotin before lysis. Cell lysates were immunoprecipitated with antibody specific to rat GP2; biotinylated proteins were further precipitated with immobilized avidin. Identical experiments were performed with parental MDCK cells to examine the kinetics of endogenous E-cadherin delivery to the plasma membrane. Section title: Truncations of E-Cadherin Cytoplasmic Domain Result in both Reduced Cell Surface Delivery and Random Sorting to Plasma Membrane Domains Educational score: 4.150740146636963 Domain: biomedical Document type: Study Language: en As shown in Fig. 7 A, 25.9 ± 1.3% of newly synthesized endogenous E-cadherin, 23.0 ± 1.8% of GP2CAD1, and 6.8 ± 3.1% of GP2CAD10 reached the cell surface. Note that the percentage of protein detected by cell surface biotinylation represented only the fraction of newly synthesized protein delivered from the ER to the plasma membrane in the 40-min chase period. Thus, the amount of GP2CAD1 delivered to the cell surface was similar to that of endogenous E-cadherin, while the amount of GP2CAD10 delivered to the cell surface was considerably lower than that of either GP2CAD1 or E-cadherin. Section title: Truncations of E-Cadherin Cytoplasmic Domain Result in both Reduced Cell Surface Delivery and Random Sorting to Plasma Membrane Domains Educational score: 4.2895731925964355 Domain: biomedical Document type: Study Language: en The difference in the amount of cell surface delivery of GP2CAD10 and GP2CAD1 (or endogenous E-cadherin) was also reflected in the steady state distribution of these proteins . GP2CAD1 and GP2CAD10 expressing cells were labeled with 35 S-Met/Cys to steady state for 24 h. Parallel cultures were either extracted immediately, or biotinylated on both apical and basal-lateral membranes. Biotinylated and non-biotinylated proteins were isolated as described above. As shown in Fig. 7 B, the amount of biotinylated cell surface GP2CAD1 was ∼90% of the total GP2CAD1 extracted from cells. In contrast, the amount of biotinylated GP2CAD10 was ∼7% of the total amount of GP2CAD10. Since the extracellular domain of GP2CAD1 and GP2CAD10 were identical, the difference in the amount of cell surface exposed proteins is unlikely to be due to a difference in the efficiency of protein biotinylation. We conclude that GP2CAD1 rapidly and efficiently exits the ER and is delivered to the basal-lateral membrane. In contrast, ∼90% of GP2CAD10 is retained in the ER, while the remainder slowly leaks out into the secretory pathway and is delivered to both apical and basal-lateral membranes. Section title: Effects of aLLN and Chloroquine on GP2CAD1 and GP2CAD10 Degradation Educational score: 4.189767837524414 Domain: biomedical Document type: Study Language: en The data presented above show that nearly all of GP2CAD10 is retained in the ER, whereas nearly all GP2CAD1 is localized in the plasma membrane. It is generally acknowledged that misfolded secretory proteins retained in the ER are degraded through the proteasome pathway . To examine degradative pathways, cells were pulse-labeled with 35 S-Met/Cys for 15 min and then chased in an excess of nonradioactive Met/Cys for 225 min in the presence of either chloroquine phosphate (a lysosome inhibitor) or N-acetyl-Leu-Leu-norleucinal (aLLN, a 26S proteasome inhibitor). As shown in Fig. 8 (upper panels), 70% of newly synthesized GP2CAD1 remained at the end of the chase period in the presence of chloroquine, compared with 40% in its absence. By comparison, ∼50% of GP2CAD10 remained in the presence of chloroquine compared with 42% in its absence. In the presence of aLLN , 82% of GP2CAD1 remained compared with 50% in its absence; 83% of GP2CAD10 remained in the presence of aLLN, compared with 60% in its absence. These results show that chloroquine phosphate suppressed degradation of both GP2CAD1 and GP2CAD10 but the effect on GP2CAD1 was greater. On the other hand, aLLN had a more inhibitory effect on the degradation of GP2CAD10 than did chloroquine, while both aLLN and chloroquine suppressed the degradation of GP2CAD1. Section title: E-Cadherin Binding to β-Catenin Is Coupled to Efficient ER Exit and Basal-lateral Membrane Delivery Educational score: 4.142350673675537 Domain: biomedical Document type: Study Language: en The GP2CAD1 mutants were designed to test whether binding of β-catenin to the cytoplasmic domain of E-cadherin and basal-lateral membrane delivery could be separated from each other. GP2CAD1, GP2CAD5, GP2- CAD7, GP2CAD9, and GP2CAD10 were predicted to bind β-catenin since they contained at least aa 83–129 of the cytoplasmic domain of E-cadherin which covers the previously defined minimal β-catenin binding domain . Section title: E-Cadherin Binding to β-Catenin Is Coupled to Efficient ER Exit and Basal-lateral Membrane Delivery Educational score: 4.5092363357543945 Domain: biomedical Document type: Study Language: en The GP2CAD1/catenin complex was coimmunoprecipitated with GP2 antibody and subjected to a high stringency wash in buffer containing 0.1% SDS, 1% sodium deoxycholate, and 1 M NaCl (High Stringency Wash Buffer, see Materials and Methods). Note that under this wash condition the stoichiometry of the coimmunoprecipitated complex of GP2CAD1/β-catenin/α-catenin was ∼1: 1:1 , similar to that of the endogenous E-cadherin/ catenin complex . Surprisingly, we found that neither GP2CAD10 nor GP2CAD7 bound catenins under these conditions , although both chimeras contained the previously mapped minimal β-catenin binding domain of E-cadherin . Similar results were obtained with GP2CAD5 and GP2CAD9 (Y.-T. Chen, unpublished results). Catenins were not coimmunoprecipitated in a complex with either GP2CAD8 in which the COOH-terminal half of the β-catenin binding domain had been deleted, or GP2CAD3 in which the β-catenin binding domain had been completely deleted. Overall, there is a strong correlation between lack of β-catenin binding to the cytoplasmic domain of E-cadherin, and retention of the majority of each GP2CAD1 mutant in the ER. Note that Fig. 9 also reveals a difference in the apparent molecular mass of GP2CAD1 and all other GP2CAD mutants. It is likely that this is due to differences in complex carbohydrate modifications between the proteins. GP2 is known to undergo extensive N-linked glycosylation . We show here that GP2CAD1 is efficiently delivered to cell surface and, thereby, receives extensive carbohydrate modifications during trafficking through the secretory pathway. In contrast, all other GP2CAD mutants are retained in the ER and, therefore, do not undergo complex carbohydrate modifications. Section title: E-Cadherin Binding to β-Catenin Is Coupled to Efficient ER Exit and Basal-lateral Membrane Delivery Educational score: 4.181954860687256 Domain: biomedical Document type: Study Language: en We also examined β-catenin binding to E-cadherin mutants in which putative basal-lateral sorting motifs has been mutated (Tyr→ Ala), and an E-cadherin chimera formed with another protein, human CD7. Stable MDCK cell lines expressing E-cadherin BL1, E-cadherin BL2, or E-cadherin BL12 were labeled with 35 S-Met/Cys to steady state for 24 h, and mutant proteins were immunoprecipitated with the monoclonal antibody KT3 under the stringent wash conditions used above. All three Tyr-to-Ala mutants of E-cadherin (BL1, BL2, and BL12) bound β-catenin and α-catenin under high stringency wash conditions ; note that these mutants were rapidly exported from the ER and sorted efficiently and specifically to the basal-lateral membrane . Section title: E-Cadherin Binding to β-Catenin Is Coupled to Efficient ER Exit and Basal-lateral Membrane Delivery Educational score: 4.503418445587158 Domain: biomedical Document type: Study Language: en We placed the putative minimal β-catenin binding domain (aa 83–129, BB1) of E-cadherin cytoplasmic domain on the COOH terminus of a unrelated type-I transmembrane protein, human CD7. The resulting chimeric protein (CD7BB1) bound α-catenin and β-catenin, whereas CD7 did not . However, quantification of the amounts of α- and β-catenins bound to CD7BB1 revealed that the stoichiometry of CD7BB1/β-catenin/α-catenin was 5:1:0.8 when normalized to the number of Met and Cys residues in CD7BB1 and catenins; this compared with the ratio of ∼1:1:1 for E-cadherin/β-catenin/α-catenin or GP2CAD1/β-catenin/α-catenin (see above). Most of CD7 and CD7BB1 was localized at the cell surface (Y.-T. Chen, unpublished results); presumably, the cytoplasmic domain of CD7 in the CD7BB1 chimera contains signal(s) for its entry into the secretory pathway regardless of whether β-catenin is bound or not. Trafficking patterns of CD7 and CD7BB1 were similar in polarized MDCK cells. Approximately 60% of the cell surface delivery of both proteins was to the basal-lateral membrane (Y.-T. Chen, unpublished results). We conclude that a domain containing aa 83–129 is a transportable β-catenin binding domain in E-cadherin, but the binding strength of β-catenin/E-cadherin, based on high stringency washes, is much lower than that of β-catenin bound to the complete cytoplasmic domain of E-cadherin. Neither the BB1 domain alone nor β-catenin binding, per se, specifies protein delivery to the basal-lateral membrane. Section title: E-Cadherin Binding to β-Catenin Is Coupled to Efficient ER Exit and Basal-lateral Membrane Delivery Educational score: 4.393768787384033 Domain: biomedical Document type: Study Language: en We also expressed a truncated E-cadherin, E-cad8 , containing the complete extracellular and transmembrane domains, and cytoplasmic domain up to aa 115 (i.e., a deletion of aa 116–151 of the cytoplasmic domain), which is identical to the COOH-terminal deletion in GP2CAD8 . This deletion covers the minimal β-catenin binding site , and β-catenin is not coimmunoprecipitated in a complex with E-cad8 (data not shown) as reported previously . E-cad8 was epitope tagged at its COOH terminus with the KT3 epitope tag, which enabled us to distinguish its distribution in cells expressing endogenous E-cadherin. In confluent monolayers of MDCK cells, in which cell–cell contacts are clear , E-cad8 is not apparent at cell–cell contacts but is localized to intracellular compartments. The staining patterns comprise bright, punctate structures that are present, although less abundant in cells expressing GP2CAD10 , and a diffuse cytoplasmic staining also similar to that of GP2CAD10 . The extracellular and membrane proximal cytoplasmic domains of cadherin have been proposed to be involved in dimer formation . To test whether E-cad8 interacted with endogenous E-cadherin, we separated proteins extracted from MDCK cells expressing E-cad8 using a Superose 6 size exclusion column. Endogenous E-cadherin eluted in fractions 13–16 , as shown previously . However, E-cad8 eluted in fractions 16–19 in a profile distinctively different from that of endogenous E-cadherin . Note that E-cad8 has an apparent molecular mass smaller than that of full-length E-cadherin, which is consistent with the cytoplasmic domain deletion. A protein band with an apparent molecular mass greater than that of the full-length E-cadherin probably represents the precursor form of E-cad8 . We also found an enrichment of higher molecular mass precursor forms of three other E-cadherin cytoplasmic domain deletion mutants when expressed in MDCK cells (data not shown). Section title: Discussion Educational score: 4.0891313552856445 Domain: biomedical Document type: Study Language: en How membrane proteins are sorted and delivered to different plasma membrane domains in polarized epithelial cells is an important question. However, to date most studies have focused on single proteins that do not form a complex with subunit membrane or cytosolic proteins during their transit through the secretory pathway. Although these studies have identified several types of intrinsic basal-lateral sorting signals in the cytoplasmic domain of a few proteins , it is not known whether they can be generalized to transmembrane proteins with heterologous binding partners. Section title: Discussion Educational score: 4.4730143547058105 Domain: biomedical Document type: Study Language: en E-cadherin is different from other type-I single-span transmembrane protein that have been used to study sorting signals, since it forms a 1:1:1 stoichiometric complex with two cytosolic proteins, β-catenin and α-catenin . Significantly, previous studies implied that assembly of the E-cadherin/β-catenin complex occurs in the very early stage of trafficking . Therefore, E-cadherin must be sorted to the basal-lateral membrane in a complex with β-catenin. Amino acid sequences in the cytoplasmic domain of E-cadherin that are superficially similar to recently described ER exit and basal-lateral sorting signals in other proteins do not have these functions in E-cadherin. Instead, assembly of the E-cadherin/β-catenin complex is the obligatory step in efficient export from the ER, and subsequent delivery of E-cadherin to the basal-lateral membrane of polarized MDCK cells. Section title: Defining the β-Catenin Binding Site in E-Cadherin Educational score: 4.276461124420166 Domain: biomedical Document type: Study Language: en To examine domains of E-cadherin important in its sorting to the basal-lateral membrane domain, we used a chimeric protein approach. The chimera was composed of the extracellular domain of GP2, which is normally attached to the membrane through a GPI-anchor, fused to the transmembrane and cytoplasmic domains of E-cadherin (GP2- CAD1). The extracellular domain of GP2, when expressed without a membrane anchor, was secreted equally from both apical and basal-lateral membranes, showing that this domain does not contain specific membrane sorting signals. Using a similar approach, we showed that the extracellular domain of E-cadherin does not contain signals that specify sorting to the basal-lateral membrane. The chimeric protein GP2CAD1, however, was delivered directly to the basal-lateral membrane domain with a rate, efficiency and fidelity similar to that of endogenous E-cadherin. Section title: Defining the β-Catenin Binding Site in E-Cadherin Educational score: 4.212541580200195 Domain: biomedical Document type: Study Language: en GP2CAD1 also formed a complex with α- and β-catenin with a stoichiometry of ∼1:1:1 that was resistant to disruption with a high stringency buffer containing 0.1% SDS, 1% deoxycholate and 1 M NaCl, similar to E-cadherin. The design of truncation mutants of GP2CAD1 used to examine sorting signals in the cytoplasmic domain of E-cadherin took into account a previously defined minimal β-catenin binding domain that had been mapped to a region of 30 aa, corresponding to aa 100–129 . However, we found that β-catenin did not bind, under these stringent conditions, to a slightly longer region containing this minimal β-catenin binding domain, aa 83–129, if regions of the cytoplasmic domain around it had been deleted (e.g., GP2CAD5, 7, 9, and 10). Section title: Defining the β-Catenin Binding Site in E-Cadherin Educational score: 4.510707378387451 Domain: biomedical Document type: Study Language: en We tested whether this difference between our results and those of Stappert and Kemler reflected the difference in the cell types used to express these mutants (L cells, MDCK cells). GP2CAD1, 6, 7, 8, and 10 were transiently transfected into L cells and HEK293 cells. By immunofluorescence in both cell types, GP2CAD1 was localized to the cell surface, whereas GP2CAD6, 7, 8, and 10 were predominantly localized to the ER (Y.-T. Chen, unpublished results), similar to their locations in MDCK cells . Another reason for the difference in results might be the methods used to assess the stoichiometry of β-catenin binding to this minimal binding site on E-cadherin. However, Stappert and Kemler , did not quantitate the stoichiometry of the complex formed between β-catenin and the minimal β-catenin binding domain (aa 100–129). We found that β-catenin bound to a chimeric protein composed of CD7 and aa 83–129 of the E-cadherin cytoplasmic domain (CD7BB1), but the stoichiometry of the coimmunoprecipitated complex was ∼5:1 after a high stringency wash. Recently, Finnemann et al. reported the expression of three COOH-terminal deletion mutants of Xenopus XB/U-cadherin in L cells. A deletion of the most COOH-terminal 19 amino acids, which would contain the minimal β-catenin binding domain intact, bound β-catenin but the adhesion function of the truncated cadherin was interpreted to be “dramatically altered.” We note that biochemical data presented in the same paper revealed a decrease in the avidity of β-catenin binding to the mutant cadherin. Interestingly, confocal microscopy of L cells expressing XB/U-cadherin revealed that full-length protein localized to cell–cell contacts, whereas the truncation mutant had a distinctly different location and appeared to have a punctate distribution. In conclusion, it seems likely that β-catenin binds to a minimal domain of E-cadherin comprising aa 100–129, but the interaction is considerably weaker than binding of β-catenin to the whole cytoplasmic domain of E-cadherin. Reduced binding of the cytoplasmic domain of cadherin to β-catenin leads to intracellular retention and reduced cell surface expression of cadherin. Section title: Defining the β-Catenin Binding Site in E-Cadherin Educational score: 4.444347858428955 Domain: biomedical Document type: Study Language: en The cadherin-binding domain in β-catenin lies within the armadillo (arm) repeats of β-catenin . The crystal structure of the arm repeats revealed a rigid cylindrical super-helix of helices that form a groove lined with positively-charged amino acids . The length of this groove could potentially accommodate a fully extended peptide of aa 25–30, or a longer peptide with local secondary structures; note that the minimal β-catenin binding site on E-cadherin is 29 aa and has a pI of 3.1 . Although structural analysis of the E-cadherin/β-catenin cocrystal is required to define the interaction in detail, our recent analysis has revealed that the cytoplasmic domain of E-cadherin is protected from proteolysis when it has formed a 1:1 stoichiometric complex with the β-catenin arm repeats (Huber, A., D.B. Stewart, W.J. Nelson, and W. Weis, in preparation). This result is compatible with the view formed from the present studies of β-catenin binding to GP2CAD1 deletion mutants in which most of the cytoplasmic domain of E-cadherin, including the previously mapped minimal β-catenin binding domain, interacts with β-catenin. Section title: β-Catenin/E-Cadherin Binding Is Coupled to Efficient Exit of E-Cadherin from the ER Educational score: 4.291259765625 Domain: biomedical Document type: Study Language: en Using a combination of results obtained by cell surface biotinylation, sensitivity to endoglycosidase H digestion, immunoprecipitation, and immunofluorescence we have shown that GP2CAD1 bound β-catenin and >90% entered the secretory pathway to the basal-lateral membrane. However, GP2CAD1 deletion mutants did not bind α- or β-catenin and >90% remained in the ER. Previous studies indicated that newly synthesized E-cadherin binds β-catenin soon after its synthesis, and that α-catenin binds to the complex when E-cadherin arrives at the cell surface . Thus, formation of the E-cadherin/β-catenin complex correlates with efficient exit of E-cadherin from the ER. Section title: β-Catenin/E-Cadherin Binding Is Coupled to Efficient Exit of E-Cadherin from the ER Educational score: 4.655076503753662 Domain: biomedical Document type: Study Language: en It is possible that E-cadherin has a specific ER export signal that is deleted from the GP2CAD1 mutants. Recently, it was reported that the COOH-terminal acidic residues of VSV-G protein are involved in efficient exit of VSV-G protein from ER , and that deletion of the most COOH-terminal two valine residues of proTGFα resulted in the ER accumulation of the protein . It has been suggested that COOH-terminal acidic amino acid residues constitute an ER export signal . In this regard, it is striking that our results showed that a deletion of the COOH-terminal three acidic amino acids of GP2- CAD1 resulted in the accumulation of the protein (GP2- CAD10) in the ER. However, our data are not consistent with the possibility that these, or other acidic amino acids at the COOH terminus constitute a minimal ER exit signal. Internal deletions of the cytoplasmic domain of E-cadherin (e.g., GP2CAD3) resulted in retention of the protein in the ER, even though COOH-terminal acidic amino acids were intact. In general, we cannot separate the roles of different regions/motifs as ER export signals from the dominant requirement for high affinity binding of β-catenin to the whole cytoplasmic domain of E-cadherin. It seems more likely that the complex of E-cadherin/β-catenin presents a conformation or motif in the cytoplasmic domain of E-cadherin that allows efficient export of the complex from the ER. Definitive proof of this mechanism will require the expression of E-cadherin in a cell line lacking both β-catenin and plakoglobin. ER retention of GP2CAD1 proteins containing a truncated cytoplasmic domain is likely to be a consequence of missfolding, due to inhibition of β-catenin binding . These misfolded proteins are targeted subsequently for degradation in either lysosomes or the 26S proteasome . Finally, our analysis of the distribution of the simple deletion mutant E-cad8 , which was not a chimeric protein, showed that this mutant was also localized intracellularly. E-cad8 did not form a complex with endogenous E-cadherin despite an intact extracellular and membrane proximal cytoplasmic domain, which have been identified as potential sites for dimer formation . Thus, E-cadherin dimerization may not occur in the absence of β-catenin binding, or is dependent on E-cadherin leaving the ER or arriving at the plasma membrane. Section title: β-Catenin Binding to E-Cadherin and Delivery of E-Cadherin to Sites of Function in the Basal-lateral Membrane Domain of the Cell Surface Educational score: 4.458339691162109 Domain: biomedical Document type: Study Language: en Previous studies have emphasized that sorting of transmembrane proteins to either the apical or basal-lateral membrane occurs in the TGN . For example, early studies showed that influenza virus HA and VSV-G protein appeared together in vesicles and membrane compartments until the TGN where they appeared to be segregated into separate vesicle populations . Our data show that complex formation between E-cadherin cytoplasmic domain and β-catenin in the ER is not only coupled to efficient export of E-cadherin but also direct delivery of E-cadherin to the basal-lateral membrane. Note that in the absence of complex formation with β-catenin, a small amount (<10%) of GP2CAD1 deletion mutants leaked out of the ER and were then delivered randomly to both the apical and basal-lateral membranes. As in the case of ER export, we suggest that binding of β-catenin to E-cadherin induces a conformation state in the cytoplasmic domain of E-cadherin that reveals a sorting determinant(s) that is recognized by the basal-lateral sorting machinery. Thus, β-catenin may function as a chauffeur to facilitate E-cadherin transport through the secretory pathway. At present, we cannot rule out an alternative possibility that β-catenin itself contains basal-lateral membrane targeting information, and that this information is recognized by the sorting machinery for delivery of the E-cadherin/β-catenin complex to the basal-lateral membrane. Section title: β-Catenin Binding to E-Cadherin and Delivery of E-Cadherin to Sites of Function in the Basal-lateral Membrane Domain of the Cell Surface Educational score: 4.705269813537598 Domain: biomedical Document type: Study Language: en That binding of β-catenin to the cytoplasmic domain of E-cadherin plays an essential role in the efficient delivery of E-cadherin to the cell surface raises the interesting possibility that ER exit is a potential regulatory point for E-cadherin function. For example, limited availability of β-catenin for binding to E-cadherin, or post-translational modifications to β-catenin that reduce the affinity of E-cadherin/β-catenin binding might lead to the retention of un-complexed E-cadherin in the ER. Since the half-life of E-cadherin is quite short , retention of newly synthesized E-cadherin in the ER would quickly result in reduced cell surface expression of E-cadherin and, consequently, reduced cell–cell adhesion. We note that β-catenin is a component of the Wnt/Wingless signaling pathway in which regulation of β-catenin levels is critical . Significantly, there is evidence that changes in β-catenin levels leads to changes in steady state and cell surface levels of cadherins, and increased cadherin-mediated cell–cell adhesion . These effects on cadherin could be mediated by the availability of β-catenin for binding to cadherin in the ER, which regulates cadherin entry into the secretory pathway and, therefore, the amount of cadherin available for adhesion functions at the cell surface. | Other | biomedical | en | 0.999994 |
10037791 | Section title: Anti–cytochrome c mAbs Educational score: 3.7252068519592285 Domain: biomedical Document type: Study Language: en The mouse anti–rat cytochrome c mAbs used in this study have been described . They were purified from ascites by affinity chromatography using rabbit cytochrome c–coupled Sepharose beads as in Urbanski and Margoliash . Cytochrome c ( Sigma Chemical Co. ) was coupled to cyanogen bromide–activated Sepharose 4B ( Sigma Chemical Co. ). Section title: Anti–cytochrome c mAb Staining on Ovaries Educational score: 4.189099311828613 Domain: biomedical Document type: Study Language: en Ovaries were dissected into 1× PBS. Tips of the ovariole were separated from one another to facilitate adequate penetration of the fixative and mAbs. Fixation was done by agitating the ovaries in a mixture of 1 vol 4% paraformaldehyde in 1× PBT (1× PBS + 0.1% Tween 20) and 3 vol of heptane for 30 min at room temperature. Samples were then washed four times in 1× PBT for 10 min each, then preincubated for 1 h in 1× PBTB (PBT + 1.5% BSA) at room temperature. The ovaries were incubated overnight at 4°C with the mouse anti–cytochrome c mAb (22 μg/ml) in 1× PBTB, washed four times in 1× PBTB followed by a 2-h incubation in 1:200 dilution of goat anti–mouse IgG-FITC (Jackson ImmunoResearch Laboratories) in 1× PBTB, washed three times in 1× PBT, and mounted in 50% glycerol in 1× PBS. Section title: Terminal Transferase Method (TUNEL) for Detection of Apoptotic Nuclei Educational score: 3.454226016998291 Domain: biomedical Document type: Study Language: en Hand-dissected ovaries were fixed as before and washed four times in 1× PBT. Section title: Terminal Transferase Reaction. Educational score: 4.058500289916992 Domain: biomedical Document type: Study Language: en Buffer (1× TdT) ( Boehringer Mannheim Corp. ) was mixed with 2.5 mM COCl 2 , 60 μM biotin-16dUTP, 200 nM dNTPs, and 15 U TdT to a final reaction volume of 50 μl. The ovaries were incubated in this mixture for 3 h at 37°C, rinsed and washed four times, 10 min each in 1× PBT. Section title: Detection. Educational score: 3.9742112159729004 Domain: biomedical Document type: Study Language: en The ovaries were preincubated in 1× PBTB for 1 h at room temperature. Incorporated biotin-dUTP was detected by incubation in streptavidin-TRITC (Jackson ImmunoResearch Laboratories) in 1× PBTB (1:1,000) at room temperature in the dark for 2 h. Ovaries were mounted in 50% glycerol in 1× PBS and viewed under the rhodamine channel. Section title: Rhodamine-phalloidin Staining Educational score: 3.741697072982788 Domain: biomedical Document type: Other Language: en Rhodamine-phalloidin (Molecular Probes Inc.) was diluted (1:200) in 1× PBTB. Egg chambers were incubated in the solution for 20 min at room temperature in the dark, and then rinsed in 1× PBT. They were then mounted in 50% glycerol/50% 1× PBS. Section title: Cell Culture and Treatments Educational score: 4.119280815124512 Domain: biomedical Document type: Study Language: en Mt-rpr and Mt-grim are expression vectors that permit conditional expression of reaper or grim , respectively, in cell culture . The genes are placed downstream of the Drosophila metallothionein promoter and conditional expression in Schneider L2 (SL2) cells can be induced either in stably or transiently transfected cells. After transfection of Mt-rpr and Mt-grim, alone or in combination with the pMt-p35 plasmid , induction was achieved by exposing the cells to 700 μM CuSO 4 . Section title: Cell Culture and Treatments Educational score: 4.0799126625061035 Domain: biomedical Document type: Study Language: en Transient expression assays were done as in Chen et al. and Nordstrom et al. . Typical transfection efficiencies ranged from 40 to 60%. 48 h after transfection, cells from each well were split into two wells, and copper was added to one of the two wells. For ceramide treatment, SL2 cells were plated at 1 million/ml and incubated with 25 μM C2-ceramide (Biomol) for up to 24 h before being processed for antibody staining. Under these conditions, ∼50% cells were killed as measured by trypan blue exclusion . Section title: Construction of pMt-(Δ1–33)-dcp-1 Educational score: 4.150653839111328 Domain: biomedical Document type: Study Language: en A forward primer (5′ATCAGGGAGCTCGGATCCATATGGCCAAGGGCTGTACGCCG3′) and a reverse primer (5′GGGGTACCGTCGACTAATGATGATGATGATGATGGGATCCGCCAGCCTTATTGCCGTTCGG3′) were used together with a dcp-1 cDNA to produce a PCR product. This fragment was gel purified, digested with the appropriate enzymes (Sac1 and Kpn1), and then ligated to a Sac1/Kpn1 digested pRmHa.3 vector . The resulting plasmid, pMt-(Δ1– 33)-dcp-1, expresses a truncated version of dcp-1 that is deleted for the prodomain (residues 1–33). Transient transfection was done as described above. Section title: Anti–cytochrome c mAb Staining of Cultured Cells Educational score: 4.174856185913086 Domain: biomedical Document type: Study Language: en Cells were plated at 25°C on coverslips, at a density of 10 6 cells/ml, and the following day apoptosis was induced. After an appropriate time of incubation in CuSO 4 (∼6 h for rpr -transfected cells and ∼2.5 h for grim -transfected cells), cells were washed with 0.1 M sodium phosphate buffer (pH 7.5). Cells were then fixed at room temperature for 20 min with 3% paraformaldehyde in PBS containing 3 mM each of trinitrophenol, KCl, and MgCl 2 . Cells were then washed in PBS and incubated in 100 mM NH 4 Cl in PBS for 10 min. After washing three times with PBS for 5 min each, cells were permeabilized with 0.1% Triton X-100 for 5 min at room temperature. Section title: Anti–cytochrome c mAb Staining of Cultured Cells Educational score: 4.082285404205322 Domain: biomedical Document type: Study Language: en Cells were then sequentially incubated with 0.8% BSA ( Sigma Chemical Co. ) at room temperature for 30 min, anti–cytochrome c mAb (22 μg/ml in 0.8% BSA) overnight at 4°C, and finally with 20 μg/ml goat anti–mouse Ig G-FITC (Jackson ImmunoResearch Laboratories) for 1 h at room temperature. Cells were washed twice with 0.4% BSA, twice with 0.1 M sodium phosphate buffer, and once in distilled water and mounted in fluoromount-G ( Fisher Scientific Co. ) containing 2.5% 1,4-diazabicyclo- (2.2.2)octane (DABCO; Sigma Chemical Co. ). Photomicrographs were recorded on conventional and confocal microscopes using appropriate filters. Section title: Rhodamine 123 Staining Educational score: 4.056087493896484 Domain: biomedical Document type: Study Language: en Cells stably transfected with pMt-grim were induced for apoptosis as described above. After 2.5 h, samples were incubated in 50 nM rhodamine 123 for 15 min at 37°C to label mitochondria . Cells were then rinsed twice in 1× PBS, fixed, and stained as described above. Section title: Use of Caspase Inhibitors Educational score: 4.009475231170654 Domain: biomedical Document type: Study Language: en Cells transfected stably with pMt-rpr and pMt-grim were treated with 50 μM each of caspase inhibitors DEVD-fmk and ZVAD-fmk (both purchased from Calbiochem ). After appropriate times, induced and control cells were stained with anti–cytochrome c mAb as described before. Section title: Immunoprecipitation Educational score: 4.145272731781006 Domain: biomedical Document type: Study Language: en Immunoprecipitation was performed using protein A/G PLUS–Agarose ( Santa Cruz Biotechnology ) according to the manufacturer's instructions with a few modifications. Briefly, SL2 cells and apoptotic grim -expressing cells were pelleted, and lysed in lysis buffer (25 mM Hepes-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl 2 , 1 mM sodium EDTA, 1 mM sodium EGTA, 1 mM dithiothreitol, 5% glycerol, 0.1% Triton X-100, supplemented with 5 μg/ml pepstatin, 10 μg/ml leupeptin, 2 μg/ml aprotinin, 0.1 mM PMSF) on ice for 30 min at a protein concentration of 0.5 mg/ml. The lysates were centrifuged at 3,000 g for 10 min, and 1 ml supernatant from each sample was precleared by incubating with 2 μg normal mouse IgG and 60 μl agarose conjugate for 30 min. Then 0.5 ml of precleared lysates was incubated with 2 μg anti–cytochrome c mAb for 2 h before 30 μl of agarose beads was added, and rotated at 4°C overnight. The agarose beads were washed three times with lysis buffer, resuspended in 30 μl 2× SDS sample buffer, resolved on a 15% polyacrylamide gel, transferred to PVDF membrane, and probed with anti–cytochrome c mAb, clone 7H8.2C12. Section title: Subcellular Fractionation and Caspase Activity Assay Educational score: 4.161362648010254 Domain: biomedical Document type: Study Language: en Subcellular fractionation was done as described . 8 × 10 7 cells were plated and, after 18 h, induced with copper as above. Cells were harvested 4 h later by centrifugation at 3,800 g for 5 min at room temperature. The pellets were washed once with ice-cold PBS and resuspended with 5 vol of buffer A (20 mM Hepes-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl 2 , 1 mM sodium EDTA, 1 mM sodium EGTA, 1 mM dithiothreitol, supplemented with 5 μg/ml pepstatin, 10 μg/ml leupeptin, 2 μg/ ml aprotinin, 0.1 mM PMSF) containing 250 mM sucrose for 15 min. Then cells were homogenized with 10 strokes in a Teflon homogenizer, and the homogenates were spun twice at 750 g for 10 min at 4°C. The supernatants (referred to as total cell lysates) were centrifuged at 10,000 g for 15 min at 4°C, and the resulting heavy membrane pellets were washed three times with cold buffer A containing sucrose, resuspended in the same buffer and stored at −80°C. The supernatants of the 10,000 spin were further centrifuged at 100,000 g for 1 h at 4°C, and the resulting supernatants (S-100) were aliquoted and stored at −80°C until use. Section title: Subcellular Fractionation and Caspase Activity Assay Educational score: 4.159258842468262 Domain: biomedical Document type: Study Language: en 16 μg of S-100 cytosol from indicated cells was incubated with 150 ng of poly ADP-ribose polymerase (PARP) (Biomol) in a total volume of 25 μl in buffer A containing 250 mM sucrose for 1 h at room temperature. The reactions were stopped by adding 10 μl of 4× SDS sample buffer. PARP was resolved on an 8% SDS polyacrylamide gel, transferred to a PVDF membrane, and probed with an anti-PARP mAb (C-2-10 from Biomol). For mixing experiments, 20 μg of heavy membrane fractions from indicated cells were incubated with SL2 cytosol for 1 h at room temperature, then the heavy membrane fractions were spun down, and the supernatants were used for PARP cleavage activity as above. To ensure that caspase activation associated with mitochondrial fractions did not result from contaminating cytosolic components, heavy membranes were washed in a 20-fold excess volume of buffer A (with sucrose) at least five times. Also, in parallel experiments, we found that mitochondrial fractions from pre-apoptotic cells retained activity even after exposure to 50 μM irreversible caspase-inhibitor peptides (DEVD-fmk or zVAD-fmk) before washing. Section title: Immunodetection of Drosophila Cytochrome c Educational score: 4.16752815246582 Domain: biomedical Document type: Study Language: en Although two cytochrome c genes, DC4 and DC3, have been described in Drosophila melanogaster studies at the level of protein and at the level of RNA suggest that DC4, which shows >86% identity with its rat counterpart, is either the predominant or only form of actively expressed product. We screened an existing panel of mAbs, directed against mammalian versions of cytochrome c, as possible probes for in situ analyses of the fly counterpart. The potential utility of these mAbs was assessed by immunoprecipitations of SL2 cell lysates, probed with a third anti–cytochrome c mAb, 7H8, to detect denatured cytochrome c in Western blots. The results in Fig. 1 show that two mAbs, 6H2 and 2G8 , recognized Drosophila cytochrome c. Both antibodies detected a doublet that comigrated with mammalian cytochrome c at ∼13 kD. While mAb 2G8 preferentially precipitated the upper band, mAb 6H2 had about equal affinity for both forms of cytochrome c. No obvious correlation between the relative abundance of the two cytochrome c bands and apoptosis was observed (see below). The same banding pattern was also observed when immunoprecipitation was performed using purified Drosophila cytochrome c (Liu, J., and R. Jemmerson, unpublished observation). Section title: Immunodetection of Drosophila Cytochrome c Educational score: 4.208371162414551 Domain: biomedical Document type: Study Language: en These bands clearly represent distinct cytochrome c species since they were immunoprecipitated with mAbs to the native protein, and were detected by Western blot using a different mAb that recognizes a distinct epitope on the denatured protein. While it is formally possible that these two bands correspond to the polypeptides encoded by the Drosophila cytochrome c genes, DC-3 and DC-4, earlier studies suggest that only DC-4 encodes a functional polypeptide and furthermore, the expected Drosophila cytochrome c products (DC-3 and DC-4) differ by only three amino acids, which is not likely to be resolved in our gels. Although the biochemical nature of these different forms is unresolved, our data indicate that mAb 2G8 preferentially recognized the higher molecular weight form of the doublet. This product occurs in relatively small amounts that were not overtly affected by the extent of apoptosis in the cultures . Section title: Altered Cytochrome c Anticipates Programmed Cell Death Educational score: 4.021749973297119 Domain: biomedical Document type: Study Language: en Between stages 11 and 13 of Drosophila oogenesis, programmed cell death eliminates nurse cells which nourish the developing egg . The apoptotic nature of nurse cell death is indicated by two distinct markers, acridine orange staining and TUNEL labeling . These readily identifiable cells offer a unique opportunity to examine pre-apoptotic events before their eventual demise. Section title: Altered Cytochrome c Anticipates Programmed Cell Death Educational score: 4.177212715148926 Domain: biomedical Document type: Study Language: en To directly demonstrate the involvement of cytochrome c during apoptosis, Drosophila ovaries were stained with the anti–cytochrome c mAbs described above and egg chambers at all stages of development were analyzed. In preliminary experiments, mAb 2G8 detected cytochrome c in apoptotic cells prompting extensive studies with this mAb. As shown in Fig. 2 , A and B, only nurse cells at stage 10B exhibited pronounced cytochrome c immunoreactivity distributed as characteristically punctate labeling of the cytoplasm in a pattern consistent with localization to mitochondria . Such staining was not seen in nurse cells before this stage, nor was this overt immunoreactivity seen in any other cell type of the Drosophila egg chamber. Moreover, this staining was specific for cytochrome c immunoreactivity because no labeling of nurse cells was observed if the antibody was first preadsorbed with cytochrome c covalently bound to Sepharose 4B . Section title: Altered Cytochrome c Anticipates Programmed Cell Death Educational score: 4.259050369262695 Domain: biomedical Document type: Study Language: en To determine the chronology of cytochrome c display relative to other apoptotic changes, we compared the onset of mAb binding with other degenerative changes known to occur in these cells. Though nurse cells at stage 10B showed pronounced exposure of the epitope for mAb 2G8 no signs of apoptosis were apparent in either the cytoplasm or the nuclei of these cells . Egg chambers were stained with rhodamine-conjugated phalloidin which detects filamentous actin at the cortices of the nurse cells. As shown in Fig. 2 D, this marker substantiates the integrity of the cytoplasmic membrane and shows that nurse cell dumping has not yet begun at a time when cytochrome c labeling is already very conspicuous. Similarly the nuclei of nurse cells at stage 10B were negative for TUNEL labeling , a method that detects a nuclear hallmark of apoptosis. By stages 12–13 (at least 0.5 to 4 h later) the nuclei of the dying nurse cells adopt characteristic apoptotic features as evidenced by the TUNEL assay and acridine orange staining (data not shown). These results demonstrate that cytochrome c display precedes overt signs of apoptosis in intact organs. Section title: Altered Cytochrome c Is Provoked by Apoptosis Activators Educational score: 4.151803970336914 Domain: biomedical Document type: Study Language: en Previous studies on Drosophila SL2 cells have shown that conditional expression of rpr or grim triggers apoptosis in cultured cells and in transgenic animals . Transiently transfected SL2 cells were induced for rpr or grim and, at various time intervals after induction, the preparations were examined for cytochrome c immunoreactivity with mAb 2G8. As seen in Fig. 3 , apoptotic cultures exhibited profound staining with the antibody. In contrast, uninduced healthy cells and cells transfected with the vector (pMt) alone in the absence or presence of the inducing agent copper showed no signs of cytochrome c display. As before, mAb 2G8 preadsorbed with cytochrome c–Sepharose 4B validated specificity of immunoreactivity for cytochrome c. It should also be emphasized that routine labeling of apoptotic SL2 cells (or egg chambers) with a variety of antibodies directed against different antigens does not behave like mAb 2G8, and we can readily exclude the possibility that the pre-apoptotic condition somehow promotes enhanced immunodetection. Section title: Cytochrome c Is Retained in Mitochondria during Apoptosis Educational score: 4.160761833190918 Domain: biomedical Document type: Study Language: en To test the possibility that cytochrome c might be released into the cytosol during apoptosis, we fractionated healthy SL2 cells and apoptotic rpr - or grim -expressing cells, and assayed for cytochrome c in the mitochondrial (heavy membrane) and cytosolic fractions. Surprisingly, these cells showed no difference in cytochrome c distribution and we found no evidence for the transit of cytochrome c to the cytosol as a correlate to apoptosis . It is worth mentioning that our experimental design is set up such that equal numbers of cells are plated ∼18 h before induction. Therefore, differences between parental and transfected samples in the levels of cytochrome c (in total and heavy membrane samples) result solely from differences in growth rate and cell number at the time of harvest . Note that reduced levels of immunogen detected are proportional to the reduced total levels of immunogen . In addition, we should emphasize that nothing about our procedure would have prevented us from observing a genuine change in the subcellular location of this protein because in several preparations we could, in fact, cause cytochrome c to be released and detected in the cytosol simply by omitting sucrose from the preparation (see Materials and Methods). Section title: Cytochrome c Is Retained in Mitochondria during Apoptosis Educational score: 4.19338321685791 Domain: biomedical Document type: Study Language: en Biochemical data indicating retention of cytochrome c in mitochondria during apoptosis is consistent with our cytological studies. For instance, under all circumstances the protein was compartmentalized in a punctate distribution which, in double labeling experiments, was coincident with the mitochondrial marker, rhodamine 123 . These observations, together with our biochemical data, indicate that appreciable efflux of cytochrome c from mitochondria does not occur during apoptosis in Drosophila cells. Section title: Mitochondria Isolated from Apoptotic Cells Trigger Caspase Activation In Vitro Educational score: 4.2951250076293945 Domain: biomedical Document type: Study Language: en Studies described in the previous section established that, although clearly altered, Drosophila cytochrome c is retained in the mitochondrial compartment during apoptosis. Since cell-free studies in vertebrate systems implicate mitochondrial factors (cytochrome c and/or other proteins) in the activation of some caspase enzymes we sought to determine whether mitochondria from our insect model might exhibit similar properties. To test this possibility, we measured caspase activation in L2 cell cytosol that had been coincubated with mitochondria isolated from parental L2 cells or from pre-apoptotic cells (induced either for rpr or grim ). Fig. 5 illustrates detection of caspase activation, as measured by signature cleavage of a bovine substrate, PARP. Cleavage of PARP in this assay is indistinguishable from the signature activity reported in many mammalian systems and is readily detected in the cytosol of pre-apoptotic cells but not in cytosol from parental L2 . Lanes 5 and 6 of Fig. 5 show that mitochondria isolated from rpr - or grim -expressing cells trigger the appearance of PARP cleavage activity in the otherwise silent L2 cell cytosol. In contrast, mitochondria from parental L2 cells did not provoke similar cleavage of PARP . These observations emphasize the importance of one or more mitochondrial factors in the activation of caspase function triggered by rpr or grim. Section title: Altered Cytochrome c Display Is Blocked by Caspase Inhibitors In Vivo Educational score: 4.207821369171143 Domain: biomedical Document type: Study Language: en The Drosophila death activators, rpr and grim , activate one or more caspases to elicit apoptosis . To study the temporal relation of cytochrome c display with respect to caspase activity, we cotransfected SL2 cells with pMt-rpr and pMt-p35. 6 h after induction, cells induced for rpr alone showed pronounced labeling with mAb 2G8 whereas cells expressing rpr together with p35 were prevented from apoptosis and did not bind the mAb . These observations suggested that apoptogenic cytochrome c display required caspase activity, a presumption that was further substantiated when rpr -expressing cells were treated with the peptide caspase inhibitors zDEVD-fmk and zVAD-fmk. As seen for p35-blocked cells, these inhibitors similarly prevented mAb 2G8 labeling and subsequent apoptosis . Parallel results were observed in grim -expressing cells (not shown). Section title: Altered Cytochrome c Display Is Provoked by the Drosophila Caspase, dcp-1 Educational score: 4.196847915649414 Domain: biomedical Document type: Study Language: en The data above demonstrate that caspase activity is required for apoptogenic cytochrome c display. To determine if caspase function is sufficient to trigger this change, we induced apoptosis in SL2 cells by conditional expression of an activated version of the Drosophila caspase, dcp-1. If deleted for its prodomain, this caspase provokes considerable apoptosis in mammalian cells and SL2 cells (this paper). When labeled with mAb 2G8, cells transfected and induced for pMt-(Δ1–33)-dcp-1 expression exhibited profound punctate cytochrome c staining with features indistinguishable from those associated with expression of the death activators. Section title: Discussion Educational score: 4.408780097961426 Domain: biomedical Document type: Study Language: en Several compelling lines of evidence implicate a role for cytochrome c in apoptotic physiology. First, release of cytochrome c from mitochondria occurs as an early response to a variety of death stimuli, and this release is prevented by overexpression of bcl-2 . Second, cytochrome c triggers activation of caspases in several in vitro models . Third, direct injection of cytochrome c into cultured cells promotes death by apoptosis . Finally, reconstitution of a purified caspase-3 activation complex was found to require cytochrome c as an obligate binding factor . Section title: Discussion Educational score: 4.235471725463867 Domain: biomedical Document type: Study Language: en We examined the status of cytochrome c during programmed cell death in an animal model. As part of this effort, we characterized mAbs directed against rat cytochrome c for binding Drosophila cytochrome c in situ. One mAb, 2G8, was of particular interest because it exhibited specific immunoreactivity to cytochrome c only in the mitochondria of cells that are committed to undergo apoptosis. For instance, pronounced labeling for cytochrome c was observed in mitochondria of cells expressing the apoptosis activators, rpr or grim , whereas healthy control cells were not labeled. Similar results were observed in intact ovarian tissues where cytochrome c display occurred specifically in nurse cells at a stage before any overt sign of apoptosis. To our knowledge, apoptogenic cytochrome c is the earliest reported molecular indicator of pre-apoptotic events in these cells and could reflect the point at which nurse cells become irreversibly committed to programmed cell death. Section title: Discussion Educational score: 4.277441501617432 Domain: biomedical Document type: Study Language: en Although mAb 2G8 was originally produced against rat cytochrome c, we conclude that the immunoreactive signal corresponds to a specific configuration of the Drosophila counterpart for the following reasons. First, the immunogen for mAb 2G8 shares >86% identity to Drosophila cytochrome c (DC4) including the immunodominant aspartic acid at position 62 . Second, all labeling was abolished by pre-absorption with antigen. Therefore, mAb 2G8, rather than a contaminant, is responsible for the in situ signal we have observed. Third, in biochemical fractionation assays and immunofluorescence studies, the signal was localized to the mitochondrial compartment where cytochrome c resides. Fourth, mAb 2G8 immunoprecipitated products of the correct size (∼13 kD), which cross-reacted with a different cytochrome c antibody specific for a distinct epitope. These studies establish the ability of mAb 2G8 to recognize cytochrome c in crude fly cell extracts and were validated in similar assays showing that mAb 2G8 could immunoprecipitate purified Drosophila cytochrome c (not shown). Section title: Discussion Educational score: 4.5059661865234375 Domain: biomedical Document type: Study Language: en Selective labeling of pre-apoptotic cells by mAb 2G8 indicates that apoptotic signaling triggers specific alterations in the configuration of cytochrome c that uncover an otherwise hidden epitope. Moreover, routine labeling of apoptotic SL2 cells with a variety of antibodies directed against different antigens does not behave like mAb 2G8, and we can readily exclude the possibility that the pre-apoptotic condition somehow promotes enhanced immunodetection. Based on numerous precedents, exposure of an otherwise hidden epitope through immunodetection could pave the way for developing reagents that probe specific protein conformations . In the case of fly cytochrome c, the precise nature of this change is not yet clear, but the alteration could reflect either a modified conformation and/or changes in associated proteins that permit immunoreactivity with the 2G8 antibody. In contrast to several mammalian models of apoptosis , the Drosophila protein was not released to the cytosol during apoptosis but instead continued to cofractionate with mitochondria. Therefore, the fly version of this protein could be more tightly tethered to its resident organelle than its mammalian counterpart. Also, since no covalent differences between healthy and pre-apoptotic samples of cytochrome c have yet been detected (either in our system or in mammalian models), our studies implicate a conformational and/or contextual change in cytochrome c which specifically occurs in cells that are committed to apoptotic death. Section title: Discussion Educational score: 4.492100715637207 Domain: biomedical Document type: Study Language: en One possibility is that antibody binding to the reactive epitope could be hindered by mitochondrial components in live cells. In cells committed to apoptosis, fly cytochrome c might translocate from the intermembrane space, but remain tethered to the outer membrane, and in the process, also expose the epitope for mAb 2G8. Except for the fact that the protein is not physically released, this scenario suggests features in common with mammalian models, where cytochrome c is liberated from mitochondria. Alternatively the polypeptide may simply undergo a conformational change detected by mAb 2G8 that does not affect its location. Consistent with this possibility, Jemmerson et al. have obtained evidence that mouse cytochrome c does assume a different conformation early in apoptosis of a T cell hybridoma. The altered conform was detected using yet a different mAb than the one used here, which does not recognize native cytochrome c but does recognize nonnative forms of the protein, such as large peptide fragments. These observations are entirely consistent with at least some mammalian cell models of apoptosis where cytochrome c remains in the mitochondria but is inactivated by death signals . While the precise nature of the change remains to be determined, the alteration is evidently specific for apoptotic forms of cell death. For instance, 2G8 immunostaining was not observed when SL2 cultures were killed by exposure to levels of ceramide that provoke a mode of cell death that is distinctly necrotic (see Materials and Methods). Section title: Discussion Educational score: 4.460077285766602 Domain: biomedical Document type: Study Language: en We authenticated a role for cytochrome c in apoptosis by examining staged ovarian tissues where the programmed death of identifiable cell types is well described. In a single egg chamber, one oocyte is connected through cytoplasmic bridges to 15 sister nurse cells. Over a period of slightly >3 d, the egg chamber develops through a sequence of well-defined stages before reaching maturity at the final stage of oogenesis, stage 14. Between stages 10B and 13 of oogenesis, the nurse cells undergo considerable changes that include a reorganization of the actin cytoskeleton and a permeabilization of nuclear membranes . These changes are thought to facilitate the transfer of nurse cell components into the developing oocyte and once completed, this process is quickly followed by the programmed death of the nurse cells themselves . Dying nurse cells display physiological changes common to apoptotic cells, such as elevated caspase RNAs and a requirement for caspase function . Similarly, diverse morphological criteria such as TUNEL labeling and acridine orange staining and ultrastructural features all point toward an apoptotic mode of death for late staged nurse cells. Section title: Discussion Educational score: 4.191324234008789 Domain: biomedical Document type: Study Language: en In our studies with the 2G8 antibody, only pre-apoptotic nurse cells were positive for cytochrome c staining. Furthermore, in double-label analyses the immunoreactive signal was detected long before overt signs of apoptosis were visualized either with a cytoplasmic marker (cortical actin visualized with phalloidin) or with a nuclear marker (TUNEL labeling). To our knowledge, these observations are the first demonstration that changes in cytochrome c actually anticipate programmed cell death within intact tissues and, since these observations occur in an invertebrate model, they also argue for widespread conservation of cytochrome c–associated functions in apoptotic physiology. Section title: Discussion Educational score: 4.5937347412109375 Domain: biomedical Document type: Study Language: en In Drosophila , at least three genes, rpr , grim , and hid function as potent activators of the apoptotic pathway and, collectively, these genes are required for cell death in the embryo . Generally, expression of each precedes programmed cell death, and each is sufficient to elicit apoptosis in cultured cells and in transgenic animals . Work presented here offers a cytological demonstration that signaling by rpr and grim trigger events which ultimately engage cytochrome c and is consistent with recent observations that rpr can provoke cytochrome c release in heterologous extracts from Xenopus . The Drosophila death activators engage caspase function and therefore we examined the effects of the viral caspase inhibitor, p35, and peptide-based inhibitors, ZVAD-fmk and DEVD-fmk, upon cytochrome c display. We and others had shown previously that caspase inhibitors prevent apoptosis induced by rpr and grim and, as demonstrated here, coexpression of p35 or incubation with anticaspase peptides similarly prevented immunodetection of cytochrome c that would otherwise occur in the presence of rpr or grim alone. These results demonstrate that display of apoptogenic cytochrome c requires caspase activity. To further test this idea, we examined the status of cytochrome c in cells where apoptosis was directly triggered by expression of an activated caspase, dcp-1. As expected, this protein promoted apoptogenic cytochrome c display, with features that were indistinguishable from labeling provoked by the death activators. Taken together, our data establish that caspase action is both necessary and sufficient to elicit pre-apoptotic alterations in cytochrome c. Section title: Discussion Educational score: 4.263317108154297 Domain: biomedical Document type: Study Language: en Cell-free models of apoptosis from vertebrate sources have implicated an important role for mitochondrial factors in the activation of some caspase enzymes . The cell-free experiments presented here are consistent with these reports, and with our in vivo studies indicating that signaling by rpr and grim engage one or more resident mitochondrial proteins. Is the apoptogenic form of cytochrome c solely responsible for caspase activation in our cell-free system? We tested this possibility in two ways, both of which were inconclusive. First, we found that the 2G8 anti–cytochrome c antibody did not interfere with the ability of apoptotic mitochondria to provoke caspase activation and second, we found that purified cytochrome c (from Drosophila , horse, and rabbit) was not sufficient on its own to promote caspase activity when added to cytosol from L2 cells. These data suggest that the 2G8 mAb might recognize, but not inhibit, cytochrome c in association with other proteins as component of a preformed apoptosome mitochondrial complex . It is also entirely possible that other mitochondrial factors might play important roles during caspase amplification in this system. Further studies will be required to address this issue. Section title: Discussion Educational score: 4.661498069763184 Domain: biomedical Document type: Study Language: en Two potential explanations reconcile our in vivo observations on apoptogenic cytochrome c with reports from mammalian cell-free systems that cytochrome c can trigger caspase activation. One possibility is that the order and/or nature of cytochrome c apoptotic function is not conserved between mammals and insects and thus, relative to caspase action, cytochrome c is upstream in the former case and downstream in the latter case. This scenario, however, seems unlikely given the widespread conservation of apoptotic components, the fact that display of fly cytochrome c in the animal significantly precedes all signs of programmed cell death, and reports from mammalian systems that upstream caspases can trigger cytochrome c release . Therefore, a more likely interpretation of our results is that cytochrome c propagates apoptotic physiology by functioning together with caspases in a feed-forward amplification loop. In this scenario, altered cytochrome c and caspase activity exert positive and reciprocal feedback upon each other, similar to observations recently reported for caspase 8 . Thus, agents that restrain caspase action (p35) are also predicted to suppress pro-apoptotic display of cytochrome c, which behaves as an amplifier of caspase function. This interpretation is also consistent with recent studies on Fas signaling in type II cells, where molecular ordering studies found that activation of an initiator caspase (caspase 8/Flice) occurs upstream of changes associated with cytochrome c . | Study | biomedical | en | 0.999998 |
10037792 | Section title: Strains and Genetic Techniques Educational score: 3.2192299365997314 Domain: biomedical Document type: Study Language: en Yeast strains used in this study are listed in Table I . Culture and genetic analysis of yeast were performed by standard procedures . Semisynthetic lactate medium was prepared as described . Plasmid DNA was prepared from Escherichia coli strain DH5α. Section title: Isolation of mdm17 Educational score: 4.221491813659668 Domain: biomedical Document type: Study Language: en The mdm17 mutant was isolated in a screen for novel alleles of another mitochondrial inheritance gene, mdm13. Strain MYY900, containing the mdm13 lesion, was crossed to the SL collection of temperature-sensitive strains which were derived from strain MYY290, and the resulting diploids were tested for growth at 37°C on yeast extract/peptone/glucose (YPD) medium. Diploids that failed to grow were sporulated and the meiotic progeny were analyzed by backcrossing and allelism tests. The recovered mdm17 spores were backcrossed three times to the wild-type parental strain, and the temperature-sensitive growth and mitochondrial distribution defects were shown to cosegregate in a 2:2 pattern. The backcrossed strain no longer displayed nonallelic noncomplementation with mdm13 and that property apparently depended on the presence of a third, unlinked mutation in the original mdm17 mutant strain. The MDM13 gene has yet to be isolated. Section title: Gene Cloning and Mapping Educational score: 4.153863906860352 Domain: biomedical Document type: Study Language: en mdm17 cells were transformed with a yeast genomic DNA library in the centromere-based LEU2 vector p366 (M. Hoekstra, ICOS Inc.). 10,000 Leu + transformants were screened for complementation of the temperature-sensitive growth defect by replica plating to YPD medium and to yeast extract/peptone/glycerol (YPG) medium at 37°C. Four different complementing clones were isolated, and restriction analysis demonstrated that they contained overlapping yeast DNA inserts. The identity of the inserts was determined by nucleotide sequence analysis of the ends of the inserts and comparison to sequences in the Saccharomyces Genome Database. Section title: Gene Cloning and Mapping Educational score: 4.182213306427002 Domain: biomedical Document type: Study Language: en A full-length version of the MGM1 gene was synthesized using PCR. The high-fidelity polymerase, Pfu (Stratagene), was used with primers 5′-CTCTCTAGAGTTCTTCTGCTCGCTAATGGTAAATG-3′ and 5′-CTCCTCGAGGCAAGAAGATGAGTTGGATGAAGG-3′ to amplify the MGM1 open reading frame together with ∼500 bp of flanking DNA sequences. The PCR product was phosphorylated with T4 DNA kinase and ligated into vector pRS313 that had been digested with SmaI and EcoRV and treated with calf intestinal phosphatase. The resulting plasmid was designated pRS313-MGM1. Section title: Gene Cloning and Mapping Educational score: 4.165600776672363 Domain: biomedical Document type: Study Language: en For integrative mapping, a 2.6-kb HindIII fragment adjacent to the MGM1 locus was subcloned into the YIp5 vector . The plasmid was linearized by digestion with HpaI and the DNA was transformed into strain MYY290. Ura + transformants were crossed to strain MYY971 containing the mdm17 mutation. The meiotic progeny of the resulting diploid consisted of 19 parental ditype and 1 tetratype, mapping the mdm17 lesion to within 2.5 cM of the MGM1 locus. Section title: Gene Replacement Educational score: 4.159056663513184 Domain: biomedical Document type: Study Language: en A gene replacement cassette was created by PCR as described by Baudin et al. . The oligonucleotides 5′-ATGAGTAATTCTACTTCATTAAGGGCCATCCCAAGAGTGGATTGTACTGAGAGTGCACC-3′ and 5′-TCATAAATTTTTGGAGACGCCCTTGTAGCTTTTCTTGAAAGTGCGGTATTTCACACCGC-3′ were used as PCR primers, and the LEU2 gene in plasmid pRS305 served as the template. The resulting PCR product was used to transform the diploid strain MYY298. Leu + transformants were screened by PCR to identify strains containing a replacement of one of two copies of MGM1 with LEU2. The heterozygous diploid strain was sporulated to obtain haploid segregants with the mgm1::LEU2 ( mgm1 -null) mutation. Section title: Phenotypic Analysis Educational score: 4.091710567474365 Domain: biomedical Document type: Study Language: en For phenotypic analysis, cultures were first grown overnight in YPD-liquid medium at 23°C and diluted to 0.5 A 600 /ml before incubation at 37°C for varying times. To evaluate respiration competence, cells were plated on YPD-agar medium, cultured at 23°C, and replica-plated to YPG medium. Colonies that failed to grow on YPG at 23°C were scored as having lost respiratory function. At least 700 colonies were scored for each time point. Section title: Phenotypic Analysis Educational score: 4.032699108123779 Domain: biomedical Document type: Study Language: en To characterize mitochondrial distribution and morphology in living cells, mitochondria were stained with the membrane potential–sensitive dye 2-(4-dimethylaminostryl)-1-methylpyridium iodide (DASPMI), and cells were examined by fluorescence microscopy . Indirect immunofluorescence microscopy was performed on fixed yeast cells as described . Section title: Antibody Preparation Educational score: 4.02166223526001 Domain: biomedical Document type: Study Language: en Antibodies were raised against a peptide, CGGYKGVSKNL, of which the last eight residues correspond to the extreme COOH terminus of Mgm1p. Peptide synthesis, coupling of peptide to keyhole-limpet hemocyanin, and immunization of rabbits was carried out by Research Genetics, Inc. The antibodies were purified on an affinity column prepared by coupling the peptide antigen to Affigel 10 (Bio-Rad Laboratories). Section title: Subcellular Fractionation and Analysis Educational score: 3.991420030593872 Domain: biomedical Document type: Study Language: en Cells were grown in semisynthetic lactate medium , converted to spheroplasts, homogenized, and fractionated by differential centrifugation as previously described . Mitochondrial subfractions were isolated as described . Mitochondria were subjected to trypsin treatment or extraction with sodium carbonate or sodium chloride as previously described . Section title: Pulse–Chase Analysis Educational score: 4.106838226318359 Domain: biomedical Document type: Study Language: en Wild-type cells were grown in minimal medium lacking methionine and cysteine to an A 600 = 0.5–0.8. Cells were harvested, resuspended in a solution of 40 mM KPi, pH 6.0, 1% glucose, and labeled for 5 min at 30°C by addition of 0.1 mCi [ 35 S]-Translabel (ICN) per ml. The chase was initiated by adding unlabeled methionine and cysteine to 20 μM and, in some cases, cycloheximide to 1 μg/ml. Cells were collected and processed for immunoprecipitation as previously described . Section title: Gel Filtration Analysis Educational score: 4.128383159637451 Domain: biomedical Document type: Study Language: en Gel filtration analysis of Mgm1p was similar to that described by Rapaport et al. . Briefly, 5 mg of mitochondria was pelleted and resuspended in 1 ml buffer A (150 mM potassium acetate, 1% Triton X-100, 4 mM magnesium acetate, 0.5 mM EDTA, 0.5 mM PMSF, and 30 mM Tris-HCl, pH 7.4). The resuspended mitochondria were sonicated for 15 min in an ice-water bath, and the mixture was centrifuged for 15 min at 90,000 g. The supernatant was loaded onto a 100-ml Sephacryl-300 column ( Pharmacia Biotech Inc. ) that had been equilibrated with buffer A and fractionated with an FPLC system ( Pharmacia Biotech Inc. ) at a flow rate of 0.5 ml/min. 1-ml fractions were collected, proteins were precipitated with TCA, and analyzed by SDS-PAGE and immunoblotting. Protein bands on the immunoblots were scanned and quantified using NIH Image software. Section title: Characterization of the mdm17 Mutation Educational score: 4.081782341003418 Domain: biomedical Document type: Study Language: en The mdm17 gene was amplified by PCR from genomic DNA isolated from mutant cells using Taq polymerase. DNA products were cloned into the TA vector (Invitrogen Corp.) and sequenced by the dideoxy method . Additionally, portions of mdm17 were amplified from genomic DNA using Pfu polymerase (Stratagene) and sequenced directly using the CircumVent Kit ( New England Biolabs, Inc. ). Section title: In Vitro Mutagenesis of MGM1 Educational score: 4.168746471405029 Domain: biomedical Document type: Study Language: en Site-directed mutagenesis to create point mutations in MGM1 was carried out using the “PCR SOEing” technique . The PCR products containing the mutations were used to replace corresponding wild-type sequences in pRS313-MGM1 via standard cloning techniques. DNA sequence analysis confirmed the presence of the novel mutations and the absence of any PCR-generated errors. Plasmids containing the mutant genes were transformed into the heterozygous ( MGM1/mgm1::LEU2 ) diploid strain MYY973. Transformants were sporulated, tetrads dissected, and mgm1 -null spores harboring each of the mutant constructs were identified and analyzed. Section title: mdm17 Cells Display Conditional Defects in Mitochondrial Inheritance, Mitochondrial Morphology, and Maintenance of the Mitochondrial Genome Educational score: 4.520595550537109 Domain: biomedical Document type: Study Language: en The mdm17 mutant was isolated in a screen for novel alleles of mdm13 , an uncharacterized gene that is required for normal mitochondrial inheritance and morphology. This screen involved crossing a haploid strain harboring the temperature-sensitive mdm13 lesion to a collection of temperature-sensitive strains and analyzing growth of the resulting diploids at the nonpermissive temperature (37°C). One diploid strain displayed temperature-sensitive growth, and analysis of its meiotic progeny indicated that one of two mutations conferring temperature-sensitive growth mapped to a genetic locus unlinked to mdm13. Therefore, rather than being an allele of mdm13 , the new mutation defined a distinct gene. Analysis of cells harboring only the new mutation (after its genetic isolation from mdm13 ) by staining with the mitochondria-specific, vital dye DASPMI and fluorescence microscopy revealed defects in mitochondrial distribution and morphology after incubation at the nonpermissive temperature . Complementation and allelism tests indicated that the new mutation was unlinked to previously characterized mdm mutations. Genetic analysis further demonstrated that the defects in growth at 37°C, mitochondrial distribution, and mitochondrial morphology were caused by a single nuclear mutation. This novel mutation was designated mdm17. Section title: mdm17 Cells Display Conditional Defects in Mitochondrial Inheritance, Mitochondrial Morphology, and Maintenance of the Mitochondrial Genome Educational score: 4.150495529174805 Domain: biomedical Document type: Study Language: en To quantify the effect of mdm17 on mitochondrial distribution and morphology, cells were stained with DASPMI and examined by fluorescence microscopy. In wild-type cells incubated at either 23°C or 37°C, mitochondria appeared as extended, tubular structures, distributed throughout the cytoplasm of both mother and bud portions of the cell . Mitochondria in cells harboring the mdm17 mutation appeared very similar to wild-type at 23°C but displayed dramatic changes in mitochondrial distribution and morphology after incubation at 37°C . In particular, mitochondrial aggregation and empty daughter buds were apparent in many cells. These alterations in mitochondrial distribution and morphology were confirmed by indirect immunofluorescence microscopy (data not shown). Section title: mdm17 Cells Display Conditional Defects in Mitochondrial Inheritance, Mitochondrial Morphology, and Maintenance of the Mitochondrial Genome Educational score: 4.170877456665039 Domain: biomedical Document type: Study Language: en To examine the development of mutant phenotypes at 37°C, populations of mutant cells were analyzed by indirect immunofluorescence microscopy at various times after shifting to the nonpermissive temperature . After 1 h at 37°C, most cells (87%) contained aggregated mitochondria, and a significant fraction of cells (18%) possessed buds devoid of mitochondria. Some of the cells with empty buds still possessed mitochondria with normal tubular morphology (data not shown). By 2 h, 90% of cells displayed aggregated mitochondria and a majority (56%) possessed empty buds. These results demonstrate that the mdm17 mutation rapidly causes defects in mitochondrial distribution and morphology after shifting cells to the nonpermissive temperature. Section title: mdm17 Cells Display Conditional Defects in Mitochondrial Inheritance, Mitochondrial Morphology, and Maintenance of the Mitochondrial Genome Educational score: 4.188473224639893 Domain: biomedical Document type: Study Language: en The fraction of cells in the mutant population that showed mitochondrial staining with the potential-dependent dye DASPMI decreased during incubation at the nonpermissive temperature, suggesting that the cells were becoming respiration deficient. To analyze this phenotype, mdm17 cells were incubated at 37°C for various times, plated on glucose (YPD) medium, and cultured at 23°C. Colonies were tested for respiratory competence by replica plating onto glycerol (YPG) medium. At early times, almost all cells were respiration competent . Significant numbers of respiration-deficient cells began appearing in the population only after 4 h at 37°C. After 24 h, >95% of the cells were respiration deficient . When cells from the 16-h time point were stained with the DNA-binding dye 4,6-diamino-2-phenylindole and examined by fluorescence microscopy, mitochondrial DNA staining was no longer evident in a majority of cells (data not shown), indicating that the respiration deficiency reflected a loss of mitochondrial DNA. Section title: mdm17 Is Allelic to MGM1, a Gene Implicated in Maintenance of the Mitochondrial Genome Educational score: 4.30852746963501 Domain: biomedical Document type: Study Language: en To identify the molecular basis for the mdm17 phenotypes, the wild-type MDM17 gene was cloned by complementation of the temperature-sensitive growth defect of mdm17 mutant cells. From 10,000 transformants, four strains harboring complementing plasmids were isolated. Restriction mapping of the yeast genomic DNA inserts in these plasmids indicated that all contained overlapping clones. DNA sequence analysis and comparison with sequences in the Saccharomyces Genome Database indicated that the genomic inserts in the plasmids corresponded to a region of chromosome XV. Deletion analysis of the plasmids localized complementing activity to a single open reading frame corresponding to the previously identified MGM1 gene . Transformation of mdm17 cells with a centromere-based plasmid containing only a single copy of MGM1 complemented all of the mutant phenotypes. Finally, the mdm17 mutation was localized to the MGM1 locus by integrative transformation and mapping (described in Materials and Methods). These results demonstrate that mdm17 is a mutant allele of MGM1. Section title: mdm17 Is Allelic to MGM1, a Gene Implicated in Maintenance of the Mitochondrial Genome Educational score: 4.142660617828369 Domain: biomedical Document type: Study Language: en Previous studies reported that mgm1 mutant cells readily lost mitochondrial DNA and contained aggregated mitochondria but showed normal mitochondrial distribution to buds . This latter property differs from the phenotype of the mdm17 mutant, where a large fraction of cells exhibited buds devoid of mitochondria. Accordingly, the phenotype of a null mutant in mgm1 generated in the same strain background as the mdm17 mutant was examined. Indirect immunofluorescence microscopy revealed many mgm1 -null cells with buds devoid of mitochondria, even in cells cultured at 23°C . As previously described , mitochondria in the mgm1 -null strain were extensively aggregated. Section title: Mgm1p Is Localized to the Mitochondrial Outer Membrane Educational score: 4.177464485168457 Domain: biomedical Document type: Study Language: en Previously reported phenotypes of the mgm1 mutant, together with the resemblance of the protein's predicted NH 2 terminus to a mitochondrial targeting sequence, suggested that Mgm1p is a mitochondrial protein . However, direct evidence of the protein's subcellular location was lacking. To determine the functional location of Mgm1p, antibodies were generated that were specific for a peptide corresponding to the extreme COOH terminus. These antibodies were used to detect the protein in subcellular fractions. Immunoblotting of total cellular extracts indicated that the affinity-purified antibodies recognized two polypeptides of ∼100 and 90 kD . Neither of these species was apparent in mgm1 -null cells , indicating that both polypeptides are products of the MGM1 gene. Furthermore, both species displayed substantially increased levels in cells harboring a multicopy (2 μ) plasmid encoding MGM1 . These increased levels had no apparent effect on mitochondrial distribution and morphology (data not shown). Section title: Mgm1p Is Localized to the Mitochondrial Outer Membrane Educational score: 4.169985771179199 Domain: biomedical Document type: Study Language: en The exact relationship between the two Mgm1p species is unclear, but the 90-kD form appears to be derived from the 100-kD form by proteolysis. The relative amounts of the two species varied randomly in different experiments, and pulse–chase analysis in intact cells failed to reveal a biosynthetic precursor-product relationship (data not shown). However, prolonged incubation of subcellular fractions in vitro (even at 0°C in the presence of a cocktail of protease inhibitors) led to conversion of the larger species to the smaller form (data not shown). These results suggest that the 90-kD form is a product of the partial proteolytic degradation of the 100-kD species. Section title: Mgm1p Is Localized to the Mitochondrial Outer Membrane Educational score: 4.222133159637451 Domain: biomedical Document type: Study Language: en Earlier characterization of MGM1 failed to identify which of five NH 2 -terminal methionine residues in the predicted open reading frame actually constituted the translational start of the protein . To resolve this uncertainty, in vitro mutagenesis was used to generate versions of mgm1 in which individual methionine codons were converted to alanine codons. These mutant versions were tested for their complementation of the phenotypes of mgm1 -null cells. No complementation was observed (and the protein was not detected) when methionine-2 was changed to alanine (data not shown). Mutation of the remaining four methionines did not affect complementation of the mutant defects, and both 100- and 90-kD forms of Mgm1p were detected in cells harboring these genes. These results indicate that the authentic translational start residue is methionine-2 (referred to henceforth as residue 1 of the coding region). Section title: Mgm1p Is Localized to the Mitochondrial Outer Membrane Educational score: 4.200331687927246 Domain: biomedical Document type: Study Language: en The subcellular distribution of Mgm1p was characterized by immunoblotting of proteins extracted from subcellular fractions isolated from wild-type cells. Mgm1p was localized predominantly to a subcellular fraction enriched in mitochondrial proteins . Analysis of purified submitochondrial fractions revealed that Mgm1p was associated with the mitochondrial outer membrane . Furthermore, Mgm1p was susceptible to protease treatment of intact mitochondria under conditions where internal proteins, such as the matrix-localized Mas2p, were protected . Section title: Mgm1p Is Localized to the Mitochondrial Outer Membrane Educational score: 4.255801200866699 Domain: biomedical Document type: Study Language: en To examine the nature of Mgm1p association with the outer membrane, isolated mitochondria were extracted with sodium chloride and with sodium carbonate. The latter treatment strips peripheral proteins but leaves integral proteins embedded in the membrane . Mgm1p was resistant to extraction with 1 M NaCl , indicating that the protein was tightly associated with the membrane. After carbonate treatment, the 90-kD form of Mgm1p was recovered in the soluble fraction, while the 100-kD form remained associated with the membrane . This result indicates that the full-length Mgm1p is an integral membrane protein. The behavior of the 90-kD fragment suggests that Mgm1p is embedded in the membrane via the putative membrane-spanning domain in the NH 2 terminus, since both Mgm1p species are recognized by the antibody specific for the extreme COOH terminus. Section title: Mgm1p Is Localized to the Mitochondrial Outer Membrane Educational score: 4.229793548583984 Domain: biomedical Document type: Study Language: en The state of Mgm1p in the mitochondrial outer membrane was further characterized by gel filtration analysis of mitochondrial proteins solubilized in 1% Triton X-100. About 60% of Mgm1p eluted in a peak corresponding to a molecular mass of ∼400 kD, while the remainder of the protein eluted in a peak appropriate to the monomeric size (90–100 kD) . The 90-kD (fragment) form of the protein eluted in a distinct peak with an apparent molecular mass of ∼120 kD (data not shown). The elution behavior of Mgm1p was distinct from that of two other integral proteins of the mitochondrial outer membrane, Tom70p and OM45 . These results suggest that Mgm1p is part of a high molecular mass complex either in a homo-oligomeric state or complexed with other proteins on the mitochondrial surface. Section title: Mgm1p Function Depends on the Conserved GTP-binding Domain Educational score: 4.469132423400879 Domain: biomedical Document type: Study Language: en To explore further the function of Mgm1p, the identity of the mdm17 mutation was determined. The mutant gene contained a single change: a transition of G to A at nucleotide 880, resulting in a change of Glu 294 to Lys. This lesion mapped near the conserved, tripartite GTP-binding motif, suggesting a role for this site in Mgm1p function. To test the importance of the GTP-binding site, a mutant form of MGM1 incorporating a change in a conserved residue, S224N, was created (the numbering of this residue is based on the designation of the second methionine in MGM1 as the NH 2 -terminal residue as described above). The equivalent mutation in the H-ras-GTPase (S17N) eliminates the protein's binding of GTP , and a corresponding mutation in rat dynamin (S45N) blocks the protein's function in endocytosis . Immunoblot analysis of cellular extracts detected Mgm1p in mgm1 -null cells harboring the mutant gene . However, the mgm1 -S224N gene failed to complement any of the mutant phenotypes of a cell containing an mgm1 -null lesion . In addition, the mgm1 -S224N gene did not complement the growth or mitochondrial distribution and morphology defects of the mdm17 mutant cells . These results indicate that an intact GTP-binding domain is essential for Mgm1p function. Section title: Mgm1p Function Depends on the Conserved GTP-binding Domain Educational score: 4.4161882400512695 Domain: biomedical Document type: Study Language: en Overexpression of dynamin family members mutated in critical residues of the GTP-binding site was previously found to cause dominant-negative effects in wild-type cells . To test whether this is also true for MGM1 , high-copy plasmids encoding either wild-type MGM1 or mgm1 -S224N were transformed into wild-type cells. Only a small number of transformants harboring the mutant gene was recovered, and Western blot analysis indicated that the mutant protein was not overexpressed in these cells (perhaps reflecting a regulatory or suppression phenomenon). However, both wild-type and mutant proteins were overexpressed in mgm1 -null cells harboring the respective plasmids (data not shown). These transformed cells were mated to wild-type cells, and the effect of overexpression in the resulting zygotes was analyzed by fluorescence microscopy. High levels of wild-type MGM1 had no apparent effect on mitochondrial distribution or morphology , but zygotes with elevated levels of the mutant protein displayed defects very similar to those caused by the original mdm17 mutation . In particular, 90% of the latter zygotes displayed aggregated mitochondria , and 52% of the buds produced by these zygotes contained no mitochondria . These results demonstrate that the mgm1 -S224N mutant gene product confers dominant-negative effects on mitochondrial distribution and morphology. Section title: Discussion Educational score: 4.423183917999268 Domain: biomedical Document type: Study Language: en We have identified a role for Mgm1p in mitochondrial inheritance through the analysis of cells harboring mdm17 , a mutation causing temperature-sensitive growth and alterations in mitochondrial distribution and morphology. Genetic analysis revealed that mdm17 is a mutant allele of MGM1 , a nuclear gene originally identified by its role in maintenance of the mitochondrial genome . Two key observations indicate that the primary functions of Mgm1p are to facilitate the maintenance of mitochondrial morphology and to mediate mitochondrial inheritance and that mitochondrial genome maintenance is a secondary role of the protein. First, analysis of mdm17 cells revealed that alterations in mitochondrial morphology and distribution appeared very quickly after a shift to elevated temperature, whereas loss of respiratory competence and the mitochondrial genome occurred only much later. Second, the location of Mgm1p on the mitochondrial surface is consistent with a primary function of the protein in morphology and distribution, but not with a direct role in maintenance of the matrix-localized mitochondrial DNA. In addition, several other mutations that cause aberrant mitochondrial morphology also cause increased loss of mitochondrial DNA . This loss appears to be a secondary effect of the morphological changes. Section title: Discussion Educational score: 4.516697883605957 Domain: biomedical Document type: Study Language: en Mgm1p is one of a small group of outer membrane proteins that have been shown to be required for mitochondrial inheritance. Mdm10p, Mdm12p, and Mmm1p are also integral proteins of the outer membrane whose loss leads to dramatic alterations in mitochondrial morphology and defects in transmission of mitochondria into developing daughter buds . Although the specific molecular activities of these other components are unknown, their location in the mitochondrial outer membrane and their exposed cytoplasmic domains suggest two potential models of function. One possibility is that these proteins serve as binding sites or anchor points for interaction with cytoskeletal structures or molecular motors. Such interactions might pull mitochondria into tubular structures and mediate mitochondrial movement into buds. A second possibility is that the outer membrane components regulate structural properties of the outer membrane, such as membrane fluidity, which are themselves essential for normal mitochondrial morphology and inheritance. Additionally, mitochondrial inheritance may depend on a tubular morphology that is maintained by these outer membrane proteins. Mgm1p may function in concert with the other outer membrane proteins, or it may fulfill a unique role. This latter possibility is suggested by the observation that phenotypes caused by mdm10 , mdm12 , and mmm1 are suppressed by the SOT1 mutation but defects caused by mutations in mgm1 are not (data not shown). Section title: Discussion Educational score: 4.613099098205566 Domain: biomedical Document type: Study Language: en Mgm1p is a member of the dynamin family of large GTP-binding proteins. The best characterized member of this family is dynamin itself, which plays an essential role in clathrin-mediated endocytosis in animal cells . Although S. cerevisiae does not have a true dynamin orthologue, this yeast possesses two other dynamin-like proteins, Vps1p and Dnm1p . Vps1p is localized to the Golgi apparatus and participates in the vesicular transport of proteins to the vacuole . Dnm1p may function in the endosomal pathway , but was recently found to play an important role in mitochondrial distribution and morphology . In particular, cells deleted for Dnm1p displayed mitochondrial tubules collapsed along one side of the cell and extending from the mother portion of the cell into the daughter bud. A related phenotype, the collapse of mitochondrial tubules into perinuclear aggregates, was observed in an independent study in which a mutant form of the human dynamin-like protein, Drp1, was expressed in cultured mammalian cells . These observations suggest that Dnm1p and its mammalian homologue Drp1 may mediate the lateral distribution or branching of mitochondrial tubules. Section title: Discussion Educational score: 4.541692733764648 Domain: biomedical Document type: Study Language: en Many molecular details of dynamin's function in endocytosis remain to be clarified, but key features of the protein's activity include GTP hydrolysis and assembly of dynamin into oligomeric structures . Dynamin has been proposed to act as a mechanical constrictor for the neck of an invaginating coated pit , or as a regulator or recruiter of the constriction machinery . Although it is not apparent that a constricting activity is necessary for the maintenance of mitochondrial morphology and distribution, our results suggest that Mgm1p shares two key functional features with dynamin. First, mutational analysis revealed that the GTP-binding site is essential for Mgm1p function, suggesting the importance of GTP hydrolysis. Second, the dominant-negative effect of the S224N mutation and the wild-type protein's gel filtration profile suggest that Mgm1p functions as part of an oligomeric complex. Mgm1p might play a structural role in locally influencing the properties of the outer membrane. For example, Mgm1p could assemble into a structure that promotes a tubular mitochondrial morphology. Alternatively, Mgm1p might fulfill a regulatory function in controlling the activity of other membrane components. More details of the protein's function should emerge from identification of interacting proteins. | Study | biomedical | en | 0.999996 |
10037793 | Section title: Buffers and Reagents Educational score: 4.324542045593262 Domain: biomedical Document type: Study Language: en HB: 50 mM K-Hepes, pH 7.6, 1 mM MgCl 2 , 1 mM EGTA, 1 mM β-mercaptoethanol (β-ME) and protease inhibitor stock (1:200 final dilution; see below). HB100: HB plus 100 mM NaCl; HB200: HB plus 200 mM NaCl; and HB500: HB plus 500 mM NaCl. EB200: HB200 plus 100 μM GTP and 1 mg/ml DrosC17 peptide; EB500: HB500 plus 100 μM GTP and 1 mg/ml DrosC17 peptide; HB block: 50 mM K-Hepes, pH 7.6, 100 mM KCl, 1 mM MgCl 2 , 1 mM EGTA, 10 mg/ml bovine serum albumin (fraction V; Sigma Chemical Co. ). Homogenization buffer: HB100 plus 10% glycerol, 1 mM PMSF and protease inhibitor stock (1:100 final dilution). Protease inhibitor stock: 10 mM benzamidine-HCl, 1 mg/ml aprotinin, 1 mg/ml leupeptin, and 1 mg/ml pepstatin A in ethanol. LPC: 10 mg/ml leupeptin, 10 mg/ml pepstatin A, and 10 mg/ml chymostatin dissolved in DMSO. Mounting medium: 20 mM Tris-Cl, pH 9.0, 90% glycerol, and 0.1% p -phenylenediamine. BRB80: 80 mM K-PIPES, pH 6.8, 1 mM MgCl 2 , 1 mM EGTA; 4× sample buffer: 250 mM Tris, pH 6.8, 12% SDS wt/vol, 20% β-ME vol/vol, and 40% glycerol vol/vol. GTP stock: 100 mM GTP ( Boehringer Mannheim Corp. ). Tubulin was purified from the bovine brain and labeled with tetramethylrhodamine as described ( http://skye.med.harvard.edu ). Drosophila embryo extract was prepared by homogenizing 0–3.5-h Drosophila embryos in homogenization buffer as described . Clarified extract was prepared by centrifugation of crude extract for 10 min at 15,000 rpm (SS34 rotor; Sorvall) at 4°C. The supernatant was transferred to a new tube, and centrifuged a second time at 50,000 rpm in a rotor (50.2 Ti or SW55; Beckman) for 1 h at 4°C. Section title: Sucrose Gradient Sedimentation and Gel Filtration Chromatography Educational score: 4.121204376220703 Domain: biomedical Document type: Study Language: en Sucrose gradients (5–20 or 5–40%) were poured as step gradients (five steps of equal volume) in HB containing 100 or 500 mM NaCl plus nucleotide and allowed to diffuse into continuous gradients. Gradients were fractionated from the top by hand with cutoff pipet tips. Fractions from standards gradients run in parallel were separated by 10% PAGE and stained with Coomassie blue. Gels were scanned, band intensities were quantitated (Adobe Photoshop; Adobe Systems Inc.) and peak fractions were assigned (Kaleidagraph Synergy Software Inc.). Standard curves of peak fraction versus sedimentation coefficient (S 20,w ) were used to estimate S values of protein complexes. Section title: Sucrose Gradient Sedimentation and Gel Filtration Chromatography Educational score: 4.112409591674805 Domain: biomedical Document type: Study Language: en Gel filtration chromatography was carried out on a column (Superose-6; Pharmacia Biotech Sverige) in HB plus 100 μM GTP, and 100 or 500 mM NaCl as indicated. The column was calibrated with standards of known Stokes radii. Molecular weights and Stokes radii of protein complexes were estimated as described . Fractions were separated by SDS-PAGE on 10% gels and γ-tubulin was detected by Western blotting. Section title: Immunoisolation of γ-tubulin–containing Complexes from Drosophila Embryo Extract Educational score: 4.25025749206543 Domain: biomedical Document type: Study Language: en PEG was added to a final concentration of 2% (from a 30% stock in HB100) to clarified Drosophila embryo extract from 20-g embryos. The mixture was incubated on ice for 20 min, spun at 17,000 rpm for 10 min in a SS34 rotor and the supernatant was discarded. The pellets were resuspended in 20 ml of HB200 plus 0.05% NP-40, and 100 μM GTP by gentle Dounce homogenization and clarified at 35,000 rpm for 30 min in a 50.2 Ti rotor. γ-tubulin complexes were immunoprecipitated from the supernatant by adding 190 μg of DrosC17 antibody and incubating at 4°C for 1 h with gentle rotation. The immunoprecipitate was collected by slowly (over 1 h) pumping the antibody-extract mixture over a 350-μl column of protein A–agarose ( GIBCO /BRL) in a disposable Bio-spin column housing (Bio-Rad). The column was washed with 15 ml of HB200 plus 0.05% NP-40 and 100 μM GTP, and 15 ml of the same buffer without NP-40. 400 μl of EB200 was loaded onto the column and the column was sealed with parafilm and incubated for 16–18 h at 4°C. γ-tubulin complexes were collected by loading an additional 400 μl of EB200 onto the column and collecting the flow through. For the sucrose gradient fractionation described in Fig. 2 , 150 μl of isolated complexes was loaded onto a 2.1-ml 5–40% sucrose gradient, poured in HB100 plus 100 μM GTP, and sedimented at 50,000 rpm for 4 h in an TLS55 rotor at 4°C. Section title: Electron Microscopy Educational score: 4.197972774505615 Domain: biomedical Document type: Study Language: en Negative stain electron microscopy of peptide-eluted complexes and sucrose gradient purified γTuRC was performed as described , except that grids of sucrose gradient fractions were rinsed with water before staining. For cryo-electron microscopy fresh peptide-eluted γ-tubulin complexes were applied to glow discharged, holey carbon films supported on copper EM grids. Excess liquid was removed by blotting and the resulting thin film was rapidly frozen by plunging into liquid ethane slush. Frozen grids were stored in liquid nitrogen. Grids were examined using a Gatan cryo-transfer holder in an electron microscope (CM120; Philips Electron Optics). During transfer, examination, and imaging, the grid was maintained at ≤−180°C. Underfocused images (−1.2 to −2.0 μm) of layers of frozen solution spanning holes in the support film were recorded using low dose methods ( Kodak SO163 film). Positive prints were made for further examination of the complexes. Section title: Immunoisolation of the γTuSC Educational score: 4.29647159576416 Domain: biomedical Document type: Study Language: en PEG was added to 3% (from a 30% stock in HB100) to clarified extract corresponding to 40 g of embryos. The mixture was incubated on ice for 20 min, centrifuged at 17,000 rpm for 10 min in the SS34 rotor at 4°C, and the supernatant was discarded. Pellets were resuspended by gentle Dounce homogenization in 40 ml ice-cold HB500 plus 100 μM GTP and clarified at 35,000 rpm for 30 min at 4°C in the 50.2 Ti rotor. γ-tubulin small complex (γTuSC) was immunoprecipitated by adding 1.46 mg of DrosC17 or DrosC12 antibody and incubating at 4°C for 1 h with gentle rotation. The immunoprecipitate was collected by pumping the antibody-extract mixture over a 500-μl column of protein A–agarose preequilibrated in HB500. The column was washed with 60 ml HB500 plus 100 μM GTP. Alternatively, the immunoprecipitate was collected by mixing the resin in a batch with the antibody-extract mixture at 4°C for 1 h. Afterwards, the resin was washed six times in batches with 5 ml of HB500 plus 100 μM GTP and loaded into a Bio-spin column. 500 μl of EB500 was loaded onto the column, the column was sealed with parafilm, and incubated for 4 h at 4°C. The γTuSC was collected by loading an additional 550 μl of EB500 onto the column and collecting the flow through. 500 μl of this eluate was fractionated on a 4.5-ml 5–20% sucrose gradient in HB500 plus 100 μM GTP at 45,000 rpm in an SW55 rotor for 10 h at 4°C. 300-μl fractions were collected from the top. For coverslip and solution nucleation assays, 100 μl of each fraction was dialyzed against HB100 plus 100 μM GTP at 4°C for 6 h. The concentration of γ-tubulin was estimated after dialysis and, in general, was not significantly altered. Section title: Solution Nucleation Assays to Quantitate Microtubule Nucleation Educational score: 4.1786370277404785 Domain: biomedical Document type: Study Language: en 5-μl reactions of identical final buffer composition (0.5× BRB80, 0.5× EB200, 500 μM GTP) containing 4 mg/ml tubulin and varying concentrations of peptide-eluted complexes were incubated at 37°C for 4 min and fixed at room temperature for 3 min by addition of 45 μl of 1% glutaraldehyde in BRB80. 10 μl was removed to a new tube and diluted by addition of 1 ml ice-cold BRB80. MT spindowns and tubulin immunofluorescence were performed as described ( http://skye.med.harvard.edu ). Varying amounts of each sample were pelleted depending on the concentration of γ-tubulin in the reaction. 20 random fields were photographed with a 60× objective (1.4 NA; Nikon Corp. ) using a cooled CCD camera ( Princeton Instruments ) and the MTs were counted. The fraction of total γ-tubulin in the peptide-eluted complexes present as the γTuRC was determined by densitometry of Coomassie-stained gels after sucrose gradient fractionation. Section title: Solution Nucleation Assays to Quantitate Microtubule Nucleation Educational score: 4.109402656555176 Domain: biomedical Document type: Study Language: en To compare nucleating activity, peptide-eluted γ-tubulin complexes containing 0.65 μM γ-tubulin in the γTuRC (0.87 μM total γ-tubulin) and isolated γTuSC containing 0.74 μM γ-tubulin were assayed in parallel as above. The following exceptions were made: the incubation at 37°C was for 3 min; the final buffer composition of the γTuSC was 0.5× BRB80, 0.5× HB100 plus 100 μM GTP, 500 μM GTP; after fixation, instead of sedimentation, samples were diluted with 200 μl of BRB80 + 70% glycerol and 3 μl were squashed and sealed under 18-mm square coverslips. Section title: Coverslip Nucleation Assay Educational score: 4.140275001525879 Domain: biomedical Document type: Study Language: en Polylysine-coated 12-mm diameter coverslips were placed on parafilm inside a humidified Petri dish kept in a 30°C water bath. The coverslips were rinsed 2× with filtered water and blocked for 5 min with 60 μl HB block. The HB block was removed by aspiration and replaced with 20 μl of the sample. After 10 min the coverslips were washed 2× with 60 μl of BRB80 + 10 mg/ml BSA + 1 mM GTP, and incubated with 20 μl 6 mg/ml tubulin (1:4 rhodamine labeled/unlabeled) in BRB80 + 1 mM GTP. After 10 min the tubulin was removed by aspiration and replaced with 60 μl 1% glutaraldehyde in BRB80 (warmed to 30°C) for 3 min, followed by 3 min postfixation with −20°C methanol. The coverslips were rehydrated, mounted, and sealed with nail polish. Section title: Cloning and Sequencing of Dgrip84 and Dgrip91 Educational score: 4.269651412963867 Domain: biomedical Document type: Study Language: en The Drosophila gamma ring proteins (Dgrips) were immunoisolated as described below and internal peptide sequences were obtained for Dgrip84 and Dgrip91. The following peptides were obtained for Dgrip84: KILRTGK, KDAQQLIIG LVRK, DRSLTH, DELPEHY, DIHTHL, DLVTQM S, DAEVLTYL, DEQIPSFLA, RHREFL, DFTMQ, ERRTYTLR, DTTPVVFVRRGP, DRHRE, DEYRTSLL, DEQIPSFLAKY, DVNSAAGSVPTTLAIAST, and DLVTQMSKIMKKEENXQAQ. For Dgrip91 the peptides obtained were: KDVVTGRF, KGVYGLTN, KTVSDH, KHMEFVLS, DIMVGPHK, DFNEYY, KLSELGYY, DATKMLP(OR L)E, DRVVKFS, DVIVQRPFNGG, EMIIC IKGKQMPE , DVVTGRIFPY, ELSKIV, DATQSSIGLXKQSLPNY, DDPNLQLFGTR, DQSRFYK, and DVS TGFNAI G. For Dgrip84, degenerate primers corresponding to the forward peptide KDAQQL and reverse peptide DLVTQM (underlined above) specifically amplified a band of ∼700 bp. A second round of PCR was performed with a primer corresponding to the forward peptide QQLIIG and the same reverse primer. For Dgrip91, primers corresponding to the forward peptide IKGKQM and reverse peptide TGFNAI (underlined above) specifically amplified a band of ∼800 bp. A second round of PCR was performed with a primer corresponding to the forward peptide GKQMPE and the same reverse primer. Both PCR products were cloned, sequenced, and used to screen a Drosophila cDNA library. Section title: Antibodies Educational score: 4.116230010986328 Domain: biomedical Document type: Study Language: en Synthetic peptides (Stanford University Medical Center) corresponding to the COOH-terminal 12 (QWSPAVEASKAG; DrosC12) or 17 amino acids (QIDYPQWSPAVEASKAG; DrosC17) of the maternal form of Drosophila γ-tubulin were used to raise and affinity purify rabbit polyclonal antibodies . Antibodies to Dgrip84 and Dgrip91 were raised in rabbits against fusions of glutathione-S-transferase with amino acids 89–199 and 29–143 of the two proteins, respectively. Specific antibodies were purified as described . Section title: Embryo Fixation and Immunofluorescence Educational score: 4.107848167419434 Domain: biomedical Document type: Study Language: en Embryos were fixed in methanol and immunofluorescence was performed as described . Embryos were double-labeled with mouse anti–γ-tubulin ( Sigma Chemical Co. ) and rabbit anti-p91 or anti-p84 followed by FITC anti–rabbit and Cy-5 anti–mouse secondary antibodies (Jackson ImmunoResearch Laboratories, Inc.). Three-dimensional images were obtained on a wide field microscope (DeltaVision; Applied Precision Inc.). 512 × 512 pixel optical sections were taken at 0.2-μm intervals using an Olympus 60×, 1.4 NA objective and deconvolved. Appropriate Z-sections were projected. Section title: GTP Cross-linking Experiments Educational score: 4.140191078186035 Domain: biomedical Document type: Study Language: en 120-μl peptide-eluted complexes were isolated as above, except GTP was omitted from the column wash and elution buffers. The isolated complexes were loaded onto a 2-ml 5–40% sucrose gradient in HB100, centrifuged at 55,000 rpm in a TLS55 rotor for 4 h at 4°C, and fractionated into 17 130-μl fractions. In a 96-well plate, 30 μl of each fraction was incubated for 90 min on ice with 10 μ Ci of [α- 32 P]GTP (400 Ci/mmol; Amersham Pharmacia Biotech Inc. ), cross-linked for 5 min on ice in a cross-linker (Stratalinker UV; Stratagene) at a distance of 10 cm, and analyzed by 10% SDS-PAGE followed by autoradiography. For competition experiments, the [α- 32 P]GTP was premixed with a 200-fold excess of cold nucleotide competitor before being added to the fraction. Section title: Determination of γTuSC Nucleotide Content Educational score: 4.199334144592285 Domain: biomedical Document type: Study Language: en γTuSC was prepared as described above with the following modifications: 1:1,000 LPC was used in place of 1:200 protease inhibitor stock; pellets from the PEG precipitation were resuspended in buffer containing 100 μM of either GTP or GDP; and the wash, elution buffers, as well as sucrose gradients contained 20 μM of either GTP or GDP. Control gradients loaded with EB500 were fractionated in parallel to generate control buffers. αβ-tubulin samples were prepared by diluting bovine brain tubulin into control buffers and incubating 1 h on ice before desalting. Free nucleotide was removed by rapid desalting into 20 mM Tris, pH 7.5, 100 mM NaCl, 1 mM MgCl 2 on a 800-μl Fast Desalting column (PC 3.2/10) mounted on a SMART™ system ( Pharmacia Biotech, Inc. ). 100 μl of sample was loaded per run and the column was eluted at a flow rate of 400 μl/ min. One 100-μl fraction containing the protein peak was collected from each desalting run. Two separate runs were pooled, generating a total desalted sample volume of 200 μl. Under these conditions, desalting was complete in ∼45 s and ≥99.9% of free nucleotide was removed. 20 μl of the desalted sample was used for protein quantitation (see below). The concentration of γ- or αβ-tubulin loaded onto the desalting column was usually between 0.5 and 1 μM; protein recoveries were ∼40–50%. Section title: Determination of γTuSC Nucleotide Content Educational score: 4.203180313110352 Domain: biomedical Document type: Study Language: en To extract nucleotide from the remaining desalted sample, 90 mg of solid urea was added and the sample was vortexed and heated to 50°C for 3 min to denature the protein and release bound nucleotide. The denatured protein was removed by filtration through a 10,000 mol wt cutoff filter . The filter was washed with 150 μl of water; eluate and wash were combined and frozen in liquid nitrogen. Nucleotide content was determined by chromatography on a 100-μl Mono Q (PC 1.6/5; Pharmacia Biotech, Inc. ) column mounted on the SMART™ system. Nucleotides were eluted with a gradient of ammonium bicarbonate (0.1–1 M in 30 column vol), quantified by peak integration, and compared with standard curves generated by processing nucleotide standards of known concentration in an identical fashion. The amount of γ-tubulin and αβ-tubulin was quantified using 10% SDS-PAGE, Coomassie-staining, and densitometry of the 20-μl aliquot of sample reserved after desalting relative to a standard curve of αβ-tubulin dimer on the same gel. Section title: Drosophila Contains Two Related γ-tubulin Complexes: The γTuSC Is a Subunit of the γTuRC Educational score: 4.290081977844238 Domain: biomedical Document type: Study Language: en Drosophila embryo extracts contain two γ-tubulin–containing complexes that can be separated by gel filtration chromatography or sucrose gradient sedimentation. In the presence of 500 mM KCl or NaCl, γ-tubulin is found exclusively in the smaller complex, indicating that the larger complex has been disrupted, and that the smaller complex is likely to be a structural subunit of the larger complex . We named the large complex, Drosophila γTuRC (see below), and the small complex the γTuSC. To obtain size estimates for each complex, we performed gel filtration and sucrose gradient sedimentation under low salt conditions in buffers that were supplemented with magnesium and GTP to reduce aggregation that occurs in nucleotide-free buffers. Under these conditions, the γTuSC has an S value of 9.8 and a Stokes radius of 7.0 nm, while the γTuRC has an S value of 35.5 S and a 15-nm Stokes radius. Based on these values, we estimate the molecular masses of the γTuSC and γTuRC to be 280,000 and 2,200,000 D (Table I ), respectively. Section title: Purified Drosophila γ-tubulin Complexes Nucleate Microtubules In Vitro Educational score: 4.155389785766602 Domain: biomedical Document type: Study Language: en To purify Drosophila γ-tubulin complexes, we used an immunoaffinity strategy based on antibodies raised against a COOH-terminal peptide of Drosophila γ-tubulin. Immunoprecipitated protein complexes were eluted from the antibody with buffers containing competing peptide . The peptide-eluted mixture of γTuRC and γTuSC nucleates MTs in solution . The number of MTs nucleated is directly proportional to the concentration of γ-tubulin complexes . At the highest concentrations tested (370 nM or ∼0.02 mg/ml γ-tubulin), γ-tubulin complex–containing reactions nucleated ∼100-fold more MTs than control reactions. The relative proportions of γTuRC and γTuSC vary between preps. For the experiments shown in Fig. 2 , 67% of the γ-tubulin was present as γTuRC, 22% was present as γTuSC, and 11% was in complexes intermediate in size. Section title: The Protein Profile of the Drosophila γTuRC Is Similar to That of the Xenopus γTuRC Educational score: 4.32517147064209 Domain: biomedical Document type: Study Language: en To determine the protein compositions of the γTuSC and γTuRC, we fractionated the peptide-eluted complexes on a 5–40% sucrose gradient . For clarity, γTuSC and γTuRC are shown side by side in Fig. 3 B. The protein profile of the Drosophila γTuRC is reminiscent of the Xenopus γTuRC . Therefore, by analogy to the Xgrips , we name Drosophila γTuRC proteins Dgrips and designate them by their apparent molecular weights. Like the Xenopus γTuRC, the Drosophila γTuRC is composed of two high molecular mass proteins (Dgrip163 and Dgrip128), two prominent proteins near 100 kD (Dgrip91 and Dgrip84), and a group of three or four proteins with molecular masses near 75 kD (Dgrip75s). The protein below γ-tubulin (between the 56- and 38.5-kD markers) has been identified as actin. It is not clear whether actin is a specific component of γTuRC, or if it fortuitously copurifies. Depending on the purification protocol, varying amounts of α- and β-tubulin copurify with Xenopus γTuRC . In contrast, we have been unable to detect α- or β-tubulin copurifying with Drosophila γTuRC. Consistent with the idea that γTuSC is a structural subunit of γTuRC, γTuSC is composed of the three most prominent proteins in γTuRC: γ-tubulin, Dgrip84, and Dgrip91 . Section title: The Drosophila γTuRC Nucleates MTs In Vitro Educational score: 4.225433826446533 Domain: biomedical Document type: Study Language: en To determine which of the Drosophila γ-tubulin complexes were able to nucleate MTs, we separated them by sucrose gradient sedimentation and tested them using a coverslip nucleation assay . In this assay, the sample to be tested is allowed to bind to a preblocked coverslip, unbound protein is washed away, and the coverslip is incubated with purified bovine brain tubulin containing a small amount of rhodamine-labeled tubulin. Unincorporated tubulin and spontaneously nucleated MTs are removed by aspiration, whereas MTs nucleated and tethered to the coverslip by the γ-tubulin complexes remain and are fixed and viewed by fluorescence microscopy. Although this assay is not quantitative, it has two advantages over the conventional solution nucleation assay: (a) the background of spontaneously nucleated MTs is removed by aspiration, allowing detection of very low levels of γ-tubulin dependent nucleation; (b) buffer components are washed away before exposure to tubulin. The latter is useful when directly assaying sucrose gradient fractions because of the strong interfering effects of varying sucrose concentrations on tubulin nucleation and elongation. Section title: The Drosophila γTuRC Nucleates MTs In Vitro Educational score: 4.17723274230957 Domain: biomedical Document type: Study Language: en The results of a coverslip assay on the sucrose gradient fractions in Fig. 3 A are shown in Fig. 3 D. The top two rows are equivalent exposures for fractions 3–14. The bottom row shows longer exposures (either 40× or 5× longer) for the indicated fractions. We observe a clear peak of activity corresponding to the fractions that contain γTuRC (fractions 10–14). Under these conditions, no activity is seen in the fractions containing γTuSC (fractions 4–6). Similar to the peptide-eluted complexes, gel filtration or sucrose gradient–fractionated Drosophila embryo extracts tested in this assay show only one peak of activity, corresponding to the peak of γTuRC (data not shown). To distinguish between nucleation and capture of spontaneously nucleated MTs, we performed the coverslip nucleation assay with peptide-eluted material and observed unfixed samples in real time using video enhanced DIC microscopy (data not shown). MTs initiated at the coverslip surface and elongated, while maintaining a fixed orientation with one end anchored to the coverslip. Capture of a MT from solution by the surface was never observed. Cumulatively, these results indicate that the Drosophila γTuRC has MT nucleating activity. Section title: Cryo-electron Microscopy of the γTuRC Reveals a Modular Structure Educational score: 4.236456871032715 Domain: biomedical Document type: Study Language: en Negative stain electron microscopy of the peptide-eluted complexes , and of the γTuRC after sucrose gradient sedimentation reveals an open ring structure with a diameter of ∼25 nm. In side-by-side pictures of comparable preparations, the structure of the Drosophila γTuRC is indistinguishable from that of the Xenopus γTuRC (Wiese, C., and Y. Zheng, unpublished observations). To get a more detailed view of the γTuRC, we examined the structure of the purified Drosophila γTuRC by cryo-electron microscopy. A gallery of cryo-EM images reveals a modular structure . The γTuRC appears to have ∼13 structural repeats arranged in a radial symmetric pattern with a diameter of 25 nm. Some internal structures are also visible. A more detailed view will require single particle reconstructions. Section title: γTuRC Is a More Potent Microtubule Nucleator than γTuSC Educational score: 4.231593132019043 Domain: biomedical Document type: Study Language: en The absence of nucleation activity of sucrose gradient–isolated γTuSC in the coverslip assay can be explained in several ways: γTuSC (a) might not have nucleating activity; (b) might not bind to the coverslip under the assay conditions; (c) might become inactivated upon binding to the coverslip, or (d) might be too dilute to exhibit activity. To distinguish between these possibilities we developed a protocol to prepare more concentrated γTuSC, taking advantage of the disruption of γTuRC into γTuSC by high salt . γTuRC was disrupted by isolating γ-tubulin–containing complexes in the presence of 500 mM NaCl. The resulting γTuSC was eluted with peptide containing buffer in 500 mM NaCl. The peptide-eluted material was further fractionated on a 5–20% sucrose gradient in 500 mM NaCl . This gradient separated γTuSC from residual larger complexes and from non-γTuSC components of γTuRC. This resulted in highly concentrated, relatively pure γTuSC . Inclusion of 500 mM salt in the sucrose gradient was important to prevent any reassociation of γTuSC with nonγTuSC components of γTuRC. Typically, the peak γTuSC sucrose gradient fraction contained ∼700 nM γ-tubulin (as judged by densitometry of Coomassie-stained bands relative to αβ-tubulin standards). For comparison, after sucrose gradient fractionation, the peak γTuSC-containing sucrose gradient fraction in the mixed complex preparation contained ∼70 nM γ-tubulin. Section title: γTuRC Is a More Potent Microtubule Nucleator than γTuSC Educational score: 4.250870227813721 Domain: biomedical Document type: Study Language: en Concentrated γTuSC fractions were dialyzed to remove salt and tested for nucleating activity. In contrast to the robust activity of the peptide-eluted complexes, nucleation by isolated γTuSC in solution was weak and slightly variable between preparations. A direct comparison between the nucleating activity of the peptide-eluted complexes (containing 0.87 μM total γ-tubulin, 0.65 μM γ-tubulin in γTuRC) and isolated γTuSC (0.74 μM γ-tubulin) in a solution nucleation assay is shown in Fig. 6 . In nucleation reactions containing between 350–450 nM γ-tubulin and 4 mg/ml tubulin, the nucleating activity of the peptide-eluted complexes was typically 80–100-fold above the level of spontaneous nucleation; under these conditions the level of nucleation for isolated γTuSC was only two- to threefold above background . Therefore, it is likely that the nucleating activity of the peptide-eluted complexes is due to primarily the activity of γ-tubulin in γTuRC. Based on those data, we estimate that per mole of γ-tubulin γTuRC is ∼25 times more active than γTuSC in promoting nucleation. Since there are ∼12 γ-tubulin molecules in γTuRC and only 2 in γTuSC (see below for stoichiometry estimates), γTuRC is ∼150 times more active than γTuSC per mole of complex. We also tested the nucleating activity of γTuSC in the coverslip nucleation assay. The concentrated γTuSC was also able to nucleate MTs in this assay . Section title: γTuRC Is a More Potent Microtubule Nucleator than γTuSC Educational score: 4.086814880371094 Domain: biomedical Document type: Study Language: en The activity of γTuSC could be explained at least two ways: (a) γTuSC might reassemble into a γTuRC-like higher-order structure, or (b) γTuSC itself might have intrinsic nucleating activity. To begin distinguishing between these possibilities, we examined dialyzed γTuSC for evidence of formation of higher-order structures using gel filtration chromatography. Undialyzed γTuSC migrated in a position typical of that for the smaller complex . The dialyzed sample migrated in the same position , suggesting that dialysis did not induce detectable assembly of γTuSC into larger structures. Section title: Dgrip84 and Dgrip91 Are Homologous to the Spc97/Spc98 Family of Proteins Educational score: 4.291415214538574 Domain: biomedical Document type: Study Language: en To characterize the molecular nature of γTuSC, we cloned and sequenced its non–γ-tubulin components, Dgrip84 and Dgrip91. Dgrip84 and Dgrip91 are homologous to each other and to the Spc97/98p family of proteins identified in S. cerevisiae. This family also includes two proteins identified in humans, hGCP2 and hGCP3 . The homology between the Drosophila proteins and the other members of this family extends over the entire length of the proteins (data not shown). In comparisons of Dgrip84 and Dgrip91 with the corresponding human proteins, a one-to-one correspondence emerges. Dgrip84 is 32% identical (46% similar) to hGCP2 and only 21% identical (32% similar) to hGCP3; in contrast, Dgrip91 is 31% identical (45% similar) to hGCP3 and only 24% identical (37% similar) to hGCP2. These results suggest that Dgrip84 and hGCP2, and Dgrip91 and hGCP3 may be functionally homologous pairs. The sequences of the Drosophila proteins are available from GenBank/EMBL/DDBJ under accession numbers AF118379 (Dgrip84) and AF118380 (Dgrip91). Section title: Stoichiometry of Proteins in γTuSC Educational score: 4.242780685424805 Domain: biomedical Document type: Study Language: en To estimate the stoichiometry of γTuSC proteins, we performed densitometry of Coomassie-stained SDS-PAGE gels of purified γTuSC. After correcting for the predicted molecular weight of each protein, we estimated that the ratio of Dgrip91 to Dgrip84 to γ-tubulin in the γTuSC is 1:1:2. Since our estimate of the molecular mass of purified γTuSC from sucrose gradient sedimentation and gel filtration is 280,000 D (Table I ), we suspect that γTuSC contains 1 molecule of Dgrip91, 1 molecule of Dgrip84, and 2 molecules of γ-tubulin. Interestingly, this corresponds to estimates of the stoichiometry of proteins in the S. cerevisiae 6 S γ-tubulin complex . If we assume that γTuRC contains only one molecule of each non-γTuSC component, and use our estimates for the molecular weights of γTuRC and γTuSC, then γTuRC would contain approximately six γTuSCs. Section title: Dgrip84 and Dgrip91 Cofractionate with γ-tubulin on Sucrose Gradients and Colocalize with γ-tubulin in Embryos Educational score: 4.345027446746826 Domain: biomedical Document type: Study Language: en If γ-tubulin in Drosophila embryos primarily exists associated with Dgrip84 and Dgrip91 in either γTuSC or γTuRC, we would expect these three proteins to cofractionate on sucrose gradients of embryo extract and to colocalize in embryos. To test this hypothesis, we raised and affinity-purified rabbit polyclonal antibodies that recognize Dgrip84 and Dgrip91. Each antibody recognizes a band of the expected molecular weight on Western blots of embryo extract . As expected, both Dgrip84 and Dgrip91 comigrate with γ-tubulin in γTuSC and γTuRC when embryo extract is fractionated on sucrose gradients . In addition, the localizations of Dgrip84 and Dgrip91 in Drosophila embryos are indistinguishable from that of γ-tubulin. Each antibody recognizes the centrosome throughout the cell cycle and shows some spindle staining during mitosis with enrichment at the spindle poles , regardless of its cognate antigen. We propose that Drosophila γ-tubulin is stably associated with Dgrip91/84. Interestingly, we found no evidence for a non–γ-tubulin associated pool of either Dgrip84 or Dgrip91. Section title: γ-tubulin in γTuRC and γTuSC Can be Cross-linked to GTP Educational score: 4.226101398468018 Domain: biomedical Document type: Study Language: en The homology between γ-, α-, and β-tubulins extends into domains that are involved in GTP binding by α- and β-tubulin . Thus, it is tempting to speculate that γ-tubulin can bind, and possibly hydrolyze, GTP. To determine if γ-tubulin binds guanine nucleotide, we immunoisolated γ-tubulin–containing complexes in the absence of GTP. The isolated complexes, either before or after sucrose gradient sedimentation, were incubated with [α- 32 P]GTP and UV cross-linked. In the peptide-eluted complexes, γ-tubulin is the only protein that cross-links to GTP . Furthermore, γ-tubulin in both the γTuRC and γTuSC cross-links to GTP . Competition experiments showed that the cross-link can be competed by addition of excess cold GTP, GDP, and GTPγS but not GMP-PNP, ATP, or CTP . Section title: γ-tubulin in γTuSC Preferentially Binds GDP Educational score: 4.175577640533447 Domain: biomedical Document type: Study Language: en To characterize the nucleotide binding properties of γ-tubulin, we compared the nucleotide content of γ-tubulin in γTuSC to that of similarly treated αβ-tubulin dimer. γTuSC was isolated in buffers containing either 20 μM GDP or 20 μM GTP. To remove free nucleotide, we used a rapid (within 45 s) microscale desalting procedure. For comparison, pure αβ-tubulin dimer was diluted into a buffer identical to that containing γTuSC and desalted in parallel. Nucleotide was extracted from the desalted samples and analyzed by mono Q chromatography. To estimate the stoichiometry of bound nucleotide to protein, the amount of γ- and αβ-tubulin in each desalted sample was quantitated by densitometry of Coomassie-stained gel bands relative to αβ-tubulin standards, and the nucleotide concentration was determined by peak integration and comparison with nucleotide standards processed in an identical fashion. Section title: γ-tubulin in γTuSC Preferentially Binds GDP Educational score: 4.365522384643555 Domain: biomedical Document type: Study Language: en Each αβ-tubulin dimer has two guanine nucleotide binding sites, one on each tubulin subunit. Exclusively GTP is bound to α-tubulin at the nonexchangeable or N-site; this nucleotide does not exchange with GTP/GDP in solution and does not undergo hydrolysis. In contrast, β-tubulin binds guanine nucleotide in an exchangeable fashion at the E-site. Both GTP and GDP bind to the E-site with GTP having a three- to fourfold higher affinity than GDP . GTP bound to the E-site does not undergo significant hydrolysis in the absence of polymerization but gets hydrolyzed soon after incorporation into the MT lattice, resulting in GDP that is locked in the lattice and can only exchange after depolymerization . These properties predict that if αβ-tubulin dimer is isolated from buffers containing GDP, then there will be 1 mol GTP (N-site) and 1 mol GDP (E-site) per mole of αβ-tubulin dimer. In contrast, if αβ-tubulin dimer is isolated from buffers containing GTP, under conditions where there is no polymerization, then there will be 2 mol GTP (1 N-site GTP and 1 E-site GTP) per mole of αβ-tubulin dimer. Consistent with these predictions, we recovered 1.1 mol of GTP and 0.8 mol GDP per mole of αβ-tubulin dimer isolated from GDP buffer ; in contrast, we recovered exclusively 2.0 mol of GTP per mole of αβ-tubulin dimer isolated from GTP buffer . These results establish the validity of our assay for comparing the nucleotide-binding properties of γTuSC to those of αβ-tubulin dimer. Section title: γ-tubulin in γTuSC Preferentially Binds GDP Educational score: 4.20980978012085 Domain: biomedical Document type: Study Language: en When γTuSC was similarly analyzed, the nucleotide recovered from γTuSC incubated in GDP buffers was exclusively GDP . Approximately 0.7 mol GDP was recovered per mole of γ-tubulin . The exclusive presence of GDP could be explained at least three ways: (a) the guanine nucleotide binding site on γ-tubulin subunits of γTuSC is freely exchangeable; (b) GDP is locked nonexchangeably into γ-tubulin subunits of γTuSC, analogous to GTP bound at the N-site in α-tubulin; or (c) GDP is locked nonexchangeably into γTuSC as the product of earlier GTP hydrolysis, much like β-tubulin bound GDP within the body of a polymerizing MT. Section title: γ-tubulin in γTuSC Preferentially Binds GDP Educational score: 4.294883728027344 Domain: biomedical Document type: Study Language: en To distinguish between these possibilities, we isolated γTuSC from GTP-containing buffer. Surprisingly, we recovered a greatly reduced amount of nucleotide . Only 0.2 mol guanine nucleotide was recovered per mole of γ-tubulin, indicating that ∼80% of the γ-tubulin was empty at its nucleotide binding site . To ascertain that the GTP in the buffer had not been degraded, we removed an aliquot before desalting and analyzed its nucleotide content. This sample contained the expected amount of GTP and a trace amount of GDP (∼3% of total guanine nucleotide). This amount of GDP was also recovered from a similarly processed control buffer, indicating that it did not arise from hydrolysis by γTuSC or a contaminating GTPase (not shown). The low recovery of guanine nucleotide bound to γTuSC isolated from GTP buffer indicates that GDP is bound exchangeably to γ-tubulin in γTuSC. This result also argues against the theory that the GDP bound to γ-tubulin in γTuSC, isolated from GDP buffer, is being generated by earlier GTP hydrolysis. The recovery of nearly 1 mol GDP per mole of γ-tubulin from GDP buffer and the nearly equivalent amounts of GTP and GDP in the 0.2 mol nucleotide recovered per mole of γ-tubulin from GTP buffer, despite a GTP/GDP ratio ≥30 before desalting, strongly suggest that γ-tubulin in γTuSC has an exchangeable guanine nucleotide binding site that has a much higher affinity for GDP than GTP. To test this further, we isolated γTuSC in buffer containing 20 μM GTP and, 1 h before desalting, added 20 μM GDP. Consistent with our interpretation, we recovered 0.44 mol GDP and 0.02 mol GTP per γ-tubulin monomer (Table II ). Section title: γ-tubulin Complexes in Eukaryotes Educational score: 4.3645243644714355 Domain: biomedical Document type: Study Language: en The γ-tubulin in Drosophila embryo extracts exists in two related complexes of ∼280,000 (γTuSC) and 2,200,000 D (γTuRC). In contrast, when extracts of Xenopus eggs , Xenopus XTC cells , human 293 cells , or mouse fibroblasts were fractionated, only one complex was observed sedimenting at ∼32 S . The significance of this finding is not clear, but might reflect a difference between γ-tubulin–containing complexes isolated from different organisms and cell types. Interestingly, γ-tubulin in the polarized human intestinal epithelial cell line Caco-2 was present in both 10 S and 29 S complexes . In Caco-2 cells, γ-tubulin localizes both to centrosomes and to a diffuse layer beneath the apical membrane . However, it is not yet clear whether the presence of an apical layer of γ-tubulin correlates with the existence of a smaller 10 S complex. Further experiments will be required to determine if the differences in γ-tubulin–containing complexes present in cellular extracts are caused by different extraction conditions, or if they reflect real diversity between systems and cell types in the nature and function of γ-tubulin complexes in vivo. Section title: γ-tubulin Complexes in Eukaryotes Educational score: 4.256619453430176 Domain: biomedical Document type: Study Language: en The protein profile of the purified Drosophila γTuRC is very similar to that of the previously purified Xenopus γTuRC . Indeed, the protein profiles of γ-tubulin complexes immunoprecipitated from a number of sources, including mouse cells after metabolic labeling and sheep brain tubulin preparations , bear a strong resemblance. In addition to molecular similarities, the Drosophila γTuRC also resembles the Xenopus γTuRC in its structure (both complexes appear as open rings when visualized by negative stain electron microscopy). Section title: γTuSC: a Conserved Subcomplex of the γTuRC Educational score: 4.691248893737793 Domain: biomedical Document type: Study Language: en Drosophila γTuRC can be converted to γTuSC by treatment with high salt, suggesting that γTuSC is a structural subunit of γTuRC. A similar dissociation by high salt has been reported for human and Xenopus large γ-tubulin complexes . The hypothesis that γTuSC is a subunit of γTuRC is supported by our finding that purified γTuSC is composed of the three proteins most prominent in γTuRC: γ-tubulin, Dgrip84, and Dgrip91. Dgrip84 and Dgrip91 are members of the Spc97/98p family of proteins. This family includes hGCP2 and hGCP3/ HsSpc98p from humans and Xgrip109 from Xenopus . Homologous ESTs have also been identified in mouse, zebrafish, and rice . Genetic evidence in S. cerevisiae suggests that this family of proteins interacts directly with γ-tubulin . Based on molecular weight estimates and densitometry of Coomassie stained gels, we estimate that Drosophila γTuSC is a heterotetrameric complex containing one Dgrip84, one Dgrip91, and two molecules of γ-tubulin. This stoichiometry is identical to the proposed composition of the S. cerevisiae 6 S complex (Sc γTuSC). Immunoprecipitation experiments with tagged proteins indicate that Sc γTuSC contains one Spc97p, one Spc98p, and two or more molecules of γ-tubulin . Together, these results support the hypothesis that the organization of γ-tubulin into γTuSC is likely to be conserved among all organisms where γ-tubulin is found. One question of fundamental importance that needs to be addressed in the future is how the two γ-tubulin molecules within γTuSC are arranged. Are they arranged in a head to tail dimer as would be suggested by the model proposing that γTuRC is a protofilament of γ-tubulin , or are they arranged in a side-by-side configuration ? Section title: γTuSC: a Conserved Subcomplex of the γTuRC Educational score: 4.2452712059021 Domain: biomedical Document type: Study Language: en In metazoa, γTuSC is further assembled into γTuRC. Based on our hydrodynamic analysis of Drosophila complexes, we estimate that γTuRC contains approximately four to six γTuSCs. An exact determination awaits a more accurate appraisal of the molecular weight of γTuRC, currently being attempted using scanning transmission electron microscopy. It will also be interesting to identify the structural correlate of γTuSC within γTuRC. When viewed by cryo-electron microscopy, γTuRC has a modular structure with ∼13 structural repeats organized in a radial symmetric pattern. Based on our current estimates, we speculate that one γTuSC might correspond to two of the radial symmetric structural repeats visible by cryo-electron microscopy. Section title: Nucleation Activity of γTuSC and γTuRC Educational score: 4.3595476150512695 Domain: biomedical Document type: Study Language: en An important issue with respect to the in vivo roles of γTuSC and γTuRC is their relative MT nucleating activity. The fact that S. cerevisiae does not appear to contain a γTuRC-like complex raises the question of whether Sc γTuSC has nucleating activity or whether it must assemble into a larger structure at the spindle pole body to become active. Conversely, in metazoa it is possible that γTuRC is a storage form for γ-tubulin and it could be γTuSC that nucleates MTs at centrosomes . To begin to address this question, we compared the nucleating activity of peptide-eluted complexes (in which ∼75% of γ-tubulin is γTuRC) to isolated γTuSC at similar concentrations of γ-tubulin. Using both solution and coverslip nucleation assays, we found that both preparations had nucleating activity. However, whereas the solution nucleating activity of the peptide-eluted complexes was robust, typically 80–100-fold above the level of spontaneous nucleation, under similar conditions the level of nucleation for isolated γTuSC was only two- to threefold above background. Thus, per mole of γ-tubulin γTuRC is ∼25 times more active than γTuSC in promoting nucleation. Combining these data with our stoichiometry measurements, we estimate that per mole of complex γTuRC is ∼150 times more active than γTuSC, suggesting that organization of γTuSC into γTuRC facilitates MT nucleation activity. Section title: Nucleation Activity of γTuSC and γTuRC Educational score: 4.34785270690918 Domain: biomedical Document type: Study Language: en We emphasize that the nature of the nucleating activity of γTuSC is still unclear. γTuSC may have intrinsic nucleating activity or it may need to assemble into larger γTuRC-like complexes in order to nucleate MTs. In Xenopus extracts, high-salt dissociated γTuRC components can be reassembled by desalting. This reassembly is blocked by depleting Xgrip109, suggesting that intact γTuSC is required for assembly of γ-tubulin into a γTuRC-like structure . Here we separate γTuSC from the remaining components of γTuRC and do not find any evidence for assembly of larger structures after desalting. This occurrence suggests that γTuSC is required but not sufficient for assembly of a γTuRC-like structure. We cannot exclude the possibility that γTuSC, like αβ-tubulin dimer, assembles into larger complexes when the temperature is raised during nucleation assays. Because the level of activity of γTuSC is very low compared to that of γTuRC, we also cannot rule out the possibility that the nucleating activity of γTuSC depends on trace levels of other γTuRC components that contaminate our γTuSC preps. Expression and purification of the γTuSC will be necessary to further characterize γTuSC activity. Section title: Nucleotide Binding Properties of γ-tubulin Educational score: 4.588440895080566 Domain: biomedical Document type: Study Language: en Cross-linking experiments showed that γ-tubulin in both γTuSC and γTuRC can bind guanine nucleotide. To investigate how γ-tubulin compares to other members of the tubulin family in its nucleotide binding properties, we compared the nucleotide bound to γ-tubulin in γTuSC to that bound to αβ-tubulin dimer after desalting from buffers containing GDP or GTP. We found, like β-tubulin , γ-tubulin in γTuSC binds guanine nucleotide exchangeably. However, in contrast to γ-tubulin, which has about a threefold higher affinity for GTP than GDP , γ-tubulin in γTuSC strongly prefers binding GDP to GTP. Our results suggest that the affinity of γ-tubulin for GTP is much lower than that of β-tubulin, based on nucleotide recovery after desalting under similar conditions . Determination of the absolute affinities of γ-tubulin for GDP and GTP will be important to know whether the affinities of γ- and β-tubulin for GDP are similar. If they are, this will suggest that the strong preference of γ-tubulin for GDP is primarily because of a reduction in its affinity for GTP relative to β-tubulin. These experiments will require a reliable supply of γTuSC, currently limited by antibody availability for immunoisolation and by lack of an expressed source. A structural comparison of the nucleotide binding pocket of γ-tubulin to those of α- and β-tubulin should also be revealing. Development of procedures to prepare more concentrated and highly purified γTuRC should allow a comparison of nucleotide binding by γ-tubulin in γTuRC to that in the γTuSC. If the nucleotide binding properties of γ-tubulin in γTuRC are similar to those of γ-tubulin in γTuSC, it will suggest that GTP hydrolysis by γ-tubulin may not be important for its function in vivo. | Study | biomedical | en | 0.999995 |
10037794 | Section title: Cell Culture Educational score: 4.17070198059082 Domain: biomedical Document type: Study Language: en SV-40 transformed rat embryo cells (REF-2A) were maintained in DME containing 10% newborn calf serum in an atmosphere of 5% CO 2 and 95% air at 37°C. CHO cells were maintained in F12 medium containing 10% fetal bovine serum. REF-2A cells at mitotic and later stages of cell division were prepared as described previously . Briefly, cells were first treated for 3 h with 0.25 μg/ml nocodazole, and mitotic cells (prometaphase) were collected. After washing with ice-cold DME to remove nocodazole, cells were plated in fresh culture dishes and incubated at 37°C in DME containing 10% newborn calf serum to allow cell cycle progression. Mitotic cells recovered spindles at 10–20 min after release of nocodazole arrest, and underwent cytokinesis at 40–60 min. In some experiments, REF-2A cells at each mitotic stage were labeled with 32 P-orthophosphoric acid as described previously . Section title: Antibodies Educational score: 3.959547281265259 Domain: biomedical Document type: Study Language: en The following antibodies against MYPT were used: a polyclonal antibody (pAb; rabbit) raised against the NH 2 -terminal 38 residues of chicken gizzard MYPT, termed Ab 1–38 ; pAb (rabbit) raised against residues 1–296 of gizzard MYPT, affinity purified using the MYPT fragment 1–296, termed Ab 1–296 ; and mAb (IgG1) raised against the gizzard phosphatase holoenzyme . Other antibodies were mAb against the type 1 phosphatase catalytic subunit, PP1c (Transduction Labs.), and mAb against the polyhistidine tag ( Sigma Chemical Co. ). Section title: Protein Preparations Educational score: 4.17955207824707 Domain: biomedical Document type: Study Language: en Trimeric myosin phosphatase was purified from chicken gizzard according to the method of Alessi et al. . Nonmuscle myosin II was purified from bovine lung as described . For the preparation of Ser 19-phosphorylated myosin, purified myosin was incubated for 30 min at 25°C with 10 μg/ml myosin light chain kinase (MLCK) and 5 μg/ml calmodulin in 30 mM Tris-HCl (pH 7.5), 0.1 M KCl, 1 mM MgCl 2 , 0.1 mM CaCl 2 , and 0.1 mM ATP (with or without 0.1 mCi/ml [γ- 32 P]ATP). Phosphorylated myosin was dialyzed extensively against 20 mM Tris-HCl (pH 7.5), 1 M KCl, 1 mM MgCl 2 , and 0.1 mM DTT. Recombinant protein phosphatase type 1 catalytic subunit, α isoform (PP1cα), was purchased from Calbiochem-Novabiochem . Section title: Immunoprecipitation and Immunoblotting Educational score: 4.126550197601318 Domain: biomedical Document type: Study Language: en Immunoprecipitation of MYPT was performed using two different buffers (buffer I and II). Buffer I contains 30 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.5% NP-40, 25 mM NaF, 100 mM sodium pyrophosphate, 50 mM β-glycerophosphate, 1 mM sodium vanadate, 1 mM DTT, 1 mM benzamidine, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 1 mM PMSF. Due to the high salt concentration of buffer I, MYPT was immunoprecipitated without the catalytic subunit. Immunoprecipitation of MYPT with buffer I was used for the myosin binding experiments. Buffer II was a lower ionic strength and was the same as buffer I except for omission of NaF and sodium vanadate, and reduction 25 mM sodium pyrophosphate and 20 mM β-glycerophosphate. Using buffer II, MYPT was immunoprecipitated in the myosin phosphatase complex and was used to determine myosin phosphatase activity. Section title: Immunoprecipitation and Immunoblotting Educational score: 4.145266532897949 Domain: biomedical Document type: Study Language: en Mitotic or interphase REF-2A cells were lysed in either immunoprecipitation buffer I or II. Cells were homogenized with a Dounce homogenizer and clarified by centrifugation at 16,000 g for 15 min. Cell lysates were stored at −80°C. After thawing quickly, the lysates were again centrifuged at 16,000 g for 15 min. Ab 1–296 or Ab 1–38 was added to the supernatants and incubated for 2 h at 4°C. The immunocomplex was precipitated with protein A–Sepharose ( Pharmacia Biotech, Inc. ) during a 1-h incubation. The immunocomplex was washed three times with each buffer, once with PBS, and analyzed by SDS-PAGE followed by Western blotting. The immunoprecipitated MYPT also was used for myosin binding or phosphatase assays. Section title: Immunoprecipitation and Immunoblotting Educational score: 3.8976473808288574 Domain: biomedical Document type: Study Language: en Immunoblotting was performed as follows. Polyvinylidene difluoride (PVDF) membranes were blocked with 5% nonfat dried milk in PBS, and then incubated with the primary antibody (1:1,000 dilution) containing 0.3% BSA in PBS. Immunoreactive bands were detected with peroxidase-conjugated secondary antibody (1:1,000 dilution) using a chemiluminescence method ( New England Nuclear ). Section title: Protein Phosphatase Treatment of MYPT Educational score: 4.122066497802734 Domain: biomedical Document type: Study Language: en Rat MYPT was prepared by immunoprecipitation using buffer I as described above. Half of the immunoprecipitate was treated for 30 min at 30°C with one unit of recombinant serine/threonine phosphatase (PP1cα from rabbit skeletal muscle; Calbiochem-Novabiochem ) in 20 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM MnCl 2 , 2 mM MgCl 2 , 1 mM DTT, 0.1 mg/ml BSA, and 1 mM PMSF. Both treated and untreated samples were analyzed by SDS-PAGE followed by Western blotting. Section title: Phosphorylation of MYPT with Xenopus Egg Extracts Educational score: 4.13892126083374 Domain: biomedical Document type: Study Language: en Mitotic or interphase extracts of Xenopus eggs were used to reconstitute cell cycle-dependent phosphorylation of MYPT. Mitotic extracts were prepared from Xenopus unfertilized eggs in an XB buffer containing 20 mM Hepes (pH 7.7), 0.1 M KCl, 2 mM MgCl 2 , 0.1 mM CaCl 2 , 5 mM EGTA, and 0.1 mg/ml cytochalasin D as described . Interphase extracts were prepared from mitotic extracts by the addition of 0.5 mM CaCl 2 followed by incubation at 20°C for 30 min to inactivate MPF. Section title: Phosphorylation of MYPT with Xenopus Egg Extracts Educational score: 4.174466133117676 Domain: biomedical Document type: Study Language: en Rat MYPT was prepared from interphase REF-2A cells by immunoprecipitation with Ab 1–296 -conjugated Sepharose beads (cross-linked with dimethylpimelimidate; Pierce Chemical Co. ). Buffer I and II were used for myosin binding studies and myosin phosphatase assays, respectively. Purified chick gizzard myosin phosphatase was also bound to the same antibody-conjugated beads. MYPT-bound beads were washed once with XB buffer (without cytochalasin), mixed with an equal volume of mitotic or interphase extracts, and incubated at 25°C for 30 min in the presence of 1 mM ATP (with or without 1 mCi/ml [γ- 32 P]ATP). The beads were washed extensively with buffer I or II, and then subjected to SDS-PAGE, immunoblotting, two-dimensional tryptic phosphopeptide mapping, myosin binding, or phosphatase assays. Section title: Phosphorylation of MYPT with Xenopus Egg Extracts Educational score: 4.164379119873047 Domain: biomedical Document type: Study Language: en A peptide (NH 2 -ISPKEEERKDESPASWRLGLRKC-COOH) corresponding to residues 421–442 of rat MYPT (which corresponds to Val 416-Lys 437 of chicken MYPT) was commercially synthesized (Bio-Synthesis Inc.). The peptide (20 μg) was phosphorylated with Xenopus mitotic extracts in the presence of 1 mCi/ml [γ- 32 P]ATP as described above. Trichloroacetic acid was added to 10% to precipitate proteins and the phosphorylated peptide was recovered by centrifugation in the supernatant. The peptide was then separated by Tricine-SDS-PAGE . The phosphorylated peptide was detected by autoradiography, excised from Tricine-SDS gels, and digested with TPCK-treated trypsin followed by two-dimensional phosphopeptide mapping. Section title: Construction of Mutants of MYPT Educational score: 4.14723539352417 Domain: biomedical Document type: Study Language: en cDNA encoding chicken MYPT304–511 was subcloned into a pQE32 vector (QIAGEN, Inc.) with a hexahistidine tag at the NH 2 terminus as described . NH 2 - and COOH-terminal truncations were made by PCR amplification with pQE32-MYPT304–511 as a template. The sense and antisense primers were designed to contain BamHI and SalI sites at 5′ and 3′ ends, respectively, to ligate the PCR products unidirectionally into the pQE32 vector. After digestion of the PCR products with BamHI and SalI, they were inserted into the BamHI- and SalI-digested pQE32 vector. The truncation mutants obtained were MYPT304–410, MYPT304–444, MYPT421–511, and MYPT432–511. These proteins were expressed in Escherichia coli and purified by a metal affinity column ( Sigma Chemical Co. ) as described . Section title: Construction of Mutants of MYPT Educational score: 4.151509761810303 Domain: biomedical Document type: Study Language: en Site-directed mutagenesis was performed using a QuickChange site-directed mutagenesis kit (Stratagene). For the mutation of Ser 427 to Asp, a forward primer, 5′-GAAGAGAGGAAAGATGAAGATCCTGCTTCGTGGAGGTTAG-3′, and a reverse primer, 5′-CTAACCTCCACGAAGCAGGATCTTCATCTTTCCTCTCTTC-3′, were used. For the mutation of Ser 430 to Glu, a forward primer, 5′-GAAAGATGAATCTCCTGCTGAGTGGAGGTTAGGTCTTCG-3′, and a reverse primer, 5′-CGAAGACCTAACCTCCACTCAGCAGGAGATTCATCTTTC-3′, were used. PCR was performed with pQE32-MYPT304–511 as a template with Pfu polymerase, according to the manufacturer's instructions (Stratagene). Section title: Myosin Binding Assay Educational score: 4.148048400878906 Domain: biomedical Document type: Study Language: en This assay was performed as follows. Rat MYPT was immunoprecipitated from mitotic or interphase cells using Ab 1–296 with buffer I, and eluted from the Sepharose beads by incubating for 2 min with 0.1 M glycine, pH 2.3, containing 0.1 mg/ml BSA, followed by immediate neutralization with 2 M Tris base. The eluted MYPT was mixed with phosphorylated myosin (0.1 μM) in 30 mM Tris-HCl (pH 7.5), 50 mM KCl, 1 mM MgCl 2 , 0.1 mg/ml BSA, and 0.5 mM ATP. Samples were incubated at 4°C for 10 min, and myosin was precipitated by centrifugation at 16,000 g for 10 min. Both pellet and supernatant were added to an equivalent volume of SDS sample buffer and subjected to SDS-PAGE followed by immunoblotting analysis. The amount of MYPT was estimated densitometrically by scanning immunoreactive bands using purified chicken MYPT as a standard. Section title: Myosin Binding Assay Educational score: 4.062808036804199 Domain: biomedical Document type: Study Language: en Myosin binding was also examined with in vitro phosphorylated MYPT. Rat MYPT was immunoprecipitated from interphase cells using buffer I and phosphorylated in vitro with Xenopus mitotic or interphase extracts as described above. Phosphorylated MYPT was eluted from the immunocomplex and used for myosin binding, as described above. Section title: Myosin Phosphatase Assay Educational score: 4.1764373779296875 Domain: biomedical Document type: Study Language: en Rat MYPT was immunoprecipitated from interphase cells using Ab 1–38 with buffer II and phosphorylated (without radioactive ATP) in vitro using Xenopus mitotic or interphase extracts as described above. After extensive washing, immobilized myosin phosphatase was incubated at 30°C with 32 P-labeled myosin (0.5 μM) in 30 mM Tris-HCl (pH 7.5), 0.1 M KCl, 2 mM MgCl 2 , and 0.1 mg/ml BSA. The reaction was terminated by the addition of trichloroacetic acid and BSA to final concentrations of 10% and 3 mg/ml, respectively. After centrifugation, the radioactivities of the supernatants were determined by Cerenkov counting. The reaction time was adjusted so that ∼10–20% of the substrate was dephosphorylated. In some experiments, the gizzard myosin phosphatase was used to examine effects of phosphorylation on myosin phosphatase activity. Section title: Other Procedures Educational score: 4.114519119262695 Domain: biomedical Document type: Study Language: en Two-dimensional tryptic phosphopeptide mapping was performed using cellulose thin layer plates as described in Boyle et al. . Phosphopeptides were detected by autoradiography. SDS-PAGE was performed as described by Blatter et al. using 12.5% polyacrylamide gel and the Laemmli buffer system . Protein concentrations were determined by the method of Bradford using BSA as standard. Section title: Mitosis-specific Phosphorylation of MYPT Educational score: 4.138797283172607 Domain: biomedical Document type: Study Language: en Initially a mitosis-specific modification of MYPT was observed by immunoblotting the mitotic cell lysates with the mAb specific for MYPT. As Fig. 1 a shows, the mAb did not detect a band in the total cell lysates of mitotic cells (lane 4) but the same mAb reacted strongly with MYPT from interphase cells (lane 3). The lack of reactivity in mitotic cell lysates was not due to degradation of MYPT during mitosis because the pAb, Ab 1–38 , reacted equally well with MYPT from either interphase (lane 1) or mitotic (lane 2) cells. In addition, in cells rounded by trypsin treatment the reactivity of MYPT with the mAb was retained (data not shown). Thus it is suggested that the loss of recognition of the mAb for MYPT was due to a modification of MYPT incurred during mitosis. Section title: Mitosis-specific Phosphorylation of MYPT Educational score: 4.2088847160339355 Domain: biomedical Document type: Study Language: en One possibility was that the lack of reactivity to the mAb was due to mitosis-specific phosphorylation. To test this idea, mitotic and interphase MYPT were immunoprecipitated, and divided into two aliquots. One aliquot was used as a control and the other was incubated with PP1cα. The phosphatase treatment completely restored the reactivity of mitotic MYPT with the mAb , indicating that the lack of reactivity to the mAb is due to mitosis-specific phosphorylation. It should also be noted that MYPT from mitotic cells showed a subtle but significant upward shift of mobility on SDS-PAGE (compare lanes 1 and 2 of the lower panel), and that the same phosphatase treatment eliminated this shift (see lanes 2 and 4 of the lower panel). In contrast, incubation with phosphatase of MYPT from interphase cells did not affect its reactivity with the mAb . These results indicate that serine/threonine phosphorylation is responsible for the mitosis-specific modification of MYPT. Section title: Mitosis-specific Phosphorylation of MYPT Educational score: 4.170905113220215 Domain: biomedical Document type: Study Language: en Next, a time course of the mitosis-specific phosphorylation during cell division was examined. Total cell lysates were prepared from interphase cells, mitotic cells, and cells at different stages of cell division (see Materials and Methods). MYPT was analyzed in each preparation by immunoblotting using the same mAb and pAb. Immunoblots with the mAb clearly demonstrated that a mitosis-specific phosphorylation of MYPT occurred and was dependent on the cell cycle stage. As Fig. 1 c (upper panel) shows, mitotic MYPT (lane M) exhibited complete loss of reactivity against the mAb, whereas interphase MYPT (lane I) showed a strong reaction to the same antibody. The lack of reactivity continued until 40 min after release of mitotic arrest and then was recovered at 60 min, at which point cytokinesis occurred. At later phases when the cell was involved in postmitotic spreading (80–180 min), the reactivity of MYPT to the mAb was similar to that of interphase cells. Section title: Mitosis-specific Phosphorylation of MYPT Educational score: 4.095457077026367 Domain: biomedical Document type: Study Language: en The lack of reactivity to the mAb parallels the mobility shift observed with the pAb. An immunoblot with the pAb reveals that MYPT showed a slight upward shift in mobility during mitosis (compare lanes I and M). The upward shift was apparent until 40 min and then was reversed at 60 min. These results again indicate that MYPT was dephosphorylated during cytokinesis. We have also observed similar modifications of MYPT during mitosis of other cells including CHO cells (data not shown). Section title: The Sites of Mitosis-specific Phosphorylation Differ from Those Observed during Interphase Educational score: 4.105630874633789 Domain: biomedical Document type: Study Language: en To examine whether net phosphate incorporation into MYPT was increased during mitosis, MYPT was immunoprecipitated from interphase and mitotic cells after in vivo labeling with 32 P-orthophosphate. As Fig. 2 a shows, the level of phosphorylation of mitotic MYPT (lane 2) is similar to that of interphase MYPT (lane 1). One explanation for the lack of reactivity to the mAb could be that different sites were phosphorylated in the two stages, rather than a net increase in phosphorylation during mitosis. Section title: The Sites of Mitosis-specific Phosphorylation Differ from Those Observed during Interphase Educational score: 4.190939903259277 Domain: biomedical Document type: Study Language: en To examine this possibility, two-dimensional phosphopeptide mapping was performed. MYPT was again immunoprecipitated from mitotic and interphase cells labeled in vivo with 32 P-orthophosphate, digested with trypsin, and analyzed by peptide mapping. Fig. 2 b shows the phosphopeptide maps generated with interphase (I) and mitotic (M) MYPT. To identify which spots are mitosis-specific, a mixture of mitotic and interphase MYPT samples was subjected to peptide mapping (Mix). The map of mitotic MYPT (M) revealed four mitosis-specific phosphopeptide spots (indicated by M1–4). In addition, one spot (M5) showed a considerably higher intensity in mitotic MYPT. On the other hand, the interphase map (I) gives two interphase-specific spots (indicated by I). There are four spots (indicated by C) which were observed commonly in both mitotic and interphase MYPT maps. These results demonstrate that the sites of phosphorylation were different between mitotic and interphase MYPT. We also examined the phosphopeptide pattern of MYPT prepared from cells at 120 min after the release of mitotic arrest. This pattern was identical to that shown with interphase cells (data not shown). This is consistent with the result that the reactivity of MYPT to the mAb at 120 min was similar to that of MYPT from interphase cells. Section title: In Vitro Reconstitution of Mitosis-specific Phosphorylation MYPT Educational score: 4.215060234069824 Domain: biomedical Document type: Study Language: en To further characterize mitosis-specific phosphorylation, an attempt was made to reconstitute the mitosis-specific phosphorylation in vitro. Mitotic Xenopus egg extracts were used as a kinase fraction. MYPT was immunoprecipitated from interphase cultured rat cells using buffer I, and aliquoted into two. One-half was incubated with mitotic Xenopus extracts in the presence of Mg-ATP. As a control, the other half was incubated with interphase Xenopus extracts that had been prepared from mitotic extracts following the addition of Ca 2+ . The incubation with the mitotic extracts eliminated the reactivity of MYPT against the mAb . At the same time, the mobility of MYPT showed an upward shift when compared with the mobility of untreated MYPT (lane 1). In contrast, incubation with the interphase extracts did not alter the reactivity to the mAb nor did it induce the mobility shift (lane 3). These results suggest that Xenopus mitotic extracts are able to reconstitute the mitosis-specific phosphorylation. Section title: In Vitro Reconstitution of Mitosis-specific Phosphorylation MYPT Educational score: 4.15322208404541 Domain: biomedical Document type: Study Language: en To confirm the reconstitution of MYPT, the phosphorylation sites of MYPT were analyzed by two-dimensional phosphopeptide mapping. As indicated in Fig. 2 d, the phosphopeptide map of MYPT phosphorylated in vitro by mitotic Xenopus extracts (X) was similar to that phosphorylated in vivo in mitotic cells . For comparison, a mixture of in vivo and in vitro phosphorylated MYPT was subjected to phosphopeptide mapping . The map of MYPT phosphorylated by Xenopus mitotic extracts showed four (M1–3, M5) out of the five mitotic-specific spots, although two interphase specific spots (I) appeared simultaneously. The spot M4 could be seen only after prolonged exposure of the autoradiograph (data not shown). On the other hand, MYPT phosphorylated by interphase extracts yielded a map similar to that of interphase MYPT (data not shown). Section title: Identification of a Mitosis-specific Phosphorylation Site Educational score: 4.248812675476074 Domain: biomedical Document type: Study Language: en The loss of reactivity to the mAb indicated that the epitope of the mAb may contain a site of mitosis-specific phosphorylation. It was known that the epitope to the mAb was between residues 371 and 511 (Hartshorne, D.J., unpublished results) and to define more precisely the epitope a series of truncation mutants was analyzed. In Fig. 3 a it is shown that fragment 421–511 has a positive reaction with the mAb while 432–511 was negative, indicating that the epitope is localized between residues 421 and 432. There are two serines (no threonine) in this sequence, Ser 427 and Ser 430 , and these two residues were mutated to Asp and Glu, respectively. The resultant point mutants were expressed in bacteria, and the reactivities of these two mutants to the mAb were examined by immunoblotting. It was found that mutation of Ser 430 to Glu (S430E) resulted in complete loss of reactivity . In contrast, the mutant replacing Ser 427 with Asp (S427D) still showed a strong reactivity to the mAb, though the reactivity was weaker than the control . A reasonable conclusion from these results is that Ser 430 of MYPT is a site phosphorylated during mitosis, although it is possible that simultaneous phosphorylation at Ser 427 may also occur. Section title: Identification of a Mitosis-specific Phosphorylation Site Educational score: 4.259956359863281 Domain: biomedical Document type: Study Language: en To further test whether Ser 430 is one of the mitosis-specific phosphorylation sites, a peptide containing Ser 430 (from residues 421–442 of rat MYPT) was synthesized and phosphorylated in vitro using Xenopus mitotic extracts. The peptide was digested with trypsin and subjected to two-dimensional phosphopeptide mapping. A map from the phosphorylated synthetic peptide yielded several spots, apparently due to incomplete trypsin digestion (the peptide has multiple lysine and arginine residues). To examine whether any of these spots matched the mitotic spots, the map generated from the phosphorylated synthetic peptide was compared with a map from MYPT phosphorylated with Xenopus extracts (X), and with a map of a mixture from the peptide and MYPT samples (Mix). It was found that two major spots (indicated by arrows) of the map generated from the synthetic peptide correspond to two mitotic spots, M2 and M3. These results, together with the mutational analyses, indicate that the spots, M2 and M3, are derived from mitosis-specific phosphorylation at Ser 430. Again, there is the possibility that Ser 427 may also be phosphorylated. It should be noted that there are other mitosis-specific phosphorylation sites corresponding to the mitotic spots of M1, M4, and M5. Section title: Increased Myosin Binding Ability of Mitotic MYPT and Higher Phosphatase Activity Shown by Mitotic Myosin Phosphatase Educational score: 4.122422218322754 Domain: biomedical Document type: Study Language: en To explore the functional significance of mitosis-specific phosphorylation of MYPT, the myosin binding activities of MYPT from mitotic or interphase cells were compared. Mitotic and interphase MYPT were immuno-affinity purified, and their myosin binding abilities were examined using phosphorylated myosin in the presence of Mg-ATP, as described in Materials and Methods. As Fig. 4 a shows, the amount of mitotic MYPT bound to phosphorylated myosin was about threefold higher than MYPT from interphase cells. Section title: Increased Myosin Binding Ability of Mitotic MYPT and Higher Phosphatase Activity Shown by Mitotic Myosin Phosphatase Educational score: 4.186654090881348 Domain: biomedical Document type: Study Language: en Because the quantity of MYPT isolated from mitotic cells was limited, the in vitro reconstitution system was used to prepare mitotic and interphase MYPT, allowing a more extensive evaluation of the myosin-binding properties of MYPT, i.e., to use a wider range of MYPT concentrations. Rat MYPT immunoprecipitates (using buffer I) were phosphorylated by either mitotic or interphase extracts, as described above, and then MYPT was eluted from the immunocomplexes. Varying concentrations of eluted MYPT (2–25 nM) were mixed with phosphorylated myosin in the presence of Mg-ATP and their binding was examined. As Fig. 4 b shows, the amount of MYPT bound to phosphorylated myosin was two to three times higher using MYPT phosphorylated with mitotic extracts compared to the MYPT phosphorylated by interphase extracts. At 25 nM MYPT, it was found that ∼68% of MYPT phosphorylated by mitotic extracts bound to myosin while only 31% of MYPT phosphorylated by interphase extracts bound to myosin. These results support the data obtained for in vivo phosphorylation of MYPT . Section title: Increased Myosin Binding Ability of Mitotic MYPT and Higher Phosphatase Activity Shown by Mitotic Myosin Phosphatase Educational score: 4.2081499099731445 Domain: biomedical Document type: Study Language: en The enhanced myosin binding activity of mitotic MYPT suggested that myosin phosphatase activity may be increased during mitosis. To test this possibility, MYPT was immunoprecipitated using buffer II (to retain the catalytic subunit in complex with MYPT), phosphorylated with either Xenopus mitotic or interphase extracts, and used to assay phosphatase activities. Again, the MYPT phosphorylated with Xenopus mitotic extracts showed loss of reactivity to the mAb , indicating that mitosis-specific phosphorylation of MYPT occurred. It was also confirmed, by immunoblotting with the pAb against MYPT and with the mAb to PP1c , that essentially identical amounts of MYPT and the catalytic subunit were present in the immunoprecipitates treated with mitotic or interphase Xenopus extracts. As Fig. 5 b shows, the myosin phosphatase treated with mitotic extracts had approximately twice the activity than that treated with interphase extracts. Similar results were obtained with chick gizzard myosin phosphatase (data not shown). These results indicate that the enhanced myosin binding ability was accompanied by a higher phosphatase activity. Section title: Discussion Educational score: 4.283860206604004 Domain: biomedical Document type: Study Language: en In this paper we have demonstrated that MYPT is phosphorylated in a mitosis-specific way, and that phosphorylation increases its myosin binding ability, as well as myosin phosphatase activity. The activation of myosin phosphatase by mitosis-specific phosphorylation is unique as it provides a positive regulatory mechanism for myosin phosphatase distinct from previous reports on phosphorylation of MYPT. For example, phosphorylation by Rho-kinase or by endogenous kinase was reported to inhibit the activity of myosin phosphatase. Phosphorylation by protein kinase A was found to decrease the association of MYPT with lipids , which was suggested as a mechanism to regulate association of MYPT with membranes. Activation of myosin phosphatase via the cGMP-dependent protein kinase has been proposed but the mechanism of activation has not been established. Section title: Mechanism of Activation of Myosin Phosphatase by Mitosis-specific Phosphorylation Educational score: 4.424591541290283 Domain: biomedical Document type: Study Language: en Ser 430 (which corresponds to Ser 435 in rat MYPT) was identified as one of the mitosis-specific phosphorylation sites. It should be noted that the sequence surrounding Ser 430 is well conserved among different species including human, rat, and chicken, although Ser 430 is changed to Thr 435 in humans . In fact, chicken and rat MYPT have an identical sequence of 42 amino acids around this phosphorylation site. There are two genes that express MYPT, denoted MYPT1 and MYPT2 . The isoform examined in this article is MYPT1, and MYPT2 is found in heart and brain. The putative mitosis-specific phosphorylation site also is found in MYPT2 at Ser 437. The sequence around this site, 428–447 of MYPT2 is 90% identical to 421–440 of MYPT1. Thus, it is possible that phosphorylation of this site on MYPT is a general mechanism for activation of myosin phosphatase activity. It is interesting that Ser 430 does not have a consensus sequence for cdc2 kinase. Although cdc2 kinase can phosphorylate MYPT in vitro, a phosphopeptide pattern generated by cdc2 is different from the peptide map of MYPT phosphorylated in vivo during mitosis (data not shown). Likewise, NIM A kinase does not seem to be responsible because a phosphopeptide map generated by NIM A kinase is quite different from the mitotic pattern (data not shown). Clearly, identification of the kinase responsible for the mitosis-dependent phosphorylation is a priority for future studies. Section title: Mechanism of Activation of Myosin Phosphatase by Mitosis-specific Phosphorylation Educational score: 4.53242301940918 Domain: biomedical Document type: Study Language: en It is not clear at present how mitotic phosphorylation influences the binding of MYPT to myosin. One of the mitosis-specific phosphorylation sites, Ser 430 (Ser 427 may be phosphorylated simultaneously) is located toward the middle of the MYPT molecule. There is controversy regarding the location of the myosin-binding sites on MYPT. It has been reported that the ankyrin repeats at the NH 2 -terminal portion of MYPT are involved and also that a COOH-terminal sequence is implicated . Neither part of the molecule is close (in terms of linear sequence) to Ser 430. Therefore, a direct influence of Ser 430 on either of the putative myosin-binding sequences is not possible unless the MYPT molecule bends, or folds, to accommodate such an interaction. It is possible that other mitosis-specific phosphorylation sites on MYPT may be close to one of the myosin binding sites and contribute to the myosin-binding effect. Alternatively, phosphorylation of Ser 430 may induce a longer-range conformational change at the myosin-binding site. Clearly, the solution to this problem requires the identification of all of the mitosis-specific phosphorylation sites as well as the region(s) of MYPT involved in interaction with myosin. Section title: Mechanism of Activation of Myosin Phosphatase by Mitosis-specific Phosphorylation Educational score: 4.235708236694336 Domain: biomedical Document type: Study Language: en It has been reported that cdc2 kinase phosphorylates PP1c, and that phosphorylation inhibits its activity toward phosphorylase a . This seems to be contradictory to the results presented here. However, PP1c is involved in the dephosphorylation of several proteins, and the activity toward each protein is dependent on various targeting molecules. For example, the activity of PP1c toward phosphorylase a is considerably decreased in the presence of MYPT . The presence of target molecules may also affect the accessibility of PP1c for phosphorylation by cdc2 kinase. It is thus possible that phosphorylation of PP1c by cdc2 kinase may not inhibit myosin phosphatase activity during mitosis. This is based on the following two reasons. First, an activation of myosin phosphatase, rather than inhibition, was found following phosphorylation with the Xenopus mitotic extracts . If phosphorylation of PP1c by cdc2 kinase and resultant inhibition was a dominant mechanism it should have been detected in these experiments. Second, the phosphorylation of PP1c of chick myosin phosphatase by Xenopus mitotic extracts was not observed (data not shown). Section title: Physiological Significance Educational score: 4.350358009338379 Domain: biomedical Document type: Study Language: en Mitosis-specific phosphorylation of MYPT may play a significant role in the regulation of microfilament reorganization during cell division of cultured cells. The enhanced myosin phosphatase activity would increase the probability for dephosphorylation of RMLC during prophase, leading to the disassembly of stress fibers and cell rounding. This notion is consistent with our previous results showing that Ser 19 phosphorylation is decreased when cells enter mitosis . The increased activity of myosin phosphatase and resultant disassembly of stress fibers also are compatible with the localization of MYPT during mitosis. MYPT was reported to show a diffuse localization during mitosis while it is associated with microfilament structures such as stress fibers and adhesion belts during interphase . Section title: Physiological Significance Educational score: 4.655574798583984 Domain: biomedical Document type: Study Language: en In addition, the reversal of mitosis-specific phosphorylation, i.e., dephosphorylation, during cytokinesis negates the activation of myosin phosphatase and thus would favor a higher level of Ser 19 phosphorylation for the activation of contractile rings. This idea again is consistent with previous results in that the phosphorylation sites on RMLC change from S1/2 to Ser 19 during cytokinesis . This is supported by our recent immunolocalization data using a Ser 19 phosphorylation specific antibody, in which prometaphase cells showed a lower level of Ser 19 phosphorylation than cells at telophase . These changes in phosphatase activity would explain a previous observation by Fishkind et al. that microinjection of a catalytic fragment of MLCK (constitutively active MLCK) delayed the onset of anaphase but did not alter the rate of progression of cytokinesis. Perhaps, the increased activity of myosin phosphatase during prometaphase could counteract phosphorylation of Ser 19 by the catalytic fragment of MLCK before cytokinesis. The decrease in myosin phosphatase activity during cytokinesis would then be able to regulate cleavage furrow contraction. If these speculations are correct, then the mitosis-specific phosphorylation of MYPT may play a pivotal role in the control of cell division. Section title: Physiological Significance Educational score: 4.56417179107666 Domain: biomedical Document type: Study Language: en The positive regulatory mechanism of myosin phosphatase described above contrasts the negative regulation by phosphorylation of MYPT with Rho-kinase. It is possible that both mechanisms collaborate to regulate the massive reorganization of microfilaments during cell division. Our current model for regulation of myosin phosphatase during cell division is shown in Fig. 6 . This incorporates two phosphorylation steps: an activation via the mitosis-specific kinase(s), and an inhibition via Rho-kinase . It is therefore reasonable to suggest that there are at least two functional phosphorylation sites to reflect the positive and negative regulatory effects. When cells enter prophase it is proposed that the mitosis-specific phosphorylation occurs (via unknown kinase) and this causes activation of myosin phosphatase and a decrease in the level of myosin phosphorylation (at Ser 19). The result is disassembly of stress fibers and cell rounding. On exit from mitosis and before, or during cytokinesis, the activating site(s) on MYPT are dephosphorylated. At the same phase of the cell cycle it is suggested that inhibition of myosin phosphatase occurs via phosphorylation of MYPT by Rho-kinase. Here the next result would be an increase in the level of myosin phosphorylation (at Ser 19) and activation of myosin for cell division. There are important components of this scheme that must be identified before a plausible mechanism can be established, these include the kinase(s) and phosphatase(s) involved at the mitosis-specific stage. | Study | biomedical | en | 0.999995 |
10037795 | Section title: Materials Educational score: 1.5071810483932495 Domain: biomedical Document type: Other Language: en U46619 was from Cayman Chemical, thrombin, histone (subgroup f2b), monoclonal anti-myosin light chain (MLC) antibody, and fluorescein isothiocyanate (FITC)-phalloidin were from Sigma . Sp-5,6-DCl-cBIMPS and 8-pCPT-cGMP were from Biolog. Y-27632 was kindly provided by Yoshitomi Pharmaceutical Industries, Clostridium botulinum C3-exoenzyme was a donation from I. Just and K. Aktories (both from University of Freiburg, Freiburg, Germany) or was purchased from Upstate Biotechnologies, anti-pp72 syk antibodies as well as anti-phosphotyrosine antibodies were from Santa Cruz Biotechnology , and anti-pp60 v-src antibodies were from Oncogene. Antisera against G protein α-subunits have been described . Section title: Platelet Preparation and Aggregation Educational score: 4.154228210449219 Domain: biomedical Document type: Study Language: en Whole blood was collected from normal and Gα q -deficient mice anesthetized with pentobarbital by puncturing the inferior vena cava with heparinized syringes at a final concentration of 25 U heparin/ml blood. The blood from three or four Gα q -deficient mice and wild-type mice was pooled for each platelet aggregation experiment. Blood was diluted with half the volume of Hepes-Tyrode-buffer (134 mM NaCl, 0.34 mM Na 2 HPO 4 , 2.9 mM KCl, 12 mM NaHCO 3 , 20 mM Hepes, 5 mM glucose, 1 mM MgCl 2 , pH 7.3), and platelet rich plasma (PRP) was obtained by centrifugation for 7.5 min at 250 g . Thereafter, prostacyclin at a final concentration of 300 nM was added to the PRP, and platelets were pelleted by centrifugation at 1,200 g for 5 min. The platelet pellet was resuspended in Hepes-Tyrode buffer and incubated for 30 min at 37°C. Platelet suspension was adjusted to 300,000 platelets per microliter with Hepes-Tyrode buffer. Optical aggregation experiments were conducted in a four-channel aggregometer . Preincubation in Hepes-Tyrode buffer without and with cGMP and cAMP analogues and Y-27632 was performed for 20 min at room temperature. Immediately before the aggregation experiments, platelets were preincubated for 1 min at 37°C in Hepes-Tyrode buffer containing 1 mM CaCl 2 . Section title: Photolabeling of Membrane Proteins and Immunoprecipitation of Gα-subunits Educational score: 4.160181045532227 Domain: biomedical Document type: Study Language: en Platelet membranes were prepared and photolabeled as described . In brief, cell membranes (50–100 μg of protein per assay tube) were incubated at 30°C in a buffer containing 0.1 mM EDTA, 10 mM MgCl 2 , 30 mM NaCl, 1 mM benzamidine, and 50 mM Hepes-NaOH, pH 7.4. After 3 min of preincubation in the absence and presence of receptor agonist, samples were incubated for another 15 min with 10–20 nM [α- 32 P]GTP azidoanilide (130 kBq per tube). [α- 32 P]GTP azidoanilide was synthesized and purified as described . For photolabeling of G i α-subunits, 5 μM GDP was present in the incubation buffer. Samples were washed, dissolved in labeling buffer, and then irradiated as described . Photolabeled membranes were pelleted and proteins were predenatured in SDS. Solubilized membranes were preabsorbed with protein A–Sepharose beads, and immunoprecipitation was done as described . Section title: SDS-PAGE and Immunoblotting Educational score: 3.9214017391204834 Domain: biomedical Document type: Study Language: en SDS-PAGE of photolabeled proteins was performed on 10% (wt/vol) acrylamide gels. Photolabeled membrane proteins were visualized by autoradiography of the dried gels. Blotting of membrane proteins separated by SDS-PAGE, processing of immunoblots, and detection of immunoreactive proteins by chemiluminescence procedure ( Amersham ) has been described . Section title: Determination of Cellular cAMP Levels Educational score: 4.169122695922852 Domain: biomedical Document type: Study Language: en Platelets (10 8 per tube) were preincubated for 15 min with 300 μM 3-isobutyl-1-methylxanthine and 20 μM 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone and incubated for 20 min in the absence and presence of receptor agonists. The reaction was stopped by the addition of 300 μl of ice-cold 10% (wt/vol) trichloracetic acid. Samples were kept for 10 min on ice, and 180 μl of 1 M Tris, pH 9.8, was added to neutralize the sample. Cyclic AMP was determined by the competitive-binding assay . In brief, samples were incubated for 2 h with 2 pmol of [8- 3 H]cAMP (925 Gbq/mmol; Amersham ) and 62.5 μg of cAMP-dependent protein kinase purified from porcine heart ( Sigma ) in a final volume of 200 μl at 4°C. Then, 4% (wt/vol) charcoal in 5 mM EDTA and 50 mM Tris-HCl, pH 7.5, was added, and samples were immediately centrifuged for 2 min at 12,000 g . Supernatants were counted in a liquid scintillation counter, and the amount of cAMP in the test sample was calculated as described . Section title: Determination of Tyrosine Phosphorylation Educational score: 4.1056084632873535 Domain: biomedical Document type: Study Language: en Isolated platelets (1–2 × 10 7 platelets per tube) were incubated in 40 μl Hepes-Tyrode buffer at 37°C as indicated. Reactions were stopped by addition of 20 μl of 3× sample buffer containing a final concentration of 1 mM Na 3 VO 4 . Heated samples were separated by SDS-PAGE on 10% gels. Immunoblotted proteins were analyzed for phosphotyrosine with an antiphosphotyrosine antibody. Section title: Immunoprecipitation and Immune Complex Kinase Assay Educational score: 4.279313564300537 Domain: biomedical Document type: Study Language: en For immunoprecipitation of tyrosine kinases pp72 syk and pp60 c-src , platelet suspensions (0.4–1 × 10 9 platelets) were incubated in the absence or presence of 5 μM U46619 for the indicated time periods, and platelets were lysed by addition of an equal volume of ice-cold 2× radioimmunoprecipitation assay (RIPA) buffer (final concentration: 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, 150 mM NaCl, 50 mM Hepes/NaOH, pH 7.4, 3 mM EDTA, 3 mM EGTA, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin, 20 μg/ml leupeptin). After incubation for 20 min on ice, samples were centrifuged for 20 min at 15,000 g at 4°C, and incubated with 5 μg agarose conjugates of rabbit polyclonal anti-pp72 syk IgG or 8 μl of agarose-conjugated mouse monoclonal anti-pp60 v-src IgG 1 for 2 h at 4°C. Immunoprecipitates were collected by centrifugation at 15,000 g for 10 min at 4°C and were washed twice with 1× RIPA buffer, once with 1% Triton X-100, 0.3% SDS, 600 mM NaCl, and 50 mM Tris-HCl, pH 7.4, and once with 300 mM NaCl, 10 mM EDTA, 100 mM Tris-HCl, pH 7.4. For detection of pp72 syk phosphorylation, precipitated proteins were eluted with 40 μl of 1× SDS sample buffer and separated by 10% polyacrylamide gels. Tyrosine phosphorylation of pp72 syk and pp72 syk protein were analyzed by immunoblotting. The anti-pp60 c-src immunoprecipitates were divided into two aliquots; one was analyzed by anti-pp60 c-src immunoblotting, and the other was subjected to in vitro kinase assay. To examine in vitro kinase activity, precipitates were incubated for 5 min at 25°C in kinase buffer containing 25 mM Hepes/ NaOH, pH 7.4, 10 mM MnCl 2 , 1 μM ATP (7 μCi of [γ- 32 P]ATP/tube), and 0.25 mg/ml histone. Reaction was terminated by addition of 2× sample buffer, and samples were subjected to SDS-PAGE. Phosphorylation of histone was analyzed by autoradiography of dried gels. Section title: Scanning Electron Microscopy Educational score: 4.170424461364746 Domain: biomedical Document type: Study Language: en Isolated platelets were preincubated under the indicated conditions. Thereafter, platelets were incubated in the absence or presence of 1 U/ml thrombin or 5 μM U46619 for 5 s at 37°C and then fixed for 10 min with 3% paraformaldehyde, 3.75% glutaraldehyde, 0.06 mM cacodylate buffer, and 3.4 mM CaCl 2 . The fixed platelets were suction filtered onto polycarbonate filters (0.45 μm; Nucleopore) which had been preincubated with 10 μg/ml polylysine. Filters were washed three times with 0.9% NaCl and dehydrated stepwise in aqueous ethanol. After exchange of ethanol for hexadimethyldisilazane, samples were air-dried and sputtered with gold. Scanning electron microscopy was carried out on a Zeiss -Gemini instrument using a beam voltage of 5 kV. Section title: MLC Phosphorylation Educational score: 4.168922424316406 Domain: biomedical Document type: Study Language: en MLC phosphorylation was determined as described . Isolated platelets (1–2 × 10 7 platelets per tube) were incubated in 30 μl Hepes-Tyrode buffer at 37°C as indicated. Reactions were stopped by addition of 30 μl of 40% (vol/vol) perchloric acid. Precipitated samples were kept on ice for 20–30 min. After centrifugation (10 min at 15,000 g at 4°C) pellets were washed twice with acetone containing 10 mM DTT. 30 μl of SDS sample buffer was added to dried samples, and proteins were solubilized by sonication for 30 min. Separation of proteins on urea/glycin gels was done as described , and MLC was detected after immunoblotting with an anti-MLC antibody. Section title: Determination of F-actin Content Educational score: 4.126839637756348 Domain: biomedical Document type: Study Language: en For actin filament content measurements, platelets (10 8 ) were incubated as indicated and fixed in 2% paraformaldehyde for 30 min at 37°C. Fixed platelets were permeabilized with 0.1% Triton X-100, incubated with 10 μM fluorescein isothiocyanate (FITC)-phalloidin ( Sigma ) for 30 min at room temperature and were then washed. Bound FITC-phalloidin was quantified using a fluorescence spectrophotometer ( Perkin-Elmer ) (excitation at 495 nm; emission at 519 nm). Section title: ADP Ribosylation of Platelet Lysates by C3 Exoenzyme Educational score: 4.105961322784424 Domain: biomedical Document type: Study Language: en Washed platelets were incubated with the indicated concentrations of C3 exoenzyme in Hepes-Tyrode buffer. Platelets were lysed by addition of an equal volume of lysis buffer (1.5% Triton X-100, 0.8% DOC, 0.2% SDS, 145 mM NaCl, 20 mM Hepes, pH 7.4, 3 mM EGTA, 0.3 mM phenylmethylsulfonyl fluoride, 5 μM leupeptin, 5 μg/ml aprotinin). ADP-ribosylation using [ 32 P]NAD was performed as described , and ribosylated samples were separated on 12% polyacrylamide gels. Section title: Results Educational score: 4.481020927429199 Domain: biomedical Document type: Study Language: en We have recently shown that Gα q -deficient platelets do not aggregate and secrete their granule contents in response to various stimuli indicating that G q -mediated activation of phospholipase C represents the central early signal transduction process leading to full platelet activation. However, G q -deficient platelets were still able to undergo ligand-induced platelet shape change. This suggests that G proteins other than G q mediate the platelet shape change response. Shape change induced by the TXA 2 analogue U46619 could be observed in Gα q -deficient platelets by scanning electron microscopy of single cells as well as by measuring the light transmission of a platelet suspension . Shape change induced by U46619 in Gα q -deficient platelets and wild-type platelets was blocked by the cAMP analogue Sp-5,6-DCl-cBIMPS but not by the cGMP analogue 8-pCPT-cGMP, whereas both cyclic nucleotides blocked aggregation in wild-type platelets . Similar results were observed with thrombin-activated wild-type and Gα q -deficient platelets (data not shown). Preincubation of platelets with the recently described Rho-kinase inhibitor Y-27632 blocked U46619-induced shape change both in wild-type and Gα q -deficient platelets . To assess the role of Rho in agonist-induced platelet shape change we preincubated platelets for 2 h with 50 μg/ml C3 exoenzyme which ADP ribosylates and inactivates the small GTPase Rho . This C3 exoenzyme concentration and preincubation time resulted in ADP-ribosylation of 70–75% of endogenous Rho as determined by the inability of C3 exoenzyme to [ 32 P]ADP-ribosylate Rho in subsequently prepared cell lysates . Longer preincubation times and higher C3 exoenzyme concentrations further increased the ADP-ribosylated fraction of Rho , but resulted in preactivation of platelets (data not shown). C3-pretreated platelets showed markedly reduced shape change in response to U46619 with only partial spheration and occasional filopodia formation . Section title: Results Educational score: 4.151824951171875 Domain: biomedical Document type: Study Language: en Since platelet shape change including protrusion of filopodia and lamellipodia is accompanied by actin polymerization we measured F-actin content in wild-type and Gα q -deficient platelets . U46619 induced an increase in F-actin content of both, wild-type and Gα q -deficient platelets, which could be completely blocked by Y-27632. Reduction of the amount of functional Rho by pretreatment with 50 μg/ml C3 exoenzyme for 2 h markedly reduced the effect of U46619 in wild-type and Gα q -deficient platelets. Section title: Results Educational score: 4.488315105438232 Domain: biomedical Document type: Study Language: en To identify the G proteins mediating receptor-induced platelet shape change we studied the coupling of TXA 2 and thrombin receptors to heterotrimeric G proteins in wild-type and Gα q -deficient mouse platelets. Receptors for both, thrombin and TXA 2 , have been shown to be able to couple to members of the G q , G 12 , and G i families . In membranes of human platelets, receptors activated by thrombin couple to G q , G 12 , G 13 , and G i , whereas TXA 2 receptors only activate G q , G 12 , and G 13 . Photolabeling of receptor-activated G proteins in mouse platelet membranes and subsequent immunoprecipitation of individual G protein α-subunits showed that in wild-type mouse platelets, activated TXA 2 and thrombin receptors couple to G q , G 12 , and G 13 , whereas G i was only activated through the thrombin receptor . Similarly, in membranes from Gα q -deficient platelets, only G 12 and G 13 were activated through the TXA 2 receptor, whereas activated thrombin receptors coupled to G 12 , G 13 , and G i . Coupling of thrombin receptors to G i in murine platelets corresponded with the ability of thrombin to decrease cAMP levels in wild-type as well as in Gα q -deficient platelets, whereas activation of TXA 2 receptors had no effect on adenylyl cyclase activity in wild-type or Gα q -deficient platelets (data not shown). These data clearly demonstrate that in Gα q -deficient platelets thrombin-receptors couple to G 12 , G 13 , and G i , whereas only G 12 and G 13 are activated through TXA 2 receptors. Consequently, effects which can still be induced by TXA 2 receptor agonists in Gα q -deficient platelets like the shape change response are mediated by G 12 and/or G 13 . TXA 2 -activated Gα q -deficient platelets therefore represent a model to study of G 12 /G 13 -mediated signaling processes. Section title: Results Educational score: 4.346728324890137 Domain: biomedical Document type: Study Language: en Agonist-induced platelet activation results in tyrosine phosphorylation of multiple proteins . Phosphorylation of these proteins occurs in three temporal phases which have been experimentally distinguished. Early tyrosine phosphorylation occurs by an integrin-independent mechanism, whereas the second and third wave of tyrosine phosphorylation depends on the aggregation of platelets through binding of fibrinogen to α IIb β 3 -integrin (glycoprotein IIb-IIIa) . In Gα q -deficient platelets that do not aggregate in response to thrombin or U46619, only a subset of proteins became tyrosine phosphorylated upon exposure of platelets to both stimuli compared with wild-type platelets . Most prominently, a rapid tyrosine phosphorylation of a protein of ∼72 kD could be observed in Gα q -deficient platelets activated with thrombin and U46619. In contrast, several proteins with relative molecular masses of 40 and 95–130 kD which were tyrosine phosphorylated in wild-type platelets did not show increased tyrosine phosphorylation in activated Gα q -deficient platelets . Section title: Results Educational score: 4.373139381408691 Domain: biomedical Document type: Study Language: en Platelets contain several tyrosine kinases among which pp72 syk and pp60 c-src are rapidly activated after stimulation of platelets in an aggregation-independent manner . To test whether the 72-kD protein that was tyrosine phosphorylated in response to U46619 and thrombin in Gα q -deficient platelets represented pp72 syk , we immunoprecipitated pp72 syk from lysates of platelets exposed to U46619. Anti-phosphotyrosine immunoblots of pp72 syk immunoprecipitates demonstrated increased tyrosine phosphorylation of pp72 syk in response to U46619 in Gα q -deficient platelets as well as in wild-type platelets . Autophosphorylation of pp72 syk on tyrosine has been demonstrated to increase its enzymatic activity . Fig. 6 C shows that incubation of wild-type and Gα q -deficient platelets with U46619 also resulted in a rapid increase in the activity of pp60 c-src . Increases in tyrosine kinase activity could be observed within 10 s after addition of U46619 and were not affected by pretreatment of platelets with Y-27632 or C3 exoenzyme (data not shown). These data indicate that TXA 2 receptor-mediated activation of G 12 /G 13 leads to rapid activation of the tyrosine kinases pp72 syk and pp60 c-src in mouse platelets. Section title: Results Educational score: 4.5135087966918945 Domain: biomedical Document type: Study Language: en MLC phosphorylation has been suggested to be involved in early processes during platelet activation . To test whether TXA 2 receptor-G 12 /G 13 – mediated signaling in Gα q -deficient platelets resulted in MLC phosphorylation, we activated platelets with U46619 for different times and separated phosphorylated and unphosphorylated MLC on urea/glycin polyacrylamide gels. Separated proteins were blotted onto nitrocellulose filters and MLC was detected using a specific antiserum. Fig. 7 A shows that U46619 caused phosphorylation of the total detectable pool of MLC in wild-type platelets within 10 s. Interestingly, a rapid and apparently complete phosphorylation of MLC was also observed in Gα q -deficient platelets activated by U46619. Chelation of extracellular Ca 2+ by EGTA or preincubation of platelets with various tyrosine kinase inhibitors had no effect on U46619-induced MLC phosphorylation in wild-type and Gα q -deficient platelets (data not shown). Although the cAMP analogue Sp-5,6-DCl-cBIMPS completely inhibited MLC phosphorylation in wild-type and Gα q -deficient platelets the cGMP analogue 8-pCPT-cGMP was without effect . In smooth muscle cells and fibroblasts, the phosphorylation state of MLC has been shown to be under dual control of the Ca 2+ /calmodulin-activated myosin light chain kinase (MLCK) as well as of myosin-phosphatase . Myosin-phosphatase has been demonstrated to be regulated by Rho/Rho-kinase . Since U46619-induced platelet shape change was blocked by the Rho-kinase inhibitor Y-27632 and was greatly inhibited after reduction of the amount of active Rho by C3 exoenzyme we tested the effect of C3 exoenzyme and Y-27632 on U46619-induced phosphorylation of MLC. Fig. 7 , D–F shows that Y-27632 blocked and C3 exoenzyme markedly inhibited U46619-induced MLC-phosphorylation in wild-type as well as in Gα q -deficient platelets. Incomplete inhibition of MLC phosphorylation by C3 exoenzyme was most likely due to incomplete inactivation of Rho by C3 exoenzyme . Y-27632 exerted its inhibitory effect on receptor-induced MLC phosphorylation with higher potency in Gα q -deficient platelets compared with wild-type platelets . Similarly, the effect of C3 exoenzyme appeared to be more pronounced in the absence of Gα q . These data indicate that activation of G 12 /G 13 through the TXA 2 receptor results in MLC phosphorylation and that this process involves Rho/Rho-kinase. The data also provide further evidence for the concept that MLC phosphorylation underlies platelet shape change. Section title: Discussion Educational score: 4.587472915649414 Domain: biomedical Document type: Study Language: en Full platelet activators like TXA 2 and thrombin function through G protein–coupled receptors which activate G q , G 12 , G 13 , and G i type G proteins . Gα 11 , a close homologue of Gα q and coexpressed with Gα q in most cells, is not present in platelets . In Gα q -deficient platelets, the TXA 2 mimetic U46619 and thrombin fail to induce platelet aggregation and degranulation. This is accompanied by a lack of phospholipase C activation and Ca 2+ mobilization after TXA 2 and thrombin receptor activation supporting the concept that G q -mediated phospholipase C activation represents the main signaling process leading to full platelet activation . Lack of Gα q -mediated phospholipase C activation did not interfere with the ability of U46619 and thrombin to induce platelet shape change as shown by scanning electron microscopy of activated Gα q -deficient platelets and measurement of light transmission through a suspension of Gα q -deficient platelets . Thus, induction of platelet shape change through receptors of different platelet stimuli is mediated by G proteins other than G q , and Gα q -deficient platelets provide a good model to study the mechanisms underlying receptor-induced shape change independently of secondary processes involving secretion and aggregation. Section title: Discussion Educational score: 4.375861644744873 Domain: biomedical Document type: Study Language: en To identify the G proteins mediating platelet shape change we studied the coupling of TXA 2 and thrombin receptors to G 12 family members and G i -type G proteins. Studies in human platelets have provided evidence that thrombin receptors but not TXA 2 receptors couple to G i -type G proteins resulting in an inhibition of adenylyl cyclase . Similarly, in membranes from wild-type and Gα q -deficient mouse platelets, thrombin increased incorporation of GTP-azidoanilide into G i , whereas U46619 was without effect . Only thrombin was able to decrease cAMP-levels in wild-type and Gα q -deficient platelets (data not shown). The fact that thrombin but not TXA 2 -receptors couple to G i in mouse platelets clearly demonstrates that G i -mediated processes do not play a significant role in the regulation of platelet shape change. Both activated TXA 2 and thrombin receptors (data not shown), coupled to G 12 and G 13 in wild-type and Gα q -deficient platelets. Thus, in Gα q -deficient platelets, the only G proteins found to be activated through TXA 2 receptors were G 12 and G 13 . We therefore conclude that G 12 and/or G 13 are the mediators of ligand-induced platelet shape change and that platelet shape change induced through TXA 2 receptors in Gα q -deficient platelets can be regarded as a G 12 /G 13 -regulated physiological cellular function. Section title: Discussion Educational score: 4.5237717628479 Domain: biomedical Document type: Study Language: en The signaling mechanisms regulating receptor-dependent platelet shape change are incompletely understood. Elevation of the cytosolic Ca 2+ concentration is necessary for full platelet activation including granule secretion and aggregation. However, there is good evidence that elevation of [Ca 2+ ] i alone is not sufficient to induce platelet shape change and that agonists can induce shape change without an increase in phospholipase C activity and without an increase in [Ca 2+ ] i . Tyrosine phosphorylation of various proteins has been associated with receptor-mediated induction of platelet shape change since this occurs rapidly in a Ca 2+ - and α IIb β 3 -integrin–independent manner . The mechanism of early receptor-induced tyrosine phosphorylation is not known. Tyrosine kinases like pp72 syk and pp60 c-src , which are rapidly activated in a partially α IIb β 3 -integrin–independent manner, may be involved , and pp72 syk has been implicated in the platelet shape change response in porcine platelets . However, there is clear evidence that activation of pp72 syk alone is not sufficient for induction of shape change . We show here that TXA 2 -receptor–mediated activation of G 12 /G 13 leads to tyrosine phosphorylation of pp72 syk and activation of pp60 c-src supporting the concept that these tyrosine kinases are involved in early platelet activation. These data also indicate that G proteins of the G 12 -family can regulate tyrosine kinases. The mechanism of this regulation remains unknown. Section title: Discussion Educational score: 4.7739739418029785 Domain: biomedical Document type: Study Language: en MLC phosphorylation has been implicated in the regulation of cytoskeletal reorganization during platelet shape change . Phosphorylated myosin interacts mainly with central actin filaments in platelets, and the forming myosin–actin complex has been suggested to be involved in the granule centralization process . The phosphorylation state of MLC is under dual control of MLCK and myosin-phosphatase. It is well established that increase in [Ca 2+ ] i activates the Ca 2+ / calmodulin-dependent MLCK. MLC phosphorylation by MLCK leads to actin–myosin interaction resulting in actin-stimulated ATPase activity of smooth muscle and nonmuscle myosin . Recently, it has been shown that upstream regulation of myosin phosphatase occurs independently of the cytosolic free calcium concentration through phosphorylation and inactivation of its regulatory subunit by Rho-kinase, a specific target of the small GTPase Rho . In addition, Rho-kinase can directly phosphorylate MLC in vitro . There is increasing evidence that Rho/Rho-kinase–mediated MLC phosphorylation is involved in contractile responses in various cell types like vascular smooth muscle cells , fibroblasts , neuroblastoma cells , astrocytoma cells , or endothelial cells . It is, however, unclear how the Rho-mediated pathway is regulated through receptors. Section title: Discussion Educational score: 4.621776580810547 Domain: biomedical Document type: Study Language: en The TXA 2 mimetic U46619 caused a rapid phosphorylation of MLC in wild-type and Gα q -deficient platelets . Since U46619 does not lead to elevation of [Ca 2+ ] i in the absence of Gα q and since Rho-kinase inhibitor Y-27632 and C3 exoenzyme inhibited U46619-induced MLC phosphorylation in Gα q -deficient platelets, we conclude that a Rho/Rho-kinase–mediated pathway regulating MLC phosphorylation operates in platelets. Consistent with this, the Rho-kinase p160ROCK has been shown to be phosphorylated upon activation of human platelets in an α IIb β 3 -integrin–independent way . In addition, Rho and Rho-kinase can be coimmunoprecipitated with the myosin-binding subunit of myosin phosphatase from human platelets, and treatment of platelets with a TXA 2 -mimetic leads to rapid phosphorylation and inactivation of myosin phosphatase . Conflicting data exist with regard to the role of Rho in early platelet activation as determined by C3 exoenzyme treatment. This is most likely due to the difficulties associated with the length of incubation and the high concentration of C3 exoenzyme required to inactivate a sufficient fraction of Rho. Although partial inactivation of the RhoA pool in human platelets by C3 exoenzyme has been shown to inhibit platelet activation , a recent report showed that ADP ribosylation of ∼90% of Rho in human platelets did not affect inside-out signaling of integrin α IIb β 3 , ligand-induced aggregation and F-actin content . Our data clearly support a role of Rho in early platelet activation. Section title: Discussion Educational score: 4.440925121307373 Domain: biomedical Document type: Study Language: en In wild-type platelets in which U46619 induces an elevation of [Ca 2+ ] i and most likely leads to Ca 2+ /calmodulin-MLCK–mediated MLC phosphorylation, Rho-kinase blocker Y-27632 and C3 exoenzyme also inhibited MLC phosphorylation induced by U46619. Interestingly, both agents appeared to be less potent in wild-type platelets than in Gα q -deficient platelets . This suggests that both, Ca 2+ -mediated activation of MLCK and inhibition of myosin phosphatase through Rho/Rho-kinase may synergistically increase MLC phosphorylation in activated wild-type platelets. In contrast, receptor-mediated MLC phosphorylation in Gα q -deficient platelets depends on the Ca 2+ -independent, Rho-mediated pathway. Since shape change could be inhibited by the C3 exoenzyme as well as by Y-27632 in Gα q -deficient platelets we suggest that Rho/Rho-kinase–mediated MLC phosphorylation is involved in TXA 2 receptor-induced platelet shape change. Section title: Discussion Educational score: 4.325843811035156 Domain: biomedical Document type: Study Language: en Cyclic nucleotides like cAMP and cGMP mediate physiological inhibition of platelet activation through activation of cAMP- and cGMP-dependent kinases. Although analogues of both cyclic nucleotides can block full platelet activation, only cAMP analogues inhibit platelet shape change . Similarly, we observed that the cAMP analogue Sp-5,6-DCl-cBIMPS but not the cGMP analogue 8-pCPT-cGMP inhibited TXA 2 receptor-G 12 /G 13 –mediated shape change and MLC phosphorylation in Gα q -deficient platelets . Inhibition of MLC phosphorylation by Sp-5,6-DCl-cBIMPS but not by 8-pCPT-cGMP suggests that the Rho/Rho-kinase–mediated signaling cascade may be inhibited by the cAMP-dependent pathway. A similar role of cAMP was suggested for the inhibition of Rho/ Rho-kinase–mediated neurite remodeling and morphology change in epithelial-like cells . Section title: Discussion Educational score: 4.673239707946777 Domain: biomedical Document type: Study Language: en Rho has been shown to be regulated by the activated α-subunits of G 12 and G 13 . Since G 12 and G 13 are the only G proteins activated through TXA 2 receptors in Gα q -deficient platelets and since TXA 2 receptor-mediated MLC phosphorylation in Gα q -deficient platelets was inhibited by C3 exoenzyme and Rho-kinase inhibitor Y-27632 we suggest that TXA 2 receptor-induced G 12 /G 13 activation results in MLC phosphorylation through Rho-mediated activation of Rho-kinase. Activated Rho-kinase may phosphorylate MLC directly or act through phosphorylation and inhibition of myosin phosphatase. Additional, synergistic regulation of MLC phosphorylation in wild-type platelets occurs through G q -mediated activation of MLCK. The mechanism by which G 12 /G 13 activate Rho remains to be elucidated. Epidermal growth factor tyrosine kinase has recently been involved in the Gα 13 -induced Rho-dependent actin stress fiber formation in fibroblasts . However, various tyrosine kinase inhibitors were unable to block TXA 2 receptor-induced, G 12 /G 13 -mediated MLC phosphorylation in Gα q -deficient platelets (data not shown). This suggests that in platelets, G 12 /G 13 -induced Rho activation is not mediated by receptor- or nonreceptor-tyrosine kinases. Another possibility is that regulation of Rho by G 12 /G 13 is mediated by a Rho-specific GEF. Genetic evidence in Drosophila showed that the Drosophila RhoGEF, DRhoGEF2, functions downstream of the Drosophila G 12 /G 13 homologue concertina , and it has recently been shown that the related mammalian RhoGEF, p115 RhoGEF, can directly link Gα 13 to the regulation of Rho . Section title: Discussion Educational score: 4.361959934234619 Domain: biomedical Document type: Study Language: en Using Gα q -deficient platelets which do not aggregate and secrete but undergo shape change in response to various stimuli, we show that activation of G 12 /G 13 is sufficient to induce platelet shape change. Thus, different G protein–mediated signaling pathways appear to be specifically involved in the regulation of distinct processes during receptor-induced platelet activation. Although G q is necessary for full platelet activation including aggregation and secretion, activation of G i may counteract anti-aggregatory influences through inhibition of adenylyl cyclase, and G 12 /G 13 appear to be centrally involved in the platelet shape change response. Our data also indicate that G 12 /G 13 can link receptors to tyrosine kinases as well as to Rho/ Rho-kinase–mediated regulation of MLC phosphorylation, and we provide evidence that the latter pathway participates in the receptor-mediated induction of platelet shape change. | Other | biomedical | en | 0.999997 |
10037796 | Section title: Biological Materials Educational score: 3.225351095199585 Domain: biomedical Document type: Study Language: en HEK-293 cells (ATCC TIB202) were obtained from the American Type Culture Collection, and CCR5-transfected HEK-293 cells were kindly donated by Dr. J. Gutierrez (Dept. of Immunology and Oncology, Centro Nacional de Biotecnología, Madrid, Spain). Antibodies used include rabbit anti-JAK1 and anti-JAK2 (Upstate Biotechnology, Inc.), anti-Gα i and anti-STAT5 ( Santa Cruz Biotechnology ); monoclonal anti-PTyr (4G10; Upstate Biotechnology Inc.), anti-PTyr (PY20) (Transduction Laboratories), and anti–β 2 -microglobulin ( PharMingen ). Anti-CCR5 mAb, CCR5-02, CCR5-03, and anti-CXCR4 mAb were generated in our laboratory as described . EGF-stimulated A-431 cell lysates were obtained from Upstate Biotechnology Inc. and recombinant human RANTES and SDF-1α were from Peprotech Inc. Section title: Flow Cytometry Analysis Educational score: 4.116732597351074 Domain: biomedical Document type: Study Language: en Cells were centrifuged (250 g , 10 min, room temperature), plated in V-bottom 96-well plates (2.5 × 10 5 cells/well), and incubated with 50 μl/ well biotin-labeled mAb (5 μg/ml, 60 min, 4°C). Cells were washed twice in PBS with 2% BSA and 2% FCS, and centrifuged (250 g , 5 min, 4°C). FITC-labeled streptavidin (Southern Biotechnologies Associates, Inc.) was added, incubated (30 min, 4°C) and plates were washed twice. Cell-bound fluorescence was determined in a Profile XL flow cytometer at 525 nm (Coulter Electronics). Section title: Calcium Determination Educational score: 4.139473915100098 Domain: biomedical Document type: Study Language: en Changes in intracellular calcium concentration were monitored using a fluorescent probe (Fluo-3AM; Calbiochem ). Cells (2.5 × 10 6 cells/ml), were resuspended in RPMI containing 10% FCS, 10 mM Hepes, and incubated with 10 μl/10 6 cells of Fluo-3AM (300 μM in DMSO, 15 min, 37°C) . After incubation, cells were washed, resuspended in complete medium containing 2 mM CaCl 2 , and maintained at 4°C until just before chemokine addition to minimize membrane trafficking and to eliminate spontaneous Ca 2+ entry. Calcium mobilization in response to 10 nM RANTES or (AOP)-RANTES and 20 nM SDF-1α was determined at 37°C in an EPICS XL flow cytometer at 525 nm. It includes background level stabilization and determination of the probe loading level for each sample. Only samples with a similar load, as assessed by ionophore-induced Ca 2+ mobilization, were used (5 μg/ml ionomycin; Sigma Chemical Co. ). Section title: Cell Migration Educational score: 4.114272117614746 Domain: biomedical Document type: Study Language: en CCR5-transfected HEK-293 cells (0.5 × 10 6 cells/ml) were starved for 120 min at 37°C, 5% CO 2 in RPMI 1640 containing 0.1% BSA. Cells (0.25 × 10 6 cells in 0.1 ml) were placed in the upper well of 24-well, 8-μm pore size transmigration chambers (Transwell; Costar Corp.) and RANTES or (AOP)-RANTES (diluted in 0.6 ml RPMI containing 0.25% BSA) were added to the lower well. Plates were incubated (240 min, 37°C, 5% CO 2 ) and the cells that migrated to the lower chamber were counted, as described . Section title: Immunoprecipitation, SDS-PAGE, and Western Blot Analysis Educational score: 4.197579860687256 Domain: biomedical Document type: Study Language: en RANTES- or (AOP)-RANTES–stimulated cells (20 × 10 6 ) were lysed in a detergent buffer (20 mM triethanolamine, pH 8.0, 300 mM NaCl, 2 mM EDTA, 20% glycerol, 1% digitonin, with 10 μM sodium orthovanadate, 10 μg/ml leupeptin, and 10 μg/ml aprotinin) for 30 min at 4°C with continuous rocking and then centrifuged (15,000 g , 15 min). Immunoprecipitations were performed essentially as described earlier . Protein extracts precleared by incubation with 20 μg of anti–mouse IgM-agarose ( Sigma Chemical Co. ) or protein A–Sepharose (60 min, 4°C) were centrifuged (15,000 g , 1 min), immunoprecipitated with the appropriate antibody (5 μg/sample, 120 min, 4°C), followed by anti–mouse IgM-agarose or by protein A–Sepharose if the first antibody was derived from rabbit serum. Immunoprecipitates or protein extracts were separated in SDS-PAGE and transferred to nitrocellulose membranes. Western blot analysis was performed as described , using 2% BSA in TBS as blocking agent for the antiphosphotyrosine analyses. When stripping was required, membranes were incubated for 60 min at 60°C with 62.5 mM Tris-HCl, pH 7.8, containing 2% SDS and 0.5% β-mercaptoethanol. After washing with 0.1% Tween 20 in TBS for 2 h, membranes were reblocked, reprobed with the appropriate antibody, and developed as above. In all cases, protein loading was carefully controlled using a protein detection kit ( Pierce Chemical Co. ) and, when necessary, by reprobing the membrane with the immunoprecipitating antibody. Section title: Receptor Cross-linking Assays Educational score: 4.114319324493408 Domain: biomedical Document type: Study Language: en Serum starved CCR5 transfected HEK-293 cells (20 × 10 6 ) were stimulated with 10 nM RANTES, 10 nM (AOP)-RANTES, or 20 nM SDF-1α for 1 min at 37°C. The reaction was terminated by addition of 1 ml of cold PBS and centrifugation (30 s, 15,000 g ). After washing twice with cold PBS, 10 μl of 100 mM disuccinimidyl suberate (DSS; Pierce Chemical Co. ) was added for 10 min at 4°C with continuous rocking. The reaction was terminated by adding 1 ml of cold PBS and washing three times. The pellet was lysed for 60 min and immunoprecipitated as above. Section title: Immunofluorescence Microscopy: Polarization of Blast Cells Educational score: 4.158638000488281 Domain: biomedical Document type: Study Language: en Immunofluorescence experiments were performed essentially as described . Briefly, 2 × 10 6 T lymphoblasts in 500 μl complete medium were allowed to adhere to fibronectin (FN)-coated coverslips in 24-well plates (Costar Corp.). Cytokines and chemokines (10 ng/ml) were added, and cells were incubated (37°C, 5% CO 2 ). After 30 min, cells were fixed with 3.7% formaldehyde in PBS for 10 min at room temperature, and stained with specific mAb plus a FITC-labeled rabbit anti– mouse IgG F(ab′) 2 ( Pierce Chemical Co. ). The proportion of CCR5 polarization was calculated by direct cell count ( n = 400–500) of 10 random fields for each condition using a photomicroscope with 60× oil immersion objectives (Labophot-2; Nikon Inc. ). Preparations were photographed on Ektachrome 400 film. Images were acquired with a high performance CCD camera (Cohu) coupled to the microscope and connected to a workstation (model Q550CW; Leica Imaging Systems, Ltd.). Images were visualized, processed, and stored using QFISH software (version V1.01; Leica Imaging Systems, Ltd.) and printed on a color printer (Phaser 440; Tektronix Inc.). Section title: RANTES and (AOP)-RANTES Induce Functional Responses in CCR5-transfected HEK-293 Cells Educational score: 4.163627624511719 Domain: biomedical Document type: Study Language: en We developed anti–human CCR5-specific mAb using synthetic peptides corresponding to the NH 2 -terminal domain of this receptor (amino acids 13–28), a CCR5-specific sequence not present in other chemokine receptors. These mAbs recognize the CCR5 in human PBMC (not shown) and in CCR5 stably transfected HEK-293 cells, in flow cytometry , as well as in Western blot, and immunoprecipitation analysis . No binding is observed in mock- or CCR2-transfected HEK-293 cells, or in any other cell tested that does not express CCR5. Section title: RANTES and (AOP)-RANTES Induce Functional Responses in CCR5-transfected HEK-293 Cells Educational score: 4.187914848327637 Domain: biomedical Document type: Study Language: en In response to RANTES or (AOP)-RANTES, these CCR5-transfected HEK-293 cells mobilize calcium and are desensitized to a second stimulation. However, only RANTES triggers migration in these cells . PTX treatment abrogates both calcium release and cell migration in response to RANTES and (AOP)-RANTES , whereas no effect was observed following incubation with cholera toxin (not shown). This is consistent with other studies showing that RANTES downstream signals in other cell lines and T cells are coupled to PTX-sensitive G proteins , and with coupling of other chemokine receptors to G i in transfected HEK-293 cells , indicating the utility of this cell line in the study of signaling through GPCR. Section title: RANTES and (AOP)-RANTES Induce Functional Responses in CCR5-transfected HEK-293 Cells Educational score: 4.1744842529296875 Domain: biomedical Document type: Study Language: en We have described the ability of RANTES to polarize peripheral blood T cells, such that the CCR5 receptor migrates to the cell's leading edge while the ICAM-1, ICAM-3/ CD43, CD44 molecules localize at the uropod . Therefore, we tested the ability of both of these ligands to induce polarization in peripheral T cell blasts. Time-course studies of ICAM-3 redistribution to the cell uropod of FN-adhered T lymphoblasts showed that RANTES induced ICAM-3 redistribution in a significant proportion of the cells as early as 15 min after stimulation. Maximum redistribution, seen at 30 min, persisted until at least 90 min of incubation with the chemokine. In contrast, (AOP)-RANTES–induced ICAM-3 polarization showed a much slower, weaker response, with a peak at 30 min that vanished thereafter . Section title: RANTES and (AOP)-RANTES Induce Functional Responses in CCR5-transfected HEK-293 Cells Educational score: 4.175485134124756 Domain: biomedical Document type: Study Language: en We next studied the effects of these ligands on the polarization of CCR5 to the advancing front of migrating T lymphoblasts, a phenomenon that may be involved in the chemotaxis mechanism. We found that whereas RANTES triggered redistribution of CCR5 molecules to the leading edge of the cells, membrane CCR5 expression was not detectable in (AOP)-RANTES–stimulated polarized T lymphoblasts bearing ICAM-3 redistributed to the uropod . The downregulation of the (AOP)-RANTES– triggered chemokine receptor results in a net inhibition of CCR5 redistribution to the leading edge of T lymphoblasts stimulated with this chemokine derivative, compared to those treated with RANTES . This concurs with previous observations showing that (AOP)-RANTES induces CCR5 downregulation by blocking receptor recycling . Section title: RANTES and (AOP)-RANTES Induce Tyrosine Phosphorylation of the CCR5 Receptor and Association of the JAK1/STAT5 Complex Educational score: 4.265898704528809 Domain: biomedical Document type: Study Language: en When CCR5-transfected HEK-293 cells were stimulated with RANTES or (AOP)-RANTES, a 38-kD protein phosphorylated in tyrosine residues was initially identified as the CCR5 receptor by immunoblot (not shown). Thereafter, lysates from transfected HEK-293 cells were immunoprecipitated with anti-CCR5, and Western blots developed with anti-PTyr antibodies . An increase in CCR5 receptor phosphorylation in tyrosines is seen as early as 60 s after stimulation. At this time no differences were observed following treatment with either stimulus, indicating that (AOP)-RANTES not only binds to CCR5, but also signals through this receptor. The early, weak tyrosine phosphorylation level may occur because only one tyrosine (Tyr 126 in the DRY motif) was being phosphorylated, analogous to the case of Tyr 139 in the CCR2 receptor . Similar results are observed in the inverse experiment, in which anti-PTyr antibody immunoprecipitates are analyzed in Western blot with anti-CCR5 receptor antibody (not shown). Again, tyrosine phosphorylation is detected in CCR5 after stimulation with either RANTES or (AOP)-RANTES. Nevertheless, significant differences are observed in tyrosine phosphorylated CCR5 after longer stimulation periods. Whereas CCR5 phosphorylation persists in RANTES-treated cells after 15 min of treatment, this is not the case after (AOP)- RANTES stimulation, reinforcing the observation that there are differences in the signaling pathways activated by these chemokines. Section title: RANTES and (AOP)-RANTES Induce Tyrosine Phosphorylation of the CCR5 Receptor and Association of the JAK1/STAT5 Complex Educational score: 4.242250442504883 Domain: biomedical Document type: Study Language: en To ascertain which kinase is responsible for the rapid CCR5 chemokine receptor phosphorylation, CCR5-transfected HEK-293 cells were stimulated with RANTES or (AOP)-RANTES, and cell lysates immunoprecipitated with anti-CCR5, or with anti–β 2 -microglobulin as an isotype-matched antibody control. The use of a specific anti-JAK1 antibody identified a 130-kD protein in the anti-CCR5 immunoprecipitates . Furthermore, the JAK1 tyrosine kinase is phosphorylated on tyrosine residues after stimulation with either ligand, since anti-PTyr immunoprecipitates can be developed with anti-JAK1 antibodies in Western blot . JAK1 association to the CCR5 receptor takes place as early as 30 s after RANTES or (AOP)-RANTES stimulation . Small amounts of JAK1 were also found associated to the CCR5 receptor in the absence of added ligand, consistent with receptor phosphorylation in the absence of exogenous ligand in CCR5-transfected HEK-293 cells . Section title: RANTES and (AOP)-RANTES Induce Tyrosine Phosphorylation of the CCR5 Receptor and Association of the JAK1/STAT5 Complex Educational score: 4.199322700500488 Domain: biomedical Document type: Study Language: en Neither immunoprecipitation of cell lysates with isotype- matched control antibodies to other cell proteins such as β 2 -microglobulin nor immunoprecipitation in mock-transfected HEK-293 cells (not shown) revealed the presence of JAK1 . This rules out nonspecific protein association to membrane components under these experimental conditions. The rapid association of JAK1 to the CCR5 receptor triggered by these two chemokines, but not by others tested (MCP-1 and SDF-1α), suggests a role for this tyrosine kinase in early receptor phosphorylation following ligand stimulation. Interestingly, while MCP-1 promotes JAK2 association to the receptor in CCR2-transfected HEK-293 cells, neither JAK2 nor JAK3 associates to CCR5 after RANTES or (AOP)-RANTES activation of CCR5-transfected HEK-293 cells (data not shown). Section title: RANTES and (AOP)-RANTES Induce Tyrosine Phosphorylation of the CCR5 Receptor and Association of the JAK1/STAT5 Complex Educational score: 4.311819553375244 Domain: biomedical Document type: Study Language: en To identify the downstream signaling pathway activated by the JAK1 kinase, we tested its association to members of the STAT transcription factor family. We analyzed JAK1 kinase-activated STAT transcription factors in anti-CCR5 immunoprecipitates, and found that STAT5 associates to the receptor complex in response to both ligands . The association of STAT5 correlates in time with JAK1 phosphorylation and binding. Furthermore, the precipitated STAT5 is phosphorylated on tyrosine residues, showing it is in the activated state . Immunoprecipitates of RANTES-stimulated cell lysates with isotype-matched control antibodies do not contain STAT5. These results were further validated by the reverse experiments, in which anti-STAT5 antibodies were used for immunoprecipitation and Western blots developed with either anti-PTyr or anti-CCR5 antibodies (not shown). Therefore, binding of both RANTES and (AOP)-RANTES to the CCR5 receptor induces receptor and JAK1 phosphorylation and STAT5 binding. Independent of the fact that other STAT transcriptional factors may be implicated in CCR5-mediated signaling, our data show that no major differences are observed in RANTES or (AOP)-RANTES activation of the JAK/STAT pathway. Section title: RANTES and (AOP)-RANTES Induce CCR5 Dimerization Educational score: 4.2302398681640625 Domain: biomedical Document type: Study Language: en Similar to growth factor–induced dimerization of tyrosine kinase receptors, some GPCR, including chemokine receptors like CCR2 and CXCR4, undergo ligand-induced dimerization . We have now tested whether RANTES and (AOP)-RANTES trigger CCR5 dimerization. DSS-mediated cross-linking in CCR5-transfected HEK-293 cells was carried out after RANTES or (AOP)-RANTES stimulation. In CCR5-transfected HEK-293 cells, but not in mock-transfected cells, a high molecular mass receptor species (76 kD) was observed following both stimuli. This band corresponds to the expected molecular mass of two CCR5 molecules, as assessed by immunoprecipitation with anti-CCR5 antibodies and Western blot developed with anti-CCR5 antibodies . When CCR5-transfected HEK-293 cells were stimulated with SDF-1α, no CCR5 dimerization was observed under these experimental conditions , despite the fact that HEK-293 cells express a functional CXCR4 receptor as determined by flow cytometry and SDF-1α–induced calcium mobilization assays . When the same experiment was done using anti-CXCR4 mAb for immunoprecipitation and Western blot, a high molecular mass species (84 kD) of the CXCR4 receptor is observed only after SDF-1α, but not after RANTES or (AOP)-RANTES stimulation . Section title: RANTES and (AOP)-RANTES Induce CCR5 Dimerization Educational score: 4.3378424644470215 Domain: biomedical Document type: Study Language: en All together, these data indicate specific ligand-induced dimerization, as SDF-1α is not able to induce CCR5 dimerization, while RANTES or (AOP)-RANTES are not able to induce CXCR4 dimers. In addition, association of CCR5 with other membrane components, including other chemokine receptors, can be discarded under these experimental conditions, as cells treated with SDF-1α render only CXCR4 dimers, and those treated with RANTES and (AOP)-RANTES render only CCR5 dimers. High molecular mass species containing CCR5 and CXCR4 simultaneously are not detected, as shown when cells are stimulated with SDF-1α or RANTES and (AOP)-RANTES, and when immunoprecipitation and Western blot are performed using anti-CCR5 or anti-CXCR4 antibodies, respectively. Both RANTES and (AOP)-RANTES, like other chemokines and similar to members of the large cytokine family, can trigger early signaling events such as receptor dimerization, followed by recruitment of tyrosine kinases that phosphorylate both the receptor and the STAT transcription factors. Section title: Gα i and p 125 FAK Association to CCR5 Differ after RANTES or (AOP)-RANTES Stimulation Educational score: 4.201506614685059 Domain: biomedical Document type: Study Language: en The PTX dependence of RANTES- and (AOP)- RANTES–triggered Ca 2+ mobilization indicates Gα i involvement in this process. It has been demonstrated recently that chemokine receptor–mediated chemotaxis is triggered by the βγ subunit of Gα i . In MCP-1–mediated chemotaxis, we have shown that Gα i associates rapidly to the CCR2 receptor, releasing βγ, which is responsible for activating migration and cell polarization. Thus, we investigated whether the differences in chemotaxis triggering reflect differences in RANTES- and (AOP)-RANTES–mediated Gα i -CCR5 receptor association. Section title: Gα i and p 125 FAK Association to CCR5 Differ after RANTES or (AOP)-RANTES Stimulation Educational score: 4.337820053100586 Domain: biomedical Document type: Study Language: en CCR5- or mock-transfected HEK-293 cells were stimulated with RANTES or (AOP)-RANTES, immunoprecipitated with anti-CCR5 or isotype-matched control antibodies, followed by a Western blot with anti-Gα i antibodies. After both RANTES and (AOP)-RANTES stimulation, Gα i associated to CCR5 . Whereas association persisted for longer than 15 min when cells were RANTES-stimulated, Gα i is dissociated from the receptor after 5 min of (AOP)-RANTES treatment . The CCR5 expression level was controlled in both immunoprecipitates by stripping and reblotting membranes with anti-CCR5 antibody . These data concur with the similar Ca 2+ mobilization promoted by both of these ligands , as Gα i association to CCR5 is unaltered at the times employed for these experiments (1–3 min). Rapid Gα i dissociation from the CCR5, induced only by (AOP)-RANTES, implies a role for G i in later chemokine-triggered events such as chemotaxis. This result concurs with data showing the importance of βγ release from G i in chemotaxis . Section title: Gα i and p 125 FAK Association to CCR5 Differ after RANTES or (AOP)-RANTES Stimulation Educational score: 4.189214706420898 Domain: biomedical Document type: Study Language: en The activation of p 125 FAK and the kinase ZAP-70 has been described in T cells after RANTES activation . Therefore, we tested whether p 125 FAK activation coincides with its association to the CCR5. CCR5-transfected HEK-293 cells were stimulated with 10 nM RANTES or (AOP)-RANTES, cell lysates were immunoprecipitated with anti-p 125 FAK or an isotype-matched control antibody and analyzed in Western blot for the presence of CCR5. RANTES, but not (AOP)- RANTES, promotes the association of this kinase to the receptor, reaching maximum binding after 15 min . Even in the absence of ligand, residual association between these two proteins was observed. The lack of p 125 FAK association and the rapid Gα i dissociation from the CCR5 receptor in the case of (AOP)-RANTES suggest that association of these proteins with the receptor is crucial in triggering the signaling pathways that lead to the different effects promoted by these two chemokines. Section title: Discussion Educational score: 4.079912185668945 Domain: biomedical Document type: Study Language: en Interest is rapidly growing in a broad view of the chemokine function, not only because of significance in HIV infection, but also because chemokines and their receptors are expressed by a wide variety of nonhematopoietic cells . Because of the relevance of chemokines in numerous pathologies, it appears of the utmost importance to devise therapies based on the administration of chemokines that lack chemotactic and leukocyte-activating properties, but are able to act as receptor antagonists. These are the basic characteristics of (AOP)- RANTES, a RANTES analogue that has been chemically modified at its NH 2 terminus, although very little is known of the mechanism underlying its action. Section title: Discussion Educational score: 4.507035255432129 Domain: biomedical Document type: Study Language: en We have generated anti-CCR5 mAbs and used them to characterize the signaling pathways activated by RANTES and (AOP)-RANTES. The results show that ligand-induced CCR5 dimerization occurs when these chemokines signal target cells for activation. In the response to cytokines, receptor dimerization triggers a pathway involving tyrosine kinases and transcriptional factors. Only the JAK1 tyrosine kinase responds to RANTES and to (AOP)-RANTES by rapidly associating to CCR5 and is phosphorylated as soon as 30 s after binding, indicating that JAK1 activation is virtually simultaneous with its association to CCR5. In the cytokine receptors, activation and association of JAK kinase to the receptor creates docking sites for SH2-containing proteins such as STAT, leading to their phosphorylation, followed by activation of gene transcription . After binding of either of the chemokine ligands studied here, STAT5 is also associated to the CCR5, in accordance with the role of the JAK kinases in transducing signals from hematopoietic growth factor receptors . Our data concur with a recent report showing rapid activation of certain STAT transcription factors in T cells after RANTES and MIP-1α stimulation, although in that case association to the CCR5 receptor was not analyzed . Together, these results indicate that chemokine-mediated activation of GPCR leads to signal transduction, which invokes intracellular phosphorylation intermediates used by other cytokine receptors. These earlier events are equally activated by both RANTES and (AOP)-RANTES. Section title: Discussion Educational score: 4.409348964691162 Domain: biomedical Document type: Study Language: en Both RANTES and (AOP)-RANTES induce CCR5 dimerization, a phenomenon that requires specific interaction between a given chemokine and its respective receptor. When experiments were performed using SDF-1α as a ligand neither CCR5 dimers nor CCR5-CXCR4 heterodimers were observed, whereas CXCR4 dimers are present in anti-CXCR4 immunoprecipitates. Direct interaction between two CCR5 molecules has also been demonstrated by Benkirane et al. , even in the absence of ligand stimulation. The functional significance of dimerization was suggested by Hebert et al. using the epitope tagging approach, showing that agonist stimulation of the β 2 -adrenergic receptor stabilized the dimeric state of the receptor. The present data indicate that although dimerization is a primary event following ligand interaction with the chemokine receptor, it does not necessarily initiate identical transduction events. (AOP)- RANTES thus triggers Ca 2+ mobilization as does RANTES, but promotes neither cell migration nor CCR5 redistribution to the leading edge in cell polarization. The rapid Gα i association to CCR5 induced by both ligands explains the similar Ca 2+ mobilization response and lack of response in PTX-pretreated cells. Section title: Discussion Educational score: 4.221146106719971 Domain: biomedical Document type: Study Language: en It has been shown for a number of GPCR that different ligands of a given receptor can induce different responses. This is the case for CXCR2, from which IL-8 and NAP-2 elicit different responses, although they bind with equivalent affinity to this receptor . It is feasible that RANTES and (AOP)-RANTES induce distinct conformational changes in the CCR5. In fact, it has been shown that different structural requirements are needed in GPCR to activate different pathways . In the case of angiotensin II receptor changes have been described in internalization kinetics that do not alter other functional responses . Section title: Discussion Educational score: 4.508225440979004 Domain: biomedical Document type: Study Language: en Whereas Gα i association to CCR5 persists as long as 15 min after RANTES treatment, it dissociates from CCR5 by 5 min after (AOP)-RANTES stimulation. This variance must give rise to a clear difference in the availability of free, active βγ subunits by affecting some ligand-induced responses. In fact, G i βγ subunit release is known to be important in chemokine receptor–mediated chemotaxis . Another conspicuous difference is the lack of late tyrosine phosphorylation in CCR5 after (AOP)-RANTES treatment, in contrast to the continuous tyrosine phosphorylation of CCR5 observed following RANTES induction. This may occur because of tyrosine phosphorylation mediated by other tyrosine kinases. The activation of both p 125 FAK and ZAP-70 has been described after RANTES stimulation . Here we demonstrate association of p 125 FAK, which is related to cytoskeletal proteins and migration phenomena , to the receptor following RANTES but not (AOP)-RANTES stimulation. The failure of (AOP)-RANTES to maintain G i association for long (15 min) periods concurs with the lack of association and activation of p 125 FAK and the inactivation of subsequent events. Both activation of G i and p 125 FAK, as well as chemotaxis, occur after RANTES activation, but are absent when cells were treated with (AOP)-RANTES, indicating a possible relationship between these events. Section title: Discussion Educational score: 4.7067341804504395 Domain: biomedical Document type: Study Language: en Chemotaxis involves several phenomena including: changes in cell shape, integrin affinity, and integrin recycling at the cell's leading edge . These events appear to be mediated by G protein activation, as well as phosphorylation signals through chemokine receptors . In fibroblasts and smooth muscle cells, the regulation of intracellular FAK and its interaction with the cytoskeletal proteins α-actinin, talin, and vinculin have also been given much attention with respect to cell polarization and migration . As a consequence of RANTES stimulation, T cells undergo a polarization process in which the CCR5 receptor redistributes to the leading cell edge and p 125 FAK associates to the receptor. Both events, implicated in the late response to chemokines, are required for chemotaxis and are triggered by RANTES. Early (AOP)-RANTES–induced signaling events, such as Ca 2+ mobilization, result in weak, transient cell polarization, and redistribution of the ICAM-3 adhesion receptor to the T lymphocyte uropod. This antagonist does not mediate cellular chemotaxis. Recycling of CCR5 molecules to the cell membrane, which probably occurs at the advancing front of the cell and is inhibited by (AOP)- RANTES , appears to be critical for the mechanism of directional cell migration towards a chemokine gradient. This is further supported by our data showing the failure of CCR5 to cluster at the leading edge of (AOP)-RANTES–stimulated cells. Therefore, these results suggest that during the chemotaxis process early signals induced by chemokines through receptor dimerization may not be sufficient to induce cell migration. Signals triggered by sustained association of the G protein to the chemokine receptor may also regulate other events necessary for the chemotactic response, such as integrin affinity or actin polymerization at the leading cell edge. Our data also support the idea that p 125 FAK kinase association to the receptor is not involved in ligand-induced CCR5 internalization, since (AOP)-RANTES is as efficient as RANTES in promoting internalization , without inducing p 125 FAK association. Section title: Discussion Educational score: 4.34996223449707 Domain: biomedical Document type: Study Language: en The data presented here can be incorporated in a model in which signaling through chemokine receptors directly involves early signals that occur in the first few minutes after ligand binding, including receptor dimerization; association and activation of JAK tyrosine kinases, and activation of STAT transcriptional factors, as well as late signals such as cell polarization, activation, and association of p 125 FAK kinase to the chemokine receptor . The fact that chemokine receptors activate the JAK/STAT pathway and that dimerization may be a general mechanism for chemokine activity adds a new perspective to understanding how the multiplicity of chemokine functions are achieved. Furthermore, it suggests an interesting new objective for therapeutic intervention in chemokine-associated pathologies, including inflammation and AIDS. Section title: Discussion Educational score: 4.432060718536377 Domain: biomedical Document type: Study Language: en Internalization of chemokine receptors is a phenomenon that can be dissociated from their role as HIV-1 coreceptors . Although the ability of the coreceptor to internalize is not required for HIV entry, it contributes to the HIV suppressive effect of CC and CXC chemokines . (AOP)-RANTES is a potent inhibitor of HIV-1 infectivity, an effect that is suggested because it enhanced CCR5 internalization and inhibition of recycling . As internalization is an agonist-driven event, (AOP)-RANTES as well as other NH 2 -terminal modified chemokines should be able to activate their receptors. The differences between RANTES- and (AOP)-RANTES– induced internalization may be related to increased affinity of (AOP)-RANTES for the CCR5 receptor , although differences in the signaling pathways activated by these two ligands have not been reported. Here we correlate the known effects of (AOP)-RANTES with a signaling cascade and suggest that the dimerization of chemokine receptors is an alternative mechanism for chemokine-induced HIV-1 inhibition. Section title: Discussion Educational score: 4.498004913330078 Domain: biomedical Document type: Study Language: en Receptor dimerization is also the mechanism through which antibodies to the CCR2 NH 2 -terminal domain block HIV-1 infection. In fact, the anti-CCR2 mAb CCR2-01 that blocks HIV-1 infection , but not MCP-1–induced responses , triggers CCR2 dimerization (Mellado, M., manuscript in preparation). The concept that receptor dimerization is required for prevention of HIV-1 infection, besides including the nonsignaling mechanism indicated by (AOP)-RANTES, incorporates the model based on receptor internalization, which does not take place in the absence of dimerization. For both receptor desensitization and internalization, we have shown the requirement for GRK2 association and phosphorylation in serine/threonine residues of the COOH- terminal receptor domain. This in turn associates with arrestin and clathrin, and undergoes internalization . Dimerization is a necessary event for the formation of the receptor/GRK-2/arrestin/clathrin complex. This model consolidates all available experimental evidence, and offers novel prospects for screening new drugs that may prevent HIV-1 infection and/or inflammatory processes. | Study | biomedical | en | 0.999995 |
10037797 | Section title: Cell Lines and Reagents Educational score: 4.043166160583496 Domain: biomedical Document type: Study Language: en Human recombinant IL-2, kindly provided by Dr. D. Bron (Institut Bordet, Brussels, Belgium), was used at 20 U/ml; IL-15 was obtained from Immunex and used at 200 ng/ml. Endotoxin-free affinity-purified sCD23 was prepared in our laboratory from CSN of CHO cell line transfected with human cDNA encoding for aa 148–321 of the CD23 molecule. The concentration of 25 ng/ml sCD23 used throughout this study was selected on the basis of previously reported dose– response curves . Jurkat T (α v + β 3 + ), THP-1 (α v − β 3 − ) monocytic, Raji (α v + β 3 − ), and Bowes melanoma (α v + β 3 + ) cell lines were obtained from the American Type Culture Collection (ATCC). K562 and K562 transfected with the cDNA encoding the full-length CR2 (K562-CR2) were a generous gift from Drs. A. Masumoto and D. Fearon (Johns Hopkins University, Baltimore, MD). CD47 deficient Jurkat T cell line and 0V10 ovarian carcinoma cell line were generated in Drs. E. Brown and F. Lindberg's laboratory (Washington University, St. Louis, MO). cDNA encoding for CD51 (α v chain) was a generous gift from Dr. E. Ruoslahti (Burnham Institute, La Jolla, CA). 10G2 mAb (IgM class) was produced in our laboratory following immunization of mice with Jurkat T cells. Hybridomas producing anti-CD47 (clone B6H12) and anti-β 3 (CD61) mAbs (clone AP3) were purchased at the ATCC. Anti-α v β 3 (CD51/CD61, clone LM609), and anti-α v (CD51, clone LM142) were kindly provided by Dr. Cheresh (Scripps Research Institute, La Jolla, CA). Anti-αv (CD51, clone AMF7) and anti-CD47 (clone BRIC126, CKm1) were purchased from Immunotech, Serotec Ltd., and Accurate Chemical and Scientific Corp., respectively. RGDS and RGES peptides, Vn, Fn, and thrombospondin (TSP) were obtained from GIBCO BRL . Section title: Expression Cloning of 10G2 Antigen (CD47) Educational score: 4.120532989501953 Domain: biomedical Document type: Study Language: en COS cells were transfected using the DEAE dextran method, with an expression library derived from Jurkat cells (4 × 10 5 clones). Cells expressing the molecule recognized by the 10G2 mAb were immunoselected by indirect panning using 10G2 and anti–mouse IgM-coated plates. Specifically bound cells were lysed, plasmid DNA was isolated, amplified, and retransfected into COS cells. Following an additional round of immunoselection and plasmid purification, pools of 150 clones each were transfected into COS cells, which were subsequently screened for positivity by incubation with radiolabeled 10G2 mAb. Two rounds of screening resulted in the isolation of a single 10G2-reactive clone. Section title: Establishment of Stable CHO Transfectants Educational score: 4.226561546325684 Domain: biomedical Document type: Study Language: en CHO cells were grown at 50% confluence in 150-cm 2 culture flasks in 5% heat decomplemented FCS containing DME ( GIBCO BRL ) supplemented with 2 mM glutamine, 100 IU penicillin, and 100 μg/ml streptomycin. Cells were harvested using versene solution ( GIBCO BRL ) and washed twice with pure DME. Cells were then resuspended at 11 × 10 6 cells/ ml, and 10 7 cells were incubated in a 0.4-cm electrotransfection cuvette (BioRad Laboratories) for 10 min with 20 μg of pBJα v (plasmid containing cDNA encoding the full-length α v molecule, CD51), in order to obtain CHO cells expressing the α v molecules. Cells were then pulsed at 220 V and 960 μF using a Gene Pulser (BioRad Laboratories). After another 10 min of incubation at room temperature, electroporated cells were grown for 24 h in nonselective culture medium which was then replaced by complete DME containing 500 μg/ml active G418 ( GIBCO BRL ). After 14 d of culture, the pool of survivors was analyzed by flow cytometry, and CHO cell line expressing α v was subsequently enriched by FACS ® for the 5% highest stained cells using anti-CD51 mAb as primary antibody. After four rounds of sorting, we obtained stable cell lines, namely CHO-51, containing >99% of CD51 + cells. Section title: Monocytes. Educational score: 4.108438968658447 Domain: biomedical Document type: Study Language: en PBMC were isolated by density gradient centrifugation of heparinized blood from healthy volunteers using Lymphoprep (Nycomed). Monocytes were prepared by cold aggregation as described in Armant et al. . Monocyte purity was shown to be >95% using phycoerythrin-conjugated anti-CD14 mAb and flow cytometry ( Becton Dickinson and Co.). Cellular viability was >90% using trypan blue exclusion. Section title: T Cells. Educational score: 4.110947132110596 Domain: biomedical Document type: Study Language: en Enriched T cell populations were obtained from the monocyte-depleted PBMC by rosetting with AET-SRBC and treatment with ammonium chloride. To obtain highly purified T cells, rosette forming cells were washed and incubated for 20 min at 37°C in Lympho-Kwik T (One Lambda). Cell purity was assessed by flow cytometry (FACScan ® ; Becton Dickinson and Co.) using phycoerythrin-conjugated anti-CD3 mAb ( Becton Dickinson and Co.) and shown to be >98% in all cases. Section title: T Cells. Educational score: 4.092532157897949 Domain: biomedical Document type: Study Language: en Cultures were performed in complete serum-free HB101 medium (Irvine Scientific) supplemented with 2 mM glutamine, 1 mM sodium pyruvate, 10 mM Hepes, 100 IU penicillin, and 100 μg/ml streptomycin in the presence of polymyxin B 10 μg/ml ( Sigma Chemical Co. ). When cultured alone, monocytes were incubated in 5 ml sterile Falcon tubes ( Becton Dickinson and Co.) at 2 × 10 5 cells/ml for cytokine measurement. For coculture experiments, T cells (10 6 cells/ml) were incubated with monocytes (2 × 10 5 cells/ml) in 24-well Falcon plates. Section title: Cytofluorometric Analysis Educational score: 4.133876800537109 Domain: biomedical Document type: Study Language: en For sCD23 binding, cells or cell lines were washed in HBSS ( Gibco Laboratories ), resuspended in HB101 complete serum free medium containing biotinylated sCD23 (B-sCD23; 50 ng/ml) in the absence or presence of mAbs (CD47, CD51, CD51/CD61), soluble Vn or Fn (20 μg/ml), and RGDS peptides (20 μg/ml). After 4–6 h of incubation at 22°C, cells were washed with PBS containing 3% BSA, and further incubated with phycoerythrin-streptavidin ( Becton Dickinson and Co.). Cell viability assessed by trypan blue exclusion was >85% before staining with fluorochrome. Indirect immunofluorescence staining of cells or cell lines with different mAbs was performed according to standard techniques . Section title: Lymphokine Determinations Educational score: 3.6788108348846436 Domain: biomedical Document type: Study Language: en IFN-γ, TNF-α, IL-10, and IL-12 were measured exactly as described in Armant et al. . IL-1β, IL-8, and PGE 2 were measured by ELISA kits purchased from R & D Systems, Inc. Section title: Immunoprecipitation and Western Blot Analysis Educational score: 4.123684883117676 Domain: biomedical Document type: Study Language: en Cells were lysed in PBS containing 1% NP-40 (NP-40/PBS) supplemented with protease inhibitors. The lysate was purified on anti-β 3 affinity column, and the eluate was separated by SDS-PAGE (5%) under nonreducing conditions and transferred to PDVF membrane ( Millipore Corp. ). Nonspecific binding sites on the membrane were blocked with PBS containing 5% milk. The membrane was incubated with milk containing B-BSA, B-sCD23, B-CD51 mAbs (clone AMF7 and LM142), or B-CD61 mAb. After overnight incubation, membrane was treated with avidin-DH and biotinylated peroxidase complex (Vectastain, ABC kit, Vector Labs, Inc.) followed by ECL detection reagent (Nycomed Amersham ). Section title: Statistical Analysis Educational score: 2.678799867630005 Domain: biomedical Document type: Study Language: en Paired t tests have been used to assess levels of significance (* P < 0.05; ** P < 0.01; *** P < 0.001). Section title: 10G2 mAb, Which Inhibits sCD23 Biological Activities, Recognizes CD47 Ag Educational score: 4.279594898223877 Domain: biomedical Document type: Study Language: en sCD23 displays potent proinflammatory activity by directly triggering monokine release from purified monocytes in the absence of costimulatory signals, such as bacterial antigens (LPS or SAC) or T cell–dependent signal . Although CD21 (CR2) and CD11b (CR3) were previously described as novel CD23 counterreceptors , we also detected binding of sCD23 to several T cell lines lacking CR2 or CR3 expression . In an effort to identify sCD23 binding component, we generated mAbs to Jurkat T cells. We identified one mAb, clone 10G2, which neutralized sCD23 biological activities , and displayed similar cell reactivity as sCD23 (Table I ). Specifically, 10G2 mAb, like sCD23, did not stain the CD11b + (CR3) and CD11c + (CR4) THP-1 cell line. It recognized weakly, but with similar intensity, K562 and K562-CR2 cell lines. 10G2 mAb also stained peripheral blood T, B cells, and monocytes (Table I ). 10G2 mAb significantly suppressed sCD23 costimulation of IFN-γ production by IL-2–stimulated T cells cocultured with autologous monocytes . Section title: 10G2 mAb, Which Inhibits sCD23 Biological Activities, Recognizes CD47 Ag Educational score: 4.082053184509277 Domain: biomedical Document type: Study Language: en The molecule recognized by 10G2 mAb was identified by immunoaffinity panning of COS cells transfected with a cDNA library prepared from Jurkat T cells. After several rounds of selection, a single clone was selected which was found by DNA sequencing to encode CD47 Ag (data not shown). Results in Fig. 1 B demonstrate binding of 10G2 to COS cells transfected with CD47 cDNA clone. Section title: 10G2 mAb, Which Inhibits sCD23 Biological Activities, Recognizes CD47 Ag Educational score: 4.124996185302734 Domain: biomedical Document type: Study Language: en The effects of several anti-CD47 mAbs on sCD23 biological activity were examined . Besides the 10G2 mAb (IgM isotype), three other anti-CD47 mAbs of different Ig isotype subclasses, C1Km1 (IgG1), BRIC126 (Ig2b), and B6H12 (IgG1) inhibited IFN-γ production induced by sCD23 plus IL-2. These results support the notion that CD47 is part of the signaling complex triggered by sCD23. Section title: 10G2 mAb, Which Inhibits sCD23 Biological Activities, Recognizes CD47 Ag Educational score: 4.135890483856201 Domain: biomedical Document type: Study Language: en However, it appears that 10G2 was preferentially binding to a particular epitope on CD47. As depicted in Fig. 1 C, THP-1 cells express CD47 Ag but are not recognized by 10G2 mAb. Clone B6H12, a well-defined anti-CD47 mAb, but not 10G2, stained THP-1 cell line in a dose-dependent manner, while Jurkat T cells were recognized with similar intensity by both anti-CD47 mAbs. There was no cross-inhibition of Jurkat staining by the two clones (data not detailed). Our unpublished data also indicated that erythrocytes which expressed CD47 in the absence of integrins were stained by B6H12 but not 10G2. Section title: Anti-CD47 mAbs Suppress CD23 Costimulation of IL-12 and IFN-γ Production Via Fc-independent Pathways Educational score: 4.187232971191406 Domain: biomedical Document type: Study Language: en We investigated the mechanisms underlying the suppression of IFN-γ production by anti-CD47 mAbs. In addition to 10G2 mAb of the IgM isotype, the F(ab)′ 2 fragments of B6H12 or intact BRIC126 mAb suppressed, in a dose-dependent manner, the ability of IL-2 and sCD23 to augment the production of IFN-γ by T cells cocultured with monocytes, demonstrating that the inhibition of IFN-γ secretion by anti-CD47 mAb was not Fc-mediated . Interestingly, IFN-γ secretion by IL-2–stimulated T cells cocultured with monocytes and graded numbers of CD23-transfected CHO cells, was also abrogated by anti-CD47 mAbs . Section title: Anti-CD47 mAbs Suppress CD23 Costimulation of IL-12 and IFN-γ Production Via Fc-independent Pathways Educational score: 4.277142524719238 Domain: biomedical Document type: Study Language: en We previously reported in the presence of IL-2 or IL-15, low levels of CD40L, expressed by unstimulated T cells, were sufficient to engage CD40 on monocytes and trigger IL-12 release. This monocyte-derived IL-12 production synergized with IL-2 or IL-15 to augment IFN-γ production by T cells . The IFN-γ response could be further amplified by sCD23-induced TNF-α release . Although sCD23 did not trigger IL-12 production by purified monocytes , it costimulated IFN-γ and IL-12 release in this coculture system, and the secretion of both cytokines was strongly inhibited by anti-CD47 mAbs . Section title: Anti-CD47 mAb Suppresses sCD23-induced Monokine Release Educational score: 4.155418395996094 Domain: biomedical Document type: Study Language: en Because sCD23 directly triggers TNF-α release by purified monocytes , and monocytes express CD47 (Table I ), we examined the effect anti-CD47 mAb had on sCD23-induced monokine release by monocytes. Anti-CD47 mAb significantly suppressed the induction by sCD23 of TNF-α, IL-1β, and PGE 2 without affecting IL-8 secretion . The data (i.e., inhibition of TNF-α and IL-12 production) provide a mechanism by which anti-CD47 mAb can suppress sCD23 costimulation of IFN-γ production. Section title: Anti-CD47 mAb Suppresses sCD23-induced Monokine Release Educational score: 4.103168487548828 Domain: biomedical Document type: Study Language: en However, anti-CD47 mAb, in the absence of sCD23 or in the presence of LPS, did not modulate TNF-α production . Our unpublished observations revealed that anti-CD47 mAb, used alone or in combination with sCD23, did not induce the production of monocyte deactivators (such as IL-10 or TGF-β), nor did it modulate SAC-induced TNF-α, IL-6, or IL-10 release. These results support the hypothesis that anti-CD47 mAbs interfere with the sCD23 signaling pathway without impairing general monocyte function. Section title: sCD23 and Vitronectin Share the Same Receptor: VnR/CD47 Complex Educational score: 4.249422073364258 Domain: biomedical Document type: Study Language: en Several studies indicate that CD47 is physically and functionally associated with the vitronectin receptor, α v β 3 . Both anti-CD47 and anti-β 3 (CD61) mAbs can block binding of Vn, but not Fn, to α v β 3 , even though Vn does not bind to CD47 . Therefore, we examined the biological effects of anti-β 3 mAb and the natural ligands of VnR (Vn and Fn) on sCD23 function. It is important to note that all cultures were performed in HB101 serum-free medium to eliminate FCS as a source of Vn and Fn. The results suggested that Vn and sCD23 might share the same receptor, namely VnR/CD47 complex. Anti-β 3 mAb suppressed sCD23 function, as defined by TNF-α secretion and IFN-γ production . Furthermore, soluble Vn, but not Fn, suppressed sCD23-induced TNF-α release by purified monocytes . sCD23 and Vn most likely bound to distinct epitopes on α v β 3 , since an anti-α v β 3 mAb (clone LM609), which specifically inhibited Vn binding and function , and RGDS peptide had no suppressive activity . Section title: sCD23 and Vitronectin Share the Same Receptor: VnR/CD47 Complex Educational score: 4.248762130737305 Domain: biomedical Document type: Study Language: en We next investigated the ability of mAbs directed to the VnR complex, anti-CD47, CD61 (β 3 ) and α v β 3 (LM609) mAbs, and natural ligands of VnR, Vn, Fn, and RGDS peptides, to alter sCD23 binding to the Jurkat T cell line. Unexpectedly, anti-CD47 mAbs, alone or in combination , anti-CD61 , or anti-α v β 3 clone LM609 (not shown) did not inhibit sCD23 binding to Jurkat or monocytes (Hermann, P., unpublished observations). Note that sCD23 binding was specifically suppressed by anti-CD23 mAb . The data suggested ligation of the CD47 or β 3 chain of the trimolecular complex by mAbs could indirectly inhibit sCD23 function by providing a negative signal to the target cells, or by modifying CD47 complex configuration without displacing the sCD23 molecule. We examined whether engagement of VnR/CD47 complex by its natural ligands would modify sCD23 binding. As shown in Fig. 5 d, Vn, but not Fn or RGDS peptide (not shown), significantly inhibited sCD23 binding, strongly suggesting the VnR complex was involved in the secretion of proinflammatory cytokine via interaction with sCD23. Section title: sCD23 and Vitronectin Share the Same Receptor: VnR/CD47 Complex Educational score: 4.159971714019775 Domain: biomedical Document type: Study Language: en The possible role of the α v chain (CD51) of VnR in sCD23 binding was explored. Three anti-CD51 mAbs were tested, and we found one anti-CD51 mAb (clone AMF7) inhibited the interaction between sCD23 and α v + β 3 + Jurkat T , as well as α v + β 3 − Raji B cell lines . Note that the Raji B cell line expresses the β 5 integrin (data not shown). We postulated that sCD23 was binding to α v and not β 3 chain of the VnR, whereas β 3 and CD47 chains were likely to be involved in the signaling of the trimolecular complex in monocytes because anti-CD47 and anti-β 3 inhibited sCD23 function, but not binding. Section title: sCD23 Interacts with α v /CD51 and CD47 Coexpression Increases its Binding Educational score: 4.132336616516113 Domain: biomedical Document type: Study Language: en Next, we selected a melanoma cell line, strongly expressing α v β 3 to purify this integrin by affinity chromatography using anti-β 3 (CD61) immobilized AFFi-gel. Western blot analysis shows sCD23 (lane 6) reacted with a single band of ∼135 kD, displaying a similar migration pattern as molecular species recognized by a cocktail of anti-CD51 mAbs (lane 2). However, sCD23 did not appear to bind purified β 3 (CD61) chain which was identified by anti-CD61 mAb (lane 4), or purified recombinant CD47 (not shown). Section title: sCD23 Interacts with α v /CD51 and CD47 Coexpression Increases its Binding Educational score: 4.218016624450684 Domain: biomedical Document type: Study Language: en To further support the hypothesis that sCD23 may directly interact with α v chain of the trimolecular complex, we prepared CHO transfectants singly expressing human α v chain (CHO-CD51). The results in Fig. 7 demonstrated sCD23 strongly bound to CHO-CD51 compared to untransfected cell line. Although CHO-CD51 transfectant did not express human β 3 (CD61) chain, CHO cell lines were reported to express rodent β chain integrins which likely associated with the human α v chain underlying the successful stable expression of a single human integrin chain. Nevertheless, CHO cells also expressed hamster CD47 which might contribute to sCD23 binding to α v /CD51 on live cells as reported for Vn binding to untransfected CHO cells . To directly assess whether CD47 expression was required for sCD23 interaction with α v /CD51, we examined sCD23 binding to human CD47 deficient cell lines. As shown in Fig. 8 , sCD23 bound to OV10 ovarian carcinoma VnR + /CD47 − cell line demonstrating that CD47 was dispensable for sCD23 binding. Coexpression of CD47 further increased its binding. A similar effect was seen on transfection of CD47 − Jurkat with CD47 (data not shown). Section title: sCD23 Interacts with α v /CD51 and CD47 Coexpression Increases its Binding Educational score: 4.119001388549805 Domain: biomedical Document type: Study Language: en Given that sCD23 was not binding to CD47 + /α v − , but reacted with α v + β 3 − , we concluded that sCD23 ligated α v /CD51. The VnR complex (α v β 3 /CD47 and/or α v β x /CD47) was used as a functional receptor for sCD23 to mediate its proinflammatory activity and, as such, may be involved in the inflammatory process of the immune response. Section title: Discussion Educational score: 4.304511070251465 Domain: biomedical Document type: Study Language: en Our results can be summarized in the schematic model presented in Fig. 9 . We first postulate that sCD23 and Vn have distinct recognition sites on the α v β 3 / CD47 complex. sCD23 binds to α v (CD51) and Vn to α v β 3 (CD51/CD61) conformational site. Binding of Vn may induce structural changes in the integrin complex leading to the masking of sCD23 binding sites, and vice versa. This hypothesis is based on observations that Vn, anti-α v mAb (clone AMF7), but not clone LM609 (which specifically recognizes Vn binding site), nor RGDS peptide inhibited sCD23 binding and function , and sCD23 directly bound to α v chain . However, CD47 was not a direct ligand for sCD23 while its coexpression improved sCD23 binding to α v /CD51. sCD23 bound CD47 deficient cell lines, but failed to bind CD47 in the absence of α v /CD51 integrin . Section title: Discussion Educational score: 4.356382369995117 Domain: biomedical Document type: Study Language: en Secondly, CD47 or CD61 engagement by mAbs prevents Vn binding without displacing sCD23 molecule while these mAbs inhibit both sCD23 and Vn function perhaps by modifying β 3 conformation or signaling pathway. Lindberg et al. indicated that anti-CD47 and anti-β 3 (CD61) mAbs (directed against an epitope of β 3 located outside the Vn binding site) inhibited Vn-opsonized particle binding and function. They also proposed the CD47 molecule participated in the appropriate folding of α v β 3 , and modulated the affinity of α v β 3 for Vn . Vn-coated particles failed to bind to a CD47 deficient cell line or to cells isolated from CD47 deficient mice , while these cells expressed α v β 3 , demonstrating that CD47 is dispensable for VnR expression, but required for Vn binding and function. Using truncated forms of CD47, they also reported the interaction between the extracellular domain of CD47 (IgV) and α v integrins was sufficient for Vn binding . Section title: Discussion Educational score: 4.235032558441162 Domain: biomedical Document type: Study Language: en It has been reported that adhesion of integrins to their counterreceptors is a dynamic phenomenon regulated by intracellular signal transduction pathways . Structural changes in the extracellular domains of integrins following mAb ligation may modify ligand adhesiveness in inducing or inhibiting ligand binding (inside-out signaling), or may directly provide a negative signal to the cell (outside-in signaling), as proposed here for anti-CD47 and anti-CD61 mAb-mediated inhibition of sCD23 function. Section title: Discussion Educational score: 4.135425567626953 Domain: biomedical Document type: Study Language: en In agreement with this hypothesis, it was reported that antibodies recognizing CD81, another member of multispan transmembrane receptors family, inhibited FcεRI-mediated mast cell degranulation without affecting IgE binding, receptor-mediated Ca 2+ release, or tyrosine phosphorylation . Section title: Discussion Educational score: 4.331396102905273 Domain: biomedical Document type: Study Language: en Therefore, we propose a novel function for VnR/CD47 complex in the regulation of inflammatory response. In the absence of pathogen, ligation of VnR by sCD23 mediates monokine release such as TNF-α which enhances the inflammatory process by triggering the cascade of proinflammatory cytokine secretion , and facilitates the elimination of apoptotic cells . Both effects are negatively regulated by the presence of Vn . Vn is a glycoprotein which is synthesized in the liver and circulates in plasma at high concentration (200–400 μg/ml). The insoluble form is localized extravascularly and is associated with granulation tissue areas in rheumatoid arthritis synovia . Section title: Discussion Educational score: 4.096882343292236 Domain: biomedical Document type: Study Language: en It has been reported that β 3 complex signaling via CD47 affected β 2 (CD18/CD11b and CD18/CD11c) integrins binding to their ligands . Previous studies identified CD11b and CD11c as novel ligands for sCD23 . Our study shows that sCD23 bound CD11 − cell lines failed to stain the THP-1 (CD11 + ) cell line , and anti-CD47 mAb did not alter CD11b expression, or sCD23 binding on monocytes (data not shown), indicating that sCD23 does not interact with β 2 integrins. We currently have no explanation for these contradictory results. Section title: Discussion Educational score: 4.202572345733643 Domain: biomedical Document type: Study Language: en Interestingly, our unpublished data indicating TSP, a newly discovered CD47 ligand , also suppressed sCD23 function, without directly triggering monokine release, further supporting our present model. The absence of VnR-mediated monokine secretion following engagement by Vn, Fn, or TSP does not exclude the possibility that other ligands would share, with sCD23, its proinflammatory activity. Finally, the ability of the anti-CD47 mAbs examined to inhibit the function of sCD23 without interfering with the phagocytosis of senescent cells may help in the design of novel therapeutic strategies for chronic inflammatory disorders, such as rheumatoid arthritis, in which CD23 and TNF-α are implicated . | Study | biomedical | en | 0.999997 |
10037798 | Section title: Cells and Cell Lines Educational score: 4.270752906799316 Domain: biomedical Document type: Study Language: en DT40 cells deficient in Syk and those reconstituted with Syk have been previously established . Human M21 melanoma cells and established lymphoblastoid cell line (LCL) (JY) and lymphoid tumor cells , with adhesion and integrin expression as published were obtained from J. Wilkins (University of Manitoba, Manitoba, Canada). Clone E6 of the Jurkat cell line was obtained from American Type Culture Collection. All lines were cultured in RPMI 1640, supplemented with glutamine/geneticin ( Sigma ) and 10% fetal bovine serum ( GIBCO-BRL ). The DT40 cell lines were further supplemented with 1% chicken serum ( GIBCO-BRL ). LCL were established by outgrowth of freshly isolated, purified B cells infected with EBV-B95 . Murine γδ T cells were established from lymphocytic choriomeningitis virus (LCMV) preimmunized animals cocultured with LCMV-infected, irradiated helper cell populations as described . In brief, splenic lymphocytes were harvested from infected mice, and cytotoxic γδ T cells (CTLs) established by outgrowth in the presence of 10 U/ml murine interleukin (IL)-2 and irradiated LCMV-infected autologous helper cells. Cells were used in the assay after 5 d in culture. Sorted human lymphocytes expressing integrin αvβ3 were isolated from PBMC after ficoll-hypaque differential gradient purification followed by flow cytometry (FACStar™; Becton Dickinson ) using murine monoclonal P4C10 (5 μg/ ml) purified from the hybridoma and rabbit antisera to integrin αvβ3 (1: 1,200), detected with secondary goat anti–mouse (PE-conjugated) or goat anti–rabbit (FITC-conjugated) preadsorbed antisera, respectively (Southern Biotechnology). Populations expressing high levels of both integrins were principally monocytes and were identified and excluded by forward scatter, side scatter, and fluorescence (phycoerythrin [PE]) gating. Analysis of these populations with direct-labeled (FITC-conjugated) reagents 24 h later confirmed these cells were a mixed lymphocyte population (∼65% CD19+, ∼20% CD3+) without monocytes (CD14−) or progenitor cells (CD38−). Antibodies were purchased from PharMingen . Section title: Cell Adhesion Educational score: 4.26750373840332 Domain: biomedical Document type: Study Language: en Cell adhesion to 48-well plates precoated with substrate ligand was performed as previously described . In brief, cells were suspended at 10 6 /ml in RPMI 1640, 1.0% BSA. 200-μl aliquots of cells were added to wells which had been precoated overnight with the specified ligands in PBS, pH 8.0, at 4°C and then blocked for 1 h at room temperature with RPMI, 3% BSA ( Sigma ). Fibronectin ( GIBCO-BRL ) and pronectin (Stratagene) were purchased; superfibronectin and monomeric vitronectin were gifts of E. Ruoslahti (The Burnham Institute, La Jolla, CA) and D. Sieffert (The Scripps Research Institute, La Jolla, CA). Recombinant, pentameric penton base (PB) was expressed in insect cells as described previously . Monomeric PB integrin-binding domain was the gift of T. Muir (The Rockefeller University, New York, NY). Adhesion was allowed to proceed for 30 min at 37°C followed by washing to remove nonadherent cells and then quantitation of adherence by staining adherent fractions with crystal violet (0.1% in 0.15 M NaCl, 20% ethanol). Cell-bound stain was resolubilized in methanol and quantitated at 600 nm. Soluble fibrin was formed by the addition of 0.2 U thrombin in DME (50 U/ml stock) ( Calbiochem-Novabiochem ) to the specified fibrinogen (American Diagnostica) concentrations during coating. The addition of thrombin to BSA- or fibrinogen-coated wells served as a control and did not differ from thrombin-free coated surfaces. Molar concentrations of pentameric PB were calculated based upon the five individual molecules within each pentamer (therefore, five integrin-binding sites). To express PB concentration as an aggregate unit (pentamer), concentration may be reduced fivefold. Purified lymphocyte adhesion to substrate-coated chamber slides was quantitated by direct counting of six fields at 100× after sorting on a FACStar™ ( Becton Dickinson ). Three independent sorts were performed. Adhesion to cryosections from three different sources was performed with Cell Tracker Green-labeled LCL (5 μM for 15 min; Molecular Probes) in binding buffer (DME, 3% BSA, 100 U/ml heparin) as previously described and was quantitated similarly. Section title: Radioimmunoassay Educational score: 4.13199520111084 Domain: biomedical Document type: Study Language: en Adhesion plates (48 well) were coated exactly as described for the adhesion assay and blocked with 1.0% BSA in DME (assay buffer) for 1 h at 37°C. Wells were probed by the addition of with 0.5 μg of [ 125 I] radiolabeled mAb DAV-1, which recognizes an epitope (IRGDTFAT) within the integrin-binding domain . DAV-1 was radiolabeled with [ 125 I] (ICN) using Iodobeads (Pierce) according to the manufacturer's instructions. The specific binding of DAV-1 (< 1%) was assessed after 1 h. Specific binding was resolubilized by the addition of 1% SDS, 50 mM Tris, pH 6.8, 10% betamercaptoethanol (100°C) ( Sigma ) after extensive washing (five times) in assay buffer. Section title: Radioimmunoassay Educational score: 3.8542797565460205 Domain: biomedical Document type: Study Language: en DAV-1 binding to BSA alone was not significantly different from background, and was subtracted from all values to get specific binding. Radioactivity bound per unit area was used to calculate sites present after normalizing specific activity per mol of DAV-1 in each experiment. Section title: Integrin-binding Assay Educational score: 4.13863468170166 Domain: biomedical Document type: Study Language: en Adhesion plates (96 well) were coated exactly as described for the adhesion assay (0.5 μg/ml of fibrinogen/fibrin) and blocked with 1.0% BSA in DME (assay buffer) for 1 h at 37°C. Wells were probed by the addition of soluble, biotinylated integrin generously provided by S.L. Goodman for 2 h at 37°C, washed with PBS five times, and then bound integrin was detected by the addition of secondary horseradish peroxidase-conjugated anti-biotin secondary antibody ( Sigma ). After four further washes, the chromogenic substrate 3,3′, 5,5′-tetramethyl benzidine (Bio-Rad) was added and absorbance quantitated at 450 nm after a 10-min incubation. Section title: Immunoprecipitation and Kinase Activity Educational score: 4.260031700134277 Domain: biomedical Document type: Study Language: en LCL were washed twice in PBS, then aliquots (5 × 10 6 in 4,000 μl) were plated for 15 min in DME on surfaces coated with different substrate as indicated. Excess media was then aspirated, leaving the bulk of the cells (adherent and nonadherent) in the final 800 μl. Cells were lysed by the addition of 700 μl 1.0% Nonidet P-40, 50 mM Tris-buffered lysates, pH 7.4, containing 2× protease inhibitor cocktail ( Boehringer Mannheim ) 2 mM PMSF, 2 mM EGTA, and 2 mM sodium vanadate ( Sigma ). Specific kinases were immunoprecipitated after preclearing lysates with protein A–Sepharose (Pierce) by the addition of monoclonal antibody 4D10 (Syk) ( Santa Cruz Biotechnology ), or polyclonal antisera against phosphoinositide-3-kinase (PIK)–p85 (Grb-1) (Upstate Biotechnology) or Syk ( Santa Cruz Biotechnology ) (2–4 μg/ml) and the addition of protein A– or protein G–Sepharose, as appropriate. Alternatively, immunoprecipitations were performed with agarose-coupled polyclonal antisera to Syk or Lyn ( Santa Cruz Biotechnology ). Immunoprecipitated proteins were resolved by 10–12% SDS-PAGE and transferred to polyvinylene difluoride membranes to facilitate phosphotyrosine analysis via immunodetection with monoclonal antibodies 4G10 (Upstate Biotechnology) and PY72 (prepared from the hybridoma). Similarly, Syk or Lyn autophosphorylation activity was determined from the immunoprecipitates as described by the addition of exogenous γ 32 P-ATP (5 μCi; ICN) in kinase buffer (20 mM Hepes, pH 7.4, 10 mM MnCl 2 , 150 mM NaCl) as described . All activity was normalized by reprobing to account for small variations in protein loading of individual lanes. The p85-associated PI(3)K activity was determined after immunoprecipitation by the addition of exogenous γ 32 P-ATP and (4,5) PIP 2 substrate ( Calbiochem-Novabiochem ) followed by silica gel thin layer chromatography (1:1:1, CHCl 3 , CH 3 OH, HCl [1 M]) as previously described and subsequent visualization via autoradiography or ImageQuant phosphorimager analysis (Molecular Dynamics). Section title: Nonadherent Lymphoid Cells Spontaneously Attach to Tissue-immobilized ECM Educational score: 4.210136413574219 Domain: biomedical Document type: Study Language: en Lymphoid cells can be detected in association with the ECM of tumor and inflammatory tissues. Fibrin, a polymeric constituent of tumor ECM , is recognized by integrin αvβ3 expressed on a subpopulation of lymphoid cells . To examine lymphoid interaction with a tumor-associated ECM, nonactivated lymphoblastoid B cells (LCL) were assessed for their attachment to fibrin-rich cryosections of human breast tumors . LCL attachment localized to regions of fibrin deposition , and the observed adhesion was significantly blocked with monoclonal (mAb) LM609 directed to integrin αvβ3 , a known receptor for fibrinogen/fibrin (and other provisional ECM components) expressed on LCL. However, these tissues also contain other adhesive ligands for integrin αvβ3. Thus, fibrin does not account for all the adhesive interactions observed. Section title: Nonadherent Lymphoid Cells Spontaneously Attach to Tissue-immobilized ECM Educational score: 4.15179443359375 Domain: biomedical Document type: Study Language: en To characterize this interaction more carefully, LCL were allowed to attach to microtiter wells coated with either fibrin or fibrinogen. Although LCL attached to immobilized fibrin in a concentration-dependent manner, they failed to attach to fibrinogen at all concentrations tested . The adhesion to fibrin, in vitro, was primarily dependent upon both αvβ3 and β2 integrins, as determined using function-blocking mAbs LM609 and TS1/ 18, respectively . In contrast, adherent M21 melanoma cells readily attached to fibrinogen or its polymeric form, fibrin, in a dose-dependent manner and this was blocked by mAb LM609 (data not shown), as previously reported . Section title: Nonadherent Lymphoid Cells Spontaneously Attach to Tissue-immobilized ECM Educational score: 4.424653053283691 Domain: biomedical Document type: Study Language: en M21 cells did not appear to prefer either form of fibrinogen/fibrin, suggesting that both ligand forms offered similar affinity sites for adhesion. To confirm this, the binding of soluble biotinylated integrin to fibrin or fibrinogen-coated surfaces was tested. No difference in ligand capacity to support integrin binding was detected as both ligand forms supported half maximal integrin binding at receptor concentrations of ∼15–20 nM. These results suggested that polymerization of fibrinogen to fibrin did not result in exposure of adhesive sites with increased affinity for integrin. Importantly, preactivation of LCL by phorbol ester , which has been shown to promote integrin clustering in lymphoid cells , activated lymphoid attachment to fibrinogen, demonstrating that this ligand supported lymphoid adhesion . In contrast, attachment to fibrinogen (or BSA) was not rescued by pretreating either the plates or the LCL with thrombin, suggesting that thrombin was not itself an activator of LCL attachment (data not shown). LCL also gained the ability to bind unpolymerized fibronectin or vitronectin after exposure to PMA . However, in all cases examined, multivalent and polymerized forms of fibronectin and vitronectin, like fibrin, supported LCL adhesion without previous stimulation suggesting that LCL adhesion was inducible by polymeric integrin ligands, and that this may be a general property of lymphoid cell integrins. Section title: Multivalent Integrin Ligands Mimic Polymeric ECM and Facilitate Lymphoid Adhesion Educational score: 4.233561038970947 Domain: biomedical Document type: Study Language: en To further establish the structural basis of lymphoid-mediated integrin function, LCL were allowed to attach to one of two structurally-defined αvβ3 ligands. In this case, microtiter wells were coated with a substrate comprised of either a monovalent or pentavalent form of the adenovirus PB protein . The pentavalent form of PB contains five available αvβ3-binding sites equally separated by ∼60 Å , whereas the monomeric construct contains only a single αvβ3-binding site . As shown in Fig. 3 A, the adherent M21 cell line attached to either form of PB, whereas LCL selectively attached to the pentamer. Adhesion to multimeric PB was entirely dependent on integrin αvβ3, since either LM609 or a peptide antagonist of this integrin completely blocked adhesion while antibodies to other integrins showed no effect . The addition of soluble monomeric or multimeric PB also disrupted adhesion , indicating that either form of PB was an effective soluble inhibitor of integrin αvβ3. Section title: Multivalent Integrin Ligands Mimic Polymeric ECM and Facilitate Lymphoid Adhesion Educational score: 4.260594844818115 Domain: biomedical Document type: Study Language: en Examination of the role of ligand density in adhesion to the pentamer revealed that LCL adhesion was dose-dependent with a half-maximal coating concentration of 10 nM . In contrast, LCL adhesion to the monomer could not be achieved at up to a 500-fold increased coating concentration unless the cells were first activated . The available density of adhesive sites were determined by radioimmunoassay with a monoclonal antibody specific for the IRGDTFAT adhesive sequence within the integrin-binding domain of PB . Significant adhesion to the pentamer was observed at concentrations where only 125 adhesive sites/mm 2 were available, yet no adhesion occurred on monomer-coated wells where more than 800 sites/mm 2 were detected . The failure of LCL to attach to the monomer was therefore not due to limiting availability or absolute density of adhesive sites. These findings suggest that it is the relative spatial distribution of the integrin adhesive sites that facilitates LCL adhesion to substrate. Section title: Multivalent Integrin Ligands Mimic Polymeric ECM and Facilitate Lymphoid Adhesion Educational score: 4.363827705383301 Domain: biomedical Document type: Study Language: en Integrin organization on the cell surface may be an important factor contributing to adhesion, since the activation of LCL attachment to monomeric PB did not influence αvβ3 expression (our unpublished data). Lymphoid integrins can be activated by conformational (affinity) changes by a variety of reagents, including manganese . Manganese activated the attachment of LCL to monomeric PB similarly to PMA , and caused conformational changes in integrin αvβ3 which were detected with monoclonal antibody ligand-induced binding site (LIBS)-1 . Although LIBS-1 detects the ligand-occupied form of αvβ3, the presence of manganese elicited conformational changes which exposed this epitope on LCL even in the absence of ligand . In contrast, LCL activated with PMA did not differ from basal LIBS-1 expression . Further, PMA did not stabilize integrin binding of soluble ligand (a relative measurement of affinity) . Nonactivated or PMA-activated LCL were allowed to bind PB monomer, and LIBS-1 was used to detect ligand binding as a function of ligand concentration. No difference in half-maximal ligand occupancy was evident between PMA-activated and untreated LCL for soluble monomeric PB (∼1.5 μM) . We observed that LCL displayed the same relative affinity for ligand as determined for M21 cells and other adherent cells . Thus, activation of LCL by PMA leading to stable adhesion on monomer does not appear to result from preexisting affinity changes. Section title: Preferential Attachment to Multimeric Integrin Substrate Is Conserved in Lymphoid Cells Educational score: 4.211334705352783 Domain: biomedical Document type: Study Language: en Importantly, other LCL and nonadherent B and T lymphoid cells, such as RPMI 8866, RPMI 8226, and Jurkat, also attached selectively to multimeric αvβ3 ligands . In each case, attachment could be inhibited with monoclonal antibody LM609 (data not shown). In contrast, B cell tumor lines which do not express αvβ3 such as DT40 and Ramos, failed to attach to either form of PB . Although integrin αvβ3 has been described in tissue-associated lymphocyte populations , it is only minimally expressed on circulating lymphocytes . However, when the subpopulation of lymphocytes expressing αvβ3 were isolated from peripheral blood (< 2%) , these cells also demonstrated selective adhesion to both multimeric PB and fibrin relative to monomeric PB and fibrinogen, respectively . Thus, lymphocytes which express appropriate receptors can attach to multivalent ligands without a requirement for exogenous activation. Section title: Tyrosine Phosphorylation of p85 and Syk Follows Integrin Contact with Ligand Educational score: 4.187654495239258 Domain: biomedical Document type: Study Language: en It has been previously reported that agonists can facilitate both lymphoid and platelet “outside-in” signaling through the activation of signaling intermediates including Syk, FAK, and PI(3)K . Although nonactivated LCL are capable of recognizing monomeric integrin ligands in solution , the specific presentation of immobilized multimeric adhesive sites was required to facilitate stable adhesion. This suggests that integrin-dependent signaling may be sufficient to initiate postreceptor-binding events that lead to stable lymphoid cell adhesion. Kinetic analysis showed that LCL adhesion occurred principally (∼75%) during the initial 5–15 min (data not shown). Therefore, to investigate possible mechanisms leading to adhesion, lysates from LCL plated on monomeric or multimeric ligands for 15 min were subjected to immunoprecipitation and phosphotyrosine analysis. Section title: Tyrosine Phosphorylation of p85 and Syk Follows Integrin Contact with Ligand Educational score: 4.251894950866699 Domain: biomedical Document type: Study Language: en Although LCL failed to express detectable levels of FAK (data not shown), they expressed both Syk and PI(3)K. Attachment of LCL to pentavalent PB resulted in enhancement of Syk phosphorylation compared with Syk isolated from cells plated on the monomeric substrate or on BSA (4.5 ± 1.7 fold) . Importantly, there was a corresponding two- to threefold increase in Syk kinase activity in cells adherent to the pentamer compared with cells plated on the monomer or the nonadherent substrate protein BSA . In contrast, an increase in phosphorylation of the p85 subunit of PI(3)K and PI(3)K activity was observed in cells interacting with either pentamer or monomer relative to cells plated on BSA. These data suggest that lymphoid cell PI(3)K becomes activated with a low threshold of integrin ligation in general, whereas integrin ligation leading to (stable) adhesion is specifically associated with activation of Syk. Section title: Syk Activity Regulates Integrin-mediated Attachment by LCL Educational score: 4.191451549530029 Domain: biomedical Document type: Study Language: en As an initial approach to testing whether Syk was involved in regulating adhesion to the ECM, LCL were allowed to bind to PB in the presence of either pharmacological inhibitors known to inhibit Syk (piceatannol) or PI(3)K . Adhesion of the LCL to PB was blocked by piceatannol (IC 50 ∼5 mM), but was only slightly impacted by the PI(3)K inhibitor LY294002 . Although LY294002 treated cells attached, they exhibited reduced PI(3)K activity , failed to spread (data not shown), and were more susceptible to shear force during washing, resulting in an apparent slight inhibition of adhesion. Similar results were obtained with an alternative inhibitor of PI(3)K, wortmannin (increasing effect from 70–80 nM, data not shown). Although selective for the kinase Syk, piceatannol has pleiotropic effects on cells, including the blockade of src family kinases at increased concentrations. However, no significant inhibition of the related src family kinase Lyn (the major src kinase in LCL) was found in these studies at the concentrations of piceatannol used . Section title: Syk Activity Regulates Integrin-mediated Attachment by LCL Educational score: 4.240346908569336 Domain: biomedical Document type: Study Language: en Surprisingly, if LCL were allowed to attach and spread, no reversal of adhesion was observed after the addition of piceatannol (data not shown), suggesting that activation of Syk was important in the initial phase of adhesion of lymphoid cells to the multimeric ligands. Therefore, Syk localization was examined by immunofluorescent confocal microscopy during attachment. In nonadherent LCL cells Syk is present diffusely in the cytosol and does not significantly colocalize with actin in the microspikes present ubiquitously across the cell surface . Similarly, there is only modest colocalization of Syk and actin in adherent cells above the plane of interaction with substrate . However, we consistently observed colocalization of Syk and actin in the podosome near the cell–substrate interface during early phases of cell attachment , although this colocalization disappeared upon full cell spreading . Together, these results suggest that the activation of Syk plays a role in the initial polarization of cells leading to productive attachment on multimeric ligand. Section title: Syk Activity Regulates Integrin-mediated Attachment by LCL Educational score: 4.299923419952393 Domain: biomedical Document type: Study Language: en These results suggest that preactivation of Syk might be able to facilitate attachment to monomeric ligands. To determine whether nonintegrin mediated activation of Syk could regulate lymphoid adhesion, we examined an antigen-specific response in Syk-expressing, tissue-resident T cells. CTL cells specific for LCMV were established by coculture with LCMV-infected, irradiated helper cells, resulting in a proliferative response and the activation of Syk . In these CTL, Syk activation by antigen was sufficient to facilitate attachment to either monomeric or multimeric ligands . To confirm that Syk activation was maintained in these cells, lysates from the CTL were generated and Syk immunoprecipitation and phosphotyrosine analysis performed at the conclusion of the adhesion assay. Interestingly, Syk phosphorylation was maintained in CTL populations plated on monomeric or multimeric substrate, but this was not the case when cells were plated on the nonadhesive substrate BSA . These results indicate that Syk phosphorylation may be maintained by ongoing adhesion, and that Syk activation correlates with stable adhesion to monomeric ligand. Section title: Syk Activity Regulates Integrin-mediated Attachment by LCL Educational score: 4.371675491333008 Domain: biomedical Document type: Study Language: en To determine if Syk activity, independent of antigen activation, was sufficient to promote attachment to ECM, nonadherent B lymphoma cells (DT40) deficient in Syk were reconstituted with either wild-type Syk or activated (Y130E) Syk and then examined for their capacity to adhere to a monomeric substrate. Stable expression of Syk (SykWT) in Syk-deficient DT40 cells failed to promote attachment to a monomeric fragment of fibronectin , however, DT40 cells expressing the constitutively active form of Syk (Y130E) gained the capacity to bind to the monomeric cell-binding domain of fibronectin. Parental DT40 cells and their derivatives lack αvβ3 and do not attach to PB , but do express β1 integrins which bind this region of fibronectin. Consistent with this, adhesion was blocked by a monoclonal antibody to β1 integrin (data not shown). These data show that β1 integrin-dependent adhesion to fibronectin, like that mediated by αvβ3, can also be upregulated by Syk activation, and that Syk requires activation to promote lymphoid cell adhesion to integrin ligands. Section title: Discussion Educational score: 4.620809555053711 Domain: biomedical Document type: Study Language: en Inflammatory processes are characterized by the coordinated deposition of provisional ECM and accumulation of leukocytes within affected tissues. Lymphocyte interaction with components of the ECM plays a crucial role in the chronicity and resolution of inflammatory processes. Lymphoid adhesion resulting from integrin-mediated recognition of multimeric ligands such as fibrin, polymerized fibronectin, and vitronectin, as demonstrated, provides a mechanism whereby nonactivated lymphoid cells can become partially activated independent of exposure to antigen or other agonists. The capacity to control adhesion by the multimerization of integrin ligands provides a means to specifically promote the accumulation of lymphoid cells within inflammatory or diseased tissues. Within the tissues, lymphocytes may then undergo further activation by antigen or other agonists leading to the formation of local immune responses or alternatively egress to the draining lymph nodes. Lymphoid activation after tissue invasion exploits the known comitogenic activity of the ECM . Section title: Discussion Educational score: 4.513335227966309 Domain: biomedical Document type: Study Language: en Evidence is presented that a crucial aspect to initiating lymphoid adhesion is the functional multimerization of binding sites for integrins. The current study used the PB protein of adenovirus, a model ligand bound exclusively by integrins , which provided a structurally defined ligand to examine lymphoid interactions with different substrate forms. In fact, the intact PB pentamer is known to have five available integrin-binding sites arranged in a pentagonal array with a separation of 60 Å allowing it to accommodate the binding of five integrins simultaneously. We contend that, like polymerized ECM proteins, PB acts as a clustered ligand and exploits local avidity as a means to initiate signaling events. It is clear that the overall (general) density of interaction is not as efficient in this respect, since much higher levels of monomeric penton base did not support adhesion. In fact, monomer coated surfaces containing 800 integrin sites/mm 2 were unable to support lymphoid adhesion, whereas multimer-coated surfaces with less than 125 sites/mm 2 available supported LCL adhesion. To demonstrate that the monomer could support adhesion, PMA, an agonist which promotes lymphoid integrin clustering via a PKC-dependent pathway , was used to rescue attachment to monomer coated surfaces. Alternatively, it is possible that adhesion was observed on the multimeric proteins because the pentameric form of PB, and of ECM proteins in general, present higher affinity sites for integrin interactions upon multimerization. However, at least in the case of αvβ3/fibrinogen interaction, this does not appear to be the case . Competitive blocking studies indicate that soluble PB monomer and the pentamer have a similarly high affinity for integrin (IC 50 ∼18 and 5 nM, respectively) although pentameric PB is significantly more potent at inducing integrin-mediated signaling events (Li, E., and G. Nemerow, unpublished data). Therefore, these findings suggest that appropriately spaced clustering of integrin ligand provides a specific mechanism through which these postreceptor events promote stable adhesion. The fact that adherent M21 cells attached to either substrate suggests that lymphoid cells regulate postreceptor ligation events differently than adherent cell types. Section title: Discussion Educational score: 4.409057140350342 Domain: biomedical Document type: Study Language: en By itself, affinity modulation of integrin function did not appear to explain the observed agonist-induced adhesion, since soluble integrins maintained the same affinity for either multimeric or monomeric ligand . It remains possible that avidity changes in integrin (clustering) can result in higher affinity through local packing and lateral stabilization of ligated integrin. Such changes might not be detected by conformational/affinity measurements such as LIBS binding due to steric issues of accessibility. Further study will be necessary to differentiate pure affinity changes from local packing effects. However, in addition to activation of PKC and Rho-dependent integrin clustering , PMA activates a variety of cellular signaling pathways, and induction of integrin-mediated adhesion by this agonist has previously been suggested to occur as a result of postreceptor events . The current investigation similarly provides evidence for postreceptor signaling events inducing attachment, and suggests basic differences in the response of lymphoid cells and adherent cells to different ligand forms. These findings suggest that ligand polymerization may provide all cues necessary to facilitate stable lymphocyte attachment. Section title: Discussion Educational score: 4.524041175842285 Domain: biomedical Document type: Study Language: en Based upon these results, we propose that integrin ligation-dependent signaling impacts the ability of lymphoid cells to attach to the ECM. Several morphological (e.g., cell spreading) and signaling events were observed to occur during adhesion, including the phosphorylation of both Syk and p85. Although p85-associated PI(3)K activity increased after interaction with PB pentamer, it was similarly observed after contact with monomeric PB where no stable adhesion was detected. Inhibitors of PI(3)K did not block attachment of LCL to pentameric PB, however, they did disrupt spreading and the extension of processes . However, some low level of PI(3)K activity may be required for adhesion, and our data supports the possibility that activation of PI(3)K plays a crucial role in attachment under conditions of flow/high shear. Pretreatment with piceatannol blocked adhesion to pentamer at very low concentrations (5 μM), suggesting that activation of Syk was required to induce subsequent attachment , but was not necessarily involved in spreading or maintenance of LCL adhesion. These results are supported by the colocalization of Syk with actin during initial cell polarization/podosome formation . The activation of Syk in lymphoid cells by antigenic stimulation or by reconstitution with constitutively active mutants rescued lymphoid cell attachment to monomer-coated surfaces . Together, these results indicate that integrin postreceptor signaling events are crucial in governing LCL adhesion, and that local clustering of ligand provides a means to elicit integrin-mediated signaling resulting in stable attachment. Parallel studies confirmed that attachment to PB occurred as a result of postreceptor signaling in other cell types. For example, we were unable to observe attachment to PB with melanoma cells bearing a mutant β3 integrin tail (N744A), which is deficient in signaling but not ligand binding (our unpublished observations). Section title: Discussion Educational score: 4.512875556945801 Domain: biomedical Document type: Study Language: en The activation of the hematopoietic kinase Syk occurs via the antigen receptor, soluble agonists, and integrin interactions with immobilized substrate . It is clear that qualitative differences exist in the outcome after interaction with different ligands. Activation of Syk through the antigen receptor leads to activation of MAP kinases and proliferation , whereas our results suggest a role for Syk in regulating adhesion. Adhesion alone was not sufficient to fully activate freshly isolated lymphocytes, since attachment to multimeric integrin ligands did not lead to significant proliferation of nonactivated, sorted peripheral blood lymphocytes (our unpublished observations). However, after initial antigenic stimulation, γδ CTL adhesion to monomer and pentameric ligands was observed, and Syk phosphorylation was maintained throughout the course of the adhesion. Supporting this result, coexpression of integrin αvβ3 and the ζ subunit of the T cell antigen–receptor complex are known to be sufficient for continued γδ CTL clone proliferation and cytokine secretion . The ζ subunit consists of a transmembrane domain and an immunoreceptor tyrosine-based activation motif (ITAM)-containing docking motif which is recognized specifically by the dual SH2 domains of Syk and its homologue ζ-associated protein (ZAP)-70 , providing a mechanism by which integrin-mediated Syk activation pathways and antigen-stimulated pathways may couple. Section title: Discussion Educational score: 4.498263359069824 Domain: biomedical Document type: Study Language: en Similarities do exist in the initial signaling pathways triggered by integrins or antigen receptors, since both integrins and antigen receptors activate specific members of the src kinase family identified as activators of Syk . Moreover, cross-linking either antigen receptor or integrins results in Syk activation , suggesting that clustering of integrins by multimeric ligand leads to Syk activation. Syk, in turn, is known to phosphorylate cytosolic target proteins including Pyk-2 , α-tubulin , and the guanine nucleotide exchange factor Vav , supporting a mechanism for rapid cytoskeletal reorganization in lymphoid cells during both antigen presentation and adhesion to the ECM . However, Syk is not always required for the activation of downstream targets by G protein– coupled receptors or other agonists . Therefore, modulation of integrin function by chemokines , multimeric ECM, and/or antigenic stimulation may potentiate adhesion via somewhat distinct signaling pathways. Section title: Discussion Educational score: 4.557343482971191 Domain: biomedical Document type: Study Language: en In general, recognition of ECM by lymphocytes is critical to a variety of processes, including inflammation, lymphoid maturation, tissue retention, trafficking, and surveillance . The implication of Syk as a crucial effector of lymphocyte–ECM interactions is interesting, since Syk-deficient lymphocytes are defective in tissue entry and distribution, as well as in subsequent maturation steps in vivo . Thymic maturation of T cells requires both integrins and Syk kinases . Taken together, these observations reveal a mechanism whereby lymphoid responses are modulated by the local composition of the ECM, and specifically by the multimeric presentation of integrin binding sites. Integrin-mediated adhesion to polymeric ligands plays a key role in modulating lymphoid responses through the central kinase Syk, which in turn exerts pleiotropic effects upon lymphoid cells. This interplay may be important in the understanding of lymphoid cell behavior in vivo, and suggests a prominent role for the local ECM in governing lymphoid behavior in malignancy and inflammatory processes. | Study | biomedical | en | 0.999997 |
10037799 | Section title: Cells, Antibodies, Chemicals, and Ig Fusion Proteins Educational score: 4.150020599365234 Domain: biomedical Document type: Study Language: en Human umbilical vein endothelial cells (HUVEC) were kindly donated by Dr. Sue Adams (Molecular Parasitology Group, University of Oxford) and used at the third passage. mAbs to human CD31 (JC70), CD34 (QBEND 10), and von Willebrand factor (vWF, F8/86) were kindly donated by Dr. David Mason (Department of Cellular Science, University of Oxford). Texas red–conjugated goat anti–rabbit IgG and fluorescein-conjugated goat anti–mouse IgG reagents were obtained from Southern Biotechnologies. Biotinylated HA-binding complex (bHABC) was purified from bovine articular cartilage by extraction with 4 M guanidine-HCl and biotinylated as described previously ( 48 ). Biotin-LC-hydrazide was obtained from Pierce Chemical Co. , and high molecular weight HA (rooster comb) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) were from Sigma Chemical Co. Soluble Ig Fc (fragment-complement binding) fusion proteins corresponding to the extracellular domains of CD44 hematopoietic form (residues 1–200, CD44H Fc; see refs. 1, 37), ICAM-2 (complete extracellular domain, ICAM-2 Fc), and CD33 (complete extracellular domain, CD33 Fc) fused to the hinge, CH2, and CH3 domains of human IgG 1 were donated, respectively, by Dr. Kelly Bennett (Bristol-Myers Squibb, Seattle, WA), Dr. Sue Adams (see above), and Dr. Regis Doyonnas (MRC Molecular Haematology Unit, Institute of Molecular Medicine, University of Oxford). A soluble truncated form of CD44H (CD44 158his ) comprising residues 1–158 of the ectodomain was expressed in Escherichia coli and refolded from urea-solubilized inclusion bodies ( 3 ). Details of the CD44 158his protein which displays similar HA binding to CD44H Fc have been published recently ( 3 ). Section title: Cloning and Identification of the LYVE-1 Receptor cDNA Educational score: 4.160489559173584 Domain: biomedical Document type: Study Language: en A commercial cDNA database (Human Genome Sciences, The Institute for Genome Research) of >10 6 expression sequence tags (ESTs) obtained by random DNA sequencing of clones within 700 different human cDNA libraries was screened for ESTs bearing significant homology to the CD44 HA receptor using the program BlastSearch (Genetics Computer Group). Several overlapping ESTs with translated amino acid sequences that were at least 30% identical to that of full-length CD44 were identified in cDNA libraries constructed from tissues including umbilical vein, placenta, fetal liver, adipose tissue, lung, heart, prostate, embryo, spinal cord, bone marrow, bone, and ovarian tumor. The LYVE-1 EST described here was isolated from a HUVEC cDNA library, subcloned into the plasmid vector pBluescript, and sequenced in its entirety. Section title: Northern Blot Hybridization Educational score: 4.105640411376953 Domain: biomedical Document type: Study Language: en For Northern blot analysis, multiple tissue RNA blots (2 μg polyadenylated RNA per lane) were purchased from Clontech and hybridized (42°C, 50% formamide, 6× SSC) to a full-length LYVE-1 double-stranded DNA probe labeled with [ 32 P]dCTP by random hexamer-priming (Rediprime DNA labeling system; Amersham International plc ). Blots were washed at high stringency (0.1× SSC, 60°C, 15 min) before exposure to Kodak X-Omat RP x-ray film. Section title: RT-PCR Educational score: 4.079716682434082 Domain: biomedical Document type: Study Language: en For the detection of LYVE-1 and CD44 mRNAs in different cell lines, total RNA was isolated by extraction with guanidinium thiocyanate/sodium acetate, pH 4.0, and ethanol precipitation followed by first-strand cDNA synthesis using reverse transcriptase, and PCR using appropriate LYVE-1 or CD44 primers. Section title: RT-PCR Educational score: 4.201875686645508 Domain: biomedical Document type: Study Language: en First-strand cDNA syntheses were carried out by oligo-dT priming in 50-μl reactions containing 5 μg total RNA, 0.5 μM dNTPs, 0.1 M Tris-HCl, pH 8.3, and 2.5 mU AMV reverse transcriptase for 3 h at 42°C. Samples (1 μl) of the final products were then committed to 50-μl PCR reactions (94°C, 1 min; 55°C, 1 min; 72°C, 1 min; 40 cycles) containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl 2 , 1 mM dNTPs, 1 U Taq DNA polymerase, and the LYVE-1 primers LYVE-1F Hind/LYVE-1R Bam (see below), or the CD44 primers AMP1 (TCCCAGTATGACACATATTGC) and AMP2 (CCAAGATGATCAGCCATTCTGG). The integrity of the cDNA and input RNA was confirmed by PCR with the glyceraldehyde-3-phosphate dehydrogenase primers G3PF (TGGTCGTATTGGGCGCCTGG) and G3PR (CCAAATTCGTTGTCATACCAGG). Products were electrophoresed on 1.25% agarose gels, transferred to charged nylon membranes (Hybond N; Amersham International plc ), and hybridized with 32 P-end-labeled oligonucleotide probes to detect either LYVE-1 (CGCGGATCCCCATAAAAACTTCTGTGACAC) or CD44 (TGTACATCAGTCACAGACCTGCA). Blots were washed at 55°C in 6× SSC for 10 min before autoradiography. Section title: Preparation of Full-Length LYVE-1 cDNA in pRcCMV and Transient Transfection of COS 1 Cells Educational score: 4.14192008972168 Domain: biomedical Document type: Study Language: en For cell surface expression of full-length LYVE-1 molecules, the entire LYVE-1 coding sequence together with 11 bp of 5′ untranslated region and 40 bp of 3′ untranslated region was reamplified (94°C, 1 min; 55°C, 1 min; and 72°C, 1 min, 30 cycles) from the original pBluescript clone (see above) with the primers LYVE-1 F Hind (CGCG AAGCTT GGGTAGGCACGATGGCCAG) and LYVE-1 R Xba (GC TCTAGA GCCTCAGGTGTGTCTCCTC) using Pyrococcus furiosus (Pfu) DNA polymerase, and ligated into HindIII/XbaI cut pRcCMV. The pRcCMV construct was then used to transiently transfect COS 1 cells using DEAE dextran and chloroquine as described previously ( 17 ). Section title: Expression of LYVE-1 as a Soluble IgFc Fusion Protein Educational score: 4.254739761352539 Domain: biomedical Document type: Study Language: en A 684-bp LYVE-1 fragment encoding the predicted NH 2 -terminal leader and extracellular domain, truncating at Gly 232, was amplified (94°C 1 min; 60°C 5 min; and 72°C 10 min, 25 cycles) from the full-length LYVE-1 cDNA clone in pBluescript using Pfu polymerase and the primers LYVE-1F Hind (see above for sequence) and LYVE-1 R Bam (CGC GGATCC CCAGCAGCTTCATTCTTGAATG). After digestion with HindIII and BamH1, the product was cloned into BamH1/HindIII cut IgFc vector to yield a construct encoding amino acid residues 1–232 of the LYVE-1 sequence, fused at the COOH terminus with the 234-residue hinge, CH2, and CH3 region of human IgG 1 ( 1 ). The absence of polymerase-induced errors was confirmed by sequencing the resulting construct on both strands. For expression, the LYVE-1 cDNA was transiently transfected into SV-40–transformed African green monkey kidney cells (COS 1) using DEAE dextran. After 72 h, culture supernatants were collected, supplemented with 0.1 M Tris-HCl, pH 8.0, and the fusion protein purified by protein A–Sepharose affinity chromatography. Purity was assessed by SDS-PAGE analysis under both reducing and nonreducing conditions, and by Western blot analysis with peroxidase-conjugated anti–human IgGFc antibody. Section title: Western Blotting/Biosynthetic Labeling Educational score: 4.099114418029785 Domain: biomedical Document type: Study Language: en For detection of LYVE-1 protein expressed in transfected COS 1 cell lysates, these were boiled in SDS-PAGE sample buffer, electrophoresed on 10% polyacrylamide SDS-PAGE gels, and transferred to nitrocellulose membranes (Hybond super; Amersham International plc ) using a semi-dry transfer apparatus (Hoefer). Blots were incubated with LYVE-1 polyclonal serum (1:100 dilution in PBS, pH 7.5, 5% dried milk powder, 0.1% Tween 20), and developed with peroxidase-conjugated anti–rabbit Ig using chemiluminescent detection (ECL kit; Amersham International plc ). Section title: Western Blotting/Biosynthetic Labeling Educational score: 4.11068058013916 Domain: biomedical Document type: Study Language: en For biosynthetic labeling of LYVE-1 Fc fusion proteins, transiently transfected COS 1 cells were cultured (24 h, 37°C) in RPMI supplemented with 100 μCi/ml [ 35 S]methionine/cysteine ( Amersham International plc ) followed by absorption of the labeled fusion proteins from the supernatants on protein A–Sepharose beads. Beads were then boiled in SDS-PAGE sample buffer and electrophoresed on 10% polyacrylamide SDS-PAGE gels. After electrophoresis, the gels were fixed, stained with Coomassie blue, and impregnated with AMPLIFY ( Amersham International plc ), before drying and exposing to Kodak X-Omat x-ray film at −70°C. Section title: Binding to Immobilized HA. Educational score: 4.190163612365723 Domain: biomedical Document type: Study Language: en Binding of LYVE-1 IgFc fusion protein to immobilized HA was measured in a 96-well microtiter plate ELISA assay. In brief, microtiter plates (Nunc Maxisorp) were coated with HA (purified from rooster comb; Sigma ) at a concentration of 2 mg/ml in coating buffer (15 mM sodium carbonate and 34 mM sodium bicarbonate, pH 9.3). After overnight incubation at 25°C, blocking (2 h) in PBS, pH 7.5, 0.25% (wt/ vol) BSA, 0.05% (wt/vol) Tween 20, and washing (three times, PBS, pH 7.5), plates were incubated with purified LYVE-1 IgFc or CD44H Fc, or with the control proteins ICAM-2 Fc or CD33 Fc (15–500 ng/well) for 1 h at 25°C. After removal of unbound protein by washing (three times) with PBS, bound fusion protein was detected by successive (1 h, 25°C) incubation with horseradish peroxidase–conjugated anti–human IgG 1 antibody. Reactions were developed by the addition of O -phenylenediamine substrate and the absorbance measured at 490 nm in a Bio-Rad microplate reader. To determine the binding specificity of LYVE-1 for other glycosaminoglycans, the standard assay was modified to include either free chondroitin-4 sulfate, chondroitin-6 sulfate, or heparin (1.5–200 μg/ml) as competitor during the primary incubation step. Section title: Binding to Soluble HA. Educational score: 4.124189853668213 Domain: biomedical Document type: Study Language: en For binding assays with soluble HA, this was first biotinylated by a modification of the method of Yu and Toole ( 33 ). In brief, high molecular weight HA (5 mg/ml) in 0.1 M MES, pH 5.5, was derivatized with biotin-LC-hydrazide (1 mM) in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC, 0.16 mg/ml) in 1-ml stirred reactions overnight at room temperature, followed by dialysis against PBS, pH 7.5, to remove free biotin. Section title: Binding to Soluble HA. Educational score: 4.1276984214782715 Domain: biomedical Document type: Study Language: en To perform the standard binding assay, microtiter plates were coated with LYVE-1 Fc, CD44H Fc, or the control fusion proteins CD33 Fc or ICAM-2 Fc (1.25–10 μg/ml, 1 h, 25°C) washed (three times) to remove unbound material, and incubated with biotinylated HA (5 μg/ml) for a further 1 h. Bound HA was then detected by incubation with horseradish peroxidase–conjugated streptavidin ( Sigma ) and O -phenylenediamine substrate. Absorbances were measured at 490 nm in a Bio-Rad microplate reader as described above. Section title: Sandwich Assay for Binding to Preformed HA Receptor Complexes. Educational score: 4.1056976318359375 Domain: biomedical Document type: Study Language: en To measure binding of LYVE-1 to HA presented as a complex with CD44, microtiter plates were coated with bacterially expressed CD44 158his (5 μg/ml), before successive incubations with HA (250 μg/ml) and either LYVE-1 (5 μg/ml) or CD33 Fc (5 μg/ml, negative control), followed by detection with peroxidase-conjugated anti–human IgFc (see above). To measure binding of CD44 to HA presented as a complex with LYVE-1, microtiter plates were coated with either LYVE-1 Fc or CD33 Fc (negative control), before successive incubations with HA (250 μg/ml) and CD44H Fc (5 μg/ml) and detection with the peroxidase-conjugated CD44 mAb A3D8 (1 μg/ml). Section title: Production of LYVE-1 Polyclonal Serum Educational score: 4.1237897872924805 Domain: biomedical Document type: Study Language: en For the production of polyclonal antisera, rabbits were immunized with purified LYVE-1 Fc fusion protein (100 μg) in complete Freund's adjuvant, followed by three consecutive boosts (50 μg) in incomplete adjuvant, at intervals of 4 wk. Sera were then tested for reactivity with LYVE-1 Fc or with the irrelevant receptor-globulin controls CD33 Fc or ICAM-2 Fc, immobilized to 96-well microtiter dishes using an ELISA assay detected with peroxidase-conjugated goat anti–rabbit IgG and O -phenylenediamine. Antibodies reactive with the human IgG portion of the immunizing antigen were depleted by preabsorption on columns of human IgG Sepharose. Section title: Immunoperoxidase and Immunofluorescent Antibody Staining of Cells and Tissues Educational score: 4.158520221710205 Domain: biomedical Document type: Study Language: en For immunoperoxidase staining, normal human tissue samples were obtained from the Department of Histopathology, Oxford Radcliffe Hospital, as paraffin-embedded sections on silanized glass microscope slides. Slides were dewaxed and rehydrated by successive incubation in Citroclear (5 min), 100% ethanol (5 min), 70% ethanol (5 min), and water followed by microwave treatment (700 W, 8 min) and incubation (30 min, 25°C) with 1:100 diluted LYVE-1 serum in PBS, 5% human serum, 0.1% azide. Antigen was subsequently detected by incubation (30 min, 25°C) with an anti–rabbit Ig peroxidase conjugate, and diaminobenzidine (Envision kit; DAKO), before counterstaining (30 s) with hematoxylin and eosin. Section title: Immunoperoxidase and Immunofluorescent Antibody Staining of Cells and Tissues Educational score: 4.145347595214844 Domain: biomedical Document type: Study Language: en For standard single/double immunofluorescent staining, tissue sections on glass microscope slides were dewaxed and rehydrated as described above before incubation (30 min, 5°C) with 1:100 diluted LYVE-1 serum in PBS, 5% human serum, 0.1% azide, either alone or in combination with 1:2 diluted CD31, CD34, or vWF hybridoma supernatants, or with CD44 mAb (10 μg/ml). After washing (twice) to remove unbound antibodies, samples were then stained (30 min, 5°C) with 1:25 diluted FITC-conjugated goat anti–rabbit Ig (to detect LYVE-1), either alone or in combination with 1–50 diluted Texas red–conjugated goat anti–rabbit Ig. Samples were fixed in 2% (wt/vol) formaldehyde and viewed under a Zeiss Axioskop fluorescence microscope using epifluorescent illumination. Section title: Immunoperoxidase and Immunofluorescent Antibody Staining of Cells and Tissues Educational score: 4.172815322875977 Domain: biomedical Document type: Study Language: en Staining for HA and colocalization with LYVE-1 receptor was carried out using complexes of biotinylated bovine aggrecan G1 domain and bovine link protein as described previously ( 48 ). In brief, dewaxed and rehydrated tissue sections on glass microscope slides were blocked (30 min, room temperature) in PBS, pH 7.5, containing 50 mM glycine and 1% (wt/ vol) fat-free milk powder, before incubation (overnight, 5°C) with bHABC (5 μg/ml) with or without LYVE-1 serum (1:100 dilution) in 0.1 M sodium phosphate buffer, pH 7.4, supplemented with 1% BSA. After washing to remove unbound reagents, the sections were incubated (1 h, room temperature) with a mixture of Texas red–labeled anti–rabbit Ig and FITC-labeled avidin (Vector Laboratories, Inc.), diluted 1:50 and 1:500, respectively, in PBS, pH 7.5. Slides were mounted in Vectashield (Vector Laboratories, Inc.) for immunofluorescence microscopy. Section title: Identification of a Novel HA Receptor LYVE-1 Educational score: 4.567070960998535 Domain: biomedical Document type: Study Language: en To identify new HA receptors we searched recent databases of ESTs for cDNAs homologous to the CD44 molecule, currently thought to be the primary HA receptor on mammalian cells. A homology search of the combined Human Genome Sciences/TIGR EST databases with the amino acid sequence of full-length CD44H using the BLAST program identified a number of ESTs with predicted amino acid sequences >30% similar to CD44. 59 identical ESTs were identified in libraries prepared from fetal tissues including liver, spleen, brain, and heart; adult human placenta, bone marrow, lung, and spinal cord; glioblastoma, ovarian, and bone tumor; and HUVEC. The 2,313-bp EST from HUVEC which we have termed LYVE-1 (lymphatic vessel endothelial HA receptor) was subjected to complete nucleotide sequencing. Translation of the LYVE-1 cDNA revealed a large (966 bp) open reading frame starting with a Kozak ( 24 ) consensus ATG initiation codon at position 91 and terminating with the TAG stop codon at position 1057. The deduced amino acid sequence encodes a 322-residue polypeptide with the features characteristic of a type I integral membrane glycoprotein. These are: a sequence of 26 largely hydrophobic residues at the NH 2 terminus most likely comprising the leader peptide; a hydrophilic sequence of 212 amino acids containing seven cysteine residues, a serine/threonine-rich region (residues 145–216) and two motifs for N-linked glycosylation (NFT and NSS, centered on residues 54 and 131, respectively) corresponding to an extracellular domain; a sequence of 21 hydrophobic residues immediately followed by the dibasic motif KR and a highly charged stretch of 63 residues predicted to form the transmembrane anchor and cytoplasmic tail, respectively . The predicted transmembrane anchor is also apparent in Kyte-Doolittle ( 25 ) and Goldman hydropathy plots of the deduced amino acid sequence . The position of the mature NH 2 terminus is predicted to be Ala 27 based on the −3 +1 rule proposed by Von Heijne ( 56 ). Section title: Similarities between the Link Modules in LYVE-1 and CD44 Educational score: 4.610657215118408 Domain: biomedical Document type: Study Language: en Alignment of the LYVE-1 amino acid sequence with that of the human CD44 HA receptor illustrates the degree of homology between the two molecules which have an overall similarity of 41%. Closer inspection reveals the region of maximal homology (57% similarity, 38% identity) to be contained within the area encompassing the extended CD44 Link domain and the corresponding region (residues 36–139) in the LYVE-1 sequence . The location of the putative Link module in LYVE-1 is marked by four central cysteine residues (Cys 61, 85, 106, and 128), whose spacing (C1 - X 23 - C2 - X 20 - C3 - X 20 - C4) is almost identical in CD44, and whose intervening sequences share 57% similarity. This similarity rises to 68% (57% identity) between C2 (Cys 85) and C3 (Cys 106). These four cysteine residues are conserved in all members of the Link superfamily where they form essential disulfide bridges that stabilize the HA-binding domain, a structure comprising two α helices and two anti–parallel β sheets surrounding a large hydrophobic core ( 23 ). Interestingly the two additional cysteine residues Cys 31 and Cys 133 that uniquely flank the CD44 Link module ( 13 , 45 ) are also present at appropriately conserved locations in the LYVE-1 sequence . Downstream of the Link module, however, the region corresponding to the membrane proximal domain of LYVE-1 shows little similarity to CD44, with the exception of a moderately high proportion of serine and threonine residues (37% within residues 145–216). Section title: Similarities between the Link Modules in LYVE-1 and CD44 Educational score: 4.321033477783203 Domain: biomedical Document type: Study Language: en The presence of the LYVE-1 Link module is more clearly illustrated by sequence alignments with other human Link superfamily members including the cartilage matrix proteins Aggrecan, Versican, and Link protein itself, each of which contain tandem repeats of the Link homology unit . A novel feature apparent from these alignments is that LYVE-1 and CD44 appear to define a subgroup within the Link superfamily. These findings suggest the divergence of the cell surface HA receptors as a separate branch from the HA-binding extracellular matrix proteins during evolution. Section title: Similarities between the Link Modules in LYVE-1 and CD44 Educational score: 4.334035873413086 Domain: biomedical Document type: Study Language: en More detailed comparisons of individual amino acid residues within the putative HA-binding regions of the LYVE-1 and CD44 Link modules reveal a number of differences. For example, of the nine key residues identified by site directed mutagenesis ( 2 , 37 ) as critical for HA binding within the human CD44 Link domain, Lys 38 + 68, Arg 41 + 78, Tyr 42, 79, + 105, and Asp 100 + 101, only three of these, Lys 38, Tyr 79, and Asp 100 are conserved within the LYVE-1 molecule . Furthermore, the peripheral cluster of basic amino acids located downstream of the Link module in CD44, which includes Arg 158 and Lys 162 previously shown to be important for HA binding ( 37 ), is not conserved in the LYVE-1 sequence. Hence, despite overall similarities in Link module sequences, the specific residues involved in the predicted LYVE-1–carbohydrate interaction are likely to be different from those in the CD44 molecule. Section title: The LYVE-1 Receptor Is Expressed on the Cell Surface Educational score: 4.169405460357666 Domain: biomedical Document type: Study Language: en To confirm the identification of the LYVE-1 molecule as an integral membrane glycoprotein, COS 1 fibroblasts were transfected with LYVE-1 cDNA and stained with polyclonal LYVE-1 antibodies for immunofluorescence microscopy. The specificity of the LYVE-1 serum is characterized in detail below. As shown by the results in Fig. 3 A, the transfectants displayed intense surface staining. Western blotting of the LYVE-1 transfected COS cells using a polyclonal serum generated against soluble LYVE-1 revealed a major band which migrated with an apparent molecular mass of 60 kD, and which was absent from mock transfected cells. The discrepancy between the experimentally observed value and the size predicted from the primary sequence (35,158 D) is largely due to glycosylation, since treatment with either N-glycanase or O-glycanase/neuraminidase reduced the molecular mass by ∼20 kD (not shown). Section title: LYVE-1 Binds Both Immobilized and Soluble HA Educational score: 4.265838623046875 Domain: biomedical Document type: Study Language: en To confirm the presence of a functional HA-binding domain in the LYVE-1 receptor, we expressed the extracellular domain with its own NH 2 -terminal leader (residues 1–232) as a soluble fusion protein with the hinge, CH2, and CH3 domains of human IgG 1 (total length 440 residues). The purified protein migrated as a single 80-kD band on SDS-PAGE, similar to a CD44H Fc fusion protein . The discrepancy with the 46,900-D molecular mass predicted from the mature 440 residue primary sequence again is largely due to N- and O-glycosylation of the LYVE-1 core polypeptide (see above). Section title: LYVE-1 Binds Both Immobilized and Soluble HA Educational score: 4.397040367126465 Domain: biomedical Document type: Study Language: en Comparison of LYVE-1 Fc with CD44 Fc in a microtiter plate glycosaminoglycan binding assay revealed that LYVE-1 binds immobilized HA in a concentration-dependent manner that is similar if not identical to CD44. In contrast, neither of the two control fusion proteins CD33 Fc or ICAM-2 Fc displayed any significant binding over this range (0.25–10 μg/ml). The specificity of the LYVE-1 HA interaction was demonstrated by competition with free glycosaminoglycans, which showed that neither chondroitin-4-SO 4 , chondroitin-6-SO 4 , nor heparan sulfate blocked binding even at concentrations up to 200 μg/ml . In contrast the LYVE-1 HA interaction was extremely sensitive to inhibition by free HA (IC 50 ∼3 μg/ml). This indicates that LYVE-1 has a much higher specificity for glycosaminoglycan binding than either CD44 or the cation-dependent liver endothelial receptors. Importantly, this also distinguishes LYVE-1 from the lymph node receptors involved in the uptake and degradation of lymph fluid HA, since these are blocked by chondroitin sulfate and chondroitin sulfate proteoglycans ( 54 ). LYVE-1 Fc also bound soluble high molecular mass HA in a concentration-dependent fashion , although the binding capacity decreased dramatically with increasing levels of HA biotinylation (data not shown), a feature that was not observed with CD44. This latter result suggests there is a significant difference in the size or geometry of the HA-binding surface within these two receptors. Section title: LYVE-1 mRNA Has a Restricted Pattern of Tissue Expression Educational score: 4.248659133911133 Domain: biomedical Document type: Study Language: en Expression of the LYVE-1 gene in vivo was investigated by Northern blot hybridization to poly(A) + RNAs prepared from a range of human tissues . Two major bands of ∼2 and 2.6 kb were abundant in spleen, lymph node, heart, lung, and fetal liver RNA and to a lesser degree in appendix, bone marrow, placenta, muscle, and adult liver RNA. These were largely if not completely absent from peripheral blood lymphocytes, thymus, brain, kidney, and pancreas RNA. In addition, minor bands of 6–7 kb were detected to a variable extent in spleen, lymph node, fetal liver, heart, lung, and muscle. The 2.6-kb band is predicted to correspond to the LYVE-1 transcript shown in Fig. 1 , whereas the 2-kb band likely represents a transcript that is polyadenylated on one or other of the consensus AATAAA motifs at positions 1819 and 1905 within the 3′ untranslated region . The minor 6–7-kb bands have not yet been characterized. Hence it is possible they represent alternatively spliced LYVE-1 transcripts. Section title: LYVE-1 mRNA Has a Restricted Pattern of Tissue Expression Educational score: 4.190730094909668 Domain: biomedical Document type: Study Language: en Intriguingly, RT-PCR analyses of individual cell lines derived from lymphocytes, myeloid cells, monocytes, microvascular endothelial cells, and fibroblasts yielded no products in any cell type with the exception of late passage HUVEC, and placental tissue, used as a positive control. In contrast, each cell line yielded abundant PCR products when amplified with primers to CD44, or the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase. These results suggested an unusually specific pattern of tissue expression for LYVE-1 that was clearly distinct from that of the CD44 molecule. Section title: The LYVE-1 Molecule Is Present on the Walls of Lymphatic Vessels Educational score: 4.201778888702393 Domain: biomedical Document type: Study Language: en To explore the expression pattern of the LYVE-1 protein in more detail, we generated a polyclonal serum by immunizing rabbits with purified LYVE-1 Fc fusion protein (see above). The serum was then preabsorbed on human IgG Sepharose to remove contaminating Fc-reactive antibodies, followed by affinity chromatography on a LYVE-1–Sepharose column. The specificity of the purified antiserum was established using immobilized LYVE-1 Fc fusion protein in an ELISA assay. As shown in the top panel of Fig. 7 , the LYVE-1 antiserum was highly specific for LYVE-1 Fc at dilutions below 1:100 and reacted only weakly with the control ICAM-2 fusion protein, which in common with LYVE-1 Fc, contains the hinge, CH2, and CH3 domains of human IgG 1 . Importantly, the LYVE-1 antiserum was unreactive with a CD44H ectodomain construct CD44 158his which contains a functional Link module (residues 1–158) expressed as a histidine-tagged bacterial fusion protein ( 3 ), with CD44H Fc and with CD44-transfected COS 1 cells (not shown). Finally, the LYVE-1 antiserum (1:100 dilution) had the capacity to completely block binding of soluble bHA . These data indicate the polyclonal serum is specific for LYVE-1 and does not exhibit reactivity with CD44, its closest homologue. Section title: The LYVE-1 Molecule Is Present on the Walls of Lymphatic Vessels Educational score: 4.506228923797607 Domain: biomedical Document type: Study Language: en Immunoperoxidase staining of human tissues with the polyclonal LYVE-1 serum revealed an unusual and highly restricted expression pattern, as shown in Fig. 8 and summarized in Table I . Intriguingly the expression of LYVE-1 was mostly confined to endothelial cells lining vessels of the lymphatic system. The only other sites of prominent LYVE-1 expression were sinusoidal endothelial cells within the spleen and placental syncytiotrophoblasts . In contrast with the expression pattern of CD44, no LYVE-1 was detected on lymphocytes or other hematopoietic cells within lymphoid organs such as the tonsil and thymus . Localization of LYVE-1 within the lymphatics was most strikingly visible among the draining lymphatic vessels within the submucosae, underlying the intestinal crypts in colon and small intestine . Many small lymph vessels, including some containing large numbers of lymphocytes, showed intense endothelial staining. High levels of LYVE-1 expression were also apparent on the lacteal vessels draining individual intestinal villi, on subdermal lymphatic vessels within the skin, and on lymphatic vessels within peripheral nodes . A similar expression pattern was found in other tissues displaying prominent lymphatics, particularly the appendix and stomach (see Table I ). In contrast, no LYVE-1 was detected on blood vessels within any of the tissues tested. Interestingly, LYVE-1 protein expression was detected on the surface of cultured HUVEC , confirming the detection of LYVE-1 mRNA by RT-PCR (see above). This is most likely to be the result of culture induced de-differentiation as no vascular staining was observed in tissue sections of umbilical vein (not shown). The selectivity for lymphatic expression is underlined by a comparison of the two vessel types in sections of salivary gland , where blood vessels that are clearly distinguishable by their erythrocyte content (visible through weak nonspecific staining with rabbit IgG) are negative for LYVE-1, while the adjacent empty lymph vessels stain intensely. This distinction was confirmed by double immunofluorescence staining of small intestine sections with antibodies to LYVE-1 and to the vascular endothelial markers CD34 (sgp90) and vWF (Factor VIII–related molecule). LYVE-1 staining was mutually exclusive with both of the other markers, and was confined to irregularly shaped vessels within the submucosae that were devoid of erythrocytes. Furthermore, double immunofluorescence staining revealed little or no CD44 expression on lymphatic vessels, indicating that LYVE-1 is likely to be the major receptor for HA on lymphatic endothelial cells . Section title: Physiological Function of the LYVE-1 HA Receptor Educational score: 4.1702880859375 Domain: biomedical Document type: Study Language: en The lymphatic system is the main conduit for transport of HA from the tissues and degradation within lymph nodes. However, the molecules within lymphatic vessels which interact with lymph fluid HA have not been identified, and the mechanisms driving HA transport to the lymph nodes have yet to be elucidated. Therefore, we considered the possibilities that LYVE-1 might either sequester HA on the lymph vessel wall or promote HA-mediated rolling of leukocytes present within the lymph fluid. In the first instance, we looked for an association between LYVE-1 and its ligand HA on the luminal face of tissue lymphatics by double immunofluorescent staining, using bHABC composed of bovine Aggrecan and Link protein . Abundant quantities of HA were detected throughout the parenchyma and lining the walls of numerous vessels in all the tissues tested (data not shown). A significant amount of this HA indeed colocalized with LYVE-1 in numerous patches (visible as yellow staining), around the luminal surface of lymph vessels, particularly those in the small intestines . Although relatively high levels of HA (50 μg/ml) are normally found within lymph vessels in vivo ( 49 ), we observed some variation in the amounts retained on LYVE-1 stained vessels between and within different tissues. It is not yet clear whether this reflects variation in the amount of HA, or its recovery during the preparation of different tissues, or variation in the binding affinity of LYVE-1 in different locations. Section title: Physiological Function of the LYVE-1 HA Receptor Educational score: 4.139571189880371 Domain: biomedical Document type: Study Language: en Finally, we looked for evidence that leukocytes adhere to HA presented by LYVE-1 present on the lymph vessel wall. Our initial attempts to demonstrate such adhesion using CD44 transfected Namalwa B lymphoma cells and frozen sections of gut lymphatics in Stamper-Woodruff type assays were difficult to interpret, because of the high levels of binding to HA within the surrounding parenchyma (not shown). As an alternative, we tested the ability of LYVE-1 Fc immobilized on plastic microtiter dishes to support HA-mediated binding to CD44. As shown in Fig. 10 , LYVE-1 indeed supported HA-mediated binding of CD44, regardless of the order used for primary presentation or secondary HA binding. In conclusion, our results indicate that LYVE-1, in addition to binding HA within the lymphatics, may also bind HA-coated lymphocytes in transit to the lymph nodes. Section title: Discussion Educational score: 4.238004684448242 Domain: biomedical Document type: Study Language: en The interaction between cells and extracellular matrix HA is clearly of fundamental importance both during embryonic limb development and in processes such as wound healing and inflammation in later adult life ( 19 – 22 ). Such importance predicts that many receptors must be involved in HA adhesion, and that complex mechanisms must exist to regulate both their expression and their ligand-binding affinity. Yet at present, the list of professional cell surface HA receptors is small and includes only the CD44 molecule, RHAMM, and the LEC HA receptors. Furthermore, deletion of the gene for CD44, the most abundant and widely expressed of these, has little if any deleterious effects on the embryo ( 43 ). Thus, it has become increasingly clear to many workers in the field that additional receptors for HA are likely to exist within the genome. Section title: Discussion Educational score: 4.293083667755127 Domain: biomedical Document type: Study Language: en In this paper we have described the primary structure and biological function of one such receptor, termed LYVE-1, which is present within vessels of the lymphatic system. This new receptor is a type I integral membrane polypeptide whose extracellular domain encodes a single cartilage Link module, the prototypic HA-binding domain conserved within all members of the Link or hyaladherin superfamily ( 51 ). The central core of the LYVE-1 Link module (C2-C3) is 57% identical to that of the human CD44 HA receptor, the only other Link superfamily HA receptor to be described to date and the closest homologue of LYVE-1. Nevertheless, there are distinct differences between LYVE-1 and CD44 that suggest the two homologues differ either in the mode of HA binding or in its regulation. Section title: Discussion Educational score: 4.598914623260498 Domain: biomedical Document type: Study Language: en Firstly, in terms of HA binding, only three of nine residues identified as essential within the CD44 Link module are conserved in LYVE-1 (Lys 38, Tyr 79, and Asp 100) and none of the downstream COOH-terminal basic residues of CD44 are present. Furthermore, unlike CD44, the affinity of LYVE-1 for bHA is drastically reduced with increasing degrees of biotinylation. These features suggest HA is orientated differently within the LYVE-1 Link module and suggest it may bind a larger HA unit than the minimal HA 6 unit bound by CD44 ( 18 , 55 ). Competition experiments with defined HA fragment sizes will be required to distinguish between these possibilities. A further distinction between LYVE-1 and CD44 is their dramatic difference in substrate specificity. Unlike CD44, LYVE-1 displays exquisite specificity for binding HA and no affinity for the glycosaminoglycans chondroitin-4 sulfate, chondroitin-6 sulfate, or heparan sulfate. This remarkable ligand specificity also distinguishes LYVE-1 from the lymph node HA receptors which mediate the uptake and degradation of HA from afferent lymph, a process that is blocked by chondroitin sulfate and chondroitin sulfate proteoglycans ( 54 ). Section title: Discussion Educational score: 4.551618576049805 Domain: biomedical Document type: Study Language: en Secondly, the regulation of HA binding and its functional consequences are likely to differ significantly from CD44. Excluding the Link domain and its immediate boundaries, the LYVE-1 extracellular domain, transmembrane anchor, and cytoplasmic tail bear no similarity to those of the CD44 molecule. In the CD44 ectodomain, alternative splicing of up to 10 exons within the membrane-proximal region regulates HA binding in some cell types ( 5 , 44 ), and introduces new ligand-binding specificities ( 4 , 17 ). Although we have detected high molecular weight LYVE-1 transcripts in some tissues by Northern blotting , we have so far not observed alternatively spliced variants by RT-PCR. In the CD44 transmembrane anchor, a central cysteine residue ( 30 ) regulates HA binding in a cell-specific manner, through the formation of lipid domains within the bilayer ( 35 , 39 ). No such residue is present in the LYVE-1 transmembrane anchor. Lastly, in the CD44 molecule, the 71-residue cytoplasmic tail binds the cytoskeletal components ankyrin and ezrin ( 52 ), and is subject to serine phosphorylation that regulates cell motility on HA substrata ( 16 , 38 ). Although the lack of sequence conservation does not rule out the possibility that LYVE-1 interacts with these cytoskeletal components via different residues or that the LYVE-1 cytoplasmic tail interacts with other as yet undefined molecules, it seems unlikely that the mechanisms which regulate CD44-HA interactions are duplicated for LYVE-1, its closest known homologue. Section title: Discussion Educational score: 4.450565338134766 Domain: biomedical Document type: Study Language: en Perhaps the most striking distinction between LYVE-1 and CD44 is in their patterns of tissue expression. Intriguingly, the LYVE-1 molecule is largely if not exclusively restricted to lymph vessel endothelial cells from which CD44 is almost completely absent. Comprehensive analyses of a large panel of human tissues by immunoperoxidase staining with specific antisera revealed LYVE-1 lining the lymphatics in virtually every tissue where these structures could be distinguished, and included vessels draining gastrointestinal tissues, skin, lymph nodes, breast, and salivary gland. The greatest concentration of LYVE-1 expression was seen in submucosal lymph vessels underlying smooth muscle in the colon, and the Lacteal vessels of intestinal villi that transport dietary lipid absorbed from the small intestine. Lymphatic vessels are morphologically distinguishable from blood vessels by their reduced or missing basement membrane and lack of erythrocyte content. There are currently no specific immunochemical markers that allow identification of lymphatics, although limited studies with endothelial markers indicate differential expression of the L-selectin ligand CD34 and the coagulation Factor VIII–related protein vWF ( 32 , 42 ). Our own results using double immunofluorescence indicate that LYVE-1 defines a set of lymphatic vessels which express neither CD34 nor vWF and identify LYVE-1 as a powerful new marker for future studies of lymphatic structure and function. Section title: Discussion Educational score: 4.497725963592529 Domain: biomedical Document type: Study Language: en The absence of LYVE-1 from vascular endothelial cells, in addition to its absence from lymphocytes, macrophages, fibroblasts, and epithelial cells, implies a rather different physiological role from that of the CD44 molecule, which is expressed abundantly in each of these locations ( 41 ) but is largely absent from lymphatic vessels. The primary role of the CD44 HA receptor appears to be the regulation of cell motility ( 50 ), as evidenced by its association with the migration of mesenchymal cells during differentiation, and with the hematogenous spread of tumor cells in experimental neoplasia ( 47 ). This is also apparent in inflammation, where CD44 expression on lymphocytes promotes migration through HA-coated vascular endothelium ( 7 , 8 ), as well as the migration of lymphocytes through HA-coated reticular networks within peripheral lymph nodes ( 6 ). LYVE-1 by contrast appears to be restricted to relatively nonmotile lymphatic endothelial cells, where it seems more likely to function in the immobilization of HA in the vessel lumen, rather than in its recognition as a positional cue for migration. Section title: Discussion Educational score: 4.389192581176758 Domain: biomedical Document type: Study Language: en A prominent function of the lymphatic system is the provision of fluid drainage of immune cells and foreign antigens from the tissues to the peripheral lymph nodes, where the latter are sampled by professional antigen-presenting cells and phagocytes. Lymph fluid contains high levels (40–50 μg/ml) of HA (see ref. 12 and reviewed in ref. 10), and its flux through the lymphatics is known to increase in inflammatory diseases such as rheumatoid arthritis and psoriasis and in response to bacterial infection ( 10 , 27 ). The lymph vessels thus act as a conduit both for migrating inflammatory cells and for HA, although the precise relationship between the two is not clear. One possibility is that LYVE-1 functions as an endocytic receptor in an analogous fashion to CD44 on macrophages and the LEC HA receptors on liver endothelium. The specificity of LYVE-1 for HA does, however, appear to rule out any likelihood that it is the receptor involved in HA uptake and degradation within the lymph node. Alternatively, since inflammatory cells express CD44, we are tempted to speculate that LYVE-1 molecules may promote their adhesion to lymphatic endothelium via HA. Such a role is supported by our finding that LYVE-1 and HA colocalize to the luminal face of the lymph vessels, and that LYVE-1 can support HA-mediated adhesion to CD44. We are currently performing experiments to distinguish between these and other interesting possibilities. | Study | biomedical | en | 0.999997 |
10049938 | Section title: Lessons Learned from Mouse Tumor Models. Educational score: 4.672236442565918 Domain: biomedical Document type: Review Language: en Studies using adoptively transferred purified T cell subsets or in vivo depletion studies have firmly established an important role for tumor-specific CD8 + CTLs in antitumor immunity (for a review, see reference 6 ). By comparing the relative contribution of CD4 + and CD8 + T cells to the overall immune response, it was shown that activated adoptively transferred CD8 + T cells alone are as effective as adoptively transferred CD4 + and CD8 + T cells, provided that IL-2 is given simultaneously ( 7 – 9 ). Nonetheless, a critical contribution by tumor-specific CD4 + Th cells in the development of an effective antitumor tumor response was consistently found in several murine tumor models ( 10 – 13 ). CD4 + T cells are likely to play a diversified role in antitumor immunity that includes several distinct antitumor effector functions. The role of CD4 + T cells in priming of CTLs is well documented ( 14 ), explaining why activated CTLs, but not naive CTLs, can mediate potent antitumor effects in the absence of CD4 + T cells. Analysis of the participation of individual T cell populations in the elimination of the Friend murine leukemia virus (MuLV)–induced tumor FBL-3, revealed that tumor-specific CD4 + T cells can also exert their effect independently of CD8 + CTLs. Adoptive transfer studies showed that both the noncytolytic CD4 + subset as well as the cytolytic CD8 + subset were individually capable of eradication of disseminated leukemia in tumor-bearing mice (for a review, see reference 15 ). Thus, although the tumor-specific MHC class II–restricted CD4 + T cells are not able to recognize this MHC class II–negative tumor directly, they are able to control tumor growth via a mechanism that does not require CTLs ( 16 ). More recently, it was shown that not only adoptive transfer of CD4 + T cells, but also vaccination with an MuLV-derived Th epitope (but not a control Th peptide) induced protection against a subsequent challenge with MHC class II–negative, virus-induced tumor cells ( 17 ). In this case, the protection induced was dependent on both CD4 + and CD8 + T cells, as depletion of either subset at the time of tumor challenge abrogated the ability to control tumor outgrowth. Simultaneous vaccination with a tumor-specific CTL epitope and the tumor-specific Th epitope, rather than an unrelated Th epitope, resulted in strong synergistic protection. Taken together, these findings illustrate the relevance of identifying and using tumor-specific Th epitopes even in the case of MHC class II–negative tumors, and emphasize the importance of activating both tumor-specific CD8 + and CD4 + cells to establish optimal immunity to cancer. Section title: Orchestration of the Antitumor Immune Response by CD4 + T Cells. Educational score: 4.828131675720215 Domain: biomedical Document type: Study Language: en As evident from the experience in the MuLV tumor models, tumor-specific CD4 + T cells can mediate several functions influencing the outcome of tumor-specific immunity. Numerous studies have focussed on the role CD4 + T cells play in delivery of help for priming of tumor-specific CTLs, resulting in important mechanistic insights into this event. Accumulating evidence indicates that for induction of MHC class I–restricted tumor-specific immunity, cross-presentation of antigens that have been captured by professional APCs plays a dominant role ( 18 – 21 ). Dissection of the cellular interactions involved in CTL priming revealed that Th cells must recognize antigen on the same APC that cross-presents the CTL epitope ( 22 ). These findings explain the requirement for epitope linkage between Th cell epitopes and CTL epitopes important for induction of CTL responses ( 23 ), and could clarify the view that help for CTLs is delivered through the release of soluble factors such as IL-2 produced by Th cells in the proximity of CTLs. Recently, however, it was shown that T cell help for CTLs is critically dependent on interaction between CD40L expressed by Th cells and CD40 expressed by APCs ( 24 , 25 ). Indeed, a central role for CD40– CD40L interactions in the generation of protective T cell– mediated tumor immunity has been demonstrated ( 26 , 27 ). These interactions most likely empower the APCs to prime CTLs, since help for CTL priming can be bypassed by activation of dendritic cells (DCs) through CD40 ( 28 ). Several lines of evidence indicate that CD40 signaling is part of an important pathway in T cell–dependent APC activation. Recombinant soluble CD40L stimulates human monocytes to release proinflammatory cytokines ( 29 ), whereas ligation of CD40 on DCs or interactions between DCs and CD4 + T cells triggers the production of IL-12. In the latter case, IL-12 production by DCs was inhibited by blockade of CD40L on the CD4 + T cell ( 30 ). Moreover, CD40 ligation is a potent stimulus to upregulate the expression of intercellular adhesion molecule 1 (ICAM-1), CD80, and CD86 molecules ( 31 , 32 ). Because CD40-induced activation of professional APCs results in the expression of costimulatory molecules important for CTL priming, this activation is likely to play an important role in the delivery of T help to CTLs. In this model, the APC that cross-presents antigen to both antigen-specific Th cells and CTLs acts as an intermediary for the delivery of help to CTLs. Section title: Orchestration of the Antitumor Immune Response by CD4 + T Cells. Educational score: 4.408203125 Domain: biomedical Document type: Study Language: en Appreciation of the fundamental role of the APC activation state to tune the outcome of T cell responsiveness helps to explain why CTL responses against tumors, including those induced by noninflammatory persistent tumor viruses such as MuLV (and likely human papillomavirus and Epstein-Barr virus), are dependent on T cell help, whereas CTL responsiveness against acute disease-causing cytopathic viruses such as influenza virus is without a clear need for CD4 + Th activity ( 33 ). The difference between these two situations appears to reside in the fact that influenza virus can directly infect and activate DCs to a phenotype conducive to CTL activation in a CD4 + T cell–independent fashion ( 28 ). However, under noninflammatory conditions, such as in many allograft situations and in most cancers, CTL responses are much more Th cell dependent because DCs need to be activated first by specific CD4 + T cells before they trigger T killer responses. Section title: Orchestration of the Antitumor Immune Response by CD4 + T Cells. Educational score: 4.397939682006836 Domain: biomedical Document type: Study Language: en Besides their intimate involvement in priming tumor-specific CTLs, CD4 + Th cells participate in additional effector functions. Evidence that these other Th cell–dependent effector mechanisms play an important role in the host defence against tumors came from studies in the MuLV system in which adoptively transferred tumor-specific CD4 + T cells are implicated in the activation of tumoricidal macrophages involved in tumor clearance ( 15 ). More recently, it was demonstrated in a model involving vaccination with irradiated tumor cells, transduced to secrete GM-CSF, that cytokines produced by CD4 + T cells belonging to the Th1 or Th2 lineage can recruit and activate macrophages and eosinophils, respectively ( 13 ). Protection against tumor challenge was strongly associated with the presence of eosinophils at the tumor challenge site as well as the production of oxygen radicals by tumoricidal macrophages, since genetically modified mice disabled to produce these radicals were severely hampered in their ability to resist tumor challenge. A significant fraction of CD8 knockout, but not CD4 knockout animals, were able to successfully resist tumor challenge, indicating that the observed effects relied on CD4 + T cell–mediated effector mechanisms. Section title: Orchestration of the Antitumor Immune Response by CD4 + T Cells. Educational score: 3.9292006492614746 Domain: biomedical Document type: Study Language: en As in the MuLV system, the tumor described above did not express MHC class II molecules, emphasizing the notion that induction or propagation of CD4 + T cell–mediated immunity can be successfully applied to counteract tumors that lack or lose expression of MHC molecules. Section title: Induction of Tumor-specific T Cell Tolerance. Educational score: 4.587264060974121 Domain: biomedical Document type: Study Language: en The studies described above, together with the identification of new tumor antigens recognized by CD4 + T cells, bring fresh encouragement to the development of anticancer immune intervention schemes. However, manipulation of the immune response to tumors in tumor-bearing hosts might be actively frustrated by the tumor itself, as it has been reported that tumors can induce tumor-specific CD4 + T cell nonresponsiveness ( 34 ). The mechanism of tolerization is, as yet, not clear, but it might mimic many aspects described for tolerance induction to peripheral tissue antigens. Peripheral tolerance induction of both antigen-specific CD4 + and CD8 + T cells to antigens expressed outside the lymphoid system has been described in several models ( 35 – 37 ). In these cases, tolerance is mediated by cross-presentation of the antigen on bone marrow–derived APCs ( 36 , 37 ). As development and growth of tumors is initially not accompanied by inflammatory stimuli or stress to the immune system, antigen derived from the tumor might be shunted in the same cross-tolerizing pathway as reported for peripheral tissue antigens. In this way tumors, as close mimics of the normal tissue from which they are derived, might shrewdly use the T cell tolerizing state of certain bone marrow–derived APCs that normally guarantee tissue tolerance. This tolerization of both helpers and killers might hamper immune intervention schemes that are based on the induction or propagation of the T cell immune system in tumor-bearing hosts. Knowledge about the epitopes recognized by human tumor-specific CD4 + and CD8 + T cells will be instrumental to study whether such a scenario could explain why certain tumors—for instance, melanoma—are able to grow, despite the expression of potentially highly immunogenic tumor antigens. Section title: Induction of Tumor-specific T Cell Tolerance. Educational score: 4.002625465393066 Domain: biomedical Document type: Review Language: en In summary, tumor-specific CD4 + Th cells can orchestrate several effector functions that can cooperate in an effective antitumor response. Knowledge of the antigens and peptides recognized by human CD4 + T cells is of crucial importance for a better understanding of the behavior and role these cells play in the immune response to human tumors, as well as for optimal use of the Th arm of the immune system in the development of new anticancer vaccine modalities. The studies published in this issue describing new MHC class II–restricted melanoma antigens and new methods to identify tumor antigens recognized by CD4 + T cells point to the vital role CD4 + T cells have in immune attack directed against human tumors, and will be of great benefit in optimizing tumor immunotherapy if the rules of the murine models apply to the situation in cancer patients. | Review | biomedical | en | 0.999996 |
10049939 | Section title: Cell Cultures. Educational score: 4.212894916534424 Domain: biomedical Document type: Study Language: en T cell, B cell, and melanoma cell lines were initiated from specimens derived from patient 1558, a 48-yr-old Caucasian male who had a primary cutaneous melanoma lesion resected from the right shoulder 13 yr before the development of metastatic disease in multiple organ systems. Tumor-infiltrating lymphocytes (TIL) were cultured from a subcutaneous metastasis resected from patient 1558 according to methods described ( 12 ), and were maintained in RPMI 1640 with 10% human AB serum and recombinant IL-2 (600 IU/ml; Chiron) for up to 120 d without Ag restimulation. Under these conditions, CD4 + T cells selectively proliferated, reaching >97% after 4 wk of culture, and manifested specific recognition of autologous tumor cells which constitutively expressed MHC class II molecules. Melanoma cultures and EBV-transformed B cell lines were established in our laboratory and maintained in RPMI 1640 with 10% FCS ( 13 ). To grow large quantities of tumor cells for protein purification, the 1558-mel line, which was initiated from the same metastatic lesion used to generate TIL 1558, was maintained in 10-chamber cell factories (Nunc International). The monocytic leukemia cell line THP-1 (American Type Culture Collection) was grown in RPMI 1640 with 10% FCS; before use as APCs, these cells were cultured in the presence of IFN-γ 100 U/ml for 48–96 h to upregulate expression of MHC class II molecules ( 14 ). Section title: HLA Typing. Educational score: 4.083083629608154 Domain: biomedical Document type: Study Language: en HLA serotypes and DNA genotypes of fresh PBLs and cell lines were determined by the National Institutes of Health HLA Laboratory, as described ( 10 ). The class II genotype of patient 1558 was found to be HLA-DRβ1*0101, 1101; DQβ1*0301, 0501; DRβ3*0202. Section title: Assessment of T Cell Responses to Tumor Cells, Tumor Lysates, Protein Fractions, and Peptides. Educational score: 4.2046003341674805 Domain: biomedical Document type: Study Language: en To assess recognition of whole melanoma cells, CD4 + TIL 1558 were cultured in flat-bottomed microtiter plates at 2 × 10 5 cells/well, in the presence of irradiated tumor cells (12,000 rad) at 10 5 /well for 20–24 h ( 15 ). To assess T cell recognition of tumor lysates or fractionated tumor- derived proteins, EBV-B cells or IFN-γ–treated THP-1 cells were used as APCs. APCs cultured at 1.5 × 10 5 cells/well were pulsed for 16–24 h with freeze–thaw lysates of tumor cells (1–2 × 10 5 cell equivalents/well) or fractionated proteins derived from tumor cells (2 × 10 5 to 8 × 10 7 cell equivalents/well), after which T cells were added for 20 h ( 10 ). Peptide recognition was tested by pulsing APCs with peptides for 16–24 h, after which T cells were added to the assay for 20 h ( 9 ). Culture supernatants were then harvested, and GM-CSF secretion by CD4 + T cells was measured using a commercially available ELISA kit (R&D Systems; detection limit 8 pg/ml). In some experiments, anti-MHC mAbs were used to inhibit specific Ag recognition by T cells ( 10 ). Antibodies included L243 (against HLA-DR; IgG2a), IVA12 (HLA-DR, DP, ?DQ; IgG1), Genox 3.53 and G2b.2 , IVD12 (HLA-DQw3; IgG1), and W6/ 32 (HLA-A, B, C; IgG2a) (all purified from American Type Culture Collection hybridoma supernatants). Section title: Preparation of Cell-free Extracts. Educational score: 4.281965732574463 Domain: biomedical Document type: Study Language: en A total of 1.2 × 10 9 adherent 1558-mel cells were harvested from cell factories with trypsin/ EDTA, and cell pellets were washed twice in ice-cold PBS and stored dry at −70°C. Cell homogenization was achieved by suspending the cells in ice-cold sucrose buffer (150 mM sucrose, 2.5 mM dithiothreitol, 4 mM 4-[2-aminoethyl]-benzenesulfonyl fluoride hydrochloride) at a concentration of 10 8 cells/ml and disrupting them in a Wheaton Potter-Elvehjem tissue grinder. All successive purification steps were carried out on ice or at 4°C. Whole nuclei were separated from cytoplasm and membranes by centrifugation at 800 g for 10 min. Salts and protease inhibitors were subsequently added to minimize protein degradation in the supernatant. The mixture was adjusted to contain 50 mM Tris-HCl, 150 mM NaCl, 10% glycerol, 1 mM benzamidine, and 2.5 mM Na-EDTA, pH 7.5. Leupeptin, pepstatin, antipain, aprotinin, and chymostatin were added at 25 μg/ml homogenate. The cytosolic fraction was recovered by a high-speed centrifugation step (31,500 g for 60 min). The pellets which were recovered constituted the membrane fraction. The supernatant (cytosol) underwent addition of polyethyleneimine (PEI) 0.08% and centrifugation to precipitate nucleic acids, followed by a three-step fractionated ammonium sulfate precipitation saturating to 28, 52, and 75% with (NH 4 )2SO 4 . These fractions were dissolved in buffer A (25 mM Tris-HCl, pH 8.0, 25 mM NaCl, 2 mM dithiothreitol, 1 mM benzamidine, 1 mM Na-EDTA) and stored at −70°C if not used directly for further purification. A portion of each subcellular fraction was dialyzed against PBS for testing in bioassays. Section title: Chromatographic Purifications. Educational score: 4.253643989562988 Domain: biomedical Document type: Study Language: en The cytosolic fraction saturated to 75% with (NH 4 )2SO 4 was desalted on Sephadex G25 M columns with buffer A (described above) and loaded onto an anion exchange chromatography column (HiTrap Q, 5 ml). Protein was eluted with an increasing NaCl gradient in buffer A (0.25 mM to 1 M NaCl). Portions of the eluted protein fractions were concentrated, equilibrated in PBS on Microcon-30 membrane units (Amicon, Inc.), and tested for T cell recognition. Bioactive fractions were then pooled and concentrated on a Centriprep-30 membrane unit (Amicon, Inc.) to ∼2 mg protein/ml, and loaded onto a Superdex 75 HiLoad 16/60 gel filtration column. After testing individual eluted fractions for bioactivity, active fractions were pooled and concentrated to ∼1 mg protein/ml. For hydrophobic interaction chromatography, the solution was adjusted to 1.5 M (NH 4 )2SO 4 in buffer A by overnight dialysis at 4°C. A decreasing 1.5–0 M (NH 4 )2SO 4 gradient in buffer A was performed on a Resource Phe hydrophobic interaction column to yield an active protein fraction with sufficient purity for sequence analysis. All chromatography columns were purchased from Amersham Pharmacia Biotech and applied on the AKTAexplorer liquid chromatography system ( Amersham Pharmacia Biotech ). Individual eluate fractions were analyzed by SDS-PAGE on 4–20% acrylamide Tris-glycine precast gels (Novex) stained with Coomassie blue (Novex) or silver stain (Bio-Rad Laboratories). Section title: Nano-HPLC Microelectrospray Ionization Mass Spectrometry and Database Searching. Educational score: 4.372001647949219 Domain: biomedical Document type: Study Language: en A bioactive purified protein fraction eluted from the hydrophobic interaction column was equilibrated in 50 mM ammonium carbonate, pH 7.8, using Microcon-30 membrane concentrators, for sequence analysis. 5 μg of protein was digested with 0.5 μg of modified trypsin ( Promega ) in a 50-μl vol at room temperature for 19 h. The digest was frozen at –35°C until analyzed. An aliquot containing ∼25 ng of digested protein was analyzed by nano-HPLC microelectrospray ionization (μESI) mass spectrometry (MS). All mass spectrometric analyses were performed on an LCQ ion trap mass spectrometer (Finnigan). The ESI head supplied with the instrument was removed and replaced with a μESI source. The ESI voltage (1.5–1.8 kV) was applied via a stainless steel union placed on the HPLC pump side of the reversed phase column. The μESI tips were made from 360-μm outer diameter (OD), 75-μm inner diameter (ID) fused silica capillary (Polymicro Technologies) pulled to ∼20-μm OD, 5-μm ID using a P-2000 laser puller (Sutter Co.). The μESI tips were connected directly to a nano-HPLC column using 350-μm ID Teflon tubing (Zues). Nano-HPLC columns were prepared by packing ∼10 cm of 5-μm C18 material (YMC Corp.) inside 360-μm OD, 75-μm ID fused silica capillary. Solvents A and B for HPLC were 0.1 M acetic acid in water and 0.1 M acetic acid in 70% acetonitrile. The HPLC gradient increased from 1 to 35% acetonitrile in 11 min. Variable flow chromatography (Settlage, R.E., R.E. Christian, J. Shabanowitz, and D.F. Hunt, manuscript submitted for publication; patent pending), technology in which the mass spectrometer data system controls the rate of sample introduction, was used for the analysis of the protein digest. Flow rate for mobile phase into the mass spectrometer was set at ∼200 nl/min at the beginning of the gradient, switched to <50 nl/min shortly before peptides began to elute, and returned to 200 nl/min once peptides were no longer detected. LCQ software programs used in the above experiment included those for data-dependent data acquisition, dynamic exclusion, and isotope exclusion. For data-dependent analysis, the instrument was programmed to continuously cycle through six scan events. Scan event 1 was an MS scan from 300 to 2,000 m/z. Scan events 2–6 were MS/MS scans on the five most abundant ions detected in scan event 1. The dynamic exclusion settings were as follows: 6 amu = exclusion width; 2 min = exclusion duration; 30 s = preexclusion duration. The isotope exclusion window was set to 4 amu. MS/MS spectra were analyzed using the SEQUEST ( 16 , 17 ) search program, a database search algorithm capable of searching thousands of MS/MS spectra consecutively and automatically. SEQUEST compares experimental MS/MS spectra with theoretical MS/MS spectra of peptides derived from proteins in protein databases. Peptides from the protein databases are scored and ranked based on the similarity between the experimental and theoretical spectra. SEQUEST settings were as follows: parent ion mass tolerance = 2.0 amu; fragment ion mass tolerance = 0.4 amu; enzyme = trypsin; group scans = 40 scans; group scan parent tolerance = 0.5 amu; and no amino acid modifications. Section title: Expression Cloning of TPI. Educational score: 4.300684928894043 Domain: biomedical Document type: Study Language: en Total RNA was prepared from 1558-mel and 1558-EBV B cells using the Trizol reagent ( GIBCO BRL ), and mRNA was subsequently prepared from 1558-mel (PolyATract mRNA Isolation Systems; Promega ). First-strand cDNA was synthesized (Superscript Preamplification System; GIBCO BRL ) followed by PCR amplification of TPI . The 5′ oligonucleotide primer, 5′-AAAGGATCCTCGGCTCGGCC ATG GCGCCCTCCAGGAAGTT-3′, contained an engineered BamHI restriction site at the 5′ end (initiation codon in bold type). The 3′ primer, 5′-AAAGGTACC TTA CTTGTCATCGTCATCCTTGTAGTC TTGTTTGG-CATTGATGAT-3′, contained an engineered KpnI site as well as a sequence encoding the 8-mer FLAG epitope DYKDDDDK allowing for mAb-mediated detection of TPI on Western blots (anti-FLAG M2 mAb; Sigma ) (FLAG nucleotide sequence underlined, stop codon in bold type). Oligonucleotide primers were synthesized using an ABI 392 DNA/RNA synthesizer (Applied Biosystems). The resulting PCR fragment was ligated into the expression vector pCR3.1 (Eukaryotic TA Cloning Kit; Invitrogen), and ligation products were used to transform DH5-α Escherichia coli ( GIBCO BRL ). Bacterial colonies harboring recombinant plasmids with correct sequence orientation were detected by PCR. Plasmids were purified from bacterial clones using the QIAprep Spin Miniprep Kit (QIAGEN, Inc.). DNA sequencing of TPI inserts was performed with T7 forward and pCR3.1 reverse primers, or with the TPI sequence-specific primers described above, using the Big Dye Terminator Cycle Sequencing Kit ( Perkin-Elmer /ABI). Sequences were determined with an ABI Prism 310 Genetic Analyzer ( Perkin-Elmer ). To test CD4 + T cell recognition of TPI, plasmids were transfected transiently into COS-7 cells (gift of Warren Leonard, National Institutes of Health) for 72 h with Lipofectamine Plus ( GIBCO BRL ), after which cell lysates were prepared with repeated freeze–thaw cycles for pulsing onto APCs. Section title: Peptide Synthesis. Educational score: 3.115140676498413 Domain: biomedical Document type: Study Language: en Peptides were synthesized and analyzed for sequence and purity as described ( 9 ). Section title: HLA-DR1–restricted CD4 + T Cell Recognition of a Unique Autologous Tumor Ag. Educational score: 4.232568740844727 Domain: biomedical Document type: Study Language: en Most melanoma TIL cultures propagated in IL-2 under standard culture conditions will become predominantly CD8 + after 3–4 wk ( 18 ); however, TIL 1558 were >97% CD4 + by 26 d. When stimulated with MHC class II + whole autologous tumor cells, either fresh or cultured, these TIL specifically secreted GM-CSF, IFN-γ, and TNF-α, but not IL-2 or IL-4. As shown in Fig. 1 , TIL recognition of cultured 1558-mel, as measured by GM-CSF secretion, was inhibited by mAb directed against HLA-DR but not HLA-DQ or MHC class I molecules. TIL 1558 also recognized autologous EBV-B cells pulsed with lysates of 1558-mel, and mAb blocking experiments similar to that shown in Fig. 1 confirmed HLA-DR restriction. By using allogeneic B cell lines with known HLA types to process and present tumor lysate, it was determined that the operational restriction element was HLA-DRβ1*0101 (data not shown). The monocytic leukemia cell line THP-1, which expresses the DRβ1*0101 molecule, proved to have enhanced processing capabilities for the 1558-mel Ag relative to EBV-B cell lines, and so was used as the APCs for tumor lysates and protein fractions in all subsequent experiments. Section title: HLA-DR1–restricted CD4 + T Cell Recognition of a Unique Autologous Tumor Ag. Educational score: 4.143795967102051 Domain: biomedical Document type: Study Language: en Although CD4 + TIL 1558 recognized autologous tumor cells, they failed to react against autologous normal cells, including cultured fibroblasts, EBV-B cells, and fresh PBLs. To assess whether the melanoma Ag recognized by TIL 1558 was commonly expressed among other human tumors, lysates were prepared from 45 different tumors for processing by THP-1 cells and presentation to TIL. CD4 + TIL 1558 were not stimulated by any lysates other than those derived from fresh or cultured autologous melanoma cells, including 19 allogeneic cultured melanomas, 7 fresh melanomas, 5 colon carcinomas, 2 breast carcinomas, 3 prostate carcinomas, 1 pancreatic carcinoma, 4 sarcomas, and 4 EBV-B cell lines (data not shown). These findings suggested that the Ag recognized by TIL 1558 was in fact a neoantigen resulting from a genetic mutation unique to the autologous melanoma. Section title: Identification of the Specific Tumor Ag Recognized by CD4 + TIL 1558. Educational score: 4.165861129760742 Domain: biomedical Document type: Study Language: en The ability of TIL 1558 to recognize the 1558-mel Ag when processed by APCs through the exogenous MHC class II pathway afforded an opportunity to apply a protein purification strategy for Ag identification. Through this approach, melanoma cells could be reduced to subcellular compartments and then to sequentially purified protein fractions, which could be assayed for bioactivity by feeding them to APCs for T cell recognition. Importantly, since the recognized protein Ag would be processed into smaller peptide fragments by APCs, it would not be necessary to preserve tertiary protein structure during the purification procedure. Section title: Identification of the Specific Tumor Ag Recognized by CD4 + TIL 1558. Educational score: 4.237026214599609 Domain: biomedical Document type: Study Language: en Melanoma cells disrupted by douncing in the presence of protease inhibitors were separated into nuclear, membrane, and cytosolic fractions by differential centrifugation. The cytosol was further fractionated by a stepwise (NH 4 )2SO 4 precipitation, and each fraction was pulsed onto APCs for T cell recognition. As shown in Fig. 2 , the cytosol provided a much stronger stimulus for melanoma-specific T cells than the membrane or nuclear fractions, and most of the cytosolic activity precipitated at 75% (NH 4 )2SO 4 saturation. This finding was reproduced in two similar experiments. Titration experiments demonstrated that the 75% (NH 4 )2SO 4 precipitate recovered from the cytosol was 5–25 times more stimulatory than the other fractions tested, although the protein content of the various fractions differed only by up to twofold (not shown). None of the 1558-mel fractions were capable of stimulating CD4 + TIL from another patient, and a cytosolic fraction prepared in a similar manner from a renal cell carcinoma line failed to stimulate TIL 1558. Thus, TIL 1558 seemed to recognize a cytosolic (soluble) protein unique to 1558-mel. Section title: Identification of the Specific Tumor Ag Recognized by CD4 + TIL 1558. Educational score: 4.279207706451416 Domain: biomedical Document type: Study Language: en To isolate the 1558-mel Ag, the active cytosolic fraction was purified through a series of chromatographic separations. First, proteins that precipitated at 75% (NH 4 )2SO 4 saturation were dissolved in buffer and applied to an anion exchange column. When individual fractions eluted from the column were pulsed onto APCs for T cell recognition, it was found that the bioactivity remained in the column run-through, and therefore the protein of interest had failed to bind to the column. However, SDS-PAGE analysis of all of the eluate fractions indicated that, although the bioactive run-through contained a complex mixture of proteins, many other proteins which lacked bioactivity had bound and separated adequately on the column (not shown); thus, a significant purification of the specific tumor Ag had been achieved. Next, the bioactive material recovered after anion exchange chromatography was separated according to molecular size on a gel filtration column. The results of testing individual fractions for T cell recognition are shown in Fig. 3 , demonstrating a peak of bioactivity in eluate fractions 20–23, with GM-CSF secretion by TIL in response to fraction 21 reaching 371-fold above background secretion (29,300 compared with 79 pg/ml). Although a significant purification was achieved at this step, SDS-PAGE indicated that the bioactive fractions still contained a complex mixture of proteins. The final chromatographic purification was performed by combining the bioactive gel filtration fractions and applying them to a hydrophobic interaction column. Fig. 4 shows that this procedure yielded two highly bioactive fractions, numbers 4 and 6. Each appeared to contain a single protein band of ∼30 kD on SDS-PAGE. Section title: Identification of the Specific Tumor Ag Recognized by CD4 + TIL 1558. Educational score: 4.248871326446533 Domain: biomedical Document type: Study Language: en Fraction 6, which was eluted from the hydrophobic interaction column, was subjected to tryptic cleavage followed by nano-HPLC μESI ion trap MS. Using data- dependent data acquisition, dynamic exclusion, isotope exclusion, and SEQUEST database searching, 19 peptide sequences were obtained, representing a total of 187 amino acids, and all were identified as nonmutated human TPI. In addition, eluate fraction 4 was subjected to NH 2 -terminal sequencing with Edman degradation, yielding an 11–amino acid sequence corresponding to residues 2–12 of TPI. No other proteins were identified in these fractions. The complete TPI sequence contains 249 amino acids with a calculated molecular mass of 26,700 daltons, corresponding to the observed mass of the 1558-mel Ag on SDS-PAGE. Section title: Characterization of the Mutated TPI Epitope Recognized by CD4 + TIL 1558. Educational score: 4.122052192687988 Domain: biomedical Document type: Study Language: en To confirm that TPI was indeed the tumor-specific Ag recognized by TIL 1558, cDNA encoding TPI was amplified from mRNA derived from 1558-mel, the cell line which stimulated CD4 + TIL, as well as from the autologous B cell line 1558-EBV, which did not stimulate TIL. TPI was cloned into a eukaryotic expression vector, and recombinant plasmids derived from individual bacterial colonies were transiently transfected into COS-7 cells. COS transfectants expressing TPI protein were then pulsed onto APCs as freeze–thaw lysates, for T cell recognition. Section title: Characterization of the Mutated TPI Epitope Recognized by CD4 + TIL 1558. Educational score: 4.338935375213623 Domain: biomedical Document type: Study Language: en Table I shows the results of testing individual TPI clones derived from 1558-mel for T cell recognition. TPI encoded by three clones (designated 1B, 5B, and 11G) stimulated T cells, as manifested by GM-CSF secretion, while three other TPI clones were not stimulatory, nor was a β-actin clone used as a negative control. Western blotting with the anti-FLAG mAb, directed against a specific 8-mer sequence fused to the COOH terminus of each of these proteins, confirmed that all recombinant proteins were expressed in COS-7 cells at comparable levels (not shown). DNA sequencing demonstrated that the three TPI clones encoding a product recognized by T cells shared a missense mutation at nucleotide position 450, consisting of a C to T substitution. The effect of this mutation was to convert the wild-type threonine (A C T) at amino acid position 28 to an isoleucine (A T T). TPI clones 4B, 12A, and 12F, also derived from 1558-mel, did not contain this mutation and were not recognized by T cells. As an incidental finding, all six TPI clones derived from 1558-mel shared two silent nucleotide substitutions, at positions 652 (A to G) and 856 (C to G), presumed to represent DNA polymorphisms. Thus, the six TPI clones obtained from 1558-mel represented two alleles, one wild-type, the other containing a unique mutation. The nucleotide substitution at position 450 was not identified in six clones derived from 1558-EBV, suggesting that this was indeed a somatic mutation which had occurred in the tumor, and not an allelic polymorph. Section title: Characterization of the Mutated TPI Epitope Recognized by CD4 + TIL 1558. Educational score: 4.14259672164917 Domain: biomedical Document type: Study Language: en To confirm that the mutated TPI protein predicted by DNA sequencing was in fact present in 1558-mel cells, a novel mass spectrometric sequence analysis computer program was applied, which generated ∼5,000 mutant TPI sequences that differed from the wild-type sequence by a single amino acid. The MS/MS data from the TPI digest was then searched against the 5,000 mutant TPI sequences using SEQUEST programmed with the same settings as described in Materials and Methods. This experiment detected a new tryptic peptide corresponding to TPI residues 20–33, in which Thr at position 28 was substituted with either Ile or Leu. No other mutant peptides were detected in this analysis. Section title: Characterization of the Mutated TPI Epitope Recognized by CD4 + TIL 1558. Educational score: 4.151668548583984 Domain: biomedical Document type: Study Language: en To further investigate the effect of the TPI mutation on T cell reactivity, peptides encompassing the site of mutation were synthesized and assessed for recognition by TIL 1558. A wild-type 15-mer peptide, TPI 23–37, was synthesized with the sequence GELIGTLNAAKVPAD, as well as a mutant peptide containing Ile instead of Thr at position 28 (TPI mut ). As shown in Fig. 5 , T cell recognition of the mutated peptide was enhanced at least 5 logs compared with the wild-type. Also of note, subnanomolar concentrations of the mutant peptide were recognized consistently, reminiscent of the extremely sensitive CD4 + T cell responses typically observed against foreign microbial epitopes. This is in contrast to the CD4 + T cell recognition of nonmutated melanoma-associated tyrosinase epitopes described previously ( 9 ), which required peptide concentrations of 10 μM or more for half-maximal stimulation. Section title: Characterization of the Mutated TPI Epitope Recognized by CD4 + TIL 1558. Educational score: 4.56783390045166 Domain: biomedical Document type: Study Language: en Finally, we wished to determine the immunologic function of the TPI mutation; that is, whether the mutated amino acid constituted an MHC-binding residue or a TCR contact. A substantial amount of information is available concerning the binding motif of peptides complexed to HLA-DRβ1*0101, which is the restriction element for TPI mut 23–37 as demonstrated by mAb blocking studies and peptide presentation by allogeneic HLA-compatible APCs (not shown). The crystal structure of this HLA molecule complexed to a high-affinity peptide, influenza HA 306– 318, reveals that the peptide binding site has one large hydrophobic pocket accommodating the P1 peptide anchor residue, which is the major determinant of peptide binding ( 19 ). The crystal structure of HLA-DRβ1*0101 also demonstrates shallower, less hydrophobic pockets accommodating the P4, P6, P7, and P9 peptide anchor residues. Inspection of the sequences of peptides naturally binding to this MHC allele ( 20 ), as well as monitoring the effects of substituted analogues and truncated peptides on MHC binding ( 21 ), have confirmed the overriding importance of the hydrophobic P1 anchor and lesser roles for the other four anchor residues. In the case of the TPI mut 23–37 peptide, potential P1 anchors include the hydrophobic residues Leu 25 and Ile 26, and the mutation itself, Ile 28. A series of truncated peptides were tested for T cell recognition, and as shown in Table II , Leu 25 was unimportant whereas Ile 26 was required for stimulating CD4 + TIL 1558. If Ile 26 were indeed the P1 anchor, then Val 34 would be the P9 anchor, and serial truncations at the COOH terminus of TPI mut 23–37 confirmed that peptides lacking the Val 34 residue were not recognized. These data identify the 9–amino acid MHC binding core of the mutated TPI epitope as extending from Ile 26 to Val 34, and suggest that the site of mutation, Ile 28, is a TCR contact, as it resides in the P3 position. For peptides binding to HLA-DRβ1*0101, the role of the P3 residue as a TCR contact has been shown by crystal structure as well as by studies of substituted synthetic analogues which competitively inhibit TCR engagement ( 22 , 23 ). Section title: Discussion Educational score: 4.385013580322266 Domain: biomedical Document type: Study Language: en This study describes the isolation and characterization of a mutated MHC class II–restricted human tumor Ag through a multimodality approach that combines biochemical fractionation, mass spectrometric sequencing, and molecular cloning. Monach et al. have used a similar strategy to identify a class II–restricted murine tumor Ag ( 11 ); to our knowledge, this report is the first successful human application. This approach to Ag identification is uniquely suited to class II systems, in which Ag pulsed exogenously onto APCs can access the endosomal processing compartment ( 24 ). Since the end-point bioassay measures the presence of a processed peptide rather than an intact protein, the biochemical purification procedures need not preserve native tertiary structure. This represents a convenient advantage relative to traditional applications of similar protein purification schemes, such as those used in enzymology. Another innovation is the use of MS to sequence the purified protein. The combination of HPLC and ion trap MS has made it possible to sequence peptides at the 20–50 amol level. This technique is particularly well suited for sequencing proteins in complex mixtures and for characterizing posttranslational modifications. These features render mass spectrometric sequencing eminently suited for situations in which limited quantities of material are available, and for subtractive approaches comparing the constituents of two or more partially purified protein fractions with known bioactivities. Section title: Discussion Educational score: 4.694602966308594 Domain: biomedical Document type: Study Language: en The protein containing the mutation recognized by CD4 + TIL 1558, TPI (EC 5.3.1.1), is an enzyme that catalyzes the interconversion of dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, a critical step for generating energy in the glycolytic pathway. Its sequence is highly conserved among species, with ∼50% amino acid homology between E . coli and humans, and human TPI is considered to have evolved into a “perfect enzyme,” with one of the highest catalytic rates known ( 25 ). TPI is found among the dozen or so predominant proteins in two-dimensional gel databases generated from human tumors ( 26 – 28 ), which is not surprising in view of its energy-generating function as well as its link to nucleotide synthesis via the pentose phosphate shunt. Studies of thousands of individuals have failed to detect allelic variants of TPI ( 29 ). The several described mutant forms of the enzyme are all linked to human deficiency diseases; mutations at nucleotide position 450, associated with 1558-mel, have not been reported. However, there is evidence for a causal relationship between UV irradiation, an agent implicated in generating melanomas in vivo, and C to T nucleotide substitutions such as occurred in 1558-mel ( 30 ). Indeed, C to T mutations have been shown to generate MHC class I–restricted neoantigens recognized by CD8 + T cells from two melanoma patients. In those two instances, functional mutations in cyclin-dependent kinase 4 ( 31 ) and β-catenin ( 32 ) may have conferred an oncogenic advantage on the tumor cells, which could have counterbalanced or outweighed the negative effect of disclosing the tumor to immune recognition. The crystal structure of TPI indicates that Thr 28, the site of the 1558-mel mutation, is not located at an active catalytic site ( 33 ); in fact, deliberate attempts to enhance the function of TPI through site-directed mutagenesis of catalytic site residues have not been successful ( 34 ). However, one might speculate that the nonconservative Thr28Ile mutation could impact on the tertiary structure of the enzyme and hence its catalytic function, and the potential role of this mutation in enhancing TPI function will be explored in future studies. Section title: Discussion Educational score: 4.4488325119018555 Domain: biomedical Document type: Study Language: en By immunohistochemical analysis, the subcutaneous melanoma metastasis from which TIL 1558 were derived expressed the shared immunogenic melanoma-associated proteins tyrosinase, gp100, and melanoma antigen recognized by T cells (MART)-1 ( 35 ). Nevertheless, CD4 + TIL 1558 failed to react against these commonly expressed, nonmutated Ags, instead responding to a unique neoantigen. These TIL are representative of the majority of MHC class II–restricted melanoma-specific CD4 + T cells analyzed from nine patients, among which two recognized the commonly expressed nonmutated tyrosinase Ag, while the rest reacted against unique tumor-specific Ag (8, 10, 36, and our unpublished data). The apparent dichotomy between melanoma Ag recognized by CD4 + versus CD8 + T cells (unique versus shared Ag) may reflect events occurring during T cell development, with selective deletion of CD4 + but not CD8 + T cells reactive with lineage-specific “self” Ag expressed in melanomas. Since melanocytes do not normally reside in the thymus during development, their Ags would be represented there by migrating APCs which had ingested and processed these Ags, preferentially leading to presentation on MHC class II molecules and deletion of class II–restricted Th cells. Thus, while it is important to identify shared tumor-associated Ags recognized by both CD4 + and CD8 + T cells for devising more effective immunotherapies, it is also necessary to define optimally potent Ags which can mediate tumor rejection. These may not be one and the same, and immunotherapies incorporating unique neoantigens may be required to realize the full potential of this treatment modality. | Study | biomedical | en | 0.999997 |
10049940 | Section title: Cell Lines, Media, and Reagents. Educational score: 4.219888687133789 Domain: biomedical Document type: Study Language: en The EBV-transformed B (EBV-B) cell lines and tumor cell line MZ2-MEL.43 were cultured in IMDM ( GIBCO BRL ) supplemented with 10% FCS ( GIBCO BRL ), 0.24 mM l -asparagine, 0.55 mM l -arginine, 1.5 mM l -glutamine (AAG), 100 U/ml penicillin, and 100 μg/ml streptomycin. The PhoenixAMPHO cell line (provided by Dr. Nolan, Stanford University, Stanford, CA) is a high-titer amphotropic retrovirus–producing cell line that was generated by stable transfection of 293T cells with a Moloney GagPol-IRES-Lyt 2 construct with a Rous sarcoma virus (RSV) promoter and a pPGK hygro selectable marker. These cells were then stably transfected with the Moloney amphotropic envelope gene driven by a CMV promoter and coselected with the diphtheria toxin resistance gene (pHED-7). This producer cell line is helper virus–free. PhoenixAMPHO cells were cultured in DMEM (Life Technologies) supplemented with 10% heat-inactivated FCS, 2 mM l -glutamine, and antibiotics. Section title: Cell Lines, Media, and Reagents. Educational score: 1.5183748006820679 Domain: biomedical Document type: Other Language: en Human recombinant IL-2 was purchased from Eurocetus, IL-7 from Genzyme , GM-CSF from Schering Plough, and TNF-α from R&D Systems. Human recombinant IL-4, IL-6, and IL-12 were produced in our laboratory. Section title: MAGE-3 Protein. Educational score: 4.186453342437744 Domain: biomedical Document type: Study Language: en The recombinant MAGE-3 protein was produced by SmithKline Beecham Pharmaceuticals. The full-length MAGE-3 sequence, preceded by the amino acid (aa) 1 sequence MHHHHHHHGG, was engineered into a vector bearing the pMB1 replicon and the PL short promoter. This vector was used to transform Escherichia coli strain AR58 ( 27 ). Bacteria were grown in LB medium containing 50 μg/ml kanamycin at 30°C. After heat induction, the MAGE-3 expression product became detectable as a 46-kD band when assayed by Western blot (SDS-PAGE 12.5%; revealed by a rabbit polyclonal antiserum to MAGE-3). Protein purification was carried out at room temperature, and involved the following steps: cell lysis and centrifugation, repeated washing of the centrifugation pellet followed by solubilization of the pellet containing MAGE-3, immobilized metal ion affinity chromatography, anion exchange chromatography, concentration, and dialysis. The resulting MAGE-3 protein was >95% pure, as assessed by Coomassie blue staining. Section title: Construction of pMFG-Ii.MAGE-3. Educational score: 4.344369888305664 Domain: biomedical Document type: Study Language: en The plasmid IipSV51L, containing a cDNA encoding the human invariant chain (Ii), was provided by Dr. J. Pieters (Basel Institute for Immunology, Basel, Switzerland). The MFG plasmid was provided by Dr. O. Danos (Somatix Therapy Corp., Alameda, CA). The MFG retroviral vector is derived from the Moloney murine leukemia virus, which lacks a drug resistance marker and does not express any other potential antigenic protein except for the inserted cDNA ( 28 ). The cDNA encoding the NH 2 -terminal end (i.e., the cytoplasmic tail and the transmembrane region) of the human (hu)-Ii polypeptide (residues 1–80) was amplified by PCR using IipSV51L as the template. The following primers were used: hu-Ii sense, 5′-TTT CCATGG ATGACCAGCGCGAC-3′, and hu-Ii antisense, 5′-TTT GGATCC GGAAGCTTCATGCGCAGGTTC-3′ (the recognition sites for NcoI and BamHI are in italics). The PCR product was cloned into pCR2.1 and sequenced. The NcoI-BamHI amplification product was cloned into pMFG and opened with the enzymes NcoI and BamHI, resulting in pMFG-Ii. A BglII recognition site, replacing the ATG codon and in frame with the BamHI site at the 3′ end of the truncated Ii-cDNA, was introduced at the 5′ end of the MAGE-3 cDNA by PCR using the following primers: BglII sense, 5′-TTT AGATCT TGAGCAGAGGAGTCAGC-3′, and BglII antisense: 5′-CCC AGATCT TCACTCTTCCCCCTCTCTC-3′ (the recognition sites for BglII are in italics). The PCR product (BglII. MAGE-3.BglII) was cloned into pCR2.1 and sequenced. The recombinant plasmid, pMFG-Ii, was reopened with BamHI, and the BglII.MAGE-3.BglII amplification product was ligated to the compatible ends. Recombinant plasmids containing the MAGE-3 cDNA in frame and in the right orientation were identified by restriction fragment analysis. Section title: Production of High-titer Ii.MAGE-3–encoding Recombinant Retrovirus. Educational score: 4.357937812805176 Domain: biomedical Document type: Study Language: en The MAGE-3–encoding retroviral vector plasmid MFG-Ii.MAGE-3 was introduced into the PhoenixAMPHO packaging cells by transfection. The transfection procedure is a modification of the calcium phosphate–mediated transfection protocol of Graham and Van der Eb ( 29 ). 24 h before transfection, PhoenixAMPHO cells were plated in cell growth medium in a 75-cm 2 tissue culture flask (Falcon; Becton Dickinson Labware ). After adding the cells, the flask was gently shaken forward and backward to distribute cells evenly in the flask bottom. The cells were incubated at 37°C and 5% CO 2 . At the time of transfection, when the cells should have reached a confluence of 70–80%, the medium was removed and replaced by 14 ml fresh PhoenixAMPHO cell growth medium containing 25 mM chloroquine ( Sigma ). A transfection cocktail was prepared in a 50-ml tube by adding 40 μg retroviral vector plasmid DNA to water and diluting to 1,575 μl final vol. To this DNA solution 225 μl of 2 M CaCl 2 was added. Then, 1,800 μl of 2× HeBS (50 mM Hepes, 10 mM KCl, 12 mM dextrose, 280 mM NaCl, and 1.5 mM Na 2 HPO 4 dissolved in distilled water, filtered through a 0.2-μm filter, and stored at −20°C) was added in drops to the DNA/CaCl 2 solution by bubbling vigorously for 15 s with an automatic pipette. The DNA/ CaCl 2 /HeBS mix was added immediately in drops onto the cells, and the flask was gently swirled to ensure uniform mixing of DNA/CaPO 4 particles. The cells were incubated at 37°C/5% CO 2 for 7–9 h, and the chloroquine-containing medium was changed for fresh PhoenixAMPHO cell growth medium. Approximately 24 h before the harvest of the retroviral supernatant, the PhoenixAMPHO medium was removed and gently replaced by 9 ml of IMDM containing only 2.5% FCS. The retroviral supernatant was harvested 48 h after transfection by removing the medium from the cells and filtering through a 0.45-μm filter to remove cell debris. After harvest and filtration, the virus-containing medium was kept on ice, aliquoted in appropriate volumes in 15-ml polypropylene tubes, and stored at −80°C. Section title: Retroviral Transduction of Cell Lines. Educational score: 4.136722087860107 Domain: biomedical Document type: Study Language: en Target cells were resuspended in 60-mm tissue culture plates (Falcon) at a density of 10 6 cells in 4 ml of infection cocktail containing 50% viral supernatant in growth medium and 6 μg/ml of protamine sulfate. The plates were centrifuged for 2 h at 32°C and 1,200 rpm, followed by another 2 h of incubation in a humidified incubator at 37°C. Cells were then transferred to 4 ml of growth medium. This transduction cycle was carried out immediately after plating the cells and was repeated at 24 and 48 h. Section title: Dendritic Cells. Educational score: 4.1570353507995605 Domain: biomedical Document type: Study Language: en Blood cells were collected as buffy coat preparations from hemochromatosis patients. PBMCs were isolated by Lymphoprep (Nycomed Pharma) density gradient centrifugation. To generate autologous dendritic cells, PBMCs were depleted from T lymphocytes by rosetting with sheep erythrocytes (Bio Mérieux) treated with 2-aminoethylisothiouronium ( Sigma ). The lymphocyte-depleted PBMCs were left to adhere for 2 h at 37°C in culture flasks (Falcon) at a density of 2 × 10 6 cells/ml in RPMI 1640 medium supplemented with AAG and 1% autologous plasma (hereafter referred to as complete RPMI medium). Nonadherent cells were discarded, and adherent cells were cultured in the presence of IL-4 (100 U/ml) and GM-CSF (100 ng/ ml) in complete RPMI medium. Cultures were fed on days 2 and 4 by replacing half of the medium with fresh medium plus IL-4 (100 U/ml) and GM-CSF (100 ng/ml). On day 5, the nonadherent cell population was used as a source of enriched dendritic cells. Section title: CD4 + Responder T Cells. Educational score: 4.099760055541992 Domain: biomedical Document type: Study Language: en Rosetted T cells were treated with NH 4 Cl (160 mM) to lyse the sheep erythrocytes and washed. CD4 + T lymphocytes were isolated from rosetted T cells by negative selection using an anti-CD8 mAb coupled to magnetic microbeads (Miltenyi Biotech) and by sorting through a MACS ® , as recommended by the manufacturer. The lymphocytes were frozen and then thawed the day before the coculture with dendritic cells. Section title: Mixed Lymphocyte/Dendritic Cell Culture. Educational score: 4.2391357421875 Domain: biomedical Document type: Study Language: en Autologous dendritic cells (5 × 10 5 /ml) were incubated at 37°C, 5% CO 2 for 18–20 h in complete medium supplemented with IL-4 (100 U/ ml), GM-CSF (100 ng/ml), and TNF-α (1 ng/ml) in the presence of the recombinant MAGE-3 protein (20 μg/ml). Cells were washed and added at 10 4 per round-bottomed microwell to 10 5 CD4 + lymphocytes in 200 μl IMDM supplemented with AAG and 10% human serum (hereafter referred to as complete IMDM) in the presence of IL-6 (1,000 U/ml) and IL-12 (10 ng/ ml). The CD4 + lymphocytes were restimulated on days 7, 14, and 21 with autologous dendritic cells freshly loaded with the MAGE-3 protein and were grown in complete IMDM supplemented with IL-2 (10 U/ml) and IL-7 (5 ng/ml). Due to the limited supply of dendritic cells for melanoma patient 7002, we used only 6 × 10 3 dendritic cells instead of 10 4 and restimulations were performed on days 10 and 20 instead of 7, 14, and 21. The microcultures containing proliferating CD4 + T cells were assessed on days 35–37 for their capacity to produce TNF and/or IFN-γ when stimulated with autologous EBV-B cells loaded with protein MAGE-3. Autologous EBV-B cells were incubated for 18–20 h in the presence of 20 μg/ml of protein MAGE-3, or OVA ( Sigma ) as a negative control. Protein-pulsed EBV-B cells were washed and distributed at 5,000 cells per round-bottomed microwell together with 2,500 CD4 + T lymphocytes in 150 μl of complete IMDM supplemented with IL-2 (25 U/ml). After 20 h, the supernatant was collected and its TNF content was determined by testing its cytotoxic effect on WEHI-164 clone 13 cells ( 30 ) in a MTT colorimetric assay ( 31 , 32 ). IFN-γ released in the supernatant was measured by ELISA using reagents from Medgenix Diagnostics- Biosource . Inhibition with mAbs W6/32 (anti–HLA class I) or 2B6 (anti–HLA-DR) was performed by addition of a 1:20 dilution of ascites during the experiment. Section title: CD4 + T Cell Clones. Educational score: 4.142510414123535 Domain: biomedical Document type: Study Language: en The microcultures that recognized cells loaded with the MAGE-3 protein were cloned by limiting dilution, using as stimulating cells either the autologous EBV-B cell line loaded with the MAGE-3 protein or the autologous EBV-B cell line transduced with a retrovirus encoding Ii.MAGE-3. Allogeneic EBV-B cells (LG2-EBV) were added as feeder cells. CD4 + T cell clones were grown in complete IMDM supplemented with IL-2 (50 U/ml), IL-7 (5 ng/ml), and 0.5 μg/ml purified PHA (HA 16; Murex Diagnostics). The clones were supplemented with fresh culture medium once a week and passaged with feeder cells (1.5 × 10 6 allogeneic PBLs plus 5 × 10 5 LG2-EBV per well in a 24-well plate) at 1–2-wk intervals. Occasionally, clones were restimulated with 2 × 10 5 autologous EBV-B cells transduced with Ii.MAGE-3 as stimulating cells and 10 6 LG2-EBV. Established CD4 + T cell clones were then tested for TNF and/or IFN-γ production after stimulation with autologous EBV-B cells pulsed with protein MAGE-3 and MAGE-3– derived peptides. Section title: Recognition Assays with Peptides. Educational score: 4.111996650695801 Domain: biomedical Document type: Study Language: en Peptides were synthesized on solid phase using F-moc for transient NH 2 -terminal protection and were characterized using mass spectrometry. All peptides were >80% pure, as indicated by analytical HPLC. Lyophilized peptides were dissolved in DMSO and used at a concentration of 5 μg/ml. EBV-B cells were incubated for 2 h at 37°C in the presence of the different peptides, the indicated concentrations representing their concentrations during the incubation step. They were distributed at 5,000 cells per round-bottomed microwell together with 2,500 CD4 + T lymphocytes in 150 μl of complete IMDM supplemented with IL-2 (25 U/ml). Supernatants were harvested after 20 h and assessed for TNF and/or IFN-γ production. The results represent the average of duplicate or triplicate cultures. Section title: Recognition Assays with Cell Lysates. Educational score: 4.15776252746582 Domain: biomedical Document type: Study Language: en The MAGE-3 cDNA sequence cloned into expression vector pCEP-4 was transiently transfected into the 293-EBNA cell line by Lipofectamine ® ( GIBCO BRL ). In brief, 5 × 10 4 293-EBNA cells per flat-bottomed microwell were transfected with pCEP4-MAGE-3 and 1 μl of Lipofectamine ® in Optimem medium ( GIBCO BRL ). After 24 h, transfected 293-EBNA cells were lysed in 50 μl of complete RPMI medium by three cycles of rapid freeze–thawing. Monocyte-derived dendritic cells expressing HLA-DR13 molecules were then added (1.5 × 10 4 cells per well) to the lysates of transfected 293-EBNA cells and kept at 37°C for 24 h. The dendritic cells were then washed, and 2,500 CD4 + lymphocytes were added in 150 μl of complete IMDM supplemented with IL-2 (25 U/ml). Supernatants were harvested after 20 h and assessed for IFN-γ production. Section title: Results Educational score: 4.13946533203125 Domain: biomedical Document type: Study Language: en Blood monocytes were cultured in medium supplemented with GM-CSF and IL-4 to favor their differentiation into dendritic cells . Autologous plasma was used to avoid loading the dendritic cells with bovine or allogeneic proteins. After 5 d, the dendritic cells were incubated overnight with 20 μg/ml of a MAGE-3 protein produced in E . coli and with TNF-α to induce their maturation. 96 microcultures were set up with 10 5 autologous responder CD4 + T cells and 10 4 dendritic cells loaded with protein MAGE-3 as stimulator cells. IL-6 and IL-12 were added during the first week to activate the T cells. The responder T cells received three additional weekly restimulations with dendritic cells pulsed with protein MAGE-3 in the presence of IL-2 and IL-7. After a resting period of 2 wk, the responder cells of each microculture were tested on day 35 for TNF or, more often, for IFN-γ production after stimulation for 20 h with autologous EBV-B cells loaded with protein MAGE-3. Section title: CD4 + T Cell Clones Directed against a MAGE-3 Antigen. Educational score: 4.180510520935059 Domain: biomedical Document type: Study Language: en CD4 + T cells of hemochromatosis patient LB 1555, stimulated with dendritic cells loaded with protein MAGE-3, were tested for their ability to produce TNF upon stimulation with EBV-B cells loaded with either protein MAGE-3 or OVA. It was possible to measure the production of TNF by the T cells because, contrary to most EBV-B cell lines, that of patient LB 1555 did not produce TNF. The microculture that produced the highest level of TNF after stimulation with protein MAGE-3 was cloned by limiting dilution using autologous EBV-B cells loaded with protein MAGE-3 as stimulator cells. These stimulator cells were used for the cloning step because of the limited supply of autologous dendritic cells. A positive CD4 + clone was obtained, referred to hereafter as clone 37. It recognized autologous EBV-B cells loaded with protein MAGE-3. Even though the protein, which had been produced in bacteria, was >95% pure, we could not at this stage exclude the possibility that clone 37 recognized a bacterial contaminant. Therefore, we prepared a set of peptides of 16 aa, which overlapped by 12 and covered the entire MAGE-3 protein sequence. Each of these peptides was incubated with autologous EBV-B cells and tested for recognition by clone 37. We identified two overlapping peptides, MAGE-3 111–126 and MAGE-3 115–130 (see below), that stimulated production of both TNF (data not shown) and IFN-γ . This demonstrated unambiguously that clone 37 recognized a MAGE-3 antigen and not a contaminant. Section title: CD4 + T Cell Clones Directed against a MAGE-3 Antigen. Educational score: 3.8137710094451904 Domain: biomedical Document type: Study Language: en A CD4 + clone was obtained from another microculture which was also very well stimulated by cells loaded with protein MAGE-3 . However, here the screening with the set of overlapping MAGE-3 peptides was negative. We found that the antigen recognized by this CD4 + clone was present in an HPLC fraction other than the one containing the MAGE-3 protein (data not shown). Our tentative conclusion is that this clone recognizes a bacterial contaminant. Section title: CD4 + T Cell Clones Directed against a MAGE-3 Antigen. Educational score: 4.09572696685791 Domain: biomedical Document type: Study Language: en Additional CD4 + clones were obtained from five independent microcultures, set up with cells from another hemochromatosis patient. Their ability to recognize cells loaded with protein MAGE-3 appeared to decrease upon further purification of the protein by HPLC. Two of these clones were also tested for their response to the set of MAGE-3 peptides, with negative results, suggesting further that they recognized a contaminant in the MAGE-3 batch. Therefore, it appears that bacterial contaminants in this MAGE-3 protein batch were more likely to activate CD4 + T cells than was MAGE-3 itself, so that CD4 + clones specific for MAGE-3 constituted only a minor fraction of the clones obtained with this procedure. Section title: Clone 37 Recognizes Peptide AELVHFLLLKYRAR on HLA-DR13. Educational score: 4.236819267272949 Domain: biomedical Document type: Study Language: en CD4 + clone 37 recognized two peptides which overlapped by 12 aa, namely RKVAELVHFLLLKYRA (aa 111–126) and ELVHFLLLKYRAREPV (aa 115–130) . The recognition by clone 37 of cells loaded with protein MAGE-3 was abolished by an anti– HLA-DR antibody (data not shown). Patient LB 1555 was serologically typed DR3, DR13, and DR52. To identify the presenting HLA-DR molecule, we tested additional EBV-B cell lines expressing DR3, DR13, or DR52. All and only those expressing DR13 were able to present the MAGE-3 111–126 and MAGE-3 115–130 peptides to clone 37 (Table I ). 31 different HLA-DR13 alleles have been described to date ( 33 ). LB 1555 expresses the DRB1*1302 allele, and the other DR13 cell lines tested express DRB1* 1301 or DRB1*1302, the latter two representing 80% of the HLA-DR13 alleles. Section title: Clone 37 Recognizes Peptide AELVHFLLLKYRAR on HLA-DR13. Educational score: 4.166589736938477 Domain: biomedical Document type: Study Language: en Unlike the peptides presented by class I molecules, those presented by class II usually vary in length and tolerate extensions at both the NH 2 and COOH termini because they are not fixed by their ends in the groove ( 34 ). Therefore, it is difficult to define the length of those peptides precisely. We tested a large number of MAGE-3 peptides of different lengths (data not shown) and concluded that the shortest peptide well recognized by clone 37 is AELVHFLLLKYRAR . Half-maximum value of stimulation was obtained by incubating the stimulator cells with 10 nM of this peptide. This compares favorably with the results obtained with other epitopes recognized on HLA class II molecules by CD4 + T cells: a minimum of 30 nM of a bcr-abl peptide was needed to induce a significant proliferation of an anti–bcr-abl CD4 + cell line, whereas 330 nM of peptide p21- ras was required for anti–p21- ras CD4 + clones ( 22 , 23 ). Section title: Dendritic Cells Incubated with Cell Lysates Present the MAGE-3 Antigen. Educational score: 4.111695289611816 Domain: biomedical Document type: Study Language: en We tested dendritic cells, incubated with decreasing concentrations of MAGE-3 protein, for their ability to stimulate clone 37 . Half-maximum production of IFN-γ was obtained when 10 4 dendritic cells were preincubated in a volume of 100 μl with 30 ng of the protein, which is approximately the amount present in 2 × 10 4 MZ2-MEL.43 tumor cells (Godelaine, D., personal communication). This suggested that a dendritic cell, having endocytosed debris of just one or a few tumor cells expressing MAGE-3, would be capable of stimulating anti–MAGE-3 CD4 + T cells. To test further the ability of dendritic cells to process debris of cells expressing MAGE-3, 293-EBNA cells transiently transfected with the MAGE-3 gene were lysed and incubated for 24 h with HLA-DR13 dendritic cells. These cells stimulated clone 37 . The amount of IFN-γ released by clone 37 increased with the amount of plasmid used for the transfection. Section title: Presentation of the Antigen by Cells Expressing the Ii. MAGE-3 Construct. Educational score: 4.304851055145264 Domain: biomedical Document type: Study Language: en A melanoma cell line which expresses HLA-DR13 and MAGE-3 was unable to stimulate the release of IFN-γ by clone 37, unless it was pulsed with the MAGE-3 115–130 peptide . This suggests that the MAGE-3 protein synthesized endogenously does not reach the class II presentation pathway. It has been reported that signals within the Ii could be used to target endogenously synthesized protein to the class II antigen-processing compartments. The expression of several constructs, which encoded fusion proteins containing part of the mouse Ii followed by fragments of the OVA gene, generated OVA peptides recognized by murine CD4 + T cells on MHC class II molecules. The highest level of antigen presentation was obtained with a construct containing the sequence coding for the first 80 amino acids of the Ii ( 35 ). Accordingly, MZ2-MEL.43 cells were transduced with a retroviral construct encoding the first 80 amino acids of the human Ii fused with MAGE-3 (retro–Ii.MAGE-3). MZ2-MEL.43 cells transduced with retro–Ii.MAGE-3 stimulated a high production of IFN-γ by clone 37, indicating that the Ii.MAGE-3 protein was processed through the class II presentation pathway . HLA-DR13 EBV-B cells transduced with the same construct also stimulated clone 37 . In contrast, HLA-DR13 EBV-B cells transduced with retro–MAGE-3 alone were unable to stimulate clone 37. Section title: Two Other CD4 + Clones Recognized a Neighboring MAGE-3 Peptide on HLA-DR13. Educational score: 4.131348609924316 Domain: biomedical Document type: Study Language: en CD4 + T cells isolated from the blood of melanoma patient 7002, who had been immunized with the MAGE-3 protein, were stimulated with autologous dendritic cells loaded with MAGE-3 protein. After three restimulations, one microculture produced IFN-γ upon stimulation with autologous EBV-B cells loaded with the protein. CD4 + clone 2 was isolated, which produced IFN-γ upon stimulation with cells that were either loaded with MAGE-3 protein or transduced with Ii.MAGE-3, the latter proving that the clone was directed against MAGE-3 and not against a contaminant of the protein batch . Section title: Two Other CD4 + Clones Recognized a Neighboring MAGE-3 Peptide on HLA-DR13. Educational score: 4.168208599090576 Domain: biomedical Document type: Study Language: en When the set of overlapping MAGE-3 peptides were tested for recognition by clone 2, peptide ELVHFLLLKYRAREPV (aa 115–130) scored positive . This peptide was shown previously to be recognized by clone 37, isolated from hemochromatosis patient LB 1555. However, peptide FLLLKYRAREPVTKAE (aa 119–134) scored positive with clone 2 but was not recognized by clone 37 . Moreover, peptide RKVAELVHFLLLKYRA (aa 111–126), previously shown to be recognized by clone 37, was not recognized by clone 2. This indicated clearly that clones 2 and 37 were directed against two adjacent but distinct epitopes. Several EBV-B cell lines were tested for their ability to present peptide MAGE-3 119–134 to clone 2. All and only those expressing DR13 were capable of presenting the peptide (Table II ). Patient 7002 expresses the DRB1*1302 allele. We tested a large number of MAGE-3 peptides at different concentrations to identify the shortest peptide efficiently recognized by clone 2 (data not shown). This proved to be LLKYRAREPVTKAE . Section title: Two Other CD4 + Clones Recognized a Neighboring MAGE-3 Peptide on HLA-DR13. Educational score: 3.970201253890991 Domain: biomedical Document type: Study Language: en Interestingly, peptide MAGE-3 121–134 is also present in proteins MAGE-1, -2, and -6. Therefore, we loaded DR13 EBV-B cells with a MAGE-1 recombinant protein and used them to stimulate the CD4 + clones. Clone 2, but not clone 37, produced IFN-γ (data not shown). Section title: Two Other CD4 + Clones Recognized a Neighboring MAGE-3 Peptide on HLA-DR13. Educational score: 4.101240158081055 Domain: biomedical Document type: Study Language: en From hemochromatosis patient LB 1158, yet another anti–MAGE-3 CD4 + clone was obtained, referred to hereafter as clone 22. Patient LB 1158 expresses the DRB1* 1301 allele. Like clone 2, clone 22 was stimulated by DR13 cells presenting peptide MAGE-3 119–134 (Table II ) and did not recognize peptide MAGE-3 111–126 (data not shown). It was also stimulated by DR13 cells loaded with the MAGE-1 protein (data not shown). Section title: Discussion Educational score: 4.123076438903809 Domain: biomedical Document type: Study Language: en The procedure described here seems efficient for the activation of anti-MAGE CD4 + precursors present in the blood of individuals with or without cancer, and for the resulting isolation of anti-MAGE CD4 + permanent T cell clones. We are quite confident that it will prove to be applicable to other proteins for the identification of new antigens presented by HLA class II molecules. But our approach is not without disadvantages: a large number of the CD4 + clones obtained were apparently directed against contaminants of the protein batch. Therefore, it will be preferable to use one source of antigen for stimulation of the T cells and another to test their specificity. This may be achieved using two batches of protein produced in different organisms, such as bacteria and insect cells infected with baculovirus constructs, or, as shown here, by the use of presenting cells expressing the protein of interest fused to a truncated Ii. Section title: Discussion Educational score: 4.482031345367432 Domain: biomedical Document type: Study Language: en Antigens can be processed through two different pathways that lead to presentation on HLA class II molecules. The endogenous pathway channels some proteins synthesized in the cell towards endosomal compartments, where they are cleaved into peptides that then associate with class II molecules ( 36 ). In the exogenous pathway, the APC takes up protein by endocytosis. Early endosomes undergo a progressive transition to late endosomes and then to lysosomes. The late endosomes have a relatively low pH, contain lysosomal hydrolases, and are enriched in HLA-DM and MHC class II molecules, which are associated with Ii in nonameric complexes. Therefore, they are also called MHC class II compartments (MIIC). Ii is rapidly degraded but class II–associated Ii peptide (CLIP), a small part of it, remains bound within the groove of the MHC molecule and blocks access of other peptides until HLA-DM catalyzes its dissociation from class II molecules. This permits the subsequent loading of potentially antigenic peptides. The peptide–HLA class II complexes are then directed to the cell surface ( 37 ). Section title: Discussion Educational score: 4.25836181640625 Domain: biomedical Document type: Study Language: en Melanocyte-specific proteins such as tyrosinase contain a lysosomal targeting sequence that enables them to follow the endogenous class II processing pathway ( 38 ). Therefore, these differentiation antigens can be recognized directly by CD4 + T cells on those melanomas that express class II molecules ( 39 ). Constitutive expression of class II occurs frequently in melanoma. In line with their cytosolic and nuclear localizations, the MAGE proteins appear to lack the targeting sequences that would enable them to follow the endogenous class II pathway ( 5 , 40 , 41 ). This probably explains why the anti–MAGE-3 CD4 + clones obtained were unable to recognize an HLA-DR13 tumor cell line expressing MAGE-3. Section title: Discussion Educational score: 4.191898822784424 Domain: biomedical Document type: Study Language: en The anti–MAGE-3 CD4 + clones could be stimulated by HLA-DR13 dendritic cells loaded with extracts of cells producing a large amount of MAGE-3 protein. However, they failed to recognize dendritic cells incubated with lysates of tumor cells expressing MAGE-3 . This might be due to a lower quantity of protein MAGE-3 in these tumor cell extracts. However, this process may be more efficient in vivo, where loading of dendritic cells with apoptotic bodies from tumor cells might result in very efficient presentation of MAGE-3 epitopes ( 42 ). The notion that MAGE-3 antigens can be presented to CD4 + T cells in vivo is supported by the observation that some cancer patients produce anti-MAGE antibodies ( 11 , 43 , 44 ). This is probably due to destruction of some tumor cells followed by uptake of the debris by macrophages or dendritic cells. Section title: Discussion Educational score: 4.4517107009887695 Domain: biomedical Document type: Study Language: en In a recently completed clinical trial, 25 tumor-bearing HLA-A1 melanoma patients with advanced disease received 3 subcutaneous injections of a MAGE-3 peptide presented by HLA-A1 ( 45 ). Tumor regressions were observed in seven patients, and three of these were complete. No increase in anti-MAGE CTLs could be detected in the blood of these patients, including those whose tumor regressed. The regressions occurred very slowly, suggesting that they may have been caused by a weak immune response. A major limitation of such a class I peptide–based approach might be that the CTLs elicited by the peptide reach the tumor but fail to be restimulated properly at the tumor site so that CTL amplification does not occur. This could be due to the lack of help by tumor-specific CD4 + T cells. Vaccination strategies with MAGE-3 products may benefit from the induction of anti–MAGE-3 CD4 + T cells, despite the fact that tumor cells that express both class II molecules and MAGE-3 are unable to activate CD4 + T cells directly. Upon lysis of some tumor cells by a first CTL attack, tumor debris could be processed by tumor-infiltrating APCs. Activated tumor-specific CD4 + T cells located around the tumor could then be restimulated by these cells. These CD4 + T lymphocytes might then favor the activation and proliferation of the effector CD8 + T cells, provoke the maturation of dendritic cells via CD40–CD40L interactions, and mediate recruitment of additional immune cells at the tumor sites ( 46 – 48 ). The resulting amplification of the immune response might then lead rapidly to the complete destruction of the tumor mass. Immunization with MAGE-3 peptides presented by class I and class II molecules or with a purified protein may represent one possibility for inducing both MAGE-3–specific CD4 + and CD8 + T cells. Another possibility is the reinfusion into patients of autologous APCs loaded with MAGE-3 peptides binding to both HLA class I and class II molecules. This will require the characterization of several epitopes to cope with HLA restriction. Alternatively, autologous dendritic cells could be infected with recombinant viruses encoding an Ii.MAGE-3 fusion protein. Expression of Ii.MAGE-3 in EBV-transformed B cells was shown to result not only in the presentation of class II epitopes, but also in a very efficient presentation of a class I epitope recognized by an anti– MAGE-3.A1 CTL (Corthals, J., and K. Thielemans, manuscript in preparation). Patients could also be immunized directly with recombinant viruses encoding Ii.MAGE-3. Section title: Discussion Educational score: 4.198603630065918 Domain: biomedical Document type: Study Language: en A number of results obtained in mice support the importance of immunization against antigens presented by class II molecules. Direct injection of a recombinant vaccinia encoding a human papilloma virus E7 protein linked to the sorting signal of the lysosomal-associated membrane protein (LAMP-1) was very effective in inducing protective immunity against a challenge with an E7 + class II + tumor. In contrast, animals injected with a vaccinia coding for the normal E7 protein were not protected ( 49 ). In another mouse model, strong protection was achieved against highly aggressive tumor cells lacking MHC class II expression through a single vaccination with a tumor-specific T helper peptide encoded by the Moloney murine leukemia virus ( 21 ). In this model, the CD8 + CTLs were helped efficiently by peptide-primed tumor-specific CD4 + T cells. Section title: Discussion Educational score: 4.252788543701172 Domain: biomedical Document type: Study Language: en A clinical trial was recently initiated with a MAGE-3 recombinant protein. In this trial as in others involving recombinant viruses harboring large MAGE sequences, it will be essential to have reliable monitoring of the anti– MAGE-3 CD4 + response. Our observations that CD4 + cells directed against a minor contaminant in the protein batch can easily be activated in vitro have implications for this monitoring. To avoid possible misinterpretation of in vitro assays and of delayed-type hypersensitivity (DTH) assays, a protein produced in another organism or autologous cells expressing the antigen endogenously should be used for these assays. Another possibility, which narrows the analysis to certain epitopes, is the use of a set of relevant peptides that can be used either to select and amplify peptide-specific T cells in vitro or to label directly T cell receptors with soluble HLA tetramers presenting the relevant peptide ( 50 , 51 ). Multimeric soluble MHC class II molecules, complexed with a peptide attached covalently, were recently shown to bind with appropriate specificity and affinity to mouse-specific T cells ( 52 ). This approach requires prior identification of the antigenic peptides, as described in this report. Considering that several MAGE genes share the sequence coding for one of the two MAGE-3.DR13 epitopes reported here, these peptides are relevant to ∼16% of Caucasian melanoma patients, as 20% of Caucasians express HLA-DR13 and ∼80% of metastatic melanomas express MAGE-1 , -2 , -3 , or - 6 . | Study | biomedical | en | 0.999997 |
10049941 | Section title: Mice. Educational score: 4.0361785888671875 Domain: biomedical Document type: Study Language: en C57BL/6 mice were purchased from IFFA Credo. Mice homozygous for lpr mutation ( lpr ) and lacking the Fas (CD95) molecule were obtained from Harlan. Mice homozygous for perforin gene disruption (P 0/0 ) completely lack perforin- dependent cytotoxicity, while the Fas/FasL pathway remains fully functional ( 17 , 18 ). Mice homozygous for the gld mutation ( gld ), homozygous for the gld mutation and heterozygous for the perforin deletion (P +/0 gld ), and mice double deficient for perforin and carrying the gld mutation (P 0/0 gld , unable to generate antigen-specific CTLs ) were provided by Michael Hahne (Institute of Biochemistry, Lausanne, Switzerland). P 0/0 gld mice were obtained by mating P +/0 gld mice, and the offspring were tested for perforin deletion as described by Lowin et al. ( 18 ). Mice with a mutation in the β2 microglobulin gene (MHC class I–deficient [I 0/0 ]) or in the I-Aβ gene (MHC class II–deficient [II 0/0 ]) were provided by Christophe Benoist and Diane Mathis ( 20 , 21 ). Section title: Mice. Educational score: 3.9994590282440186 Domain: biomedical Document type: Study Language: en All mutant mice were on a C57BL/6 (H-2 b ) background (backcrossed more than eight times with C57BL/6 mice) and were used between 8 and 12 wk of age. Lpr , gld , and P 0/0 gld mice, which develop a diffuse lymphoproliferation by the age of 2 mo, which could interfere with the development of the CHS reaction, were used at the age of 6 wk, at a time when they show no clinical sign of disease and have normal sized lymphoid organs. P 0/0 , I 0/0 , and II 0/0 mice were bred at the IFFA Credo/Transgenic Alliance specific pathogen–free facility (L'Arbresle, France). Section title: Chemicals. Educational score: 2.3633127212524414 Domain: biomedical Document type: Study Language: en DNFB and its water soluble form, dinitrobenzene sulfonic acid (DNBS), were obtained from Sigma and used for in vivo and in vitro experiments, respectively. Section title: Antibody. Educational score: 1.6890071630477905 Domain: biomedical Document type: Other Language: en Ascites from the anti–MHC class I (heavy chain) hybridoma 20.8.4.S was obtained from Jean-Pierre Abastado (Institut Pasteur, Paris, France). Section title: Assay for CHS to DNFB. Educational score: 4.10599422454834 Domain: biomedical Document type: Study Language: en DNFB was diluted in acetone/olive oil (4:1) immediately before use. The procedure used for the CHS, i.e., the mouse ear swelling test (MEST), has been described elsewhere ( 22 ). In brief, 25 μl of 0.5% DNFB solution was applied to a 2-cm 2 area of shaved dorsal skin. After 5 d, test and control animals received 10 μl of 0.15% (nonirritant concentration) DNFB applied on both sides of the left ear, and the solvent (acetone/olive oil) alone on the right ear. Ear thickness was monitored using a micrometer (J15; Blet SA, France), before challenge and every day after challenge. The ear swelling was calculated as [(T − T 0 ) left ear] − [(T − T 0 ) right ear], where T and T 0 represent values of ear thickness after and before challenge, respectively. Section title: Assay for CHS to DNFB. Educational score: 2.7558958530426025 Domain: biomedical Document type: Study Language: en In each experimental group, some mice were killed at different time intervals after DNFB challenge for histological and PCR analysis. Section title: RNA Extraction and Reverse Transcription PCR Analysis of CD8 and IFN-γ mRNA. Educational score: 4.119418144226074 Domain: biomedical Document type: Study Language: en At different intervals after challenge, ear samples were collected from sensitized or unsensitized mice and frozen in liquid nitrogen. The detection of RNA was conducted as described in detail elsewhere ( 23 ). In brief, total RNA was extracted using an RNAXEL kit (Eurobio). After DNase I treatment, 1 μg of total mRNA was reverse transcribed using poly dT15 primers and Superscript II RT (90 min, 37°C; GIBCO BRL ). The amount of RNA to be used for detection was normalized using the housekeeping gene hypoxanthine phosphoribosyltransferase (HPRT) as reference. The cDNA obtained was amplified using different sets of primers: for HPRT (5′ primer, 5′-GTA ATG ATC AGT CAA CGG GGG AC-3′; 3′ primer, 5′-CCA GCA AGC TTG CAA CCT TAA CCA-3′), for CD8 (5′ primer, 5′-AGG ATG CTC TTG GCT CTT CC-3′; 3′ primer, 5′-TCA CAG GCG AAG TCC AAT CC-3′), and for IFN-γ (5′ primer, 5′-GCT CTG AGA CAA TGA ACG CT-3′; 3′ primer, 5′-AAA GAG ATA ATC TGG CTC TGC-3′). The amplifications were carried out with 29 cycles for HPRT and 33 cycles for IFN-γ and CD8 (1 min at 94°C, 1 min 30 s at 60°C, 2 min at 72°C). The PCR products were analyzed on 1.5% agarose gel. Section title: In Vitro Secondary T Cell Proliferation. Educational score: 4.17324161529541 Domain: biomedical Document type: Study Language: en Spleen cells from DNFB-sensitized C57BL/6 and P 0/0 gld mice were collected 5 d after sensitization. T lymphocytes were purified through negative selection using anti-Ig columns (Biotex) as described elsewhere ( 6 ). The resulting cell suspensions contained >90% CD3 + viable cells. CD8 + T cells were isolated from the spleen T cells by elimination of CD4 + T cells using columns coated with goat anti– mouse and goat anti–rat IgG and a rat anti–mouse CD4 + mAb (YTS191.1; Biotex). FACS ® analysis of cells eluted from the column showed <0.5% contaminating CD4 + T cells. In vivo DNFB-primed unfractionated or CD8 + T cells (2.5 × 10 5 /well) obtained on day 5 after DNFB sensitization were cocultured for 3 d at 37°C in 96-well plates with 10 6 mitomycin C–treated syngenic spleen cells from naive mice, that were either DNBS-derivatized as described ( 6 ) or left untreated. In brief, 10 7 cells were incubated for 30 min with 25 μg/ml of mitomycin C ( Sigma ), washed, and haptenated by 30-min incubation at 37°C with 4 mM DNBS, pH 8.0, in serum-free RPMI medium. The proliferative responses were assessed on day 3 of culture by [ 3 H]thymidine incorporation (1 μCi/well) for the last 6 h of culture. The results are expressed as proliferation indices: (cpm in cultured T cells + DNBS-treated spleen cells)/(cpm in cultured T cells + untreated spleen cells). Section title: IFN-γ Enzyme-linked Immunospot Assay. Educational score: 4.158790588378906 Domain: biomedical Document type: Study Language: en Inguinal and axillary LNs were harvested 5 d after DNFB sensitization. Cell suspensions were restimulated in vitro by overnight culture in complete RPMI medium supplemented with 10% FCS and containing a final concentration of 0.4 mM DNBS. Control cultures included cells cultured overnight in medium supplemented with 0.2 mM of the irrelevant hapten TNBS, or in medium alone. The number of IFN-γ–producing cells was determined using an enzyme-linked immunospot (ELISPOT) assay. In brief, 96-well nitrocellulose plates (MAHA 45; Millipore ) were coated overnight at 4°C with anti–IFN-γ antibody (R46A2) and blocked with PBS/2% BSA for 2 h at 37°C. The plates were washed three times with PBS/Tween 0.1% before use. The cell suspensions were washed, and incubated at different concentrations in duplicate wells for 4 h at 37°C, 5% CO 2 . Plates were washed three times with PBS/0.1% Tween and incubated with a biotinylated anti–IFN-γ antibody (AN18). IFN-γ spot-forming cells (SFCs) were developed using streptavidin–alkaline phosphatase ( Boehringer Mannheim ), incubated for 2 h, and extensively washed before adding the substrate (5-bromo-4-chloro-3-indolyl-phosphate; Sigma ). The number of IFN-γ SFCs present in each well was counted using a microscope, and the results were expressed as IFN-γ SFCs/10 6 cells. Section title: Production of CTLs. Educational score: 4.08843994140625 Domain: biomedical Document type: Study Language: en DNBS-specific CTLs were recovered from splenocytes of various H-2 b mice. Spleens were recovered 5 d after cutaneous sensitization with DNFB, perfused with RPMI to eliminate red blood cells. 10 7 splenocytes were used either fresh or after restimulation for 5 d in culture with 10 7 syngenic mitomycin C–treated, DNBS-derivatized spleen cells from either normal C57BL/6, I 0/0 , or II 0/0 mice. Section title: Target Cells. Educational score: 4.125321388244629 Domain: biomedical Document type: Study Language: en Target cells included MBL2 and MBL2-Fas cell lines, provided by Maries van den Broeck (Institute of Experimental Immunology, University of Zurich, Zurich, Switzerland ), and EL-4 cells, all maintained in RPMI plus 10% FBS. Target cells were simultaneously haptenated and labeled by incubating 2 × 10 6 cells in 10 μl of RPMI with or without 40 mM DNBS and 100 μCi of Na 2 51 CrO 4 (sodium chromate solution, 1 Ci/mM) for 1 h at 37°C with periodic mixing. Labeled targets were washed thoroughly before use. Section title: Cytotoxicity Assays. Educational score: 4.073261260986328 Domain: biomedical Document type: Study Language: en Various numbers of effector cells were incubated at 37°C for 4 h with 10 4 labeled target cells. Supernatants were then collected, and 51 Cr release was counted in a γ counter. The percentage of cytotoxicity was calculated using the formula: 100 × (experimental cpm − spontaneous cpm)/(maximal cpm − spontaneous cpm), where the maximal cpm and spontaneous cpm are the radioactivity released from targets exposed to 0.5 M HCl or medium, respectively. The results presented are representative of at least three independent experiments. Section title: Statistical Analysis. Educational score: 3.2295401096343994 Domain: biomedical Document type: Study Language: en Data were examined for normality and equal variance, and groups were compared by a two-tailed Student's t test. Section title: CHS Reaction to DNFB Is Impaired in Perforin and FasL Double-knockout (P 0/0 Gld) Mice. Educational score: 4.158109664916992 Domain: biomedical Document type: Study Language: en The contribution of CD8 + T cell–mediated cytotoxicity to the CHS reaction to DNFB was examined in mice double deficient for Fas/FasL and perforin (P 0/0 gld ), which are devoid of CTL activity ( 15 ). The CHS reaction was dramatically reduced in P 0/0 gld mice compared with that observed in C57BL/6 mice . No difference in ear swelling could be observed between the sensitized and unsensitized groups of P 0/0 gld mice and the unsensitized C57BL/6 mice. Similarly, none of the characteristic pathological changes occurring during a normal CHS reaction, namely edema, vascular enlargement, and mononuclear cell infiltration, was observed in P 0/0 gld mice . Section title: CHS Reaction to DNFB Is Impaired in Perforin and FasL Double-knockout (P 0/0 Gld) Mice. Educational score: 4.132633209228516 Domain: biomedical Document type: Study Language: en Spleen cells from sensitized animals were tested for their ability to lyse haptenated targets either directly or after in vitro restimulation. As shown in Fig. 1 c, T cells from DNFB-sensitized C57BL/6 mice exhibited a potent hapten-specific CTL activity after restimulation in vitro but which was already detectable directly ex vivo (data not shown). In contrast, DNFB-specific CTLs could never be demonstrated in double-deficient P 0/0 gld mice ex vivo, nor after in vitro restimulation with DNBS-derivatized cells. Section title: CHS Reaction to DNFB Is Impaired in Perforin and FasL Double-knockout (P 0/0 Gld) Mice. Educational score: 4.048379898071289 Domain: biomedical Document type: Study Language: en Thus, P 0/0 gld mice cannot produce a CHS reaction to DNFB and are unable to develop any of the pathological changes associated with CHS, suggesting that hapten-specific CTL activity is mandatory for expression of the CHS reaction. Section title: DNFB Can Prime for Specific CD8 + T Cells in the Lymphoid Organs of Perforin and FasL Double-deficient Mice. Educational score: 4.112563133239746 Domain: biomedical Document type: Study Language: en To determine whether the lack of CHS reaction in P 0/0 gld mice is secondary to an impairment in the priming of specific CD8 + T cells in the lymphoid organs, P 0/0 gld and C57BL/6 mice were sensitized with DNFB, and lymphoid cells were recovered 5 d later and tested for hapten-specific proliferative responses and IFN-γ production. Section title: DNFB Can Prime for Specific CD8 + T Cells in the Lymphoid Organs of Perforin and FasL Double-deficient Mice. Educational score: 4.180509567260742 Domain: biomedical Document type: Study Language: en Lymphocytes from P 0/0 gld mice responded vigorously to hapten-treated syngenic cells in secondary proliferative responses, with stimulation indices identical to those observed with T cells recovered from sensitized C57BL/6 mice . Similarly, CD8 + T cells from P 0/0 gld mice exhibited hapten-specific responses indistinguishable from those of control C57BL/6 cells. Thus, CD8 + T cell priming has occurred in the lymphoid organs of P 0/0 gld mice. Section title: DNFB Can Prime for Specific CD8 + T Cells in the Lymphoid Organs of Perforin and FasL Double-deficient Mice. Educational score: 4.123341083526611 Domain: biomedical Document type: Study Language: en We next used an ELISPOT assay to determine the frequency of DNFB-specific, IFN-γ–producing LN cells in primed C57BL/6 and P 0/0 gld mice. In C57BL/6 mice, the IFN-γ–producing cells were entirely contained in the CD8 + T cell subset, with a mean frequency of 3 IFN-γ SFCs/10 5 LN cells . Interestingly, P 0/0 gld mice exhibited comparable levels of hapten-specific, IFN-γ–producing cells . Section title: DNFB Can Prime for Specific CD8 + T Cells in the Lymphoid Organs of Perforin and FasL Double-deficient Mice. Educational score: 4.168857097625732 Domain: biomedical Document type: Study Language: en These data indicate that the lack of CHS in double-deficient P 0/0 gld mice is not due to impaired priming of hapten-specific CD8 + T cells nor to an altered production of IFN-γ, but rather to the deficient CTL activity. Section title: IFN-γ–producing CD8 + T Cells of P 0/0 Gld Mice Migrate to the Challenged Skin but Are Unable to Induce the Recruitment of Inflammatory Cells. Educational score: 4.421535491943359 Domain: biomedical Document type: Study Language: en Two possibilities could explain why P 0/0 gld mice did not show a CHS response despite the presence of IFN-γ–producing DNFB-specific CD8 + T cells in the lymphoid organs: (a) primed CD8 + T cells lacking cytolytic activity are unable to migrate from the blood to the skin; (b) CD8 + T cells are able to infiltrate the skin but, due to lack of cytolytic functions, are unable to induce recruitment of inflammatory cells mandatory for the full development of CHS. Since the CHS reaction to DNFB is associated with the infiltration of IFN-γ–producing CD8 + T cells in situ a few hours after challenge (Akiba, H., manuscript in preparation), we examined the presence of CD8 + T cells and the expression of IFN-γ in the skin of P 0/0 gld and C57BL/6 mice after sensitization and challenge . PCR analysis demonstrated that CD8 and IFN-γ mRNAs were not found in the skin of naive mice or in hapten-painted ears of unsensitized mice. In contrast, upregulation of both CD8 and IFN-γ mRNA occurred as early as 6 h after challenge in both C57BL/6 and P 0/0 gld mice, indicating that lack of both perforin and FasL did not affect the migration of CD8 + T cells to the skin. However, at 24 and 48 h after challenge, dramatic differences were noted. C57BL/6 mice showed a gradual increase in the intensity of the CD8 and IFN-γ mRNA expression, whereas no signal was observed in the skin of P 0/0 gld mice. These results suggest that DNFB-specific CD8 + T cells from P 0/0 gld mice are able to migrate from blood to skin, but cannot induce the specific signals necessary for the constitution of the inflammatory cellular infiltrate. Section title: DNFB-specific CHS and CTL Responses Develop in Lpr Mice, Gld Mice, and P 0/0 Mice. Educational score: 4.055032730102539 Domain: biomedical Document type: Study Language: en To determine the relative contribution of the Fas/FasL and perforin pathways to the CHS response, we tested the ability of Fas-deficient ( lpr ), FasL-deficient ( gld ), and P 0/0 mice to exhibit specific CHS and CTL responses after DNFB sensitization. Section title: DNFB-specific CHS and CTL Responses Develop in Lpr Mice, Gld Mice, and P 0/0 Mice. Educational score: 4.153044700622559 Domain: biomedical Document type: Study Language: en In marked contrast to double-deficient P 0/0 gld mice, lpr , gld , or P 0/0 mice developed a normal CHS reaction to DNFB , with intensity, kinetics, and histological characteristics comparable to those observed in C57BL/6 mice. Mice homozygous for the gld mutation and heterozygous for the perforin deletion (P +/0 gld ) also had a similar reaction to DNFB (data not shown). Interestingly, lpr , gld , and P 0/0 mice exhibited a strong hapten-specific CTL response as well as normal quantities of DNFB-specific, IFN-γ–producing LN cells (data not shown). Section title: DNFB-specific CHS and CTL Responses Develop in Lpr Mice, Gld Mice, and P 0/0 Mice. Educational score: 4.132966041564941 Domain: biomedical Document type: Study Language: en Thus, exclusion of only one cytolytic pathway did not prevent the induction of hapten-specific CTLs nor the development of a CHS reaction to DNFB, indicating that CTLs can use either the Fas/FasL or the perforin pathway to mediate CHS. Section title: Hapten-specific CTL Activity Is Found in the MHC Class I–restricted CD8 + T Cell Subset and Is Mediated by Both Perforin and Fas/FasL. Educational score: 4.076849460601807 Domain: biomedical Document type: Study Language: en The above data demonstrating that CTLs mediate the CHS reaction to DNFB were obtained in mice genetically deficient for molecules involved in CTL activity, raising the question of the nature of the cells endowed with the specific CTL activity and of the existence of similar mechanisms in normal C57BL/6 mice, which have both functional perforin and Fas/FasL pathways. Section title: Hapten-specific CTL Activity Is Found in the MHC Class I–restricted CD8 + T Cell Subset and Is Mediated by Both Perforin and Fas/FasL. Educational score: 4.291369438171387 Domain: biomedical Document type: Study Language: en Hapten-specific CTL activity was restricted to the MHC class I–restricted CD8 + T cell subset. Indeed, depletion of CD8 + cells, but not of CD4 + cells, totally abolished the CTL activity of primed C57BL/6 spleen cells (data not shown). MHC class I molecules were mandatory for the induction, expansion, and effector function of specific CD8 + CTLs, since: (a) primed spleen cells from C57BL/6 and II 0/0 , but not from I 0/0 , mice could lyse haptenated targets ; (b) hapten-specific lysis was not observed when effector cells were restimulated in vitro with haptenated APCs from I 0/0 mice ; and (c) CTL activity of C57BL/6 spleen cells was suppressed by incubation with mAbs to the K b MHC class I molecules . These results indicate that the hapten-specific CTLs are “classical” MHC class I–restricted CD8 + T cells. Section title: Hapten-specific CTL Activity Is Found in the MHC Class I–restricted CD8 + T Cell Subset and Is Mediated by Both Perforin and Fas/FasL. Educational score: 4.174177646636963 Domain: biomedical Document type: Study Language: en The contribution of perforin or Fas/FasL to the CTL activity was tested using perforin + (from C57BL/6 mice) and perforin-deficient (from P 0/0 mice) effectors and targets comprising Fas + (MBL2-Fas) and Fas-deficient (MBL2) cells. As shown in Fig. 5 d, perforin-deficient effectors could lyse hapten-treated Fas + cells, but not Fas-deficient cells, whereas perforin + CTLs were able to lyse Fas-deficient cells as efficiently as Fas-expressing cells. Thus, hapten-specific MHC class I–restricted CD8 + CTLs could use the Fas/FasL or the perforin pathway for their CTL activity, which could be abolished only by inactivation of both cytolytic pathways. Section title: Discussion Educational score: 4.242660045623779 Domain: biomedical Document type: Study Language: en This study demonstrates that CD8 + T cells require cytotoxic activity to mediate CHS. The invalidation of the two main cytolytic pathways, as observed in mice deficient in perforin and FasL (P 0/0 gld ), is responsible for the lack of generation of specific CTLs and for the abolition of the CHS reaction. Interestingly, the presence of a single cytolytic pathway, in lpr mice, gld mice, and P 0/0 mice, is sufficient for the development of a CHS reaction and for the priming of specific CTLs, indicating that the Fas/FasL and the perforin pathways could be used independently and with similar efficiency for cytolytic activity. These results are in line with recent data on influenza virus infection showing that virus clearance by CTLs could be achieved only if one of the two main lytic pathways remained functional ( 25 ). Section title: Discussion Educational score: 4.163532257080078 Domain: biomedical Document type: Study Language: en Until our study, cytokines, and especially IFN-γ and TNF-α, were thought to mediate skin inflammation through their ability to activate keratinocytes and endothelial cells ( 2 , 26 – 29 ). Recent studies excluded a pivotal role for IFN-γ in the elicitation phase of CHS, since IFN-γ receptor–deficient mice developed a normal CHS reaction ( 30 ). In addition, studies in TNF-α–deficient mice indicated that the cytokine was not involved in the elicitation phase but played an important role in the sensitization phase by inducing the emigration of LCs to the draining LNs ( 29 ). Section title: Discussion Educational score: 4.392566204071045 Domain: biomedical Document type: Study Language: en CHS develops in two phases, the sensitization (i.e., afferent) phase leading to the priming of hapten-specific CD8 + T cells and the elicitation (i.e., efferent) phase occurring after challenge and leading to the development of skin inflammation. MHC class I molecules expressed by LCs are mandatory for the priming of hapten-specific CD8 + T cells in the LNs during the afferent phase ( 6 , 31 ). These MHC class I–restricted CD8 + T cells exert CHS through CTL activity using classical cytolytic pathways. That cytotoxicity is the effector mechanism responsible for the efferent phase of CHS is supported by the observation that hapten-primed, IFN-γ–producing CD8 + T cells are present in lymphoid organs of P 0/0 gld mice and respond vigorously in secondary proliferative responses, demonstrating that the lack of CHS reaction is not due to an impairment of the sensitization phase of CHS, but rather reflects an alteration during the elicitation phase. Section title: Discussion Educational score: 4.66609001159668 Domain: biomedical Document type: Study Language: en The mechanisms involved in the development of skin inflammation upon challenge in sensitized mice, i.e., during the elicitation phase, associates hapten-specific and nonspecific steps ( 2 ). First, lymphocytes have to emigrate from the blood to the skin. Infiltration of the skin by mononuclear cells has been reported to occur within a few hours after challenge ( 32 ). Haptens are able to directly induce expression of E- and P-selectins on endothelial cells 2 h after skin painting ( 33 , 34 ). It is thought that circulating antigen-specific memory T cells which carry homing receptors (the cutaneous leukocyte-associated antigen [CLA] molecule) are able to enter the skin ( 35 ) through interaction with P- and E-selectins expressed on endothelial cells ( 36 ). Second, specific T cells are activated on hapten presentation by skin-resident cells, as revealed by detection of IFN-γ mRNA in situ between 4 and 8 h after challenge ( 37 , 38 ). Third, activation of skin-resident cells by T cell cytokines results in the amplification of the inflammatory reaction leading to the cellular inflammatory infiltrate. Upon IFN-γ activation, keratinocytes upregulate intercellular adhesion molecule 1 (ICAM-1) and Ia molecules and produce a wide array of inflammatory cytokines and chemokines such as IL-8 ( 2 , 26 , 39 , 40 ). Likewise, endothelial cell activation is followed by leukocyte migration from the blood vessels to the dermis, leading to the formation of inflammatory cellular infiltrate ( 2 , 26 ). In this scheme, the hapten-specific limb of the CHS reaction corresponds to the activation of hapten-specific T cells and occurs ∼6 h after challenge, whereas the nonspecific phase corresponds to the infiltration of the skin by the polymorphic cellular infiltrate, which peaks 24 h after challenge. Section title: Discussion Educational score: 4.422096252441406 Domain: biomedical Document type: Study Language: en It appears from our data that the failure of P 0/0 gld mice to exhibit a CHS reaction is not due to the inability of CD8 + T cells to infiltrate the skin or to be activated, since IFN-γ–producing CD8 + T cells could be detected in the skin 6 h after challenge, at levels comparable to those found in C57BL/6 mice. However, these early parameters of the CHS reaction were not followed by an increase in cellular infiltration in situ, as observed in C57BL/6 mice at 24 and 48 h. These observations suggest that cytolytic function of hapten-specific CD8 + T cells is required for the recruitment of inflammatory cells and full development of the CHS response. Both epidermal dendritic LCs and keratinocytes express MHC class I molecules and may produce upon activation (or lethal hit) a wide array of inflammatory cytokines and chemokines. Destruction of haptenated LCs by CTLs may account for the recent observation that LCs undergo apoptotic cell death in CHS ( 41 ). Alternatively, keratinocytes, which represent >90% of epidermal cells, could be the targets of antihapten CTLs, the more so since IFN-γ, produced in situ during the course of CHS, upregulates Fas expression by keratinocytes ( 42 ). Section title: Discussion Educational score: 4.158404350280762 Domain: biomedical Document type: Study Language: en In conclusion, our data demonstrate that CHS to DNFB is mediated by hapten-specific CTLs which may use either the Fas/FasL or the perforin pathway for the induction of cutaneous inflammation. The precise nature of the MHC class I–expressing skin cells able to present the hapten to CTLs in vivo during the elicitation phase is currently under investigation. | Study | biomedical | en | 0.999997 |
10049942 | Section title: cDNA Library Construction. Educational score: 3.944190502166748 Domain: biomedical Document type: Study Language: en The expression cDNA library was prepared in VR1012 plasmid (Vical Inc.) using RNA extracted from IL-2–activated polyclonal NK cells as described ( 7 , 8 ), and was divided into 10 fractions of ∼10 5 clones each. Section title: Library Screening by cDNA Expression in COS-7 Cells. Educational score: 4.0136260986328125 Domain: biomedical Document type: Study Language: en The library screening procedure was performed as described ( 7 , 9 ). Briefly, cDNA library was transiently transfected in COS-7 cells; selection of positive pools was performed by immunocytochemical staining using the specific anti-NKp44 mAb Z231 ( 3 ) and sib selection. Section title: DNA Sequencing. Educational score: 3.445598602294922 Domain: biomedical Document type: Study Language: en DNA sequencing was performed using d-Rhodamine Terminator Cycle Sequencing Kit and a 377 Applied Biosystems Automatic Sequencer ( Perkin Elmer -Applied Biosystems). Section title: Reverse Transcriptase PCR (RT-PCR) Amplification of cDNAs Encoding for NKp44 and DAP12. Educational score: 4.194188117980957 Domain: biomedical Document type: Study Language: en Total RNA extracted using RNAzol (Cinna/Biotecx) from polyclonal NK and T cell populations and clones and from different hemopoietic cell lines (Table I ) was reverse transcribed using oligo dT priming. Primers used for cDNA amplification of complete NKp44 open reading frame (ORF) (857 bp) were the following: 5′ CCA CGA GCG CAC AGG AAA AGG (NKp44 ORF UP) and 5′ TCA CAA AGT GTG TTC ATC ATC ATC ATC GCT TAT CTT AGT CC (NKp44 ORF DOWN). Amplification was performed for 30 cycles (30 s at 94°C, 30 s at 65°C, and 30 s at 72°C), followed by a 7-min incubation at 72°C, utilizing TAQ-GOLD ( Perkin Elmer -Applied Biosystems) after preactivation for 15 min at 95°C. The cDNA obtained from a polyclonal NK cell population (NKp44 ORF) was subcloned in pcDNA3.1/V5/His − TOPO vector (Invitrogen) and sequenced. Primers used for cDNA amplification of complete DAP12 ORF ( 10 ) (353 bp) were the following: 5′ TCA TGG GGG GAC TTG AAC C (DAP12 UP) and 5′ GAT TCG GGC TCA TTT GTA ATA C (DAP12 DOWN). Amplification was performed for 30 cycles (30 s at 94°C, 30 s at 60°C, and 30 s at 72°C), followed by a 7-min incubation at 72°C. The amplification product obtained from the NK cell clone K17 was subcloned in pCR3.1 vector (Invitrogen) and sequenced. Polyclonal or clonal NK cell populations and clones were obtained as previously described ( 2 , 3 , 11 ). Section title: Reverse Transcriptase PCR (RT-PCR) Amplification of cDNAs Encoding for NKp44 and DAP12. Educational score: 4.036323070526123 Domain: biomedical Document type: Study Language: en RT-PCR analysis of NKp44 and DAP12 mRNA on a panel of different cell populations of lymphoid origin and hemopoietic human cell lines was performed utilizing a semiquantitative PCR technique ( 12 , 13 ). Section title: Transient Transfections. Educational score: 4.1288161277771 Domain: biomedical Document type: Study Language: en COS-7 cells (5 × 10 5 per plate) were cotransfected with VR1012-15C and pCR3.1-DAP12 or with pcDNA3.1/V5/His − TOPO NKp44 ORF (pcDNA3.1–NKp44 ORF) and pCR3.1–DAP12 constructs by the DEAE-dextran method as described ( 11 ). Cells were stained with different anti-NKp44 mAbs (Z231, IgG1; AZ140, IgG1; KS38, IgM) followed by a phycoerythrin-conjugated goat antibody to mouse IgG1 or IgM and analyzed by flow cytometry using a FACSort ® ( Becton Dickinson ). Section title: Expression of NKp44 and DAP12 mRNA in Transfected COS-7 Cells by RT-PCR and by Dot Blot Analysis. Educational score: 4.147315979003906 Domain: biomedical Document type: Study Language: en Total RNA was extracted from COS-7 cells (untransfected, transfected with pcDNA3.1–NKp44 ORF, or co-transfected with pcDNA3.1– NKp44 ORF and pCR3.1–DAP12) utilizing CsCl gradient. Poly A + fraction was purified from total RNA using oligo dT Dynabeads ( Dynal , Norway) following the manufacturer's instructions. cDNA was obtained by RT reaction using oligo dT priming starting from 200 ng poly A + . PCR amplification of NKp44 ORF and DAP12 ORF was carried out with the primers described above for 25 cycles, each consisting of 30 s at 95°C, 30 s at 60°C, and 30 s at 72°C. A 228-bp β-actin fragment was amplified as control using the following primers: 5′ ACT CCA TCA TGA AGT GTG ACG (β-actin UP) and 5′ CAT ACT CCT GCT TGC TGA TCC (β-actin DOWN). PCR amplification was carried out for 25 cycles, each consisting of 30 s at 95°C, 30 s at 58°C, and 30 s at 72°C. As a negative control we included for each set of primers poly A + without RT reaction step. PCR products were run on a 1.5% agarose gel and stained with ethidium bromide. Section title: Expression of NKp44 and DAP12 mRNA in Transfected COS-7 Cells by RT-PCR and by Dot Blot Analysis. Educational score: 4.114591598510742 Domain: biomedical Document type: Study Language: en For the dot blot analysis, 200 ng poly A + were denatured in 60 μl 20× SSC and 40 μl 37% formaldehyde at 60°C for 15 min and spotted onto a positively charged nylon membrane (GeneScreen plus; Dupont-NEN ) in 15× SSC using a microfiltration apparatus (Bio-dot; BioRad). The dot blot was hybridized with the following probes: 857-bp NKp44 ORF fragment, 353-bp DAP12 fragment, and 228-bp β-actin fragment. cDNA probes were 32 P labeled by random priming ( 14 ). Blot was washed at high stringency conditions as described ( 8 ). Section title: Biochemical Characterization of the NKp44 Molecule. Educational score: 4.088969707489014 Domain: biomedical Document type: Study Language: en Cyanogen bromide Sepharose ( Pharmacia , Sweden) –coupled Z231 mAb was used to immunoprecipitate NKp44 molecules from 1% NP-40 lysates of 125 I surface labeled cells ( DuPont-NEN ) as previously described ( 2 , 3 ). Immunoprecipitates were analyzed by discontinuous SDS-PAGE under reducing conditions (5% 2-mercaptoethanol). Section title: Biochemical Characterization of the NKp44-associated KARAP/ DAP12 Molecules. Educational score: 4.152309417724609 Domain: biomedical Document type: Study Language: en NK cells were treated with sodium pervanadate ( 3 ) or with KS38 (IgM, anti-NKp44) mAb (3 × 10 6 , 5 μg, 30 min, 4°C) followed by affinity purified F(ab′) 2 rabbit anti–mouse IgM (1.5 μg, 1 min, 37°C, ZYMED). Untreated and treated cells were lysed in 1% digitonin and immunoprecipitated with various mAbs as previously described ( 3 ). Samples were analyzed in SDS-PAGE, transferred to Immobilon P ( Millipore Corp. ) and the membranes probed with PY20-HRPO (anti-phosphotyrosine mAb; Transduction Laboratories) or with anti-FcεRIγ rabbit antiserum (provided by E. Vivier, INSERM, Marseille, France) or anti-DAP12 rabbit antiserum (SI-28) followed by donkey anti–rabbit HRPO (Amersham, UK). The Renaissance Chemiluminescence Kit ( Dupont-NEN ) was used for detection. SI-28 antiserum was obtained as previously described ( 11 ), using the COOH-terminal peptide (SDVYSDLNTQRPYYK) of DAP12 molecule ( 10 ). Section title: Analysis of NKp44 Transcript Expression by Northern Blotting. Educational score: 4.111440658569336 Domain: biomedical Document type: Study Language: en 5 μg of total RNA prepared from LCL721.221 and Jurkat cell lines, NK cell clones and polyclonal NK cell populations using CsCl gradient was size-fractionated by denaturing agarose gel electrophoresis and transferred onto a positively charged nylon membrane (GeneScreen plus; DuPont-NEN ). Northern blot was performed under high stringency conditions as described ( 8 ). The NKp44 cDNA probe (857-bp fragment NKp44 ORF) and the 228-bp β-actin probe were 32 P labeled by random priming ( 14 ). Section title: Southern Blotting and Chromosomal Localization of NKp44 Gene. Educational score: 4.117107391357422 Domain: biomedical Document type: Study Language: en 10 μg of genomic DNA extracted from human, monkey, and mouse was digested with EcoRI, HindIII, or SacI restriction enzymes. DNA was size-fractionated by electrophoresis on a 0.8% agarose gel, transferred onto a positively charged nylon membrane ( DuPont-NEN ), hybridized as described ( 8 , 14 ), and washed at low stringency conditions (0.5× SSC at 65°C). Section title: Southern Blotting and Chromosomal Localization of NKp44 Gene. Educational score: 4.135892868041992 Domain: biomedical Document type: Study Language: en The Somatic Cell Hybrid blot (BIOS Laboratories) was used to assign NKp44 gene to a specific chromosome by Southern blotting. The NKp44 ORF probe was used for high stringency hybridization; washes were performed as previously described ( 8 ). Chromosomal assignment was further confirmed by PCR on DNA from human–hamster monochromosomal hybrids (BIOS Laboratories) utilizing the following primers: 5′ CCA CGA GCG CAC AGG AAA AGG (NKp44 ORF UP) and 5′ GTA GAT TCT ACA CCA GTA ATG (15C-REV2). These primers allowed us to discriminate, by the size of the amplified products, between genomic and cDNA amplified sequences. Section title: Molecular Cloning of the cDNA Encoding the NKp44 Receptor. Educational score: 4.446447372436523 Domain: biomedical Document type: Study Language: en The availability of different mAbs specific for NKp44 molecules allowed us to attempt the isolation of the cDNA encoding NKp44 by an expression cloning strategy. To this end, a cDNA library was prepared from human NK cells and divided into 10 fractions of ∼10 5 independent recombinant clones. Individual fractions were transiently transfected in COS-7 cells and analyzed by immunocytochemical staining using the prototype NKp44-specific mAb Z231 ( 3 ). The first screening yielded 2 (out of 10) positive fractions. By subsequent screening of progressively smaller cDNA library pools, we obtained positive fractions, each containing ∼3 × 10 3 recombinants. Further attempts aimed at obtaining positive pools of smaller size (e.g., 500 recombinants/pool) were unsuccessful. However, cotransfection of pairs of these small pools allowed us to identify two pools (termed C67 and C68) yielding Z231 + COS-7 cells. Since individual transfection of C67 and C68 pools gave negative results, a possible explanation was that NKp44 expression at the surface of COS-7 cells required cotransfection with other molecule(s). On the basis of this assumption, the C67 fraction was transfected with progressively smaller pools of C68. A single clone (clone 15C) was isolated that could direct surface expression of the NKp44 protein when cotransfected with the C67 fraction. Since we recently demonstrated that NKp44 is associated with KARAP/DAP12 ITAM-bearing molecules ( 3 ), we analyzed whether these molecules were required for NKp44 expression at the cell surface. Indeed, cotransfection of clone 15C and DAP12 cDNAs resulted in surface expression of NKp44 (not shown). This clearly indicated that clone 15C encoded the NKp44 molecule. Importantly, in agreement with these results, DAP12 cDNA could be amplified by PCR from the C67 library fraction, thus confirming that, at least in COS-7 cells, DAP12 is required for the surface expression of NKp44. Section title: Molecular Cloning of the cDNA Encoding the NKp44 Receptor. Educational score: 4.156652450561523 Domain: biomedical Document type: Study Language: en Next, COS-7 cells were either cotransfected with NKp44 ORF and DAP12 cDNAs or transfected separately with the individual constructs and stained with different anti-NKp44 mAbs (including Z231, AZ140, and KS38). Fig. 1 A shows that COS-7 cells were efficiently stained by the anti-NKp44 mAbs only when cotransfected with both NKp44 ORF and DAP12 cDNAs. On the contrary, COS-7 cells transfected with NKp44 ORF cDNA alone either failed to express NKp44 or (in some experiments) displayed a low level of NKp44 surface expression. As illustrated in Fig. 1 B, the expression of NKp44 and DAP12 mRNA in COS-7 cell transfectants was checked both by RT-PCR and by dot blot analysis. Surface molecules reacting with anti-NKp44 mAb were immunoprecipitated from 125 I surface labeled COS-7 cells transfected with NKp44 ORF and DAP12 cDNAs. As shown in Fig. 1 C, the surface molecules immunoprecipitated from COS-7 cell transfectants (lane e) displayed a molecular size similar to that of molecules isolated from activated NK cells (lane b). Section title: Molecular Cloning of the cDNA Encoding the NKp44 Receptor. Educational score: 4.3766865730285645 Domain: biomedical Document type: Study Language: en Notably, cotransfection of COS-7 cells with NKp44 ORF and cDNAs encoding for other ITAM-bearing molecules, such as CD3ζ or FcεRIγ ( 15 ), did not result in NKp44 surface expression (not shown). Thus, we can conclude that neither CD3ζ nor FcεRIγ can substitute KARAP/DAP12 to allow surface expression of NKp44. In this context, we previously showed that, in activated NK cells, NKp44 molecules do not associate with CD3ζ molecules ( 3 ). In Fig. 2 A, we show that NKp44 do not associate with FcεRIγ chains either. On the other hand, FcεRIγ associated with NKp46. Remarkably, the use of an antiserum specific for the recently cloned DAP12 molecules formally demonstrates that DAP12 is identical to the previously described NKp44-associated KARAP molecules ( 3 , 16 ). As shown in Fig. 2 B, in NKp44 immunoprecipitates derived from activated NK cells treated with sodium pervanadate, the DAP12-specific antiserum recognized both nonphosphorylated and tyrosine-phosphorylated KARAP molecules. Moreover, as shown in Fig. 2 C, tyrosine phosphorylation of the NKp44-associated KARAP/DAP12 molecules also occurred when cross-linking of NKp44 molecules was induced by a specific mAb. Taken together, these data may suggest a direct involvement of KARAP/ DAP12 molecules in the signal transduction leading to the NKp44-dependent NK cell activation. Section title: Molecular Cloning of the cDNA Encoding the NKp44 Receptor. Educational score: 4.755603313446045 Domain: biomedical Document type: Study Language: en Nucleotide sequence analysis showed that the 1,192-bp isolated cDNA (clone 15C) contained a 828-bp ORF preceded by a 338-bp 5′ untranslated region. The ORF encoded a 276–amino acid (aa) type I transmembrane protein belonging to the Ig-SF. This protein is characterized by a 21-aa leader sequence, a 169-aa extracellular region, a 23-aa transmembrane portion, and a 63-aa cytoplasmic tail . Two cysteines in the extracellular region (positions 19 and 88 of the mature protein) determine the formation of a single Ig-related domain of the V type. Thus, the NKp44 V domain displays the typical long spacing (68 aa) between the two conserved cysteines as well as an arginine at position 59 and an aspartic acid at position 82 characteristic of Ig V domains ( 17 ). It also displays an additional pair of cysteines at positions 34 and 42, possibly involved in the formation of a second disulphide bond similar to that described in the NH 2 -terminal V domain of the poly Ig receptor ( 18 ). The membrane-proximal region of the NKp44 receptor contains a high proportion of proline (20%), serine (16%), and threonine (13%). By analogy with other Ig-SF members, this region is predicted to display an extended open conformation typical of hinge-like sequences ( 19 ). Computer search ( 20 ) revealed seven serine and six threonine residues in the hinge-like region as putative O-glycosylation sites and an asparagine at position 159 as a potential N-linked glycosylation site. The predicted molecular mass of the NKp44 polypeptide is ∼29 kD. Thus, glycosylation of the molecule may account for the apparent molecular mass (44 kD) of the glycoprotein isolated from normal NK cells ( 3 ). The transmembrane portion contains the positively charged amino acid lysine. This may be involved in the association with the negatively charged residue aspartic acid contained in the transmembrane region of KARAP/DAP12 protein ( 10 ). The cytoplasmic tail of NKp44 contains the amino acid sequence ILYHTV, which fits the consensus sequence for immunoreceptor tyrosine-based inhibitory motif (ITIM) (i.e., I/LxYxxL/V) ( 21 ). Remarkably, this motif is typical of all the HLA class I–specific inhibitory receptors, while none of the activating NK receptors identified so far (including CD16, p50, and NKp46) display ITIM in their cytoplasmic region ( 1 , 21 ). On the other hand, it should be stressed that other surface receptors that are involved in cell activation, such as the erythropoietin receptor, the stem cell factor receptor, and the IL-3 receptor β chain, are characterized by a cytoplasmic tail containing ITIM sequences ( 22 – 24 ). The homology of NKp44 with known proteins was confined to the Ig-like V domain and limited to few members of the Ig superfamily. In particular, a low degree of homology was observed between the Ig-like V domain of NKp44 and that of the human poly-Ig receptor ( 18 ) (25% identity) and of the human CMRF35 protein ( 25 ) (29% identity). Section title: Cellular Distribution of NKp44 Transcripts. Educational score: 4.202495574951172 Domain: biomedical Document type: Study Language: en Northern blot analysis was performed on RNA isolated from clonal and polyclonal NK cells as well as from LCL721.221 and Jurkat cell lines. As shown in Fig. 4 , hybridization with the NKp44 ORF probe yielded two transcripts of ∼3.7 and 1.2 kb. Since the cloned 1.2-kb cDNA encodes for a 44-kD protein that is specifically recognized and immunoprecipitated by anti-NKp44 mAbs, it is likely that the long 3.7-kb transcript may use a different polyadenylation site located 3′ with respect to the 1.2-kb transcript. Moreover, analysis of the cDNA sequence 5′ to the ATG initiation codon showed that there are four in-frame stop codons, making it unlikely that the 3.7-kb transcript may encode for a longer protein (not shown). Whereas β-actin hybridization showed that similar amounts of RNA were loaded in each lane, the amount of NKp44 mRNA varied considerably among different NK cell clones or populations analyzed. This is consistent with previous data indicating that the amount of NKp44 expressed at the cell surface differs among NK cell clones ( 3 ). Section title: Cellular Distribution of NKp44 Transcripts. Educational score: 4.412261486053467 Domain: biomedical Document type: Study Language: en Further analysis of the expression of NKp44 and DAP12 mRNA in different cell populations of lymphoid origin and in hemopoietic human cell lines was performed by a semiquantitative RT-PCR technique. Table I shows that all IL-2–cultured NK cell populations and clones analyzed (all reactive with anti-NKp44 mAbs) expressed both NKp44 and DAP12 transcripts. On the contrary, fresh PBL expressed DAP12 but not NKp44 transcript, in agreement with the lack of surface reactivity with anti-NKp44 mAb. This is consistent with previous data indicating that only activated NK cells express surface NKp44 ( 3 ). Likewise, all TCR-α/β + T cell clones were not stained by anti-NKp44 mAb and did not express the NKp44 transcript. The DAP12 transcript was only expressed in one of the clones analyzed; notably, this clone (R3.2) expressed the p50.2 activating receptor, known to associate with KARAP/ DAP12 molecules ( 16 ). Among TCR-γ/δ + T cell clones, most did not express surface NKp44 molecules. However, two of them expressed DAP12 transcripts. Two TCR-γ/δ + T cell clones (17.12 and 17.31), isolated from a single donor, displayed low levels of surface NKp44 and expressed both NKp44 and DAP12 transcripts. In both clones, the NKp44 receptor was functional, as revealed by redirected killing assays in the presence of anti-NKp44 mAbs (not shown). We also analyzed several human cell lines of different histotype. All these lines lacked surface expression of NKp44 and did not display NKp44 transcripts; on the other hand, the DAP12 transcript was also expressed in cells of nonlymphoid origin. Unlike normal activated NK cells, the two NK cell lines analyzed (NK3.3 and NKL) displayed neither surface NKp44 molecules nor NKp44 transcripts. Section title: Conservation Across Species and Chromosomal Localization of the NKp44 Gene. Educational score: 4.2005815505981445 Domain: biomedical Document type: Study Language: en Southern blot analysis of the NKp44 gene was performed on genomic DNA extracted from human, monkey, and mouse cells and digested with three different restriction enzymes . Hybridization performed with the NKp44 ORF probe under low stringency conditions revealed a relatively simple hybridization pattern for the NKp44 gene. Interestingly, cross-species hybridization could be observed with both monkey and mouse genomic DNA. These data suggest that the NKp44 ORF probe hybridizes with a single or relatively few human genes and that the NKp44 gene may be conserved between humans and rodents. Section title: Conservation Across Species and Chromosomal Localization of the NKp44 Gene. Educational score: 4.157490253448486 Domain: biomedical Document type: Study Language: en Chromosomal localization of the NKp44-encoding gene was determined by two different approaches. First, we performed Southern blot analysis, using genomic DNA derived from a panel of polychromosomal human–hamster cell hybrids. Hybridization with the NKp44 ORF cDNA probe showed segregation of the NKp44 gene on human chromosome 6. The same conclusion was achieved by PCR analysis of a panel of genomic DNA extracted from monochromosomal human–hamster cell hybrids by using NKp44 specific primers (not shown). Taken together, these results indicate that the NKp44 gene is localized on human chromosome 6. Section title: Discussion Educational score: 4.250080108642578 Domain: biomedical Document type: Study Language: en The present study reports the molecular cloning of NKp44, a NK-specific triggering receptor involved in non-MHC–restricted natural cytotoxicity ( 3 ). Different from NKp46 ( 2 , 7 ), which is expressed by all resting and activated NK cells, NKp44 is selectively expressed by activated human NK cells. In this context, it is well known that culture in IL-2 greatly enhances the NK-mediated anti-tumor cytotoxicity both in vitro ( 4 ) and in vivo ( 5 , 6 ). It is conceivable that the increased cytolytic activity mediated by activated NK cells may be consequent, at least in part, to the de novo expression of triggering receptors. NKp44 may well represent one of these receptors since it is involved in triggering of activated NK cells in the process of tumor cell lysis ( 3 ). Section title: Discussion Educational score: 4.357553482055664 Domain: biomedical Document type: Study Language: en Molecular cloning revealed that NKp44 is a type I glycoprotein belonging to the Ig-SF, which does not display any major amino acid sequence homology with known proteins. NKp44 is characterized by an extracellular region containing an Ig-like domain of the V type. The transmembrane portion contains the charged amino acid lysine, possibly involved in the association with KARAP/DAP12 molecules. Indeed, biochemical data confirmed that NKp44 associates with KARAP/DAP12 molecules ( 3 ). Moreover, molecular cloning of NKp44 demonstrated that the association with KARAP/DAP12 molecules is required for the surface expression of NKp44. Section title: Discussion Educational score: 4.209893703460693 Domain: biomedical Document type: Study Language: en Interestingly, the cytoplasmic tail of NKp44 contains a classical ITIM ( 21 ). In this context, it is of note that, under the experimental conditions resulting in a strong tyrosine phosphorylation of the KARAP/DAP12 molecules associated to the NKp44 receptor ( 3 ), no tyrosine phosphorylation of the NKp44 molecule itself could be detected. Moreover, under the same experimental conditions, no evidence has been obtained so far that NKp44 may associate with phosphatases such as SHP-1, SHP-2, or SHIP, which have been reported to associate with ITIM-bearing receptors ( 21 ). The functional role of this ITIM is presently under investigation. Section title: Discussion Educational score: 4.337653160095215 Domain: biomedical Document type: Study Language: en Remarkably, unlike most of the genes encoding Ig-SF receptors involved in the regulation of NK cell–mediated cytolytic activity (including KIR , KAR , NKp46 , and ILT/LIR ), which are on human chromosome 19, the NKp44 gene is on chromosome 6. Southern blot analysis of human genomic DNA revealed a restriction enzyme digestion pattern that is compatible with the existence of a single NKp44 gene. Moreover, the NKp44 gene appears to be conserved across species since the human NKp44 ORF cDNA probe cross-hybridized with genomic DNA from monkey and mouse. It will be of interest to analyze whether the murine counterpart of NKp44 exists and whether its expression is restricted to NK cells and to a subset of TCR-γ/δ + T lymphocytes as it occurs in humans. | Study | biomedical | en | 0.999996 |
10049943 | Section title: Mice and Antibodies. Educational score: 2.1173245906829834 Domain: biomedical Document type: Study Language: en BALB/cJ and C57BL/6 mice were obtained from The Jackson Laboratory . Antibodies to CRT and protein disulfide isomerase (PDI) were purchased from Affinity BioReagents , and antibodies to gp96 were obtained from NeoMarkers. A rabbit serum to a synthetic peptide derived from the sequence of human Erp57 was a gift from Peter Cresswell (Yale University, New Haven, CT). Section title: Tumor Cell Lines. Educational score: 3.595839738845825 Domain: biomedical Document type: Study Language: en Methylcholanthrene-induced fibrosarcomas Meth A and CMS5 (BALB/c) have been described previously ( 2 ). Meth A was maintained in ascites formed in BALB/cJ mice by weekly intraperitoneal passage of cells. Section title: Purification of gp96 and CRT. Educational score: 4.17941427230835 Domain: biomedical Document type: Study Language: en A 40-ml Meth A cell pellet (or equivalent wet weight of mouse liver) was homogenized in 160 ml of hypotonic buffer (30 mM NaHCO 3 , 1 mM PMSF, pH 7.1) and a 100,000 g supernatant was obtained. Solid ammonium sulfate was added to bring the solution to 50% saturation. This was centrifuged at 14,000 rpm for 30 min. The precipitate was discarded and the supernatant was subjected to subsequent fractionation at 80% ammonium sulphate. After centrifugation at 14,000 rpm for 30 min the precipitate was solubilized in PBS containing 2 mM Ca 2+ and 2 mM Mg 2+ . This was applied to a Con A–Sepharose column ( Pharmacia ). The Con A–bound proteins were used for purification of gp96 as previously described ( 2 ). The buffer of Con A unbound material was changed to 25 mM Na citrate buffer, pH 5.3, by PD-10 column (Sephadex G-25; Pharmacia Biotech .). It was then applied to the CM-Sephadex C-50 column. The buffer of unbound of CM-Sephadex was then changed to 19 mM Na-phosphate buffer, pH 6.1 by PD-10 columns. It was then applied to the DEAE-sephacel column and eluted with a linear gradient of 0.15–0.5 M NaCl in 20 mM Na-phosphate buffer. The CRT-containing fractions were identified by immunoblots. Section title: Tumor Rejection Assay. Educational score: 4.031946182250977 Domain: biomedical Document type: Study Language: en 6-wk-old female BALB/cJ mice were immunized subcutaneously with gp96 or CRT or PBS in a 200 μl vol twice at weekly intervals. Mice were challenged intradermally 1 wk after last immunization with 100,000 live tumor cells. Tumor diameter was measured every 2–3 d. Section title: Complexing In Vitro of Peptide to gp96 or CRT. Educational score: 4.099247455596924 Domain: biomedical Document type: Study Language: en Gp96 or CRT was mixed with vesicular stomatitis virus (VSV)19 peptide in a 50:1 peptide/protein molar ratio in 0.7 M NaCl in Na-phosphate buffer (for gp96) or 20 mM Na-phosphate buffer (for CRT) and heated at 50°C for 10 min, then incubated at room temperature for 30 min. Excess free peptide was removed with PBS using microcon 10 (Amicon, Inc.). Section title: Induction of Cytotoxic T Cells. Educational score: 4.091368675231934 Domain: biomedical Document type: Study Language: en C57BL/6 mice were immunized intraperitoneally with 50 μg of gp96 or 27.5 μg of CRT complexed with VSV19 peptide. 10 d later, recipient spleens were removed and splenocytes were stimulated with VSV8 synthetic peptide at 1 μM concentration. After 5 d, mixed lymphocyte tumor cultures were tested for cytotoxicity in a Cr-release assay using EL4 cells alone and EL4 cells pulsed with VSV8 peptide as targets. Section title: Tumor-specific Immunogenicity of Tumor-derived gp96 and CRT Preparations. Educational score: 4.226128101348877 Domain: biomedical Document type: Study Language: en Apparently homogenous preparations of gp96 and CRT were obtained from Meth A cells. The two proteins were recognized by the respective antibodies on immunoblots . As CRT has a mol wt very similar to two other chaperones, PDI ( 18 ) and Erp57 ( 28 ), CRT preparations were immunoblotted with antibodies to each, as described in Materials and Methods. Although the antibodies recognized the cognate proteins in control blots, neither antibody detected a corresponding protein in purified CRT preparations . BALB/cJ mice were immunized twice at weekly intervals with a 20-μg dose of purified gp96 or an equimolar dose (11 μg and a lower dose of 5.5 μg) of CRT and were challenged with 100,000 live Meth A cells 1 wk after the second immunization. Tumors grew progressively in all buffer-immunized mice, but mice immunized with the Meth A–gp96 or Meth A–CRT preparation were equally protected from tumor growth. The immunogenicity of CRT was dose dependent, as mice immunized with half the effective dose succumbed to tumor challenge. Furthermore, mice immunized with Meth A–CRT were found to be not resistant to challenge with the antigenically distinct BALB/cJ fibrosarcoma CMS5, indicating the tumor-specificity of immunogenicity of Meth A–CRT. In a further demonstration of specificity, immunization with homogenous preparations of gp96 or CRT from BALB/cJ mouse liver did not elicit immunity to challenge with Meth A sarcoma at any dose tested . Section title: Tumor-specific Immunogenicity of Tumor-derived gp96 and CRT Preparations. Educational score: 4.126684665679932 Domain: biomedical Document type: Study Language: en Because Con A chromatography was used in purification of both gp96 and CRT and because Con A is known to stimulate monocytes and T lymphocytes, the possibility that the immunogenicity of CRT and gp96 preparations observed here derives from a Con A contamination of our preparations should be considered. Three observations rule out this possibility: the specificity of immunogenicity of tumor-derived HSP preparations, as has been demonstrated repeatedly in several studies from our laboratory ( 1 , 2 ) and by other groups ( 11 ) argues against such a nonspecific effect. Furthermore, in a routine, periodic screening of gp96 preparations with anti–Con A antibodies over the years, such contamination has not been observed, nor has inclusion of α-methyl pyranoside in the immunizing preparations reduced the immunogenicity of HSP preparations (data not shown). Section title: Association of CRT Molecules with Peptides In Vivo and In Vitro. Educational score: 4.310247898101807 Domain: biomedical Document type: Study Language: en The immunogenicity of tumor-derived but not normal tissue–derived CRT is reminiscent of similar observations with the HSPs gp96, hsp90, and hsp70. In those instances, the immunogenicity was demonstrated to derive from HSP-associated peptides and not the HSPs per se. The possibility that CRT molecules are associated with peptides was considered. Because CRT is an ATP-binding protein ( 19 , 20 ), and as hsp70–ATP interaction has been shown to result in dissociation of peptides from hsp70 ( 6 ), the ability of ATP to dissociate peptides from CRT was tested. It was observed that treatment of homogeneous preparations of CRT with ATP did not dissociate peptides from CRT under conditions that led to dissociation of peptides from hsp70 . Interestingly, treatment of gp96 with ATP also did not result in elution of associated peptides, although gp96 is an ATP-binding protein ( 21 ). These observations must be considered with the caveat that ATP binding by CRT might require conditions other than those used for hsp70, hsp90, or gp96. Although this possibility is considered unlikely because of the conserved ATP-binding domains present in ATP-binding proteins ( 22 ), our observations do not rule it out. Section title: Association of CRT Molecules with Peptides In Vivo and In Vitro. Educational score: 4.281308174133301 Domain: biomedical Document type: Study Language: en The possibility that the peptides bound to CRT in vivo can be exchanged with exogenous peptides at high temperatures was tested. The procedure shown to be effective with other HSPs ( 13 ) was tested. CRT preparations, and gp96 preparations as a positive control, were incubated with radio-iodinated peptide VSV19 (NH 2 -Ser-Leu-Ser-Asp-Leu- Arg-Gly-Tyr-Val-Tyr-Gln-Gly-Leu -Ly s-Ser-Asn-Val-Ser-COOH which is extended on the NH 2 and COOH termini of K b -binding VSV nucleocapsid protein epitope VSV8, underlined) at 25°C, 37°C, or 50°C, followed by 30 min at room temperature. Equimolar quantities of each protein were used so that the molar ratio of peptide to protein was the same in each lane. The samples were analyzed by SDS-PAGE followed by Coomassie staining (to determine protein content) and by autoradiography (to determine the bound peptide) . It was observed that CRT associated with exogenous radiolabeled peptides in a temperature-dependent manner and that CRT–peptide complexes remained stable under conditions of SDS-PAGE. Quantitative analysis of bound peptides indicated that under the conditions used for peptide- exchange in vitro, ∼2% of the CRT molecules received exogenous peptides at 50°C, and at a lower temperature such as 37°C only half as many CRT molecules could associate with the exogenous peptide. When gp96 and CRT were used together, such that they had to compete for the same pool of peptides under the same conditions, gp96 was observed to bind peptides two to three times more efficiently than CRT. Section title: Generation of VSV-specific CTLs in Mice Immunized with CRT–peptide Complexes Reconstituted In Vitro. Educational score: 4.210916519165039 Domain: biomedical Document type: Study Language: en The ability of CRT to act as a CD8 + response-eliciting adjuvant was tested. The VSV19 peptide was complexed in vitro with either gp96 or CRT at 50°C for 10 min, followed by incubation at room temperature for 30 min. C57BL/6 mice were immunized with the reconstituted complexes (50 μg of gp96 or 27.5 μg of CRT complexed with VSV19), or VSV19 alone, or gp96 or CRT alone without complexing. Spleen cells of immunized mice were cultured with the VSV8 and tested for cytotoxic activity on 51 Cr-labeled EL4 cells or EL4 pulsed with the VSV8 peptide as targets. It was observed that spleen cells of mice immunized with CRT– peptide complexes showed peptide-specific CTL activity, whereas splenocytes of mice immunized with peptide or CRT alone showed no cytotoxic activity . Similar results were obtained with gp96–peptide complexes. Based on the quantity of peptides bound to CRT, these results suggest that ∼20 ng of VSV19 or 5.7 × 10 12 molecules of VSV19 coupled to CRT are sufficient to elicit a CD8 + CTL response. The in vitro complexing studies were carried out with two variations: those where the CRT used for complexing with peptides was derived from syngeneic C57BL/6 mice and those where it was derived from allogeneic BALB/cJ mice. It was observed that CRT of either haplotype could be complexed to VSV19 and could be used to immunize C57BL/6 mice to elicit an H-2 b – restricted CTL response against VSV8 . Section title: Discussion Educational score: 4.301745414733887 Domain: biomedical Document type: Study Language: en CRT is a calcium-binding protein of the ER and has been shown to be involved in the folding and assembly of MHC I molecules. Recent observations by Spee and Neefjes ( 18 ) demonstrate that peptides transported into the lumen of the ER by the TAP molecule associate with gp96 as well as with PDI and CRT. That study showed the general ability of CRT to bind to proteins as well as to glycosylated peptides of specific sequences inside lumen of the ER. Association of CRT with peptides as well as the evidence of CRT to be associated with MHC I molecule during its folding and assembly ( 23 – 25 ) makes CRT a potential candidate of the relay line of HSPs during antigen processing and presentation by MHC I molecules, as we had suggested previously ( 17 ). Section title: Discussion Educational score: 4.306816577911377 Domain: biomedical Document type: Study Language: en In our studies, CRT and gp96 bind peptide with comparable efficiency in vitro, when each is tested alone. In a competition assay, gp96 binds to the longer peptide more efficiently than CRT, but when gp96 is not present in the assay, CRT binds to the peptide as efficiently as gp96. Although Spee and Neefjes ( 18 ) demonstrated that only glycosylated peptides are accepted by CRT in their assay, the peptide complexed in vitro in the present study is unglycosylated. This suggests either that the requirement for glycosylation of peptides for their acceptance by CRT in the lumen of the ER is not absolute, or that such apparent requirement is a peculiarity of the assay used in the study of Spee and Neefjes ( 18 ). The possibility should also be entertained that although CRT has the capacity to bind both glycosylated and unglycosylated peptides, only glycosylated peptides are routed to it in vivo, due to the intricacies of the peptide trafficking from the cytosol to the ER. The fact that calnexin, a protein related to CRT, is a chaperone for a wide array of glycosylated integral membrane protein, and has been defined as a lectin ( 26 ), is consistent with this possibility. This line of reasoning also raises the question of whether calnexin, as an integral membrane protein or in a soluble luminal form, has the capacity for binding peptides in vivo and the capacity to immunize. Section title: Discussion Educational score: 4.134538650512695 Domain: biomedical Document type: Study Language: en The mechanism by which CRT–peptide complex elicits immunity is presently unknown. It is conceivable that it is similar to the mechanism by which gp96–peptide complexes elicit immunity, which includes a receptor through which gp96 is taken up by the professional APCs and the peptides are re-presented. A crucial question in this regard is whether this is a common receptor for the HSPs or whether the mechanisms are different. This is particularly intriguing since gp96, hsp70, or CRT molecules have no structural homologies. Section title: Discussion Educational score: 4.334023475646973 Domain: biomedical Document type: Study Language: en Binding of peptides by CRT provides evidence of the generality of the peptide-binding property of HSPs and of the immunogenicity of such HSP–peptide complexes. From an evolutionary perspective, considering the broad chaperoning function of HSPs and the association of HSPs with the degradative machinery of cells, this is not surprising. However, it is interesting to note that the hsp70, hsp90, gp96 and CRT molecules do not share any obvious sequences or other structural homology. The possibility that their peptide binding pockets may share conformational similarities despite the lack of structural homology, clearly exists and will be instructive with regard to evolution of peptide-binding proteins including the MHC molecules ( 27 ). Our previous observation that complexes of non-HSPs, such as serum albumin with peptides, do not elicit peptide-specific CTLs ( 13 ) suggest that the immunogenicity does not result solely and inevitably from peptide binding and that the immunogenicity of HSP–peptide complexes is unique, deriving perhaps from specific interaction between HSPs and APCs ( 16 ). | Study | biomedical | en | 0.999997 |
10049944 | Section title: Mice. Educational score: 4.062753677368164 Domain: biomedical Document type: Study Language: en BALB/c mice were purchased from Bomholtgaard Breeding & Research Centre, Denmark. IL-4-deficient mice (IL-4 −/− ) were generated by targeted disruption of the IL-4 gene in embryonic stem cells derived from BALB/c mice ( 26 ) and produced in a manner such that they can be considered IL-4–congenic to BALB/c. The deficiency of the IL-4 gene was confirmed by PCR of tail DNA as previously described ( 27 ). All mice used in the experiments were 6–12-wk-old and were sex matched. Heterozygous control littermates were also included in some vaccination experiments and showed no significant differences in tumor rejection compared with BALB/c mice that were commercially obtained (data not shown). Section title: Cell Lines. Educational score: 4.163398742675781 Domain: biomedical Document type: Study Language: en TS/A is a spontaneous mammary adenocarcinoma cell line ( 28 ) and CT-26 is an N -nitroso- N -methylurethane–induced colon carcinoma ( 29 ), both syngeneic to BALB/c. Both tumor cell lines express MHC class I but not MHC class II molecules (data not shown). NIH-3T3-IL-4 cells were generated by transfection of NIH-3T3 with an expression vector encoding the cDNA coding region of murine IL-4. 10 6 NIH-3T3-IL-4 cells produce 130 U/ml within 24 h of culture (data not shown). Biological activity of IL-4 was determined by a proliferation assay with the IL-4–dependent cell line CT.4S ( 30 ). 1 U of IL-4 was defined as the amount of IL-4 required to obtain half-maximal proliferation. YAC-1 is an MHC-deficient NK cell target cell line ( 31 ). All cell lines were cultured in RPMI-1640 with 10% FCS. Section title: Tumor Vaccination Experiments. Educational score: 4.0763397216796875 Domain: biomedical Document type: Study Language: en For vaccination experiments, cells of the indicated lines were washed twice in Dulbecco's PBS, irradiated (100 Gy), and injected subcutaneously in a vol of 0.2 ml Dulbecco's PBS in the middle of the left flank. 2 wk after immunization, viable tumor cells were injected contralaterally. Mice bearing tumors of 1 cm in diameter were scored as tumor positive. Tumor incidence is expressed as percentage of tumor-free mice. Section title: Serum Ig Detection and Analysis of Tumor-specific IgG2a. Educational score: 4.114299774169922 Domain: biomedical Document type: Study Language: en Mice were immunized twice with 10 6 irradiated (100 Gy) CT-26 cells on days 0 and 21. Serum Ig levels before and 14 d after the second immunization were determined by ELISA. Relative serum levels of IgG1 and IgG2a were measured using the Ig isotyping kit according to the manufacturer's recommendation ( PharMingen , Germany). For IgE detection, a pair of commercially available mAbs and the appropriate mouse IgE standard were used (all from PharMingen ). For detection of tumor-reactive IgG2a, 10 6 CT-26 cells were incubated with sera (1:20 dilutions), rat anti– mouse IgG2a ( PharMingen ), and PE F(ab′) 2 goat anti–rat Ig (Immunotech, France), followed by flow cytometric analysis using Coulter EPICS-XL (Coulter Electronics GmbH, Germany). Fold above background was determined by dividing the median fluorescence of a stained sample by that of cells treated identically but without serum. Section title: Cellular Cytotoxicity. Educational score: 4.132266044616699 Domain: biomedical Document type: Study Language: en For the generation of CT-26–specific CTLs, mice were immunized twice with 10 6 irradiated (100 Gy) CT-26 cells on days 0 and 21. 14 d after the second injection, single cell suspensions of splenocytes were prepared and red blood cells were removed by NH 4 Cl treatment. Splenocytes were cultured at 2 × 10 6 /ml with irradiated (100 Gy) CT-26 cells at a responder/stimulator cell ratio of 30:1 in RPMI-1640 plus 10% FCS, penicillin/streptomycin, MEM, 2-ME (50 μM), and 50 U/ml IL-2 ( Boehringer Mannheim , Germany). After 5 d of culture, responder cells were harvested, washed twice, and incubated with 51 Cr (1 mCi/ml; NEN)-labeled CT-26 or YAC-1 cells at different E/T cell ratios. After an incubation period of 4.5 h, supernatants were assayed for radioactivity on a gamma counter (Top Count; Packard). The percentage of specific lysis was calculated as [(sample cpm − spontaneous cpm) / (maximal cpm − spontaneous cpm)] × 100%. Spontaneous release was <19%. Section title: IFN-γ Detection. Educational score: 4.117471218109131 Domain: biomedical Document type: Study Language: en For IFN-γ detection, mice were immunized as described above. Splenocytes of immunized mice were cultured in 24-well plates at 2 × 10 6 /ml in the presence of Con A (2.5 μg/ml) for 3 d and supernatants were collected. CD8 + T cells were depleted by negative selection with anti-CD8a (53-6.7), followed by separation using magnetic beads coated with sheep anti–rat IgG ( Dynal A.S., Norway). Depletion was checked by cytofluorimetric analysis using PE-conjugated anti-CD8a (53-6.7) ( PharMingen ). Culture supernatants were assayed for IFN-γ by ELISA using commercially available reagents (all from PharMingen ). The detection limit was 30 pg/ml. Section title: In Vivo Neutralization of IL-4. Educational score: 4.0354814529418945 Domain: biomedical Document type: Study Language: en Groups of IL-4 +/+ mice were immunized with 5 × 10 5 irradiated (100 Gy) TS/A cells and challenged 14 d later with 10 5 of viable TS/A cells. Treatment with the IL-4–neutralizing mAb 11B11 was done by intraperitoneal injections of 4 mg in 0.2 ml every 2 d from day −2 to 6 of immunization or challenge, respectively. Animals of the control group received Dulbecco's PBS. Section title: Induction of Tumor Immunity Is Impaired in IL-4 −/− Mice. Educational score: 4.299830913543701 Domain: biomedical Document type: Study Language: en The ability to induce tumor immunity with nonproliferating tumor cells can depend on several variables, one of which is the number of tumor cells. To titrate the number of immunizing cells capable of inducing tumor immunity against the mammary adenocarcinoma TS/A, IL-4 +/+ and IL-4 −/− mice were immunized with increasing numbers of irradiated cells. 2 wk later, the mice were challenged with a constant number of TS/A cells sufficient to give rise to tumors in 100% of naive mice . At the lowest immunization dose, 100% of IL-4 +/+ and IL-4 −/− mice developed tumors. With increasing vaccination doses, IL-4 +/+ mice rejected the challenge tumor, whereas IL-4 −/− mice were consistently less protected. These results were confirmed with a second tumor . 100% of challenge tumors grew in mice of both genotypes after immunization with the lowest cell number of CT-26. Increasing immunization doses induced protection in the majority of IL-4 +/+ mice, whereas almost all IL-4 −/− mice were unable to reject the challenge tumor. The defect of IL-4 −/− mice to reject the challenge tumor was not due to generally enhanced tumor growth, because TS/A and CT-26 cells grew with similar kinetics in naive IL-4 +/+ and IL-4 −/− mice . Therefore, IL-4 is required for the generation of protective tumor immunity. Section title: IL-4 −/− Mice Have a Defective CTL Response. Educational score: 4.153644561767578 Domain: biomedical Document type: Study Language: en Immunization with CT-26 induces tumor-reactive CTLs that are able to confer protection against challenge tumors ( 32 ). To find whether the defective tumor immunity in IL-4 −/− mice was associated with reduced CTL activities, IL-4 +/+ and IL-4 −/− mice were immunized with CT-26 cells and tumor-specific lysis was measured. CTL activity of IL-4 −/− splenocytes was undetectable, whereas splenocytes of IL-4 +/+ mice contained substantial CTL activity . Cytolytic activity against the NK target YAC-1 was negligible in spleen cells from both mouse strains, suggesting that lysis of CT-26 by IL-4 +/+ CTLs was specific. Additionally, immunization with β-galactosidase–expressing TS/A cells resulted in clearly reduced β-galactosidase-specific CTL-activity in IL-4 −/− mice (data not shown). Section title: Tumor Immunity in IL-4 +/+ Mice Is Associated with a Th1 Response. Educational score: 4.262599468231201 Domain: biomedical Document type: Study Language: en Changes in serum Ig isotype levels are an indication for ongoing Th1 or Th2 responses in vivo. We have shown that immunity to TS/A cells requires CD4 + T cells to be present during the priming phase ( 8 ). Similarly, immunization with recombinant vaccinia virus encoding β-galactosidase elicited maximal therapeutic effects to CT-26–β-galactosidase cells through the involvement of CD4 + T cells ( 33 ). Therefore, we analyzed total serum levels of different Ig isotypes before and after immunization of IL-4 +/+ mice with CT-26 to evaluate if tumor immunity was associated with a dominant cytokine response . Amounts of IgE and IgG1 remained largely unaltered, whereas IgG2a was significantly increased. To detect IgG2a antibodies reacting with tumor cells, CT-26 cells were stained with the same sera and the binding efficiency was measured by FACS ® analysis. As shown in Fig. 4 b, sera of immunized mice showed, to varying extents, elevated amounts of tumor-reactive IgG2a compared with sera of naive mice indicating IFN-γ production in response to CT-26 cells. These data show that the immunization of IL-4 +/+ mice with a sufficient amount of CT-26 cells initiated a typical Th1-associated response. Section title: IL-4 −/− Mice Fail to Generate a Th1-associated Antitumor Response. Educational score: 4.251881122589111 Domain: biomedical Document type: Study Language: en Next, Ig levels in IL-4 −/− mice before and after injection of irradiated CT-26 cells were measured to evaluate if impaired T helper cell responses might account for reduced tumor immunity in IL-4 −/− mice . As reported previously, IgE is undetectable in naive IL-4 −/− BALB/c mice ( 27 ) and was not induced by the injection of CT-26. The amount of IgG1 was not significantly altered in response to CT-26. Surprisingly, serum concentrations of IgG2a were only slightly enhanced after vaccination, although basal levels of IgG2a were elevated in comparison to IL-4 +/+ mice. These results indicate a reduced IFN-γ production upon immunization in the absence of endogenous IL-4 . To confirm this assumption, we cultured splenocytes of immunized IL-4 +/+ and IL-4 −/− mice in the presence of Con A and measured IFN-γ in culture supernatants . Spleen cells of IL-4 −/− mice produced twofold lower amounts of IFN-γ than those of IL-4 +/+ mice. Similar results were obtained with CD8 + -depleted spleen cells. These data are in accordance with the measurements of serum Ig isotypes, again suggesting that endogenous IL-4 is required for the induction of a Th1 antitumor immune response. Section title: The Establishment of Tumor Immunity Requires IL-4 Production during the Priming Phase. Educational score: 4.161075592041016 Domain: biomedical Document type: Study Language: en IL-4 is produced by naive CD4 + T cells under Th1 polarizing conditions in vitro within the first 24 h of antigen contact ( 34 ). Therefore, we questioned at which time IL-4 was required for the generation of tumor immunity. IL-4 was neutralized in IL-4 +/+ mice by anti–IL-4 mAb during either the immunization or the challenge period. After immunization and challenge with TS/A cells, 50% of control mice but none of the animals treated with the anti–IL-4 mAb during the immunization phase rejected the challenge tumor . IL-4 neutralization during the challenge phase did not impair tumor rejection. In fact, fewer mice developed a challenge tumor, the significance of which is not clear. These data indicate that memory effector cells do not require IL-4 to exert their tumoricidal activity, although their development is dependent on early IL-4 production. Section title: Exogenous IL-4 Restores Tumor Immunity in IL-4 −/− Mice. Educational score: 4.244548797607422 Domain: biomedical Document type: Study Language: en The data presented thus far do not clearly exclude that unknown developmental defects in IL-4 −/− mice, such as reduced precursor frequencies of potential tumor reactive lymphocytes, rather than the absence of IL-4 during the priming phase, diminished vaccination efficiencies. To address this, IL-4 −/− mice were immunized with different doses of CT-26 cells mixed with 10 6 IL-4–secreting NIH-3T3-IL-4 cells. As controls, IL-4 −/− mice were injected with identical doses of CT-26 and parental, non-IL-4-secreting NIH-3T3. 2 wk later, mice were challenged with CT-26 cells and tumor incidence was measured. Depending on the number of CT-26 cells used for immunization, 40–70% of mice developed protective tumor immunity after immunization in the presence of exogenous IL-4, whereas none of the control animals was protected. These results show that tumor immunity can be induced in IL-4 −/− mice, as long as IL-4 is provided during the immunization phase. As demonstrated for IL-4 +/+ mice , protection efficiency in IL-4 −/− mice could also be modulated by the amount of antigen used for immunization. Therefore, these experiments suggest that IL-4 −/− lymphocytes are functionally intact and emphasize the necessity of IL-4 during the T cell priming stage for the induction of tumor immunity. Section title: Discussion Educational score: 4.661985397338867 Domain: biomedical Document type: Study Language: en We have shown that IL-4 −/− mice have a severe defect in the development of tumor immunity. Furthermore, the absence of tumor immunity in IL-4 −/− mice was associated with the absence of a Th1 response to the tumor as shown by reduced serum IgG2a levels and IFN-γ production. In addition, we showed that IL-4 was required for the induction but not for the maintenance of tumor immunity. To understand this unexpected role of IL-4, the sequence of events leading to tumor immunity needs to be considered. Antigens derived from the immunizing tumor cells which are MHC class I + /II − must be taken up and processed by host APCs and presented by MHC class I and class II molecules to CD8 + and CD4 + T cells, respectively. Because CD4 + T cells are critical for the development of tumor immunity, the initial event is the activation of CD4 + T cells, which provide help for activation of CD8 + CTLs ( 3 , 8 ). Like IL-4, CD4 + T cells are necessary in the priming phase. In the TS/A model, we have shown previously that once CTLs have been generated, CD4 + T cell depletion (at the time of challenge) did not impair CTL mediated tumor rejection ( 8 ). Therefore, it is likely that the defective CTL response in IL-4 −/− mice is the result of defective help by Th1 cells. This help has been explained previously by providing cytokines to CTLs, probably Th1 cytokines like IL-2 or IFN-γ ( 17 ). Recently, however, it has been shown that CD4 + T cell help for CTL activation does not consist of a direct interaction between CD4 + and CD8 + T cells but rather by activation of APCs, perhaps dendritic cells ( 13 – 15 ). The cross-talk between CD4 + T cells and APCs is mediated by CD40–CD40 ligand interaction and T cell help for CTL generation can be replaced by in vivo application of an agonistic antibody that acts on APCs other than B cells ( 15 ). This mechanism not only obviates the need of CD4 + and CD8 + T cells to meet each other at the same APC but also the need of Th1 cytokines for CTL generation. It is possible that a similar mechanism is operative in our tumor models, a possibility that is supported by the finding that IL-4 −/− mice can mount an undiminished CTL response to some viral antigens ( 35 ), which at least partially occurs independently of CD4 + T cell help ( 36 , 37 ). Th1-independent CTL responses to viral antigens correlate with the observation that viruses can directly infect dendritic cells. Therefore, at the present time we cannot distinguish whether IL-4 (which is produced for a short period under Th1-polarizing conditions, see below) acts on the APC or the CD4 + T cells, or directly contributes to CTL generation during priming. Section title: Discussion Educational score: 4.526989936828613 Domain: biomedical Document type: Study Language: en Although our results apparently contradict the Th1/Th2 paradigm, they do not if the time of IL-4 activity that is required for development of a Th1 response is considered. The inhibitory effect of IL-4 on Th1 development has been most clearly observed in vitro under conditions of prolonged addition of exogenous IL-4 or in vivo under conditions of chronic antigen exposure ( 18 ). Our results do not exclude the possibility that IL-4 has opposing effects in later stages of an antitumor response, e.g., in tumor-bearing animals. However, the fact that the development of a Th1 response and concomitant tumor immunity requires IL-4 at the time of immunization only (when T cell priming should happen) is reminiscent to the finding that CD4 + T cells cultured under Th1-polarizing conditions express IL-4 for the first 24 h ( 34 ). Also, the artificial expression of IL-4, either by tumor cells transfected to secrete IL-4 or in a transgenic mouse model, leads to induction of IFN-γ in vivo, in both T cell–competent and –deficient mice ( 23 , 38 ). The importance of IFN-γ for the development of T cell–dependent tumor immunity ( 39 ) and for tumor growth inhibition of IL-4–secreting tumors has been demonstrated ( 38 ). Recently, it has been shown that IL-4 −/− mice are impaired to resolve Candida albicans in the late stage of infection, which has been explained by the missing IL-4 to induce neutrophils for IL-12 production and subsequent Th1 development ( 40 ). The fact that the induction of an antitumor response induced by tumor cell–produced IL-4 is critically dependent on neutrophils ( 41 ) raises the possibility that a similar mechanism is operating during the generation of tumor immunity, although CTLs to C . albicans had not been measured. Section title: Discussion Educational score: 4.519998550415039 Domain: biomedical Document type: Study Language: en The immunogenicity of tumor cells depends on a number of factors related to the host, e.g., the inhibitory effect of B cells ( 8 ), or to the tumor, e.g., the expression of immunosuppressive cytokines ( 42 ) or the amount of tumor cells used for immunization . The failure to induce tumor immunity with low numbers of tumor cells could mean that it is simply the limiting amount of tumor- derived antigens available for the appropriate APCs, yet IL-4 is produced in a manner unrelated to the immunizing tumor cells ( 43 ), or that a certain number of tumor cells is required to induce IL-4. We favor the latter assumption, because Bosco et al. showed in the TS/A tumor model that in mice challenged with a low number of viable tumor cells which did not lead to a measurable antitumor response that tumor immunity could be induced by IL-4 injection around the tumor-draining lymph node where T cell priming probably occurs ( 44 ). IL-4 was injected for a period of 10 d, which is comparable to the estimated time of IL-4 production by the NIH-3T3-IL-4 cells, which when added to the immunizing tumor cells restored tumor immunity in IL-4 −/− mice . It is important to note that very low amounts of recombinant IL-4 (0.1 pg/d) were most effective ( 44 ), showing that not only the time point but also the amount of IL-4 required for induction of tumor immunity is critical. We also show that the lack of IL-4 is not compensated for by other cytokines such as IL-13 ( 45 , 46 ) for the generation of tumor immunity. Our results are surprising because a number of studies in other experimental models with IL-4 −/− mice showed that IL-4 was not necessary for a normal immune response, e.g., against parasites ( 47 ), viruses ( 35 ), or even diminished immune reactions, e.g., in retrovirus-induced immunodeficiency syndrome (MAIDS; reference 48 ). Whether this difference is due to Th-dependent CTL generation often required for tumor immunity remains to be established. In conclusion, our study reveals a novel role of IL-4 for the generation of tumor immunity that contributes to a better mechanistic understanding as a rationale for immunotherapy. | Study | biomedical | en | 0.999998 |
10049945 | Section title: Experimental Animals and Cell Lines. Educational score: 3.9640896320343018 Domain: biomedical Document type: Study Language: en BALB/cByJ, CByD2F1, C3H/J, and C57BL/6j mice were purchased from The Jackson Laboratory . Nude mice (nu/nu) were obtained from The National Cancer Institute. 6–18-wk-old male mice were used for the experiments. Plasmacytoma J558 was cultured in RPMI 1640 containing 5% FCS. J558 cells transfected with either pSRα (J558-Neo) or pSRα–πlnB7-1 (J558-B7) have been described ( 34 ). Anti-P1A CTL line P1CTL ( 35 ) was provided by Dr. Lieping Chen (Mayo Clinic, Rochester, MN). Section title: Analysis of P1A Expression by RT-PCR. Educational score: 4.209502220153809 Domain: biomedical Document type: Study Language: en Total RNA was isolated from testis, liver, lung, spleen, and thymus of normal BALB/c mice as well as J558 tumor cells. The first strand DNA was prepared using GAATTCTCGAGTCTAGATTTTTTTTTTTTTTTTTTT as a primer. In brief, the reaction mixture consisted of: 30 μl of total RNA (5 μg), 10 μl 5× first strand buffer, 3 μl 10 mM dNTP, 2 μl primer (100 μM), 2.5 μl H 2 O, and 2.5 μl RT ( GIBCO BRL ). Reverse transcription reactions were carried out at 42°C for 1 h. The RT was then inactivated by heating. The first strand DNA was denatured at 96°C for 1 min, and the P1A cDNA was then amplified by 40 cycles of PCR. Each cycle consisted of: 94°C for 1.5 min, 55°C for 1.5 min, and 72°C for 2 min. The following primers were used. P1A: TGGCCTGGATAGCCAGGCAAAGCAAGCGCA as forward primer and CTTCCGCTGTCCATTTCTTTCTTCTGGGCT as reverse primer; glyceraldehyde 3-phosphate dehydrogenase (GAPDH): ATGGTGAAGGTCGGTGTGAACGGATTTGGC as forward primer and CATCGAAGGAAGAGTGGGAGTTGCTGT as reverse primer. The P1A primers used should amplify a 500-bp product from properly spliced P1A RNA. PCR product from genomic DNA should be 3.1 kb. The size of amplifed GAPDH cDNA should be 900 bp. The PCR products were separated on agarose gels and then transferred onto nylon membrane as described ( 36 ). P1A or GAPDH products were detected by 32 P- labeled specific probes. Section title: Cytotoxic T Cell Assays. Educational score: 4.115468978881836 Domain: biomedical Document type: Study Language: en Cytotoxic T cell line P1CTL ( 35 ) specific for P1A35–43:L d were used as effector cells and BALB/c spleen cells that had been stimulated with LPS (10 μg/ml), Con A (2 μg/ml), or anti-CD3 (0.25 μg/ml) for 48 h were used as target cells. As control, P388D1 (H-2 d ) pulsed with 1 μg/ml of P1A or control peptides were also used as targets. The lysis of target cells was determined by release of 51 Cr after 6 h of coincubation with P1CTL. Data presented were specific lysis of target cells calculated according to the following formula: Specific lysis % = (cpm sample − cpm medium)/(cpm max − cpm medium) × 100. In other experiments, transgenic spleen cells were stimulated in vitro for 3 d with 1 μg/ml of P1A peptide and used as effectors. Section title: Production of P1A Transgenic Mice. Educational score: 4.1568403244018555 Domain: biomedical Document type: Study Language: en We designed synthetic oligonucleotide primers corresponding to the ends of the P1A–open reading frame; the forward primer contained a HindIII restriction site and the reverse primer a ClaI site. These primers were used to amplify the P1A–open reading frame from pBS–P1A. We used the restriction sites at the end of the amplified fragment to clone it into the E μ mb–Id2 vector, a gift from Dr. Xiao-Hong Sun (New York University Medical Center, New York) which contains the IgM heavy chain enhancer (E μ ), the mb-1 -promoter, an intron from the small T antigen gene, and an SV40 polyadenylation signal ( 37 ). The construct was linearized by digestion with NotI, and the fragment containing the P1A sequence was purified and used for microinjection into fertilized eggs of (B6 × D2)F1 origin. Section title: Transgenic Mice Expressing TCR from a P1A-specific CTL Line. Educational score: 4.182103633880615 Domain: biomedical Document type: Study Language: en Identification and cloning of the anti-P1A TCR and production of transgenic mice follow the basic procedure established by Kouskoff et al. ( 38 ). In brief, we cloned the VJ fragment of the TCR α chain, and DNA sequencing confirmed that the CTL line utilized Vα8 and J10. We used linked PCR to generate a PCR fragment containing the full length Vα8 and J10 as well as a small leading intronic sequence to ensure proper splicing of the TCR mRNA. This fragment was then inserted into a PTR-α vector provided by Dr. Mathis (Institut de Génétique et de Biologie et Cellulaire, Strasburg, France; 38). Using published primer sequences, we found that P1CTL express only Vβ1, a 10-bp D segment, Jβ2, and Cβ1. Transgenic vectors were produced as described ( 38 ). After removing the prokaryote DNA sequences, the transgenic vectors were injected into fertilized eggs from Swiss Webster (SW) mice. Section title: Proliferation of Transgenic T Cells. Educational score: 4.138850212097168 Domain: biomedical Document type: Study Language: en To test whether antigen-presenting spleen cells express P1A antigenic epitope, spleen mononuclear cells from BALB/c mice were depleted of T cells by two rounds of treatment with anti-Thy1.2 mAb plus complement. The T-depleted APC were cultured with either LPS (10 μg/ml) or medium alone for 16 h; after five washes with PBS, the viable spleen cells were irradiated for 2,000 rads and used as stimulator cells (2 × 10 5 /well). T cells (6 × 10 4 /well) purified from transgenic mice as described ( 36 ) were used as responders. After 66 h, the cultures were pulsed with 3 H-TdR (1.25 μCi/ well) for 6 h. To test antigen specificity of the transgenic T cells, we added varying concentrations of P1A peptide or control NP peptide to 2 × 10 5 /well of transgenic or nontransgenic spleen cells for 42 h and pulsed with 3 H-TdR. Section title: Tumorigenesis Assay. Educational score: 4.096177101135254 Domain: biomedical Document type: Study Language: en Because the tumor cell lines used in this study originated from BALB/c mice, we used mice that carried at least one copy of the BALB/cByJ gene in all in vivo protection experiments to avoid immune responses against minor histocompatibility antigens. In brief, transgenic founder SW mice were crossed with BALB/cByJ mice for three generations, and the N2 mice were typed by flow cytometry for expression of the transgenic TCR-α chain using PE-conjugated anti-Vα8 mAb. Transgenic mice and their littermate controls were injected with 5 × 10 6 J558-B7 and J558-Neo subcutaneously in the left inguen. In other experiments, both types of tumor cells were injected into the same mice but on different sides. The tumor incidence was recorded every other day. Palpable tumors with a diameter of >3 mm were scored as positive. Section title: Tumor Antigen P1A Is Expressed at Low Levels in Normal Tissue. Educational score: 4.224966049194336 Domain: biomedical Document type: Study Language: en To determine if the P1A gene is expressed in normal tissues, we isolated RNA from a number of normal mouse organs, including spleen, thymus, liver, lung, and testis, and measured P1A mRNA by both Northern blot and RT-PCR. Northern blot analysis did not reproducibly detect significant levels of P1A RNA expression in most tissues except in the testis (data not shown), which is consistent with an earlier study by Uyttenhove et al. ( 24 ). We therefore used the more sensitive method of RT-PCR to detect the expression of P1A RNA. The PCR products were detected by Southern blot using 32 P-labeled probes. This procedure not only ensured the identity of the products amplified but also enhanced the sensitivity of the assay. The forward and reverse primers are in separate exons, and the products amplified from genomic DNA should be 3.1 kb, whereas those amplified from mRNA should be 500 bp. As shown in Fig. 1 a, a significant amount of P1A RNA was detected in most tissues by RT-PCR. The mRNA level was highest in the testis as has been reported ( 24 ). Lung and spleen tissues expressed more P1A RNA than liver tissue. Although P1A RNA can be detected in the thymus in some experiments, its thymic level was always at least 10-fold lower than in the spleen (data not shown). To compare the levels of P1A mRNA in spleen and tumor cells, we titrated the amount of cDNA prepared from either spleen or J558 tumor cells. As shown in Fig. 1 b, the relative abundance of the P1A cDNA in the spleen was ∼100– 1,000-fold lower than levels found in the tumor cells. Section title: Tumor Antigen P1A Is Expressed at Low Levels in Normal Tissue. Educational score: 4.141960144042969 Domain: biomedical Document type: Study Language: en We used a P1A:L d -specific CTL line, P1CTL, to determine if the P1A35–43:L d epitope is presented on the normal lymphoid cells. The P1CTL was generated after immunization with P815 cells and has been restimulated in vitro for almost two years ( 35 ). All cells in the P1CTL line express the Vα8 chain on cell surface (data not shown). As shown in Fig. 2 a, P1A-specific CTL failed to kill syngeneic spleen cells that had been activated by either LPS, Con A, or anti-CD3 mAb. As a result we failed to detect P1A antigenic epitope on activated T and B cells by CTL assay. However, since direct cytolysis requires antigen expression on a substantial proportion of target cells, this assay cannot rule out the possibility that a small proportion of cells may express the antigen. Section title: Tumor Antigen P1A Is Expressed at Low Levels in Normal Tissue. Educational score: 4.119882583618164 Domain: biomedical Document type: Study Language: en We have recently produced transgenic mice that express the TCR from P1CTL. We therefore used proliferation assay to determine if antigen-presenting spleen cells from syngeneic mice can induce activation of P1A-specific T cells. As shown in Fig. 2 b, unstimulated spleen APC failed to induce proliferation of transgenic T cells. However, after stimulation with LPS, weak but significant proliferation of transgenic T cells could be detected. Thus, activated APC can present P1A epitope. The relatively weak proliferation could be due to either low frequency of APC that present P1A epitope or a low level of P1A epitope expression on all APC. Section title: Normal Development and Induction of P1A-specific CTL. Educational score: 4.342158794403076 Domain: biomedical Document type: Study Language: en Given the fact that the P1A gene is expressed in hematopoietic cells, it is possible that endogenous expression of the P1A gene may cause either clonal deletion or clonal anergy of T cells. To analyze the development of P1A-specific T cells, we cloned the T cell receptor from a P1A-specific CTL line, P1A CTL. Using the cassette vector provided by Kouskoff et al. ( 38 ), we produced mice with integration of both α and β chains. Five independent founders were obtained that have cointegration of α and β chains, three of which are shown in Fig. 3 a. After breeding with BALB/c mice, all F1 mice that carried the transgenes expressed Vα chain on the surface of >50% of PBL . Lacking a mAb specific for Vβ1, we were unable to directly measure expression of the β chain; however, expression of the β chains to which mAbs are available has been excluded, presumably due to transgenic expression of Vβ1 (data not shown). To determine whether endogenous expression of P1A impairs development of P1A-specific T cells, we analyzed the cellular composition of T cells in the thymus. As shown in Fig. 3 c, the transgenic mice contain a large proportion of CD4 + CD8 + T cells and CD8 + CD4 − T cells. A skewing toward CD8 + CD4 − subset is consistent with the fact that transgenic TCR is restricted by MHC class I ( 39 , 40 ). Moreover, essentially all CD8 + CD4 − T cells in the thymus express a high level of Vα8 on the surface. These results demonstrate that P1A-specific CD8 + T cells are not subject to clonal deletion in normal mice. Section title: Normal Development and Induction of P1A-specific CTL. Educational score: 4.15809440612793 Domain: biomedical Document type: Study Language: en To test whether endogenous P1A chronically stimulates P1A-specific T cells, we measured expression of L-selectin on T cells from the TCR-transgenic mice. L-selectin is expressed at a high level in naive T cells and is downregulated upon T cell activation ( 41 , 42 ). Because antigen-experienced T cells remain L-selectin low for a long period, this phenotype has been used as a marker for antigenic exposure of T cells. As shown in Fig. 3 d, spleen CD8 T cells that expressed the transgenic α chain are of L-selectin high phenotype. In contrast, in the same spleen, a substantial proportion of Vα8 − CD8 T cells is of L-selectin low phenotype. These results demonstrated that the transgenic T cells were not activated by the endogenous P1A antigen. This conclusion is consistent with the fact that in vitro syngeneic APC cannot stimulate transgenic T cells without LPS activation . Section title: Normal Development and Induction of P1A-specific CTL. Educational score: 4.240264892578125 Domain: biomedical Document type: Study Language: en To test whether the P1A-specific T cells are anergic, we stimulated the spleen cells from P1A-transgenic mice and their littermate controls with either P1A peptide or a control peptide from influenza virus. As shown in Fig. 4 a, spleen cells from P1A-transgenic mice mounted a vigorous, proliferative response to a low dose of P1A peptide. Maximal proliferation of T cells occurred with a dose of 10 ng/ml of peptide, indicating that the transgenic TCR must have high avidity for the tumor antigens. Moreover, after in vitro restimulation, transgenic T cells exhibited strong cytotoxic activity against P1A-expressing plasmacytoma . Interestingly, B7-1 + tumor cells are more susceptible than their B7-1 − counterparts, which is consistent with our previous observation of ex vivo, P1A-reactive CTL ( 34 ). Since J558-B7 and J558-Neo expressed comparable amounts of MHC class I ( 34 ) and P1A antigen ( 20 ), it is likely that after 4 d of in vitro stimulation, the effector function of transgenic T cells is dependent on B7 presence on the target cells. Section title: Clonal Deletion of P1A-specific T Cells in Transgenic Mice Overexpressing P1A Gene in the Thymus. Educational score: 4.1744585037231445 Domain: biomedical Document type: Study Language: en The lack of clonal deletion and anergy of P1A-specific T cells is most likely due to low expression of the P1A gene in hematopoietic cells. We produced transgenic mice in which the expression of the P1A gene is under the control of mb-1 promoter and Eμ enhancer. We prepared cDNA from total thymic RNA and compared the amount of P1A mRNA in normal versus transgenic thymus by titration of cDNA. P1A mRNA was detectable in the thymi of transgenic mice after the cDNA reached 1:270 dilution, whereas 1:10 dilution of cDNA from nontransgenic littermates failed to yield any P1A product. Thus, P1A transgenic mice significantly overexpressed P1A. In fact, the levels of P1A mRNA in transgenic mice are comparable to those in J558 tumor cells . Section title: Clonal Deletion of P1A-specific T Cells in Transgenic Mice Overexpressing P1A Gene in the Thymus. Educational score: 4.348528861999512 Domain: biomedical Document type: Study Language: en We bred the TCR-transgenic mouse (TG) with the P1A-TG to generate TCR/P1A–TG and analyzed the cell numbers and cell surface markers of TCR-TG and TCR/ P1A-TG thymi . The total number of TCR/ P1A–TG thymocytes was ∼40% of that of TCR-TG (data not shown). In addition, a discrete population of CD8 + CD4 − T cells in the TCR–TG is replaced by a population of cells that has downregulated both CD4 and CD8 coreceptors. This progressive downregulation of CD8 receptors is more obvious among the thymocytes that have expressed the highest level of transgenic TCR. Moreover, the relative number of CD4 + CD8 + T cells is greatly reduced, and, as a result, an increase in the percentage of CD4 − CD8 − T cells is observed. These results demonstrate that P1A-specific T cells are being deleted in the TCR/P1A–TG. Consequently, the CD8 + Vα8 + T cells are reduced by >80% in the TCR/P1A–TG compared to TCR-TG. Thus, transgenic T cells specific for unmutated tumor antigen are susceptible to clonal deletion in the thymus. Under normal circumstances, these T cells must have escaped clonal deletion because of low or no P1A expression in the thymus. Section title: The Effector Function of P1A-specific CTL Is Restrained In Vivo. Educational score: 4.1257524490356445 Domain: biomedical Document type: Study Language: en To test whether the transgenic, P1A-specific cytotoxic T cells can efficiently reject the J558 tumor cells, we bred the transgenic founder mice with BALB/cByJ for three generations and tested the tumorigenicity of B7-transfected (J558-B7) or vector-transfected (J558-Neo) tumor cells in the N2 mice. Since all N2 mice carry at least one copy of the BALB/c genes, minor histoincompatibility should not cause tumor rejection. As shown in Table I , despite the fact that an overwhelming majority of the T cells in the transgenic mice express the P1A-specific T cell receptor, TCR-TG + mice were no more resistant to the plasmacytoma than their TCR-TG − littermates. The rejection of J558-B7 is mediated by T cells, as J558-B7 and J558-Neo gave similar tumor onset (not shown) and incidences (Table I ) in nu/nu mice. Section title: The Effector Function of P1A-specific CTL Is Restrained In Vivo. Educational score: 4.137884140014648 Domain: biomedical Document type: Study Language: en As the J558-Neo tumor lacks costimulatory activity that is essential for T cell activation, it is possible that lack of tumor rejection is due to poor activation of the cytotoxic T cells in vivo. To rule out this possibility, we injected J558-B7 in the right inguens and J558-Neo in the left inguens of the same mice and monitored the tumor incidence. As shown in Table II , most of the mice that rejected J558-B7 tumors developed J558-Neo tumors. Both incidence (Table II ) and growth kinetics (data not shown) of the J558-Neo tumor were unaffected by the J558-B7 injected into the same mice. Thus, the failure of the TCR-TG mice to reject J558-Neo tumors cannot be attributed to a lack of T cell activation in vivo, and the results presented in this section indicate that the effector function of the transgenic T cell is restrained in vivo. Section title: Discussion Educational score: 4.406806945800781 Domain: biomedical Document type: Study Language: en Successful identification of tumor antigens marks the first important step toward the development of tumor vaccines. However, much of our current understanding of vaccination is based on experience with infectious agents that are immunologically distinct from self antigens ( 22 ). Even though such knowledge may be directly applicable to mutated tumor antigens ( 4 – 10 ) that are, by definition, nonself, a major issue is whether such approaches can be directly applicable to unmutated antigens, many of which may turn out to be self antigens ( 11 – 19 ). To address this important issue, we used P1A as a model tumor antigen and took a transgenic approach to study the development and effector function of P1A-specific T cells. We have made two major observations. First, endogenous expression of tumor antigen does not interfere with the development and induction of P1A-specific CTL. P1A was the first unmutated tumor antigen to be identified. Although it was originally described as expressed only in mastocytoma, later studies indicated that it is expressed in testis ( 24 ) and in other lineages of tumors ( 20 ). Here we used RT-PCR and were able to reproducibly detect P1A mRNA in normal tissues, including hematopoietic tissues. Two earlier studies based on Northern blot analysis and RT-PCR analysis of P1A RNA have concluded that in tissues other than testis, the level of P1A mRNA is <1% of that in P815 cells ( 24 ). Our finding that P1A RNA in the spleen is ∼100-fold lower than that of J558 tumor cells does not contradict findings in earlier reports. Moreover, though resting APC fail to activate P1A-specific T cells, significant proliferation of transgenic T cells is induced by activated syngeneic APC. Nevertheless, P1A is unlikely to be expressed on a large proportion of spleen cells. If so, it would have been detected by CTL assay using anti-P1A CTL, which generally requires less ligand but a wider distribution of antigen among the target population than is required for proliferation or IL-2 production ( 43 ). Section title: Discussion Educational score: 4.316051959991455 Domain: biomedical Document type: Study Language: en Given the expression of the unmutated tumor antigen in normal tissues, it is possible that a T cell repertoire specific for such unmutated tumor antigens may have been selected so that the high affinity TCR are eliminated as in the case of T cells for some self antigens such as myelin basic protein ( 44 ). Although this is an attractive hypothesis, clonal deletion of T cells specific for unmutated tumor antigens has not been directly addressed due to lack of an experimental model. To our knowledge, this is the first report that has evaluated clonal deletion of a transgenic TCR specific for a natural tumor antigen. Our data clearly demonstrate that despite expression in normal tissues, P1A product does not induce clonal deletion of P1A-specific T cells. Since the transgenic T cells require <10 ng/ml of P1A peptide for maximal proliferation, it is likely that the TCR–L d / P1A35–43 interaction is of high avidity. It follows that endogenous P1A epitope does not cause clonal deletion of T cells with high avidity for P1A:L d complex, in contrast to the epitope on myelin basic protein ( 44 ). Our conclusion is supported by the fact that P1A-specific CTL clones characterized by other researchers also have high avidity for the peptide–L d complex ( 39 ). Our results suggest that TCR with high affinity for unmutated tumor antigen are not eliminated from the T cell repertoire. Section title: Discussion Educational score: 4.105824947357178 Domain: biomedical Document type: Study Language: en The transgenic T cells maintain a naive phenotype for >6 mo (the age at which mice were killed for the study), although a substantial proportion of T cells utilizing endogenous α chain have experienced antigenic stimulation from the environment during the same period. It is therefore most likely that the P1A epitope is essentially ignored by T cells under physiological conditions. In this regard, it should be emphasized that although P1A mRNA is detected in normal tissue, we have not analyzed expression of P1A protein in these tissues due to lack of reagents. The P1A epitope is detected on the spleen cells only after LPS activation. Section title: Discussion Educational score: 3.899135112762451 Domain: biomedical Document type: Study Language: en Clonal deletion requires less TCR ligand than T cell activation ( 45 , 46 ). Lack of a discernible effect of endogenous P1A on the development of P1A-specific T cells strengthens the conclusion that endogenous P1A epitope is not present in significant amounts, particularly in the thymus. Section title: Discussion Educational score: 4.245704650878906 Domain: biomedical Document type: Study Language: en Our second major observation was that the effector function of P1A-reactive CTL is restrained in vivo. Four lines of evidence strongly suggest that P1A can be a tumor rejection antigen. First, classic studies by Uyttenhove et al. reported that tumor cells that escaped immunity induced by the tum − P815 had lost P1A antigen ( 47 ). Second, immunization with P1A cDNA induces protection against subsequent challenge of P815 cells, although the authors noticed that the correlation between the strength of the recall CTL response in vitro and the anti-tumor immunity was poor ( 48 ). Third, adoptive transfer of P1A-specific CTL line ( 35 ) or polyclonal CTL ( 49 ) protects against subsequent challenge of P815 cells. Fourth, in mice challenged with P815 tumor, a strong correlation was observed between expansion of recurrent TCR repertoire and tumor rejection ( 50 ). Thus, although there is a strong case that P1A can induce immunity against tumors that express the antigen, it is less clear whether P1A-specific CTL generated in vivo are responsible for tumor rejection. Section title: Discussion Educational score: 4.345181465148926 Domain: biomedical Document type: Study Language: en It is intriguing that active immunization with P1A, either by adenoviruses engineered to express P1A in vivo ( 49 ) or by P1A peptide (our unpublished results) which induces anti-P1A CTL, does not confer immunity against P1A-expressing tumors. In addition, we have previously demonstrated that multiple lineages of tumors that share the P1A antigen are not crossprotected ( 20 ). A critical issue is whether the poor CTL-mediated protection is due to insufficient clonal expansion of P1A-specific CD8 T cells or to diminished effector function of these T cells. To address this issue, we have produced transgenic mice in which the overwhelming majority of CD8 T cells are specific for P1A. We report here that unless B7-1 is present on the tumor cells, no immunity is conferred by the cytotoxic T cells. Moreover, when the same transgenic mouse is injected with both B7-1 + and B7-1 − tumors, B7 + tumors are rejected but B7-1 − tumors grow progressively. Our finding supports and extends findings by Wick et al. ( 51 ), who have recently reported that a tumor expressing an alloantigen was not rejected in mice that carry transgenic T cells specific for the antigen, although the same mice could reject skin grafts with the same alloantigen. Even though both studies highlight a restrained T cell effector function, our study emphasized that such restriction can be overcome by expressing B7-1 on the tumor cells. Section title: Discussion Educational score: 4.450259685516357 Domain: biomedical Document type: Study Language: en Theoretically, at least four mechanisms can be proposed to account for the apparent restraint of the effector function of P1A-specific CTL. First, T cells specific for unmutated tumor antigen may be of intrinsically low affinity because high affinity P1A-reactive CTL may have been deleted due to endogenous expression of the tumor antigen. We show here that the high avidity, P1A-specific T cells were not deleted, unless P1A is overexpressed in the thymus as a transgene, which would rule out this possibility. Second, endogenous P1A may have rendered P1A- reactive T cells anergic. This is also unlikely, as we have shown that transgenic P1A-reactive T cells remain fully responsive to low concentrations of P1A and exhibit strong cytotoxicity against P1A-expressing tumors after in vitro stimulation. Third, it is possible that endogenous P1A has prevented full maturation of P1A-reactive CTL. The fact that fully active, P1A-specific CTL generated in vitro can be therapeutic in vivo ( 35 , 49 ) and that the effector function of ex vivo tumor-infiltrating lymphocytes recovered from P1A-expressing tumors requires B7 on target cells for optimal cytolysis ( 34 ) support this contention. Fourth, we have recently demonstrated that anti-tumor CD8 T cells do not exhibit cytotoxicity until after they have reached the tumor site ( 52 ). The requirement of B7-1 for tumor rejection in the transgenic mice can also be explained in part by the need for local activation of anti-tumor CTL. Section title: Discussion Educational score: 4.039474010467529 Domain: biomedical Document type: Study Language: en Since tissue destruction by some autoreactive transgenic T cells also requires expression of B7 in the target tissues ( 53 – 55 ), it is tempting to suggest that endogenous expression of unmutated tumor antigen may contribute to the restrained effector function of transgenic T cells. This concept can be formally tested when mice lacking endogenous P1A are generated. Section title: Discussion Educational score: 4.126497745513916 Domain: biomedical Document type: Study Language: en In summary, we have used a transgenic model to study the development, induction, and effector function of P1A-specific CTL. Our results demonstrate that the endogenous P1A antigen does not cause clonal deletion of high avidity, P1A-specific T cells in vivo. Even though P1A may well be a tumor rejection antigen, the effector function of P1A-specific T cells is restrained. Since P1A is the prototype of unmutated tumor antigen, the findings presented here have important implications: although this type of unmutated antigen can be used as a target for tumor vaccines and tumor immunotherapy, more emphasis must be placed on the strategy to induce full activation of T cells to achieve optimal effector function. | Study | biomedical | en | 0.999994 |
10049946 | Section title: Generation and Stimulation of DCs. Educational score: 4.129424571990967 Domain: biomedical Document type: Study Language: en To generate immature DCs, peripheral blood monocytes purified by centrifugal elutriation were cultured in RPMI-1640 supplemented with 10% FCS (Hyclone), 50 ng/ml GM-CSF, and 1,000 U/ml IL-4 for 6–7 d, as previously reported ( 3 , 4 ). Immature DCs were challenged with LPS (1 μg/ml, from Salmonella abortus equi ; Sigma Chemical Co. ), recombinant TNF-α (50 ng/ml; R&D Systems), poly I:C (20 μg/ ml, Sigma Chemical Co. ), and IFN-α (50–500 U/ml; Roferon-A, Roche), or infected with influenza virus strain PR8 (allantoic fluid containing 750 HAU/ml and 4 × 10 8 PFU/ml). Two neutralizing sheep antisera to human type I IFN were used: Iivari (450,000 neutralizing U/ml anti–IFN-α + 3,000 U/ml anti– IFN-β) and Kaaleppi (30,000 U/ml anti–IFN-α + 30,000 U/ml anti–IFN-β) ( 16 ). Section title: Surface and Intracellular Staining. Educational score: 4.146414279937744 Domain: biomedical Document type: Study Language: en DC maturation was evaluated by staining the cells with antibodies to HLA class I (W6/32; American Type Culture Collection [ATCC]), DR (L243; ATCC), CD86 , CD83 (HB15a; Immunotech), CD115 (3-4A4; Santa Cruz Biotechnology ), and CD38 (OKT10; ATCC), followed by FITC-labeled secondary antibodies. For intracellular staining, the cells were fixed for 30 min with 2% paraformaldehyde, and permeabilized for 30 min with PBS containing 0.5% saponin, 5% FCS, and 10 mM Hepes. The cells were stained with rabbit anti-MxA antibody ( 17 ) and biotinylated rat anti-PR8 HA mAb 1-10 ( 18 ), followed by appropriate secondary reagents (Southern Biotechnology Associates, Inc.). Intracellular staining for cytokine production was performed after stimulation of T cells for 6 h with 10 −7 M PMA and 0.5 μg/ml ionomycin ( Sigma Chemical Co. ). 2 h after stimulation monensin was added at 2 μg/ml. Cells were fixed and permeabilized as above and stained with PE-labeled anti–IL-4 and FITC-labeled anti–IFN-γ antibodies ( PharMingen ). Section title: Determination of Cell Viability. Educational score: 3.9173696041107178 Domain: biomedical Document type: Study Language: en Viability of the cells after PR8 infection was evaluated by staining cells with FITC-labeled Annexin V ( PharMingen ) and propidium iodide. The samples were analyzed on a FACScalibur ® using Cell Quest software ( Becton Dickinson ). Section title: Immunoblotting. Educational score: 4.122306823730469 Domain: biomedical Document type: Study Language: en Identical numbers of unstimulated DCs or DCs treated with different stimuli for 5 or 13 h were lysed in lysis buffer (100 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 1 mM PMSF, 10 μg/ml aprotinin, and 10 μg/ml leupeptin). Lysates were solved by 8% SDS-PAGE under reducing conditions, and transferred to nitrocellulose membranes ( Amersham ). After blocking with PBS/0.1% Tween/5% nonfat dry milk, the membranes were incubated with the rabbit anti-MxA antiserum ( 17 ), followed by horseradish peroxidase–conjugated goat anti-rabbit IgG (Southern Biotechnology Associates). Proteins were visualized by chemiluminescence using the ECL detection reagents ( Amersham ). Section title: Cytokine Determination. Educational score: 4.0330810546875 Domain: biomedical Document type: Study Language: en IL-12 p40 and p75 levels were measured by ELISA as previously described ( 19 ). Type I IFN was measured evaluating inhibition of Daudi cell proliferation ( 20 ) with reference to a standard IFN-α curve. The sensitivity of the assay was 0.2 U/ml. Section title: Class I Synthetic Rate and Stability. Educational score: 4.120359420776367 Domain: biomedical Document type: Study Language: en 5 h after stimulation or infection, DCs were labeled with 35 S[methionine]/cysteine ( Amersham ) for 1 h. Cells were chased for one additional hour or longer; identical number of cells were lysed and HLA class I molecules were immunoprecipitated using the W6/32 mAb (ATCC) followed by protein G–Sepharose ( Pharmacia Biotech ). The precipitates were resolved by SDS-PAGE and the radioactivity of specific bands was quantified by PhosphorImager (Molecular Dynamics). DCs do not divide during the assay. Total protein synthesis was estimated by counting the radioactivity incorporated by a fixed number of cells. Section title: T Lymphocyte Stimulation. Educational score: 4.117954254150391 Domain: biomedical Document type: Study Language: en Immature HLA-A2 + DCs either were pulsed for 1 h with 1 μg/ml Influenza Matrix 58-66 peptide or were infected with PR8. After 5 or 24 h, a graded number of cells were tested for their capacity to trigger proliferation of a specific T cell clone ( 21 ). In a different set of experiments, immature DCs were left untreated or were pretreated for 40 h with poly I:C, LPS, TNF-α, and IFN-α, or were infected with PR8. After irradiation (1,500 rad), a graded number of cells were cultured with allogeneic T cells and the proliferative response was measured on day 5 by [ 3 H]thymidine incorporation ( Amersham ). Naive T cells were either derived from cord blood or from adult donors lymphocytes, negatively depleted of CD8, CD14, CD20, CD56, HLA class II, CD45 RO + cells. The resulting population was 99% composed of CD4 + CD45RA + cells. Cells were stimulated with differently matured DCs, at a responder/stimulator ratio of 10:1, in the absence or presence of neutralizing anti–IL-12 antibodies ( 19 ). Section title: Susceptibility of DCs to Influenza Virus Infection Correlates with Expression of MxA. Educational score: 4.228293418884277 Domain: biomedical Document type: Study Language: en Immature DCs can be generated by culturing peripheral blood monocytes with GM-CSF and IL-4 ( 3 , 4 ). These cells are competent in antigen capture and mature when stimulated by LPS, TNF-α, or CD40L, losing their antigen-capturing capacity and acquiring T cell stimulatory capacity. Immature and mature DCs, obtained after LPS or TNF-α stimulation, were incubated with different doses of influenza virus strain PR8, and the proportion of infected cells was measured after 14 h by staining with a specific mAb. As shown in Fig. 1 , there was a dose-dependent increase in the percentage of cells expressing viral proteins as well as in the level of viral proteins expressed by individual cells. Immature and TNF-α–matured DCs were highly susceptible to influenza virus infection: at high multiplicity of infection (MOI), all cells expressed high levels of viral protein, whereas at low multiplicity viral proteins were expressed in a bimodal distribution. In contrast, LPS-matured DCs were more, and in some experiments completely, resistant to infection; at high MOI they showed a bimodal protein expression, whereas at low multiplicity only a small proportion of cells was infected, expressing low levels of viral proteins. Finally, DCs pretreated for 40 h with IFN-α were almost completely resistant to infection. The different susceptibility of DCs to influenza infection could not be explained by their different levels of endocytic activity. TNF-α–matured DCs, which had completely lost endocytic activity, were equally, if not more, susceptible to infection than immature DCs. Conversely, IFN-α–treated DCs, which were as endocytic as immature DCs (data not shown), were completely resistant. Section title: Susceptibility of DCs to Influenza Virus Infection Correlates with Expression of MxA. Educational score: 4.063975811004639 Domain: biomedical Document type: Study Language: en In search for a mechanism that might modulate the susceptibility of DCs to influenza virus infection, we investigated the expression of MxA, a protein induced by type I IFN, that is known to mediate resistance to several viruses including influenza ( 22 , 23 ). MxA was initially identified by two-dimensional gel analysis as a protein abundantly represented in LPS-matured but not immature DCs (Sakakibara, Y., unpublished data). Section title: Susceptibility of DCs to Influenza Virus Infection Correlates with Expression of MxA. Educational score: 4.255279064178467 Domain: biomedical Document type: Study Language: en Fig. 2 shows the level of MxA expression in relation to the level of production of viral HA (A–G) and to the viability of the infected cells (H–P). Immature DCs did not express MxA, but became MxA + after infection with PR8, explaining their capacity to resist the cytopathic effect of the virus. At low MOI (1:1), immature DCs could be efficiently infected (61%) with no increase in cell death over the background, whereas at higher MOI they were infected more efficiently and produced higher protein levels, but showed progressively lower viability. In contrast, LPS-matured DCs, which already expressed MxA, required ∼10-fold higher doses of virus to produce comparable amounts of proteins, but were far more resistant to the cytopathic effect even at an MOI of 100:1. Taken together, these results suggest that susceptibility to influenza virus infection is modulated in DCs by the expression of MxA, which is induced by LPS stimulation as well as by infection with influenza virus. Section title: MxA Is Induced by Autocrine Production of Type I IFN. Educational score: 4.145902633666992 Domain: biomedical Document type: Study Language: en The time course of MxA protein expression was analyzed in DCs challenged with different stimuli, either by immunofluorescence on fixed and permeabilized cells or by immunoblotting . MxA was induced with comparable fast kinetics by IFN-α, LPS, and PR8 infection, as well as by poly I:C, a synthetic source of dsRNA. In contrast, TNF-α failed to induce MxA expression, and CD40L induced it only to a low extent and with a slower kinetics. Section title: MxA Is Induced by Autocrine Production of Type I IFN. Educational score: 4.151048183441162 Domain: biomedical Document type: Study Language: en The capacity of different stimuli to induce MxA correlated with their capacity to induce production of type I IFN by DCs . Both poly I:C and LPS led to a rapid production of type I IFN that reached a plateau at 4 h, whereas PR8 infection induced an equally rapid, but more sustained and consistently higher production. In contrast, TNF-α and CD40L stimulation did not induce production of detectable levels of IFN. Section title: MxA Is Induced by Autocrine Production of Type I IFN. Educational score: 4.224798202514648 Domain: biomedical Document type: Study Language: en These results demonstrate that DCs can produce type I IFN in response to specific stimuli, which in turn leads to upregulation of MxA gene expression via an autocrine loop. To address this possibility, we evaluated the capacity of neutralizing antibodies to type I IFNs to inhibit MxA expression . The antisera we used completely abrogated MxA upregulation at 5 h induced by recombinant IFN-α, PR8 infection, and CD40L. However, they also inhibited significantly, although not completely (at least 50%), MxA induction after LPS or poly I:C stimulation. Although these results do not rule out the possibility of an IFN-α–independent MxA upregulation, they suggest that autocrine production of type I IFN by DCs represents a major mechanism to ensure the rapid build-up of a resistance state to the infecting virus. Section title: dsRNA and PR8 Upregulate Protein Synthesis in Immature and IFN-α–treated DCs. Educational score: 4.417379379272461 Domain: biomedical Document type: Study Language: en dsRNA has been shown to trigger PKR, an IFN-α–induced kinase that inactivates eIF2, thereby reducing protein synthesis ( 24 ). This mechanism may be important to ensure reduction of viral replication and elimination of infected cells. However, PKR has also been shown to activate NF-κB ( 25 ) and transcription. PKR was expressed constitutively in DCs and its level was not further increased upon IFN-α treatment (data not shown). We thus compared the effect of poly I:C and PR8 on protein synthesis in DCs and Hela cells before and after treatment with IFN-α . As expected, PR8 infection and poly I:C induced a marked decrease in the rate of total and MHC class I protein synthesis in IFN-α–pretreated, but not in untreated, Hela cells. In contrast, in DCs the same treatments resulted in a strong boost of total, and particularly MHC class I, protein synthesis, irrespective of whether or not the cells had been pretreated with IFN-α. These results demonstrate that, as compared with other cell types, DCs have a unique capacity to respond to dsRNA, a property that may be important to allow efficient presentation of viral antigens. Section title: Influenza Virus Infection Promotes and Sustains Generation of Peptide–MHC Class I Complexes. Educational score: 4.170437812805176 Domain: biomedical Document type: Study Language: en We next investigated whether stimulation of DCs by infectious virus might optimize loading of class I molecules with antigenic peptides. In PR8-infected DCs there was a strong upregulation of HLA class I synthesis (5–10-fold in four experiments) . Furthermore, the class I molecules synthesized during the initial stages of infection displayed a two- to threefold longer half-life than those synthesized in immature or in LPS-treated DCs . This increased stability may be related to the abundant supply of high affinity peptides derived from degradation of viral proteins, since it was not noticed in DCs stimulated with LPS. Section title: Influenza Virus Infection Promotes and Sustains Generation of Peptide–MHC Class I Complexes. Educational score: 4.231884002685547 Domain: biomedical Document type: Study Language: en PR8-infected immature DCs rapidly acquired a high capacity to trigger an HLA-A2–restricted T cell clone specific for the influenza matrix peptide M58-66. Furthermore, the stimulatory capacity increased with time after infection . In contrast, although immediately stimulatory, immature DCs pulsed with a high dose of M58-66 peptide lost the capacity to stimulate specific T cells by 20 h of culture, a finding consistent with the short half-life of MHC class I–peptide complexes. In addition, IFN-α–pretreated DCs, which were highly resistant to viral infection, did not stimulate the virus-specific T cell clone even at late time points. Section title: Influenza Virus Infection Promotes and Sustains Generation of Peptide–MHC Class I Complexes. Educational score: 4.125326156616211 Domain: biomedical Document type: Study Language: en Taken together, these results indicate that in PR8- infected DCs the increased synthesis and loading of MHC class I molecules results in a rapid and continuous production of large numbers of complexes containing viral peptides. These events allow presentation to T cells to be sustained over a long period of time. Section title: Influenza Virus Infection and dsRNA Trigger DC Maturation and Prime for a Th1 Response. Educational score: 4.2095208168029785 Domain: biomedical Document type: Study Language: en We compared dsRNA and influenza virus infection to known stimuli for their capacity to induce DC maturation. As a readout we analyzed the expression of costimulatory molecules and the production of Th1 polarizing cytokines (i.e., IL-12 ), and evaluated the capacity of DCs to stimulate and polarize allogeneic naive T cells. As shown in Table I , poly I:C and PR8 induced a marked upregulation of MHC class I and class II, CD80, CD86, CD83, and CD38, and downregulation of CD115, a phenotype characteristic of mature DCs ( 19 , 27 ). In addition, poly I:C and PR8 induced the production of low but significant levels of IL-12 p75 (1–2 ng/ml), although the amount detected was always lower (at least 10– 50-fold) than that induced via CD40L ( 19 ). Although TNF-α stimulation never led to production of bioactive IL-12, low levels of IL-12 p75 (0.1–0.2 ng/ml) were detectable after LPS stimulation. On the other hand, IFN-α tested over a broad range of concentrations (10–500 U/ml) did not induce DC maturation but only upregulation of HLA class I and a modest shift in the expression of CD38 and CD86 with no significant increase in CD83, HLA class II, or other costimulatory and adhesion molecules. Section title: Influenza Virus Infection and dsRNA Trigger DC Maturation and Prime for a Th1 Response. Educational score: 4.395838737487793 Domain: biomedical Document type: Study Language: en Consistent with the effect on DC maturation and cytokine production, poly I:C and PR8 increased ∼30-fold the capacity of DCs to stimulate a proliferative response by allogeneic CD45RA + CD4 + T cells, whereas IFN-α treatment did not show a significant effect. Poly I:C-matured DCs were as efficient as DCs induced to mature by TNF-α or LPS . Although the ability to trigger T cell proliferation was comparable, the capacity to polarize T cells was remarkably different, depending on the nature of the maturative stimulus applied. Alloreactive T cells expanded by TNF-α–matured DCs were poorly polarized (only 32% of cells producing either IFN-γ or IL-4) and consisted of both Th1 and Th2 . In contrast, T cells expanded by poly I:C-matured DCs were highly polarized (70–90%, in different experiments) with most of the cells producing high amounts of IFN-γ, consistent with a dominant Th1 phenotype . Addition of neutralizing antibodies to IL-12 prevented Th1 polarization, skewing the cultures towards a Th2 phenotype . In some experiments, the inhibition of Th1 development required the simultaneous neutralization of type I IFN and IL-12 (data not shown). Taken together, these results indicate that dsRNA and influenza virus infection can induce DC maturation, and confer the capacity to prime a Th1 response through the production of IL-12 and type I IFN. Section title: Protection and Maturation of DCs Induced by the Viral Pattern dsRNA. Educational score: 4.201998710632324 Domain: biomedical Document type: Study Language: en We have shown that dsRNA induces two functionally distinct responses in DC: (i) protection from the viral cytopathic effect, and (ii) maturation, leading to increased capacity to prime and polarize T cells. In the course of viral infection, the simultaneous induction of protection and maturation optimizes loading of viral antigens on MHC class I molecules. Section title: Protection and Maturation of DCs Induced by the Viral Pattern dsRNA. Educational score: 4.335151672363281 Domain: biomedical Document type: Study Language: en dsRNA is a classical inducer of type I IFN ( 28 – 30 ), which plays a critical role in antiviral responses ( 31 , 32 ). DCs represent a strategic source of type I IFN and we have shown that it is produced not only in response to dsRNA or virus infection ( 33 ), but also in response to LPS stimulation. However, IFN is not produced in response to other stimuli such as TNF-α, and only in minute amounts in response to CD40L. Type I IFN produced by DCs results in rapid induction of MxA, a cytoplasmic protein that has been shown to protect cells from the cytopathic effect of some viruses ( 22 , 23 , 34 ). Neutralizing antibodies to type I IFN completely inhibited MxA induction by recombinant exogenous IFN-α, influenza virus infection, and CD40L, but only partially inhibited MxA upregulation by poly I:C or LPS, suggesting a possible direct activation of MxA expression independent from type I IFN ( 35 ). Section title: Protection and Maturation of DCs Induced by the Viral Pattern dsRNA. Educational score: 4.239864826202393 Domain: biomedical Document type: Study Language: en dsRNA and viral infection also induced a rapid maturation of DCs with upregulation of MHC, adhesion, and costimulatory molecules. These phenotypic changes were to a large extent comparable to those induced by other maturation stimuli such as LPS, TNF-α, or CD40L, although a different pattern in cytokine production was observed. These results may well account for the increased capacity of influenza virus–infected DCs to stimulate CTL responses ( 15 ). Although a minor shift in CD38 and CD86 was reproducibly observed, IFN-α per se was unable to support a complete DC maturation. The maturation effect of type I IFN reported in other studies was observed in a different culture system and was dependent on the simultaneous presence of TNF-α, which by itself is a DC maturation factor ( 36 ). Section title: Protection and Maturation of DCs Induced by the Viral Pattern dsRNA. Educational score: 4.264494895935059 Domain: biomedical Document type: Study Language: en The signal transduction pathway triggered by dsRNA has been studied in detail. It has been shown that dsRNA activates PKR, a serine-threonine kinase that phosphorylates and inactivates eIF2, thereby shutting off protein synthesis ( 24 ). PKR can also activate NF-κB ( 25 ) and consequently may induce transcription of genes involved in DC maturation. The fact that PKR-deficient mice can still respond to dsRNA suggests that there may be additional pathways of signal transduction that may be operative in DCs ( 37 ). The relative role and function of the pathways activated by dsRNA may vary in different cells. Our experiments show that the response to dsRNA in DCs and Hela cells is considerably different. In Hela cells dsRNA induces a downregulation of protein synthesis, whereas in immature DCs dsRNA actually upregulates total protein synthesis, while fully inducing the maturation process. Section title: Protection and Maturation of DCs Induced by the Viral Pattern dsRNA. Educational score: 4.251269340515137 Domain: biomedical Document type: Study Language: en It has been suggested that recognition of conserved molecular patterns characteristic of pathogens is a property of the innate immune system, which is instrumental to initiating and regulating the adaptive immune response ( 38 ). Our results clearly indicates that dsRNA behaves as one of such molecular patterns. dsRNA can directly signal to the APCs the presence of an infectious agents and, by activating the DCs, can lead to activation of lymphocytes bearing clonally specific antigen receptors, thus triggering an adaptive immune response. The relevance of this recognition system is also underlined by the fact that many viruses specifically target the dsRNA-binding protein PKR to escape immune recognition ( 39 – 41 ). Section title: Sustained Synthesis of Viral Antigens and Class I Molecules Maximizes Antigen Presentation. Educational score: 4.175933361053467 Domain: biomedical Document type: Study Language: en We have shown that efficient presentation influenza virus is the result of a delicate compromise. On one hand, immature DCs are highly susceptible to infection and thus produce large amounts of viral proteins. On the other hand, they can rapidly build up resistance to the virus, thus limiting its cytopathic effect. The importance of synchronizing these two functions is illustrated by the fact that IFN-α–pretreated DCs, which are resistant to influenza infection, are extremely inefficient at presenting viral antigens. Section title: Sustained Synthesis of Viral Antigens and Class I Molecules Maximizes Antigen Presentation. Educational score: 4.334330081939697 Domain: biomedical Document type: Study Language: en The dramatic upregulation of class I synthesis (up to 10-fold) induced by viral infection is an important mechanism that ensures effective presentation of viral antigens. MHC class I synthesis is sustained after induction of maturation, allowing continuous accumulation of peptide–MHC complexes and thus compensating for the relatively short half-life of class I molecules (10–20 h). The importance of the sustained antigen loading is exemplified by the fact that although the capacity to stimulate class I–restricted, virus-specific T cells is rapidly lost in peptide-pulsed DCs, it actually increases with time in virus-infected DCs. This fact should be taken into account when considering the use of DCs as vaccines to induce class I–restricted responses. Section title: Sustained Synthesis of Viral Antigens and Class I Molecules Maximizes Antigen Presentation. Educational score: 4.25475549697876 Domain: biomedical Document type: Study Language: en We have previously shown that in maturing DCs the synthesis of class II molecules is transiently upregulated and subsequently stopped, while stable peptide class II complexes are retained with extremely long half-lives (>100 h) ( 5 ). In contrast, in DCs MHC class I molecules have a short half-life that is not significantly affected by the maturation process. The different regulation of class I and class II biosynthesis and stability in DCs makes good sense. Class II molecules present antigens that are transiently encountered in the surrounding environment, so it is important for a DC to be able to load antigenic peptides over a short period of time and retain the antigen as a stable complex. Instead, the short half-life of class I molecules is instrumental to allow a continuous monitoring of early and late viral antigens, as long as the cell remains infected. Section title: Flexibility in Cytokine Production Determines the T Cell Polarizing Capacity of DCs. Educational score: 4.428060531616211 Domain: biomedical Document type: Study Language: en Although DCs induced to mature by different stimuli share several common features, such as the increase in MHC, costimulatory molecules, and T cell stimulatory capacity ( 3 , 19 ), consistent differences are observed in the pattern of cytokines produced in response to the different stimuli. These in turn determine both the extent and the type of T cell polarization, suggesting that mature DCs can exist in different functional states. TNF-α–matured DCs, which produce neither IL-12 nor type I IFN, were highly stimulatory, but induced only a low percentage of fully polarized cells. In contrast, dsRNA- or LPS-matured DCs, which produced both type I IFN and IL-12 (although in different proportions) elicited strong Th1 polarization, with 70–90% of T cells producing high levels of IFN-γ. The Th1 polarizing effect was inhibited in most cases by neutralization of IL-12, although in some cases neutralization of type I IFN was also required, as previously reported for monocytes ( 30 ). These results indicate a flexibility of DCs in the use of Th1 polarizing cytokines and suggest that a multiplicity of responses can be generated by distinct environmental signals. In addition, it is possible that the relative production of IL-12 and type I IFN may play a more subtle role in modulating T cell polarization. Although both IL-12 ( 26 ) and type I IFN in humans ( 42 ) are known to drive Th1 polarization, it is possible that they may play distinct roles by differentially regulating IL-4 and IL-13 production by T cells ( 43 ). Section title: Flexibility in Cytokine Production Determines the T Cell Polarizing Capacity of DCs. Educational score: 4.1705708503723145 Domain: biomedical Document type: Study Language: en Our results also imply that the nature of the signal inducing DC maturation may determine the capacity of the DCs to generate polarized immune responses. On one hand, stimuli from viral or bacterial patterns (dsRNA or LPS) or T cell help, through CD40–CD40L interaction, generate DCs capable of priming strong Th1 responses. On the other hand, an endogenous inflammatory stimulus, such as TNF-α, generates DCs capable of inducing a more balanced response, comprising both Th1 and Th2 cells. The fact that the same DCs can mature to different functional states capable of stimulating polarized Th1 or Th2 responses makes good sense, since it allows the APCs to initiate a response that is appropriate for the signal received and has practical implications for the therapeutic use of DCs. | Study | biomedical | en | 0.999997 |
10049947 | Section title: Immunogens. Educational score: 4.207452774047852 Domain: biomedical Document type: Study Language: en For the production of isoform-specific polyclonal antisera directed against Nramp2, rabbits were immunized with fusion proteins containing glutathione S-transferase (GST) fused to a peptide segment derived from the amino terminal region of Nramp2 (residues 1–71; for amino acid numbering see reference 10 ). This peptide is in a region of the protein which is not conserved in other Nramp family members, including Nramp1 ( 18 ). The GST–Nramp2 fusion protein was constructed in the plasmid vector pGEX ( Pharmacia ) as follows: The Nramp2 sequence was amplified by PCR using oligonucleotides NF2 (5′-AA AGATCT ATGGTGTTGGATCC-3′) and NR (5′-CT GAA TTC GAACGCCCAGAGT-3′) (nucleotides 1–268), and the full-length Nramp2 cDNA as template. The PCR product was digested with BglII and EcoRI and the fragment was subcloned into pGEX digested with BamHI and EcoRI to create the in-frame GST fusion protein. Overexpression of the Nramp2–GST fusion protein was carried out in large scale cultures of Escherichia coli and the protein was purified from bacterial lysates using glutathione–Sepharose 4B ( Pharmacia ) as previously described ( 19 ). Purified proteins were analyzed by 7.5% SDS-PAGE and excised from the gel after light staining with 0.05% Coomassie blue in ddH 2 O. Section title: Production of Anti-Nramp2 Antibodies. Educational score: 4.1668291091918945 Domain: biomedical Document type: Study Language: en Polyclonal antibodies were produced in male New Zealand White rabbits as described previously ( 8 ). A system for affinity purification of the antibodies was devised using the same Nramp2 peptide fused to a second fusion partner, dihydrofolate reductase modified by the addition of eight consecutive histidine residues (his–DHFR). The fusion protein construct was made as described above, except the PCR product was digested with EcoRI and the resulting overhangs repaired using the Klenow fragment of DNA polymerase I ( Pharmacia ) before digestion with BglII. The digested PCR product was ligated into BglII- and SmaI-digested pQE40 plasmid vector (Qiagen). The in-frame his–DHFR–Nramp2 fusion protein construct was transformed into E. coli strain M15(pREP4) for expression (Qiagen). Purification was performed on Ni–NTA agarose according to experimental conditions suggested by the manufacturer (Qiagen). The polyclonal antiserum directed against the GST fusion protein was purified against the his–DHFR fusion protein by a preparative immunoblot procedure ( 20 ). The anti-Nramp1 polyclonal antiserum ( 8 ) was affinity purified against the corresponding Nramp1–GST fusion protein by the same protocol. Section title: Cell Culture. Educational score: 4.210579872131348 Domain: biomedical Document type: Study Language: en The mouse monocyte–macrophage cell lines RAW 264.7 and J774a, the mouse Sertoli cell line TM4, and the mouse kidney line mIMCD-3 were obtained from the American Type Culture Collection (ATCC). They were cultured in media and under conditions recommended by the ATCC. WEHI 3B (myelomonocyte), WEHI 231 (B lymphocyte), BI 141 (T lymphocyte), and 70Z/3 (pre-B cell) cells were cultured as described previously ( 21 ). Chinese hamster ovary (CHO) cells LR73 ( 22 ) were grown in α-MEM supplemented with 10% fetal calf serum, 2 mM L-glutamine, 50 U/ml penicillin and 50 μg/ml streptomycin. All media and media supplements were purchased from GIBCO BRL . Murine macrophages were obtained by peritoneal lavage, as previously described ( 8 ). We have previously described the production and characterization of RAW macrophages expressing a transfected wild-type Nramp1 fused to a c-myc epitope ( 9 ). The c-myc –tagged Nramp2 expression plasmid was constructed by excising the myc -tagged Nramp2 cDNA from plasmid pBluescript ( 23 ) using Spe I and Eco RV sites from the polylinker, followed by cloning into the mammalian expression plasmid pCB6 ( 24 ). For expression in CHO cells, the same insert was cloned into the expression vector pMT2 ( 25 ). CHO cells were transfected by the calcium phosphate coprecipitation method ( 26 ). RAW cells were transfected by electroporation as described previously ( 9 ). Clones of stable transfectants were selected in geneticin (G418, 1 mg crude/ml final; GIBCO BRL ) for 10–14 d, picked and expanded individually, and tested for protein expression by immunofluorescence using the anti– c-myc tag monoclonal antibody 9E10 (Babco). Section title: Immunoblotting and Immunoprecipitation. Educational score: 4.224009037017822 Domain: biomedical Document type: Study Language: en Crude membrane fractions from the various cells were prepared as described previously ( 27 ). Protein concentration of the membrane fraction was determined by the Bradford assay (BioRad). Proteins were separated on SDS–polyacrylamide gels and transferred by electroblotting to nitrocellulose membranes. For experiments where the membrane was to be stripped and reprobed, a polyvinylidene fluoride membrane was used (Westran; Schleicher and Schuell) to reduce protein loss from the membrane during stripping. Equal loading and transfer of proteins was verified by staining the blots with Ponceau S ( Sigma Chemical Co. ). The blots were blocked in TBST (10 mM Tris/Cl, pH 8, 150 mM NaCl, 0.05% Tween 20, pH 8) plus 5% skim milk powder for 1 h at room temperature. Primary antibodies used were as follows: affinity purified rabbit anti– mouse Nramp2 (1:100 dilution); affinity purified rabbit anti– mouse Nramp1 (1:200); mouse monoclonal anti c-myc –epitope tag 9E10 (Babco; 1:100), rat anti–mouse transferrin receptor ( Biosource International ; 1:200), and rat anti–mouse Lamp1 (1: 200). Anti–rabbit, anti–rat, and anti–mouse secondary antibodies conjugated to horseradish peroxidase were used at 1:10,000 ( Amersham ). Chemiluminescence was used for detection of immune complexes on the immunoblot (ECL; Amersham ). For immunoprecipitation, CHO cells and Nramp1 and Nramp2 CHO transfectants were metabolically labeled with [ 35 S]methionine by incubating overnight in 100 μCi/ml of [ 35 S]methionine ( DuPont ) in methionine-free DMEM ( GIBCO BRL ) containing 10% heat-inactivated, dialyzed fetal bovine serum, 2 mM L-glutamine, and 2 mM Hepes. Immunoprecipitation was performed exactly as described previously ( 8 ). Section title: Immunofluorescence. Educational score: 4.220276355743408 Domain: biomedical Document type: Study Language: en Cells were grown on glass coverslips and fixed with 4% paraformaldehyde in PBS for 30 min at 4°C. Immunofluorescence was performed as previously described ( 9 ) with the following modifications: incubation with the primary antibody was 1 h at 20°C for the anti– c-myc mouse monoclonal 9E10 (1:200; Babco), and the anti-Lamp1 rat monoclonal (1:200), or overnight at 4°C for the anti-Nramp2 antiserum (1:800) followed by anti–mouse, anti–rat, or anti–rabbit secondary antibodies conjugated to rhodamine (1:300) or FITC (1:200) (Jackson Immunochemicals). Immunofluorescence was analyzed with a Nikon microscope using the 100× oil immersion objective. Certain colocalization studies were carried out using a Zeiss laser confocal microscope with a 63× objective. Composites of confocal images were assembled and labeled using PhotoShop, Metamorph, and Freehand software. To label the lysosomal compartment, cells were incubated with 1 mg/ml lysine-fixable FITC–dextran (Molecular Probes) in growth medium for 4 h at 37°C in 5% CO 2 . After washing, cells were incubated an additional 30 min to chase the dextran from the early endosomal to the lysosomal compartments. For identification of the early and recycling endosomal compartment, cells were incubated in serum-free medium containing 50 μg/ml FITC–transferrin (Molecular Probes) for 30 min at 37°C in 5% CO 2 . Phagosomes were formed by incubating the cells with 3 μm latex beads ( Sigma Chemical Co. ) diluted 1:200 in complete culture medium for 15 min at 37°C in 5% CO 2 . After treatments to identify the specific subcellular compartments, cells were fixed in 4% paraformaldehyde and processed for immunofluorescence. Section title: Glycosidase Treatments. Educational score: 4.111147880554199 Domain: biomedical Document type: Study Language: en Endo–β-acetylglucosaminidase H (Endo H) and peptide N -glycosidase F (PNGase F) were obtained from New England Biolabs . Aliquots of membrane preparations from J774a and CHO cells were denatured before digestion in a buffer containing 0.5% SDS and 0.1 M β-mercaptoethanol for 2 min at 70°C. For Endo H digestion, samples were diluted twofold and incubated with 4,000 U of Endo H in 50 mM sodium citrate, pH 5.5, for 1 h at 37°C. The −Endo H controls were treated identically except an equivalent volume of ddH 2 O was added in place of Endo H. For PNGase F digestion, samples were diluted twofold and incubated in 50 mM sodium phosphate, pH 7.5, 1% NP-40, with 500 U of PNGase F for 1 h at 37°C. The −PNGase F controls were treated identically except an equivalent volume of ddH 2 O was added in place of PNGase F. Section title: RNA Isolation and Hybridization Studies. Educational score: 4.1821818351745605 Domain: biomedical Document type: Study Language: en Total cellular RNA was isolated using guanidinium–HCl solubilization and differential ethanol precipitation ( 28 ). 20 μg of total cellular RNA were denatured in a formamide–formaldehyde mixture and loaded onto denaturing agarose gels containing 0.66 M formaldehyde. Blots were prehybridized in a solution containing 10% dextran sulfate, 1 M NaCl, 1% SDS, and heat-denatured salmon sperm DNA (200 μg/ml) at 65°C for 2–16 h. Hybridization was for 24 h at 65°C in the same buffer containing the radiolabeled probe (10 6 cpm/ml of hybridization buffer; specific activity 10 9 cpm/μg DNA). Blots were washed under conditions of increasing stringency up to 0.1× SSC and 0.1% SDS at 65°C and then exposed to Kodak XR film with two intensifying screens at −70°C for 18 h to 7 d at −80°C. Section title: Phagosome Fractionation. Educational score: 4.287115573883057 Domain: biomedical Document type: Study Language: en Phagosomes were isolated from J774a cells by a modification of a method described previously ( 9 ). 10 subconfluent 150 mm dishes of each cell line were fed with a 1:200 dilution of blue-dyed latex beads (0.8 μm; Sigma Chemical Co. ) in culture medium for 1 h at 37°C in 5% CO 2 . The cells were then washed in PBS and harvested in the presence of protease inhibitors (1 μg/ml leupeptin, 1 μg/ml aprotinin, 1 μg/ml pepstatin, and 100 μg/ml PMSF; all Boehringer Mannheim ) and recovered by centrifugation (2,000 g , 5 min). The cell pellets were washed and resuspended in homogenization buffer (8.5% sucrose, 3 mM imidazole, pH 7.4) and homogenized by passage through a 22G needle until 90% of the cells were broken, as monitored by light microscopy. Nuclei and unbroken cells were pelleted and the supernatant loaded onto a sucrose step gradient as follows: the supernatant was brought up to 40% sucrose by addition of 62% sucrose and loaded on top of a 1-ml 62% sucrose cushion. Layers of 2 ml of 35, 25 and finally 10% sucrose (all sucrose solutions wt/wt in 3 mM imidazole, pH 7.4, plus protease inhibitors) were sequentially added to the top of the tube, and the gradients were centrifuged at 100,000 g for 1 h at 4°C (SW41; Beckman). Phagosomes were recovered from the 10–25% sucrose interface, washed with PBS containing protease inhibitors, and recovered by a final centrifugation at 40,000 g in an SW41 rotor at 4°C. The final pellets were resuspended in 2× Laemmli sample buffer. Phagosomes prepared by this protocol have been previously shown to be free of endoplasmic reticulum (endoplasmin, BiP, and calnexin) and Golgi apparatus (galactosyl transferase) contaminants ( 29 ). Section title: Results Educational score: 4.375773906707764 Domain: biomedical Document type: Study Language: en To generate an isoform-specific anti-Nramp2 antiserum, a protein segment derived from the amino terminus of Nramp2 was selected based on its predicted antigenicity and its sequence divergence from Nramp1 (28% identity). A GST–Nramp2 fusion protein containing the amino terminal Nramp2 segment was produced and used for immunization, and the anti-Nramp2 fraction was further isolated from the immune serum by affinity purification against a second immobilized Nramp2–DHFR fusion partner. The antiserum was tested for specificity by immunoblotting crude membrane fractions as well as by immunoprecipitation of [ 35 S]methionine–labeled cell lysates from transfected CHO cell clones expressing either c-myc –tagged Nramp1 or c-myc –tagged Nramp2 proteins . The anti– c-myc monoclonal antibody (9E10 ) specifically recognized a species of apparent molecular mass 90–100 kD in the Nramp2-transfected CHO cells, and a protein of 85–95 kD in the Nramp1 -transfected cells that were absent from extracts of untransfected CHO controls . These protein species migrated as broad bands in SDS–acrylamide gels. In membrane preparations from Nramp2-transfected CHO cells (CHON2), the affinity-purified anti-Nramp2 antiserum recognized a single protein species of apparent molecular mass 90–100 kD . Immunoblotting analysis of the same set of membrane fractions with anti-Nramp1 antiserum revealed a single protein species of 85–95 kD in membranes from the Nramp1-transfected CHO cells . These immunoreactive bands were absent from untransfected CHO cells and from transfected cells expressing the other Nramp isoform. The electrophoretic mobility characteristics of the Nramp1 and Nramp2 detected by the respective polyclonal antisera were very similar to those of the species detected by the anti– c-myc antibody in the same cell extracts . The reactivity and isoform specificity of the antibodies were confirmed by immunoprecipitation studies of [ 35 S]methionine metabolically labeled cell extracts from CHO cells or from Nramp1 and Nramp2 CHO transfectants . These data indicate that the anti-Nramp1 and anti-Nramp2 antisera produced and purified according to our protocol are isoform specific. Section title: Results Educational score: 4.344332695007324 Domain: biomedical Document type: Study Language: en Northern blotting and in situ hybridization studies have shown that, as opposed to Nramp1 , which is expressed almost exclusively in mononuclear phagocytes, Nramp2 mRNA is expressed in most tissues ( 10 – 12 ). We questioned whether the two Nramp proteins would display an overlapping or mutually exclusive expression pattern. Northern blot analysis of total cellular RNA from a panel of murine hematological cell lines revealed a readily detectable level of Nramp2 mRNA expression in the macrophage lines RAW 264.7 and J774a as well as in Friend virus– transformed erythroleukemia (MEL) cells . A much lower level of expression of Nramp2 was found in other cell lines: WEHI 231 (B lymphocyte), WEHI 3B (myelomonocyte), 70/Z (pre-B lymphocyte), and BI 141 (T lymphocyte). In comparison, Nramp1 mRNA expression was restricted to the macrophage cell lines RAW264.7 and J774a and is expressed at levels ∼50-fold higher than that of Nramp2 . Thus, Nramp1 and Nramp2 mRNAs are coexpressed in macrophages. Section title: Results Educational score: 4.34296989440918 Domain: biomedical Document type: Study Language: en To analyze Nramp2 protein expression in macrophages, membrane fractions were prepared from thioglycolate- induced primary mouse macrophages, from J774a and RAW 264.7 cultured macrophages. For comparison, membranes were also prepared from cell lines derived from tissues previously shown to express a high level of Nramp2 mRNA: the Sertoli cell line TM4 and the kidney inner medullary collecting duct line mIMCD-3 ( 17 ). Membranes were also prepared from control and Nramp2-transfected CHO cells, as well as from two cell lines expressing low levels of Nramp2 mRNA . The membranes were analyzed by immunoblotting with the anti-Nramp2 antibody . The antibody detected a major heterogeneous immunoreactive protein species of broad electrophoretic mobility with an apparent molecular mass of 80–90 kD in all cells tested, with the exception of WEHI 231 cells and untransfected CHO cells. The protein was most abundant in TM4, RAW 264.7, J774a and MEL cells. The protein was also detected in the membranes prepared from primary mouse macrophages and mIMCD-3 cells, although at a lower level. WEHI 3B membranes showed the lowest level of Nramp2 expression, whereas WEHI 231 and untransfected CHO cells were negative for Nramp2 expression. In the positive membrane samples, the electrophoretic mobility and heterogeneity of the immunoreactive species varied, possibly due to different posttranslational modification of the protein in these cell types. Thus, Nramp2 is expressed in a wide variety of tissues, including macrophages, and macrophages coexpress Nramp1 and Nramp2. Section title: Results Educational score: 4.467199325561523 Domain: biomedical Document type: Study Language: en The apparent mass of endogenous Nramp2 estimated by SDS-PAGE is considerably greater than the 62.3 kD molecular mass predicted by the primary amino acid sequence of the cDNA. Together with the broadness of the immunoreactive band, this anomalous mobility suggests that Nramp2 may be posttranslationally modified by glycosylation. To test this hypothesis, membrane fractions from J774a cells and Nramp2-transfected CHO cells were treated with endoglycosidases followed by electrophoresis and immunoblotting. Nramp2 was resistant to digestion with Endo H , which specifically cleaves high mannose and some hybrid N-linked oligosaccharides from glycoproteins. In contrast, PNGaseF, which hydrolyzes high mannose, hybrid, and complex oligosaccharides, converted the 82-kD Nramp2 species into smaller forms of approximate apparent molecular masses of 50–55 kD . PNGase treatment of membranes from the Nramp2 CHO transfectants also resulted in a shift of the apparent molecular mass of the protein from 85 to ∼56 kD . Therefore, Nramp2 is posttranslationally modified extensively by complex N-linked glycosylation. Section title: Results Educational score: 4.440079212188721 Domain: biomedical Document type: Study Language: en To gain insight into the subcellular localization of Nramp2, we first performed immunofluorescence studies on CHO and RAW 264.7 transfected cells, using an antibody directed against the c-myc epitope attached to the carboxy terminus of the transfected Nramp2 protein. The high levels of transfected protein in these cell lines facilitated nonambiguous localization of Nramp2, without possible limitation associated with low levels of expression of the endogenous protein. We have previously observed that the c-myc –tagged Nramp2 protein is functional in both CHO and RAW macrophage backgrounds and carries out active Fe 2+ transport in these cells (Govoni, G., and P. Gros, unpublished results). We initially tested whether Nramp2 localizes to the late endosomal/lysosomal compartment, as found earlier for Nramp1 ( 9 ). To label the lysosomal compartment, cells were cultured in the presence of FITC–conjugated dextran, followed by a chase period of 30 min to remove the dextran from the early endosomal compartments, before fixation and immunostaining with the anti– c-myc antibody. In Nramp1-transfected CHO cells, there was clear colocalization of the anti– c-myc staining and the dextran-loaded late endosomal/lysosomal compartment . Nramp1-stained vesicles negative for FITC–dextran were also detected. In contrast, in Nramp2-transfected CHO cells, anti– c-myc staining revealed an intracellular network of finer punctate vesicles distributed throughout the cytoplasm . This staining does not appear to colocalize with the FITC–dextran . Similar results were obtained in parallel experiments using the c-myc –Nramp1 and c-myc –Nramp2-transfected RAW cells (data not shown), indicating that Nramp2 is not expressed in the lysosomal compartment. Thus, Nramp1 and Nramp2 clearly appear to have distinct, nonoverlapping subcellular sites of expression. Section title: Results Educational score: 4.540614604949951 Domain: biomedical Document type: Study Language: en Since Nramp2 is implicated in cellular iron uptake, it appears logical that Nramp2 be present at the plasma membrane and/or in recycling endosomes. To label these compartments, CHO and RAW transfected cells were cultured in the presence of FITC–conjugated transferrin before fixation and immunostaining with the anti– c-myc antibody. Analysis by confocal microscopy indicated that, as expected, transferrin (green) stained both the plasma membrane (ring-like staining at the edge of the cells) and the recycling endosomes (subcellular punctate staining) . A very similar and overlapping pattern was observed for Nramp2, as revealed by the anti– c-myc antibody . Superimposition of the two images clearly identifies colocalization (yellow) of the two signals. Certain cells stained with FITC–transferrin but were negative for the c-myc staining, suggesting that although these cells are positive for the pSV2neo plasmid and are resistant to G418, they failed to express c-myc –tagged Nramp2. Such cells provide an internal control for the specificity of the anti– c-myc staining and for the overlapping staining with FITC–transferrin. Similarly, when untransfected CHO cells were identically processed and examined, the cells were not stained with the anti– c-myc antibody , but displayed a normal pattern of staining with FITC–transferrin . Finally, when the lysosomes of the Nramp2-transfected cells were stained with FITC–dextran and with the anti– c-myc antibody for Nramp2 , no significant overlap between the two signals was detected . A similar colocalization of Nramp2 and FITC–transferrin was noted in parallel experiments with RAW macrophages expressing c-myc – Nramp2 . These results confirm that Nramp1 and Nramp2 have nonoverlapping, subcellular localization, and that Nramp2 colocalizes with transferrin in the early recycling endosomal compartment. Section title: Results Educational score: 4.130558967590332 Domain: biomedical Document type: Study Language: en To confirm the endosomal localization of Nramp2 determined in transfected CHO and RAW cells, we performed immunofluorescence in nontransfected cell lines that tested positive (MEL, TM4) or negative (WEHI 231) for Nramp2 expression by immunoblotting . Immunofluorescence was performed with the anti-Nramp2 polyclonal antiserum and FITC–transferrin, and results are shown in Fig. 6 . In MEL and TM4 (C and D) cells, the endogenous Nramp2 protein (B and D) showed very similar staining pattern to that generated by FITC–transferrin (A and C), similar to that seen in transfected cells . Finally, in agreement with the absence of Nramp2 expression in WEHI 231 cells noted by immunoblotting , no Nramp2 staining was observed in WEHI231 cells , although the endosomal compartment of these cells could readily be labeled by FITC–transferrin . Together, these results verify data obtained in transfected CHO and RAW cells. Section title: Results Educational score: 4.445708751678467 Domain: biomedical Document type: Study Language: en The localization of Nramp2 to the plasma membrane and endosomal network raised the possibility that like Nramp1, Nramp2 may become associated with phagosomal membranes after phagocytosis. Phagosomes are initially derived from the plasma membrane and are known to sequentially interact and acquire proteins from both early and late endosomes before their final fusion with lysosomes ( 29 ). We have shown that Nramp1 is acquired during the phagosomal maturation process, using the model system of latex bead–containing phagosomes ( 9 ). Latex bead–containing phagosomes are ideal for microscopic examination and can also be purified from cell homogenates by flotation on sucrose gradients. To determine whether Nramp2 can associate with the phagosomal membrane, J774a cells were fed latex beads for 1 h at 37°C, and the phagosomal fraction was isolated from cell homogenates by fractionation on sucrose gradient. Equal amounts of the purified phagosomal fraction and of a crude total membrane fraction were separated by SDS-PAGE, and the relative amount of endogenous Nramp2 in each sample was determined by immunoblotting. As shown in Fig. 7 A (left), Nramp2 was significantly enriched in purified phagosomes as compared to the crude membrane preparation. In these experiments, the Lamp1 protein (marker of the phagolysosome, center) and the transferrin receptor (marker of the plasma membrane, right) were used as controls. As expected, significant enrichment of Lamp1 was seen in the phagosomal fraction whereas the transferrin receptor was not enriched in phagosomes even though it is readily detectable in crude membrane fractions. These results suggest that Nramp2 becomes associated with the phagosome during its maturation to phagolysosome. Possible association of Nramp2 with latex beads phagosomes was further analyzed in J774a cells by immunofluorescence and confocal microscopy. J774a cells were fed latex beads and then fixed and processed by double immunofluorescence using anti-Nramp2 and anti-Lamp1 antibodies . In J774a cells, the Nramp2 signal obtained with our antibody was weak, which limited the analysis. Nevertheless, in several of the sections analyzed a portion of the Nramp2 signal could clearly be seen at the periphery of the bead, suggesting association with the phagosome. However, this signal was much weaker than that obtained using the anti-Lamp1 antibody. Thus, results from immunoblotting and immunofluorescence suggest the possibility that a portion of the endosomal Nramp2 protein becomes associated with the phagosome during phagolysosome maturation. Section title: Discussion Educational score: 4.712950706481934 Domain: biomedical Document type: Study Language: en A large body of biochemical data supports the proposal that Nramp2 functions as a transporter for several divalent cations, including Fe 2+ ( 12 – 14 , 23 , 31 ). Nramp2 is mutated in the mk mouse and in the Belgrade rat ( 13 , 14 ), with both animals exhibiting a severe microcytic hypochromic anemia and a severe defect in iron absorption by intestinal cells ( 15 , 16 ). However, in vitro studies have shown that iron acquisition is also decreased in the peripheral cells and tissues of these animals ( 17 , 32 – 36 ) and that the anemia cannot be corrected by direct iron injections ( 37 ), suggesting a second block of iron entry into peripheral tissues. Thus, physiological consequences of Nramp2 mutations in vivo strongly suggest that Nramp2 is not only involved in iron uptake at the level of the intestinal enterocyte but also participates in iron acquisition in other cell types as well. In peripheral tissues, cellular iron uptake is through the transferrin cycle (for review see reference 38 ). Diferric transferrin binds to the transferrin receptor and is internalized, and acidification of the internalized vesicles results in release of iron from transferrin followed by alkalinization of the vesicles and recycling of the receptor to the cell surface ( 39 ). Iron escapes the acidified endosomal compartment to reach the cytoplasm, where it can be captured by mitochondria for heme biosynthesis and incorporation into heme-containing proteins, stored in the cytoplasm in the form of ferritin, and/or used directly for synthesis of nonheme-containing proteins (e.g., ribonucleotide reductase). The mechanism by which iron is extruded from the acidified endosome to enter the cytoplasm is unknown and has been a matter of considerable debate. Section title: Discussion Educational score: 4.526165962219238 Domain: biomedical Document type: Study Language: en In the current study, we have raised isoform specific anti-Nramp2 antiserum and have used it to verify a number of structural and biochemical features of Nramp2 predicted from the primary amino acid sequence deduced from the cDNA. These analyses have shown that Nramp2 is an integral membrane protein which is extensively modified by N-linked glycosylation. As opposed to Nramp1, which is macrophage-specific, Nramp2 protein was found ubiquitously expressed in a majority of cell lines analyzed. The current study has also clearly established that Nramp2 and Nramp1 localize to distinct subcellular compartments. Whereas Nramp1 colocalizes with FITC–dextran in the lysosomal compartment, Nramp2 is not detectable in this compartment but rather shows clear colocalization with FITC–transferrin both at the plasma membrane and in recycling endosomes. The demonstration of Nramp2 expression in several peripheral tissues, the colocalization of Nramp2 and transferrin in plasma membrane and recycling endosomes, the iron transport properties of the Nramp2 protein, and the effect of Nramp2 mutations on iron metabolism in peripheral tissues are strong evidence that Nramp2 is responsible for transporting Fe 2+ into the cytoplasm after acidification of the transferrin-positive endosome . Interestingly, this acidification would simultaneously provide a gating mechanism for iron transport by Nramp2 and for release from transferrin . Indeed, the pH dependence of iron transport by Nramp2 has been demonstrated in several systems, including Xenopus oocytes ( 12 ), transfected HEK293T cells ( 14 ), and CHO cells (Govoni, G., and P. Gros, unpublished results). Likewise, release of iron from transferrin and its subsequent release from endosomes is dependent on endosome acidification, which can be inhibited by bafilomycin ( 40 ) and concanamycin ( 41 ), specific inhibitors of the vacuolar H + –ATPase, but is insensitive to the Na + , K + –ATPase inhibitor ouabain ( 41 , 42 ). This suggests a critical role for the vacuolar H + – ATPase in this process. Indeed, the association of vacuolar H + –ATPase with transferrin-positive endosomal vesicles has been demonstrated by immunohistochemical means in LLC–porcine kidney epithelial 1 cells ( 43 ). Section title: Discussion Educational score: 4.476678371429443 Domain: biomedical Document type: Study Language: en The demonstration that Nramp1 and Nramp2 are coexpressed in the same cell type with distinct subcellular localizations suggests possible functional parallels between Nramp1 and Nramp2 . Both the yeast Smf1 and the mammalian Nramp2 proteins can transport Mn 2+ , with the latter also transporting Fe 2+ and other divalent cations. Nramp2 and Smf1 share approximately 40% sequence identity within the conserved hydrophobic core ( 18 ). As the mammalian Nramp1 and Nramp2 proteins share almost 80% sequence identity within their hydrophobic cores, it is likely that Nramp1 is involved in the transport of divalent cations as well. The removal of such metabolically essential ions from the phagosomal space would provide an attractive explanation for the observed pleiotropic effect of Nramp1 mutations in vivo on the replicative potential of internalized microbes that inhabit the phagosomal space in macrophages. Additionally, the observation that a portion of Nramp2 associates with latex bead phagosomes in J774a cells suggests that Nramp2 may also play a role in depleting the phagosomal space of divalent cations necessary for microbial survival. Section title: Discussion Educational score: 4.4330925941467285 Domain: biomedical Document type: Study Language: en Despite considerable efforts, transport studies in CHO and RAW cells transfected and overexpressing Nramp1 protein have so far failed to demonstrate an Nramp1-mediated transport of either 54 Mn or 55 Fe transport in these cells (Govoni, G., and P. Gros, unpublished results). The distinct, nonoverlapping distribution of Nramp1 and Nramp2 reported here provides an explanation for this apparent lack of transport activity associated with Nramp1. While the two proteins may have the same transport potential, the observed targeting of Nramp2 to the plasma membrane and recycling endosome compartment would result in a net increase in cellular accumulation of extracellularly added, radiolabeled ligand under acidic pH transport assay conditions. On the other hand, the restricted expression of Nramp1 to the lysosomal compartment would not cause a similar increased cellular uptake of a ligand presented in the extracellular milieu, although it could act on such ligand if present in the phagolysosomal space. Therefore, it is tempting to speculate that both Nramp1 and Nramp2 have similar transport function but act at different, nonoverlapping, intracellular sites . If Nramp1 and Nramp2 do indeed transport the same substrates, it is also tempting to speculate that vesicular acidification via the vacuolar H + – ATPase may provide a key common gating mechanism for the activation of both transporters, through fusion with vacuolar H + –ATPase-positive vesicles . Possible similarities and differences in the mechanism of action and regulation of Nramp1 and Nramp2 in macrophages are currently being investigated. | Study | biomedical | en | 0.999997 |
10049948 | Section title: Materials. Educational score: 3.5994789600372314 Domain: biomedical Document type: Study Language: en Human recombinant IL-1β, TNFα, and IFNγ were obtained from Endogen . Phorbol-12 myristate 13-acetate (PMA) and polymyxin B were purchased from Sigma Chemical Co. Human recombinant CD40L (rCD40L) was generated as described previously ( 44 ) and the mouse anti–human stromelysin-3 antibody 5ST-4A9 was produced in a program sponsored by Bristol Myers Squibb and is subject of an issued U.S. utility patent number 5484726 ( 45 ). Experiments employing rCD40L were performed in the presence of polymyxin B. Anti-CD40L, a rat mAb IgG2 antibody raised against mouse CD40L was prepared as described ( 46 ) and provided by Immunex Corp. Rat IgG salt-free crystalline powder ( Sigma Chemical Co. ), reconstituted in pyrogen-free normal saline and sterile filtered, was obtained from Immunex Corp. Both rat anti–mouse CD40L antibody and rat IgG contained <2 pg/μl of endotoxin. Anti–human CD40 as well as control IgG1 mAb (FITC conjugated) used for immunohistochemistry were obtained from PharMingen . Low density lipoprotein receptor–deficient (LDLR −/− ) mice (B6/129LDRr-tm1Her) were obtained from Jackson Laboratory . Section title: Cell Isolation and Culture. Educational score: 4.18931245803833 Domain: biomedical Document type: Study Language: en Human vascular EC were isolated from saphenous veins by collagenase treatment (1 mg/ml; Worthington Biochemicals) and cultured in dishes coated with fibronectin (1.5 μg/cm 2 ; New York Blood Center Reagents). Cells were maintained in medium 199 (M199; BioWhittaker) supplemented with 1% penicillin/streptomycin (BioWhittaker), 5% fetal bovine serum (FBS) (Atlanta Biologicals), 50 μg/ml heparin ( Sigma Chemical Co. ), and endothelial cell growth factor (Pel-Freez Biological). SMC were isolated from human saphenous veins by explant outgrowth ( 47 ) and cultured in DMEM (BioWhittaker) supplemented with 1% l -glutamine (BioWhittaker), 1% penicillin/streptomycin, and 10% FBS. Both cell types were subcultured following trypsinization (0.5% trypsin [Worthington Biochemicals]/0.2% EDTA [EM Science]) in 75 cm 2 culture flasks ( Becton Dickinson ) and used throughout passages two to four. Culture media and FBS contained <40 pg endotoxin/ml as determined by chromogenic Limulus amoebocyte assay analysis . EC and SMC were characterized by immunostaining with anti von Willebrand factor and anti SMC α-actin antibody (Dako), respectively. Both cell types were cultured 24 h before the experiment in media lacking FBS: vascular EC were cultured in M199 supplemented with 0.1% human serum albumin and vascular SMC cultured in insulin/transferrin medium as described previously ( 48 ). Section title: Cell Isolation and Culture. Educational score: 4.137101173400879 Domain: biomedical Document type: Study Language: en Mononuclear phagocytes were isolated by density gradient centrifugation ( 49 ), using lymphocyte separation medium (Organon-Teknika), and subsequent counterflow elutriation from freshly prepared human PBMCs obtained from leukopacs of healthy donors (provided by Steve K. Clinton, Dana-Farber Cancer Institute, Boston, MA). Mononuclear phagocytes were used directly (monocytes) for the experiments or cultured for 1, 3, or 9 d (MØ) in RPMI 1640 containing 2% human serum ( Sigma Chemical Co. ). The purity of monocytes/MØ was ≥96%, as determined by FACS ® analysis (anti–human CD68 mAb FITC; PharMingen ). For certain studies, MØ were stimulated in RPMI 1640 lacking serum. Section title: Cell Isolation and Culture. Educational score: 4.113386154174805 Domain: biomedical Document type: Study Language: en Freshly isolated human CD4 + T cells were a gift from Dr. F.W. Luscinskas (Brigham and Women's Hospital, Boston, MA). The purity of CD4 + T cells was ≥98%, as determined by FACS ® analysis (anti–human CD4 mAb FITC; Calbiochem ). Human CD4 + T cells were activated with PMA (50 ng/ml, 12 h) and CD40L cell surface expression confirmed by FACS ® analysis using FITC-labeled anti–human CD40L mAb ( Calbiochem ). Finally, activated CD4 + T cell membranes were prepared as described previously ( 42 ) and used for stimulation at a ratio of T cells to SMC/EC/MØ of 10:1. Section title: Immunohistochemistry. Educational score: 4.288313865661621 Domain: biomedical Document type: Study Language: en Surgical specimens of human carotid atheroma and aorta were obtained by protocols approved by the Human Investigation Review Committee at the Brigham and Women's Hospital. Serial cryostat sections (5 μm) were cut, air dried onto microscope slides ( Fisher Scientific ), and fixed in acetone at −20°C for 5 min. Sections were preincubated with PBS containing 0.3% hydrogen peroxidase activity. The sections were then incubated (90 min) with primary or control (mouse myeloma protein MOPC-21; Sigma Chemical Co. ) antibody diluted in PBS supplemented with 5% appropriate serum. After washing three times in PBS, sections were incubated with the respective biotinylated secondary antibody (45 min; Vector) followed by avidin–biotin–peroxidase complex (Vectastain ABC kit; Vector), and antibody binding was visualized with 3-amino-9-ethyl carbazole (Vector) according to the recommendations provided by the supplier. For colocalization of stromelysin-3 with CD40 or the respective cell type, double immunofluorescence staining was performed. The anti–human stromelysin-3 mAb (1:200) was applied for 60 min followed by biotinylated anti–mouse secondary antibody for 45 min and Texas red–conjugated streptavidin ( Amersham ). Subsequent to application of the avidin–biotin blocking kit (Vector), rabbit–anti human CD40 antibody (1:250; Santa Cruz), anti-muscle actin mAb for SMC (Enzo Diagnostics), anti-CD31 mAb for EC (1:400, Dako), or anti-CD68 mAb for MØ (1:600; Dako) were added and sections incubated overnight at 4°C. Subsequently, the appropriate secondary antibodies were applied for 30 min followed by Streptavidin–FITC ( Amersham ). To detect stromelysin-3 in mouse atherosclerotic lesions, the stromelysin-3 mAb and a specific anti– mouse absorbed rat biotinylated secondary antibody (1:500), followed by avidin–biotin–peroxidase complex, were applied. Section title: Biochemical Analysis of Human Atherosclerotic Lesions. Educational score: 4.06912899017334 Domain: biomedical Document type: Study Language: en Frozen tissue from five nonatherosclerotic arteries and seven atheromatous carotid plaques were homogenized (IKA-Labortechnik, Ultra-turrax T 25) and lysed (0.3 mg tissue/ml lysis buffer) as described previously ( 5 ). The lysates were clarified (16,000 g , 15 min) and the protein concentration for each tissue extract as well as for the cell culture samples was determined using a bicinchoninic acid protein assay according to the instructions of the manufacturer (Pierce). 50 μg total protein were applied to Western blot analysis. Section title: In Situ Hybridization. Educational score: 4.2434234619140625 Domain: biomedical Document type: Study Language: en In situ hybridization was performed according to the instructions of the manufacturer (Hyb-Probe™; Shandon/Lipshaw). Frozen tissue sections, obtained as described above, were fixed in cold acetone, air-dried, and incubated with a mixture of FITC labeled stromelysin-3–specific (5′-GGTACCGTCAACCAGGTCCTCGTCCACG-3′; 5′-CTCAGAGTCGGGTCTACTGACCGTCC-3′; 5′-CCTACTGGTCCCGTGTCTGGACGACGTCCA-3′; 5′-ACGGTCCGGTGCTTATAGTCC-GATCTCCTGG-3′), CD40L-specific (5′-TTATGGGTGTCAA-GGCGGTTTGGAACGCCCGTT-3′; 5′-TGAAAAACGACA-CATAGAAGTATCTTCCAA-3′; 5′-TACTAGCTTTGTATGTTGGTTTGAAGAGGGGCT-3′; 5′-TGCAGGAAACCGAATGAGTTTGAGACTTGT-3′), or random oligomers in hybridization buffer (30% formamide, 0.6 M NaCl 2 , 10% dextran sulfate, 50 mM Tris [pH 7.5], 0.1% sodiumpyrophosphate, 0.2% Ficoll, 5 mM EDTA) for 10 min at 65°C and subsequently for 2 h at 37°C in a moist chamber. Thereafter, slides were immersed in TBS/ Triton (50 mM Tris, 150 mM NaCl, pH 7.6/0.1% Triton X-100) to allow coverslips to float off and were washed (TBS/ Triton) three times for 3 min at 37°C. The slides were forwarded to the immunological reaction by incubation with blocking solution (10 min, rt) and subsequent addition of the alkaline phosphatase–conjugated rabbit Fab′ anti FITC (30 min, rt, moist chamber) as the primary detection reagent. Finally, the slides were washed twice in TBS (3 min, rt), covered with alkaline phosphatase substrate buffer (5 min, rt), and developed using the NBT/BCIP (nitro-blue-tetrazolium/5-bromo-4-chloro-3-indolyl phosphate) chromogen solution (1–2 h, rt, moist chamber). Section title: Western Blotting and Radioimmunoprecipitation. Educational score: 4.116772651672363 Domain: biomedical Document type: Study Language: en Cell extracts (25 μg total protein/lane) and culture supernatants were separated by standard SDS-PAGE under reducing conditions and blotted to polyvinylidene difluoride membranes (Bio-Rad) using a semidry blotting apparatus (0.8 mA/cm 2 , 30 min; Bio-Rad). Blots were blocked and first and second monoclonal antibodies were diluted in 5% defatted dry milk/PBS/0.1% Tween 20. After 1 h of incubation with the respective primary antibody, blots were washed three times (PBS/0.1% Tween 20) and the secondary, peroxidase–conjugated, goat anti–mouse antibody (Jackson ImmunoResearch) was added for another hour. Finally, the blots were washed (20 min, PBS/0.1% Tween 20) and immunoreactive proteins were visualized using the Western blot chemiluminescence system (NEN™). Section title: Western Blotting and Radioimmunoprecipitation. Educational score: 4.199478626251221 Domain: biomedical Document type: Study Language: en For radioimmunoprecipitation experiments, cells were washed and further incubated with unlabeled medium lacking methionine/cysteine. Subsequently, medium containing 10% FBS and 50 mCi/ml [ 35 S]methionine/cysteine (NEN™) was added to the cells for 24 h. Supernatants were harvested and concentrated (10×) using Centricon 3 devices (Amicon). Immunoprecipitation buffer (50 mM Tris-HCl, 0.1% SDS, 0.1% sodium deoxycholate, 1% NP-40, 150 mM NaCl, 5 mM EDTA, 20 μg/ml soybean trypsin inhibitor, 0.1% mM PMSF, 0.2 U/ml aprotinin, 0.025% sodium azide) was added to the cultures and cells were harvested by scraping. Subsequently, non immune mouse serum (Vector) was added (24 h, 4°C) to preclear the samples. Antigens in supernatants and cell extracts were immunoprecipitated with the specific anti stromelysin-3 antibody 5ST-4A9 (2 h, 4°C) and pelleted by subsequent addition of rabbit anti–mouse IgG (18 h, 4°C) as well as protein A–Sepharose beads (2 h, 4°C). The beads were washed four times in 50 mM Tris-HCl and finally 50 μl SDS-PAGE loading buffer (200 mmol/liter Tris, 5% glycerol, 0.1% SDS, 3% β-mercaptoethanol, 0.1 mg/ml bromophenol blue) was added. After heating the samples 5 min at 95°C, supernatants were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Blots were dried and exposed to x-ray film for detection of the immunoprecipitated antigen. Section title: Isolation of RNA and PCR. Educational score: 4.158265590667725 Domain: biomedical Document type: Study Language: en Total RNA from unstimulated or stimulated (24 h) EC, SMC, or MØ was isolated by a one-step preparation according to the method of Chomczynski and Sacchi ( 50 ). The cDNA was prepared by reverse transcription (RT) of total RNA (1 μg) with oligo(dT) using superscript reverse transcriptase ( GIBCO BRL ). The RT products were diluted in 480 μl twice distilled water and 10 μl of these cDNA preparations were mixed on ice with 10 μl primers (20 μM), 80 μl reaction mix (including 10 ml of PCR buffer, 2.5 mM MgCl), 4 μl dNTPs (200 μM; final concentration), and 0.5 μl Taq-polymerase (2.5 U; all GIBCO BRL ). PCR was performed for 35 cycles at 95°C (120 s), 62°C (120 s), and 72°C (180 s, 2 s prolongation per cycle) after hot start. The sequences of primers for human stromelysin-3 were 5′-CTGCAGTCATCTGGGCTGAGACTC-3′ and 5′-CCATGGCAGTTGGTGCAGGAGCAG-3′. The primers were obtained from Integrated DNA Technologies. Aliquots of the PCR products were run on 1.3% agarose gels and visualized by UV transillumination. Section title: In Vivo Analysis of CD40L-mediated Stromelysin-3 Expression. Educational score: 4.0930328369140625 Domain: biomedical Document type: Study Language: en LDLR −/− at a minimum age of 8 wk were fed a high cholesterol diet (1.25% cholesterol, 0% cholate; Research Diets) and treated for a period of 12 wk with either rat IgG (control) or rat anti– mouse CD40L (M158) antibody (both at 250 μg per mouse twice a week intraperitoneal; both provided by Immunex Corp. ) ( 51 ). Thereafter, the mice (10 per group) were killed, the aortic arches embedded, and tissue sections examined by immunohistochemistry as described below. The extent of lesion formation and the expression of vascular adhesion molecule-1 in these animals has been reported separately ( 51 ). No difference in the serum lipid profiles (total cholesterol, very low density lipoprotein, low density lipoprotein, and high density lipoprotein [analyzed by fast protein liquid chromatography] or triglycerides) or in circulating leukocytes, hematocrit, or body weight was observed among the groups. Section title: Expression of Stromelysin-3 in Human Atherosclerotic Plaques. Educational score: 4.416900634765625 Domain: biomedical Document type: Study Language: en Immunohistochemical analysis of the expression of stromelysin-3 in normal aortic specimens and human atherosclerotic fatty streaks ( n = 5; data not shown) revealed little or no expression of the enzyme. In contrast, well-developed human carotid atherosclerotic lesions ( n = 7) consistently showed strong stromelysin-3 immunoreactivity, most prominently at the luminal border and in the shoulder region of the plaque . Western Blot analysis, performed on protein extracts of the surgical specimens and using the identical antibody used for the immunohistochemistry studies, revealed barely detectable immunoreactive stromelysin-3 in control specimens but markedly increased levels of the proteinase in atherosclerotic tissue . The immunoreactive bands detected had molecular masses of ∼64, 48, 35, and 28 kD, corresponding to the zymogen, intermediate, and active forms of stromelysin-3 (9, 10, 22, 23, 52, and 53). Higher magnifications of the immunohistochemical analysis, as well as immunofluorescent double staining with respective cell-selective antibodies, localized stromelysin-3 within EC, SMC, and MØ of the plaque . Tissues showed no staining with the respective control IgG1 antibody (data not shown). Because we recently localized CD40 and CD40L in human atherosclerotic plaques and have shown that CD40 ligation induces interstitial collagenases and gelatinases in atheroma-associated cells ( 41 – 43 ), we investigated the possible colocalization of stromelysin-3 with CD40. Indeed, cells expressing stromelysin-3 also bear CD40 . Furthermore, we analyzed the cellular localization of stromelysin-3 transcripts by in situ hybridization . Human atheroma , but not normal arteries , contained stromelysin-3 mRNA. Within the atherosclerotic lesion, stromelysin-3 transcripts localized most prominently at the luminal border and the shoulder region of the plaques, areas described above as positive for the immunoreactive protein. The staining for the transcripts colocalized with smooth muscle cell- and macrophage-like cells as well as the endothelium . Furthermore, transcripts for the immune mediator CD40L showed a similar distribution on adjacent sections . In situ hybridization with negative control probes did not yield any signal . Section title: Ligation of CD40 on Human Vascular Endothelial and SMC as well as MØ Induces De Novo Expression of Stromelysin-3. Educational score: 4.267655849456787 Domain: biomedical Document type: Study Language: en To determine the mechanisms involved in stromelysin-3 expression in atheroma-associated cells, we analyzed the expression of the enzyme in human vascular EC and SMC as well as in human mononuclear phagocytes in vitro. Human vascular EC, SMC, and MØ released immunoreactive stromelysin-3 constitutively in moderate amounts. Stimulation of the cells with the classical mediators of MMP regulation, IL-1 (1 and 10 ng/ml), TNF-α (5 and 50 ng/ml), or IFNγ (100 and 1,000 U/ml), did not affect the expression of stromelysin-3, as illustrated here for IL-1 and SMC . However, the immunohistochemical studies presented above indicated possible involvement of the CD40– CD40L signaling pathway. Stimulation with both T cell– derived as well as recombinant human CD40L induced stromelysin-3 expression in all three cell types . Besides an increased intensity of the higher (∼64 kD) molecular mass band, which corresponds to the molecular mass of the stromelysin-3 zymogen, CD40 ligation induced the expression of immunoreactive proteins of lower molecular mass, corresponding to the molecular mass of the active cleavage products of the zymogen . In control experiments, addition of blocking anti-CD40L antibody during stimulation of the cells with native or recombinant ligand inhibited induction of stromelysin-3 expression. Section title: Ligation of CD40 on Human Vascular Endothelial and SMC as well as MØ Induces De Novo Expression of Stromelysin-3. Educational score: 4.262805461883545 Domain: biomedical Document type: Study Language: en Among the cell types analyzed, the immunoreactive forms of stromelysin-3 detected after CD40 ligation showed slight differences in the intensity of the respective bands but were identical in molecular mass. In vascular SMC, CD40 ligation concentration and time dependently elevated expression of the 64-kD protein, as well as induced immunoreactive proteins with molecular masses of ∼48 and ∼28 kD. The induction of the lower molecular mass forms of stromelysin-3 in cultures of SMC required stimulation with 3–10 μg/ml rCD40L . An increase in the 64-kD protein, as well as in the ∼48-kD immunoreactive protein, occurred after 1 h of stimulation, whereas detection of the 28-kD band required at least 6–12 h of exposure to CD40L, as determined by Western blot analysis (data not shown) and radioimmunoprecipitation experiments . The patterns of immunoreactive proteins detected in cultures of vascular smooth muscle cells resembled those found in fibroblasts, an established source of stromelysin-3 (data not shown). The pattern of immunoreactive bands observed with supernatants of CD40L-stimulated EC resembled that obtained with cultures of SMC, except that the 64-kD form was much less abundant in EC. Section title: Ligation of CD40 on Human Vascular Endothelial and SMC as well as MØ Induces De Novo Expression of Stromelysin-3. Educational score: 4.1713151931762695 Domain: biomedical Document type: Study Language: en Macrophages, derived from monocytes cultured for 9 d, showed an additional immunoreactive protein at ∼35 kD. Stimulation with ≥0.3 μg/ml rCD40L increased levels of immunoreactive proteins with apparent molecular masses of 64, 48, 35, and 28 kD . Induction of these immunoreactive bands required at least 6 h of stimulation (data not shown). Specificity of the antibody used was confirmed by Western blot analysis performed with the antistromelysin-3 antibody preincubated with recombinant stromelysin-3 . In contrast to monocyte-derived MØ cultured for 9 d, freshly isolated peripheral blood monocytes expressed stromelysin-3 neither constitutively nor when stimulated with rCD40L . Responsiveness of monocyte-derived cells to CD40 ligation required a minimum of three days of in vitro culture (data not shown). Section title: Ligation of CD40 on Human Vascular Endothelial and SMC as well as MØ Induces De Novo Expression of Stromelysin-3. Educational score: 4.183557987213135 Domain: biomedical Document type: Study Language: en The stromelysin-3 expression induced by CD40 ligation in human vascular EC, SMC, and MØ resulted from de novo synthesis of the protein. Metabolic labeling and immunoprecipitation experiments yielded autoradiographic bands resembling the patterns of immunoreactive proteins observed by Western blot analysis as shown here for vascular SMC . In accordance with our protein analysis, RT-PCR experiments showed increased product corresponding to stromelysin-3 transcript after CD40 ligation in EC and SMC as well as MØ (data not shown). Section title: Interruption of CD40–CD40L Signaling Diminished Stromelysin-3 Expression in Mouse Atherosclerotic Lesions. Educational score: 4.172612190246582 Domain: biomedical Document type: Study Language: en The in situ observations of stromelysin-3 expression in human atherosclerotic plaques, in combination with the in vitro findings that CD40 ligation selectively mediates the expression of stromelysin-3, suggested an in vivo evaluation of the importance of CD40–CD40L signaling for the expression of this enzyme within atherosclerotic plaques. For this purpose, an established animal model of arteriosclerosis was used: LDLR −/− mice were fed a high cholesterol (1.25%) diet to develop atherosclerotic lesions ( 51 ). Immunohistochemical analysis of lesions within the aortic arch as well as the thoracic portion of the aorta revealed a stromelysin-3 expression pattern similar to that found in human atherosclerotic plaques. All three vascular cell types—EC, SMC and MØ—within the lesions stained for the proteinase. Treatment of the mice with rat IgG ( n = 8) did not affect the stromelysin-3 expression compared to controls. In contrast, mice treated with the anti–mouse CD40L antibody ( n = 8) showed substantially reduced immunoreactivity for stromelysin-3 within the atherosclerotic lesions . Since the anti-CD40L antibody treatment also resulted in a decrease of total plaque number and area ( 51 ), lesions of similar sizes within the treatment groups were compared. No immunoreactivity was observed in tissues stained with the control IgG1 antibody (data not shown). Section title: Discussion Educational score: 4.761962890625 Domain: biomedical Document type: Study Language: en This study establishes the expression of stromelysin-3 in advanced human atheroma and furthermore provides evidence for (a) colocalization of this matrix metalloproteinase with CD40 on lesional EC, SMC, and MØ in situ and (b) regulation of de novo expression of stromelysin-3 by CD40 ligation, rather than by the classical soluble mediators of MMPs such as IL-1, TNF-α, or IFN-γ. We also demonstrated in vivo that the interruption of CD40– CD40L interaction markedly reduced the expression of stromelysin-3 in mouse atheroma. The finding that EC, SMC, and MØ express stromelysin-3 establishes atheroma-associated cells as novel sources of stromelysin-3. Thus, this report provides evidence that stromelysin-3, whose expression correlates with the invasiveness of malignancies ( 3 , 11 , 12 , 16 , 17 , 54 ), might also participate in the pathogenesis of another common human disease, atherosclerosis. In view of its unusual substrate specificity, stromelysin-3 might participate in several pathways. The pattern of stromelysin-3 expression within tissues that undergo extensive remodeling in pathological (carcinomas; 11, 12, 16, 17, 54) as well as physiological (placenta and uterus; 11, 18) events, indicates a role for stromelysin-3 in these processes. Although related by sequence to the MMP family, stromelysin-3 does not hydrolyze many of the extracellular matrix components that are substrates for other MMPs such as fibronectin, laminin, elastin, or collagen type I and type IV ( 23 ). From this perspective, the enzyme has been considered a nonmatrix–degrading metalloproteinase. Instead, stromelysin-3 appears to act as a predominant regulator of serpin function. Interestingly, a recent report postulated that extracellular matrix degradation might depend not only on an imbalance between matrix metalloproteinases and their inhibitors, but might accelerate in face of decreased levels of certain serpins such as α 2 -M, α 2 -AP, or α 1 -PI ( 33 ), preferred substrates of stromelysin-3 ( 23 ). Pathways by which this enzyme might regulate matrix degradation include (a) inactivation of matrix-degrading, enzyme-inhibiting serpins as described for cathepsins ( 32 ) or for elastolytic activity by α 1 -PI, shown to simultaneously augment the proliferative and invasive activity of cells ( 23 ); (b) activation of other members of the MMP family ( 17 ) copiously expressed in atheroma, such as interstitial collagenase, gelatinases A and B, and stromelysin-1 ( 5 ) (at sites of chronic inflammation, such as atherosclerosis, constitutively active stromelysin-3 might act proximally to promote conversion of the zymogen forms of these classical MMPs to their active forms); and (c) degradation of matrix molecules not cleaved by the classical MMPs such as extracellular proteins containing amino acids with unusual long side chains, including those generated in vivo by certain posttranslational modifications ( 55 ). Thus, matrix degradation, a critical step in the progression from the stable atheroma to one prone to rupture and capable of causing thrombotic complications, might indeed involve the action of stromelysin-3. The serpin-degrading function of stromelysin-3 may thus have significance beyond tumor invasion and metastasis. Section title: Discussion Educational score: 4.719022750854492 Domain: biomedical Document type: Study Language: en Furthermore, the action of stromelysin-3 on serpins also might affect atherosclerosis by pathways other than extracellular matrix remodeling. Serpins regulate multiple functions associated with the disease, including (a) blood pressure (e.g., the serpin α 1 –antitrypsin inhibits renin and thus renin–angiotensinogen interaction, as well as angiotensinogen itself ), (b) fibrinolysis (e.g., the serpin α 1 –antiplasmin targets plasmin, a key effector of fibrinolysis ), (c) blood coagulation (e.g., the serpins anti-thrombin III and heparin cofactor II rapidly interact with thrombin in the presence of heparin ), or (d) lipoprotein uptake (cleavage of the serpins α 1 -antitrypsin and α 2 -macroglobulin is associated with increased low density lipoprotein uptake into cells, indicating that those cleaved serpins disturb the intracellular cholesterol homeostasis [27–29]). Aside from these effects, stromelysin-3 might further affect atherosclerosis via the insulin-like growth factor–insulin-like growth factor binding protein (IGF–IGFBP) system. Human atheroma contain IGF-1 and IGFBPs ( 56 ). These mediators are associated with cardiovascular pathophysiology particularly via their critical role in vascular growth ( 57 ). IGF-1 directly accelerates arteriosclerosis in rat aorta allografts via myointimal proliferation and intimal thickening ( 58 ). The level of free, biologically active IGF-1 depends on the degree of complex formation with the respective binding proteins. A recent study identified IGFBP-1 as a potential physiological substrate for human stromelysin-3 ( 59 ). Finally, IGF also acts as a survival factor for human vascular SMC derived from normal vessels as well as coronary atherosclerotic plaques ( 60 ), suggesting stromelysin-3 may prevent apoptosis of vascular SMC by augmenting IGF-1 levels. Section title: Discussion Educational score: 4.319143772125244 Domain: biomedical Document type: Study Language: en The potential relevance of serpin degradation for the pathogenesis of atherosclerosis in humans is supported by reports showing that levels of several serpins, including AT-III ( 36 ), α 2 -M, and α 2 -AP ( 35 ), decrease in this prevalent disease. In addition, recent in vivo studies demonstrated that serpins inhibit coronary restenosis in atherosclerotic swine ( 37 ) and atherosclerotic plaque development in rabbits post injury ( 38 ). Our findings that (a) differentiated atherosclerotic lesions but not their precursor, the fatty streak, bear stromelysin-3; (b) the immunoreactive bands detected within the atheromatous plaques resembled mostly the pattern obtained with MØ in vitro; and (c) only differentiated MØ, but not peripheral blood monocytes, exhibit stromelysin-3 induction; support a role of this enzyme in the late rather than the early states of atherosclerosis. Interestingly, the Rotterdam study showed increased AT-III levels in moderate peripheral arterial atherosclerosis and decreased levels in more severe atherosclerosis ( 61 ). Section title: Discussion Educational score: 4.360436916351318 Domain: biomedical Document type: Study Language: en Future studies will have to establish whether these various functions apply to human atherosclerosis and whether stromelysin-3 is the crucial mediator in reduction of all or only certain atheroma-associated serpin activities. However, based on the recently burgeoning background information on stromelysin-3, our findings suggest functions of this proteinase in regulation of plaque progression and (in)stability by mechanisms distinct from those ascribed to members of the MMP family, including augmented extracellular matrix degradation, promotion of blood coagulation, decreased fibrinolytic activity, and dysregulation of blood pressure as well as lipoprotein catabolism. The colocalization of stromelysin-3 with CD40 and CD40L within the human atherosclerotic lesion, as well as the demonstration that CD40L, rather than the classical mediators of MMPs, induce stromelysin-3 expression in atheroma-associated cells demonstrate CD40–CD40L signaling as a new, and, as indicated by the in vivo studies, probably crucial pathway of stromelysin-3 induction. | Study | biomedical | en | 0.999996 |
10049949 | Section title: Reagents. Educational score: 3.8620615005493164 Domain: biomedical Document type: Study Language: en The protein G–Sepharose column–purified antibodies anti–λ1 L chain (Ls136) and FDC-M2 were provided by Garnett Kelsoe (Duke University, Durham, NC) and Marie Kosco-Vilbois (Serono Pharmaceutical Research Institute, Geneva, Switzerland), respectively. Both were conjugated using a biotin coupling reagent ( Boehringer Mannheim ) at a biotin/antibody molar ratio of 10:1 and purified on a Sephadex G-25 column ( Boehringer Mannheim ). Rat anti–mouse IgD and anti–mouse CD11b (Mac-1) from PharMingen were also coupled as above but with digoxigenin (DIG) rather than biotin, also from Boehringer Mannheim . Section title: Reagents. Educational score: 4.051054000854492 Domain: biomedical Document type: Study Language: en Chicken γ-globulin (CG; Sigma ) was conjugated with (4-hydroxy-3-nitrophenyl)acetyl (NP) succinimide ester ( Calbiochem ) in 0.1 M sodium borate, pH 9.2, to a NP/CG molar ratio of 13:1 (NP 13 CG) and then dialyzed against PBS. PE (Molecular Probes) was haptenated in the same fashion but in the dark at a molar ratio of 20:1 to generate NP 20 PE. BSA was also haptenated to create NP 2 BSA and NP 15 BSA. The precise degree of haptenation was not determined. Section title: Animals and Animal Challenge. Educational score: 3.61389422416687 Domain: biomedical Document type: Study Language: en Cd40l −/− mice and ltβ −/− mice are those described previously ( 26 , 35 ). Wild-type control mice were derived from original wild-type littermates of ltβ −/− mice ( 26 ). Tcr(β×δ) −/− mice were generated from breeding pairs originally purchased from The Jackson Laboratory . All mice are on a mixed background of C57BL/6 and 129/Sv and were housed in specific pathogen–free conditions. All experiments were conducted in accordance with Yale University Animal Care and Use guidelines. Section title: Animals and Animal Challenge. Educational score: 3.4857969284057617 Domain: biomedical Document type: Study Language: en Mice at 6–8 wk of age were challenged intraperitoneally with 50 μg of either CG or NP 13 CG, both of which were adsorbed to alum in 0.1 ml PBS. Some mice at various times may have received a second challenge of 200 μg NP 13 CG in 0.1 ml PBS alone. Section title: Animals and Animal Challenge. Educational score: 3.890667200088501 Domain: biomedical Document type: Study Language: en Hyperimmunized mouse serum for the purpose of anti-NP antibody standardization (see below) was obtained from wild-type mice 6 d after being boosted with 0.2 mg NP 13 CG in PBS, 27 d after the primary challenge with 50 μg NP 13 CG adsorbed to alum. Section title: MLN Histology. Educational score: 4.220554351806641 Domain: biomedical Document type: Study Language: en MLNs were frozen in Tissue-Tek OCT compound (VWR Scientific) using a dry-ice/methylbutane bath and stored at −70°C until cutting. Sections of 5-μm thickness were cut onto silanized glass slides, fixed in cold acetone for 10 min, air-dried, and then stored at −70°C until use. For staining, sections were thawed for 30 min and then rehydrated in PBS for 20 min. Endogenous peroxidase was quenched with 0.3% hydrogen peroxide for 5 min. Sections were washed in PBS for 10 min and then preblocked with PBS/3% BSA/0.1% Tween 20 for 30 min in a humidified chamber. Staining for IgD was with rat anti-IgD (Southern Biotechnology Associates) and then horseradish peroxidase–conjugated goat anti–rat IgG (Southern Biotechnology Associates). The presence of FDCs was assessed with biotin-conjugated FDC-M2 (see Reagents). Other biotin-conjugated antibodies were anti-CD23 and anti-CD24, both from PharMingen . All biotin conjugates used a secondary step of alkaline phosphatase–conjugated streptavidin ( Zymed) . Incubations were in a humidified chamber for 1 h. Washes between steps were with PBS/0.1% Tween 20. Substrates for horseradish peroxidase and alkaline phosphatase were diaminobenzidine (brown) and NBT/ BCIP (purple-blue), respectively ( Zymed) . Counter-staining was with nuclear fast red ( Zymed) . Section title: MLN Cell Preparation. Educational score: 4.192620754241943 Domain: biomedical Document type: Study Language: en MLNs were harvested into 0.5 ml cold digest buffer in a 4-well plate (Nunc) on ice. Digest buffer was Bruff's medium with 5% FCS, 0.1 mg/ml collagenase type IV ( Sigma ), and 0.1 mg/ml deoxyribonuclease type I ( Sigma ). Bruff's medium is Click's medium (Irvine Scientific) supplemented with 40 mM l -glutamine, 60 μM 2-ME, 0.7 mM sodium bicarbonate, and 58 mg/l gentamycin. The capsule of each MLN was torn and teased open with 27-gauge needles before incubating plates at 37°C for 30 min. Capsules were then further disrupted to release MLN cells by pipetting. The cell suspension was made up to 10 ml with Bruff's/5% FCS, filtered through 0.1-mm nylon mesh (Small Parts, Inc.), and centrifuged at 800 revolutions/min in a bench-top centrifuge at 4°C for 5 min. Finally, cells were resuspended in 2 ml Bruff's/5% FCS for fluorocytometry or antigen-specific antibody determination as described below. Section title: Fluorocytometry. Educational score: 4.153182029724121 Domain: biomedical Document type: Study Language: en MLN cells were prepared as described above, and aliquots of 10 6 cells were resuspended into 0.2 ml PBS/1% FCS supplemented with 5 μg/ml FcBlock ( PharMingen ). Samples were left on ice for 30 min before primary antibodies were added, and were then left on ice in the dark for an additional 1 h. Samples were washed by being made up to 1.2 ml with PBS/1% FCS before centrifugation at 800 revolutions/min at 4°C for 5 min. Secondary antibody incubation and rewashing were done as above. Four-color fluorocytometry used a FACSCalibur ® with argon and helium-neon lasers ( Becton Dickinson ). Data from 2.5 × 10 5 events were analyzed with CellQuest software by first gating on lymphocytes/lymphoblasts, based on forward and side scatter. PharMingen antibodies used included anti-CD4–allophycocyanin (APC; L3T4, RM4-5), anti-CD8α–APC (53-6.7), anti-CD90.2– APC (53-2.1), GL7-FITC, anti-CD45R–FITC, anti-CD24–biotin (M1/69), and anti-CD45R–biotin. Peanut agglutinin (PNA)- biotin, streptavidin–Cy-Chrome, and anti-digoxin–Cy5 were from Vector, PharMingen , and Jackson Laboratories , respectively. Other reagents used were anti-λ1–biotin, anti-IgD–DIG, and anti-CD11b–DIG, described in Reagents above. Section title: MLN Antigen-specific Antibody Determination. Educational score: 4.216730117797852 Domain: biomedical Document type: Study Language: en Antigen-specific antibody determination was done essentially as described by Kelly et al. ( 36 ), using antibody-secreting cells directly in an ELISA. Maxisorp 96-well plates (Nunc) were coated at 4°C for 16–20 h with 10 μg/ml NP 2 BSA or NP 15 BSA in PBS, 0.1 ml aliquot per well. Plates were then washed four times with PBS before blocking with PBS/1% FCS for 2–3 h. Meanwhile, MLN cells were prepared at 10 7 cells per ml of Bruff's/5% FCS as described above. MLN cell aliquots of 0.1 ml were then plated in wells and cultured at 37°C for 5 h alongside a twofold serial dilution of serum from a standard hyperimmunized wild-type mouse (see above; serum dilution was with PBS/1% FCS). Subsequent washes were with PBS/0.05% Tween 20. Ig detection used isotype-specific alkaline phosphatase–conjugated reagents and pNPP substrate as described (Southern Biotechnology Associates). OD 405 was determined with a microplate reader (model 550; BioRad). Typically, a 1:12,800 dilution of hyperimmunized wild-type mouse serum gave an OD 405 of ∼2.7 and 1.9 above background for IgG1 detected with NP 2 BSA and NP 15 BSA, respectively. The lower limit of detection was typically at a serum dilution of 1:1,638,400, giving an OD 405 of ∼0.05–0.10 above background. The anti-NP–specific antibody titer of samples was expressed as relative units, representing the reciprocal of the standard serum dilution giving the same OD 405 as the sample. Calculations were by four-parameter analysis using Microplate Manager III software (BioRad). Section title: Serum Antigen-specific Antibody Determination. Educational score: 4.084769248962402 Domain: biomedical Document type: Study Language: en Blood was harvested from mice for serum by cardiac puncture at the time of culling. Maxisorp plates were coated with NP 2 BSA or NP 15 BSA, blocked, and washed as described above. Serum aliquots of 0.1 ml (1:10 5 in PBS/1% FCS) were applied to wells for 1 h alongside a twofold serial dilution of serum from a hyperimmunized wild-type mouse (see above; serum dilution was with PBS/1% FCS). Antigen-specific Ig determination then proceeded as described in the previous section. Section title: Ltβ − /− Mice Form GC B Cell Clusters but Lack FDCs. Educational score: 4.211250305175781 Domain: biomedical Document type: Study Language: en We showed previously that ltβ −/− mice form PNA-binding, IgD − B220 + cell clusters in B cell areas of their MLNs ( 26 , 30 ), characteristic of GCs ( 37 ). Although IgD + B cells were also found to infiltrate T cell areas, the GC-like B cell clusters were within IgD + B cell follicles at the periphery of MLNs, as in wild-type mouse MLNs ( 26 , 30 ). These observations were extended here by staining for CD24. Primary follicle B cells are CD24 + , but GC B cells are CD24 hi . This also appeared to be true of ltβ −/− GC B cell clusters . Again, the ltβ −/− GC B cells were clustered within the location expected of wild-type GCs. Section title: Ltβ − /− Mice Form GC B Cell Clusters but Lack FDCs. Educational score: 4.226074695587158 Domain: biomedical Document type: Study Language: en Immunohistology with anti–complement receptor 1 (CR-1) and FDC-M1 suggested that ltβ −/− mice lack FDCs ( 26 ). We subsequently showed that although wild-type mouse MLNs have very large amounts of IgM immune complex on FDCs, ltβ −/− mouse MLNs did not have any such deposits ( 30 ). However, despite all of the above, it is possible that Ig Fc receptor–bearing (FcR + ) FDCs ( 3 , 4 , 9 ) constitute a distinct subset of FDCs which are negative for both FDC-M1 and CR-1. It is also conceivable that antigen–Ig complexes retained on such FcR + FDCs may support GC reaction in the absence of CR-1 + FDCs. In potential support of this, mice deficient in CR-1 still possess GCs ( 41 , 42 ), albeit somewhat smaller than wild-type. Section title: Ltβ − /− Mice Form GC B Cell Clusters but Lack FDCs. Educational score: 4.207141876220703 Domain: biomedical Document type: Study Language: en Therefore, an evaluation was made as to whether or not ltβ −/− mice possess FcR + FDC networks by staining for CD23 ( 43 ). This revealed that ltβ −/− mice completely lack FcR + FDC networks , and that the ltβ −/− GC B cell clusters were CD23 − compared with the surrounding follicle B cells, as expected of wild-type GC B cells . FDC networks clearly stained very strongly for CD23 in wild-type mouse MLNs , and were observed in every B cell follicle regardless of whether or not there was an ongoing GC reaction. Some discrete CD23 lo cells were seen in the ltβ −/− mouse MLN CD23 − IgD − GC cell clusters . These cells most likely represent GC B cells that have not yet fully downregulated CD23, but it is conceivable that they represent immature FDCs or the mouse equivalent of GCDCs ( 31 ). Section title: Ltβ − /− Mice Form GC B Cell Clusters but Lack FDCs. Educational score: 4.176582336425781 Domain: biomedical Document type: Study Language: en Finally, the lack of staining by FDC-M2 further emphasized the complete absence of FDC networks in the MLNs of ltβ −/− mice . Although FDC-M2 has not been fully characterized, it is a useful marker of FDC networks . Like CD23, FDC-M2 staining was observed in every wild-type mouse MLN B cell follicle regardless of whether or not there was an ongoing GC reaction (data not shown). As with CD23, a low level of FDC-M2 staining may well mark immature FDCs or another cell type such as the mouse equivalent of GCDCs ( 31 ). Neither immature FDCs nor mouse GCDCs have yet been defined. Section title: Ltβ − /− Mice Show Increased GC B Cells upon Intraperitoneal Challenge. Educational score: 4.165742874145508 Domain: biomedical Document type: Study Language: en Four-color fluorocytometry was adopted in order to study further the GC reaction in ltβ −/− mouse MLNs (see Materials and Methods). Most non-GC B cells were excluded as “lineage”-positive (Lin + ) cells, using a combination of antibodies against CD4, CD8α, CD90.2, IgD, and CD11b . Markers to discriminate GC B cells included PNA binding ( 37 , 38 ), anti-CD24 ( 38 – 40 ), and GL7 ( 44 ). PNA binding and CD24 are increased on GC B cells, whereas GL7 recognizes an activation antigen found on GC B cells but not naive or memory B cells. Fluorocytometry of MLNs is simple compared with spleen due to the relative lack of immature B cells, granuloid cells, and erythroid cells. After excluding nonlymphoid particles of low forward scatter (data not shown), most Lin − cells in MLNs are PNA hi CD24 hi (data not shown). This is demonstrated in Fig. 2 with MLNs 12 d after intraperitoneal challenge with CG adsorbed to alum. Section title: Ltβ − /− Mice Show Increased GC B Cells upon Intraperitoneal Challenge. Educational score: 4.165348052978516 Domain: biomedical Document type: Study Language: en As expected, both T cell–less mice and cd40l −/− mice fail to generate GC B cells , most clearly demonstrated with GL7. Both wild-type mice and ltβ −/− mice revealed substantial levels of Lin − GL7 + cells in their MLNs, although ltβ −/− mice consistently had lower levels than wild-type mice (see also below). Lin − GL7 + cells represented about half of all Lin − PNA hi CD24 hi cells and were CD45R + (data not shown; see below). Lin − cells include non-GC B cells such as antibody-secreting cells and memory B cells, which would be GL7 − . Section title: Ltβ − /− Mice Show Increased GC B Cells upon Intraperitoneal Challenge. Educational score: 4.141511917114258 Domain: biomedical Document type: Study Language: en The levels of Lin − GL7 + GC B cells at day 12 after challenge were much higher than those in unchallenged mice, and were similar to those observed in the spleen with the same antigenic challenge ( 45 ). Without intraperitoneal challenge, the levels of Lin − GL7 + cells were 0.3–0.7% of total MLN cells in both wild-type mice and ltβ −/− mice (data not shown; see below), consistent with levels in the spleen of unchallenged wild-type mice ( 45 ). Thus, intraperitoneal challenge in alum is an effective means of inducing GC B cell generation in the MLNs of both wild-type mice and ltβ −/− mice. Section title: Ltβ − /− Mice Generate Antigen-specific GC B Cells. Educational score: 4.058197498321533 Domain: biomedical Document type: Study Language: en The hapten NP has been used extensively in GC studies (40, 45– 48). The primary response to NP is dominated by antibodies bearing a λ1 light chain, recognized by the antiidiotype antibody Ls136 ( 39 , 49 – 52 ). NP-specific antibody-bearing cells can also be followed by their capacity to bind NP-conjugated PE (NP 20 PE). NP also provides a means for evaluating anti-NP–specific antibody affinity maturation (described below). Section title: Ltβ − /− Mice Generate Antigen-specific GC B Cells. Educational score: 4.107906341552734 Domain: biomedical Document type: Study Language: en Mice were challenged intraperitoneally with either CG or NP-haptenated CG (NP 13 CG), and MLNs were examined for NP-specific response 8 d later. Both wild-type mice and ltβ −/− mice showed massive numbers of λ1 + NP 20 PE-binding MLN cells in response to NP 13 CG . The percentage of CD45R + cells that were λ1 + NP 20 PE-binding were 66.1 ± 18.2 and 76.6 ± 17.2 for wild-type mouse MLNs and ltβ −/− mouse MLNs, respectively ( n = 6 each), compared with <0.1% in unchallenged mice. As expected ( 46 ), CG itself did not elicit any significant levels of λ1 + NP 20 PE-binding cells (data not shown). Also, T cell–less mice and cd40l −/− mice did not show significant levels of λ1 + NP 20 PE-binding cells . Section title: Ltβ − /− Mice Generate Antigen-specific GC B Cells. Educational score: 4.212114334106445 Domain: biomedical Document type: Study Language: en NP-specific GC B cells were also evident in MLNs from both wild-type mice and ltβ −/− mice at day 8 after challenge , defined as the Lin − GL7 + subset . Both the fraction of ltβ −/− GC B cells that were NP 20 PE-binding and the intensity of NP 20 PE-binding appeared to be relatively normal . The response of wild-type mice and ltβ −/− mice was then followed from day 8 to day 24 after challenge . As early as day 12 after challenge, ltβ −/− mouse MLN λ1 + GC B cells were greatly reduced compared with wild-type, and even fewer were NP 20 PE-binders . Having said this, the rate of decline of ltβ −/− GC B cells was not sustained at day 16 after challenge. Instead, Lin − GL7 + λ1 + ltβ −/− GC B cell levels appeared to plateau such that they were comparable to wild-type at day 20 after challenge before then falling again by day 24 . This pattern was reflected among Lin − GL7 − λ1 + non-GC cells. Section title: Ltβ − /− Mice Generate Antigen-specific Memory B Cells. Educational score: 4.173957347869873 Domain: biomedical Document type: Study Language: en Both ltβ −/− mice and wild-type mice showed anti-NP antibody secretion among MLN cells at day 6 after challenge with 50 μg NP 13 CG adsorbed to alum . IgA and IgG2a were not detected at all (data not shown). Clearly, IgG1 secretion by ltβ −/− mouse MLN cells (average relative units = 0.91) was much lower than wild-type (average = 6.03), perhaps indicating a lack of T cell and/or dendritic cell help. Nonetheless, this level of IgG1 secretion by ltβ −/− mouse MLNs was ∼20-fold higher than the lower limit of detection. Also, a primary challenge with 0.2 mg NP 13 CG in PBS alone did not result in significant anti-NP antibody secretion by either wild-type or ltβ −/− mouse MLN cells at day 6 after challenge (data not shown). Thus, the response to NP 13 CG in PBS was a good indicator of memory generated as a result of primary challenge with NP 13 CG adsorbed to alum (see below). Section title: Ltβ − /− Mice Generate Antigen-specific Memory B Cells. Educational score: 4.1253862380981445 Domain: biomedical Document type: Study Language: en To determine whether or not humoral memory was generated, mice were challenged as before and then rechallenged at various times with either 0.2 mg NP 13 CG in PBS without alum or PBS alone. 6 d later, anti-NP antibody was determined among MLN cells and in serum. Both the level of anti-NP antibody and the relative affinity for NP were determined by ELISA using two different BSA substrates. Total and relatively high affinity anti-NP antibody were determined with densely (NP 15 BSA) and sparsely (NP 2 BSA) haptenated BSA, respectively. As GC reaction proceeds, a greater proportion of anti-NP antibody becomes detectable with NP 2 BSA. The principal of this approach has been used in several other studies ( 24 , 26 , 34 ), and was recently validated with mAbs of various affinity for NP ( 45 ). Section title: Ltβ − /− Mice Generate Antigen-specific Memory B Cells. Educational score: 4.104250907897949 Domain: biomedical Document type: Study Language: en When given a secondary challenge and harvested at week 4, ltβ −/− mouse MLNs appeared to secrete more anti-NP antibody than wild-type mouse MLNs , implying that ltβ −/− mice had generated greater humoral memory than wild-type mice. This pattern was not so apparent in serum . Nonetheless, ltβ −/− mice clearly showed memory even in their serum. The substantial memory response was still evident among ltβ −/− mouse MLNs at week 10 after challenge but was less evident in serum . Section title: Ltβ − /− Mice Generate Antigen-specific Memory B Cells. Educational score: 4.147042751312256 Domain: biomedical Document type: Study Language: en Both wild-type mouse MLNs and ltβ −/− mouse MLNs showed affinity maturation in the post-GC phase between weeks 4 and 10 after challenge . Thus, the relatively low NP 2 /NP 15 ratio in ltβ −/− mouse MLNs at week 4 after challenge was almost normal at week 10. Again, this observation in MLNs was not obvious in serum . At both weeks 4 and 10 after challenge, ltβ −/− mice showed a lower NP 2 /NP 15 ratio in their serum than wild-type mice, indicating a defect in GC affinity maturation processes. Section title: Discussion Educational score: 4.307225704193115 Domain: biomedical Document type: Study Language: en Numerous lines of evidence have now shown that ltβ −/− mice do not have FDC networks (26, 27, 30, and this study). In addition, MacKay and Browning ( 33 ) have very recently shown that administration of soluble LTβR to adult mice causes regression of FDC networks and dissipation of the antigen they were retaining. Clearly however, ltβ −/− mice might still have FDC precursors. Indeed, irradiated adult ltα −/− mice generate mature FDC networks of ltα −/− origin upon reconstitution with wild-type bone marrow ( 53 ), and equivalent results have been obtained with ltβ −/− mice (data not shown). The location of FDC precursors in ltβ −/− mice is at present unknown, and it is conceivable that they will be found within B cell follicles and GCs even though they fail to develop into mature FDC networks. Others have shown that ltβ −/− mice and ltβr −/− mice do have small numbers of discrete FDC-M2 + cells in the spleen ( 27 , 28 ), leading to the suggestion that these cells may represent FDC precursors or immature FDCs. However, there is no evidence that FDC-M2 is completely specific to FDCs, although it is a useful marker of FDC networks. Unlike FDC-M2, FDC-M1 has been well characterized and is considered to be relatively FDC specific, but even this marker also stains tingible body macrophages (which are found in GCs) and some endothelial cells ( 54 ). Section title: Discussion Educational score: 3.724573850631714 Domain: biomedical Document type: Study Language: en The origin of FDCs is somewhat controversial (for a review, see reference 55 ) and will undoubtedly be further complicated by the fact that mice may also prove to have GCDCs ( 31 ), which might be the FDC-M2 + cells seen by others. Regardless of whether or not ltβ −/− mice have FDC precursors, the roles FDCs are said to fulfill largely rely on the extensive dendritic processes of FDC networks. Clearly, these structures are absent. Section title: Discussion Educational score: 4.249978065490723 Domain: biomedical Document type: Study Language: en The absence of FDC networks presumably has indirect as well as direct effects on GC reactions. For example, this study has not considered GC T cells in ltβ −/− mice. Other defects clearly exist in ltβ −/− mouse MLNs, such as the B cell infiltration of the T cell areas and reduced primary humoral response. What this study has attempted to do is highlight the processes that occur despite the defects. Most notably, ltβ −/− mice generate antigen-specific GC B cells and class-switched memory B cell responses. Having said this, antigen-specific ltβ −/− GC B cells decline rapidly at times when wild-type GC B cell numbers are still relatively high. GCs normally have a life span of a few weeks ( 10 , 56 ). It has been argued that the regression of GCs begins at a time when FDCs begin to bury their retained antigen within membrane pockets, thereby ceasing to present antigen to GC B cells ( 56 ). The decline of ltβ −/− GC B cells in the absence of FDC networks may be a premature execution of this process. On the other hand, ltβ −/− GC B cell numbers did not decline further between days 12 and 16 after challenge but instead appeared to plateau until they finally decreased further between days 20 and 24. It is conceivable that the Lin − GL7 + λ1 + cell numbers were maintained by further generation of such cells from centroblasts. Section title: Discussion Educational score: 4.1387739181518555 Domain: biomedical Document type: Study Language: en Anti-NP memory and relative affinity maturation were assessed at various times. The humoral memory response in ltβ −/− mouse MLNs was as great if not greater than that seen in wild-type mouse MLNs . Of course, this may not be a direct reflection of the actual frequency of NP-specific memory B cells. The relatively low level of NP-specific non-GC B cells (Lin − λ1 + NP 20 PE-binding) in ltβ −/− mouse MLNs late in the GC phase suggests that ltβ −/− mouse MLNs produce significantly fewer memory B cells than wild-type mouse MLNs. Section title: Discussion Educational score: 4.221492767333984 Domain: biomedical Document type: Study Language: en The degree of affinity maturation observed in serum here was similar to that previously reported for ltα −/− mice, Lyn kinase ( lyn ) −/− mice, and ltβ −/− mice ( 24 , 26 , 34 ). Affinity maturation requires somatic hypermutation and subsequent selection of higher affinity clones, suggesting that specific activated B cells had entered into a “GC B cell program” despite the complete absence of GCs in ltα −/− mice and lyn −/− mice. Indeed, somatic hypermutation is evident in ltα −/− mice and lyn −/− mice ( 24 , 34 ), and there is no reason to believe that this is not occurring in ltβ −/− mice. Certainly, somatic hypermutation is normally evident as early as day 7 after challenge ( 40 , 45 , 48 , 57 , 58 ) and, unlike ltα −/− mice ( 24 ) and lyn −/− mice ( 34 ), ltβ −/− mice generate appreciable levels of antigen-specific GC B cells upon challenge (this study). Section title: Discussion Educational score: 4.201426029205322 Domain: biomedical Document type: Study Language: en Where and how are high-affinity mutants selected in the absence of GCs? Takahashi et al. ( 45 ) recently described evidence in support of a phenomenon best described as “post-GC intraclonal competition.” The average affinity of anti-NP antibody from bone marrow antibody-secreting cells continued to increased long after the GC reaction had waned ( 45 ). Although clonal selection occurs independently in each GC and low-affinity B cells can survive the selection process within GCs if high-affinity competitors are absent ( 45 ), post-GC affinity-driven selection processes effectively constitute “inter-GC selection.” This concept is supported by the study here, where the relative affinity of anti-NP antibody at week 10 after challenge was about twofold higher than that at week 4 after challenge , in the MLNs of wild-type mice and ltβ −/− mice. Section title: Discussion Educational score: 4.252650260925293 Domain: biomedical Document type: Study Language: en Thus, mutants with higher affinity for the antigen are generated and selected even in ltβ −/− mice, but the highest possible affinity for antigen is not achieved in ltβ −/− mice , presumably because further rounds of somatic mutation cannot occur in the post-GC phase. Hence, despite post-GC intraclonal competition, the benefit of the GCs is as an environment in which repeated rounds of somatic mutation and selection can occur rapidly in order to achieve the highest possible affinity for antigen. Indeed, although substantial somatic hypermutation was observed in ltα −/− mice, the degree of mutation among V186.2 genes was only about half that seen in wild-type mice ( 24 ). Section title: Discussion Educational score: 4.183886528015137 Domain: biomedical Document type: Study Language: en It should also be borne in mind that the apparent degree of affinity maturation by post-GC intraclonal competition will vary substantially depending on the nature of the antigen. In some experimental instances, random mutations lead to higher affinity clones at a relatively high frequency ( 59 ). The NP hapten used here and by others ( 24 , 34 , 45 ) would appear to be another such example, since a frequently occurring single point mutation in the V186.2 gene ( 24 , 60 ) is associated with greatly increased affinity for NP ( 61 ). Thus, the studies here showing defective NP-specific affinity maturation in ltβ −/− mice can only be interpreted to suggest that affinity maturation within GCs has failed in these mice. Section title: Discussion Educational score: 4.250165939331055 Domain: biomedical Document type: Study Language: en Recirculating memory B cells migrate between secondary lymphoid organ follicles where they can respond to antigen held on FDC networks ( 62 , 63 ). Marginal zone memory B cells are said to be long-lived and non-recirculating ( 64 – 66 ), and it is difficult to conceive of how their maintenance could be dependent on antigen retained by FDC networks. As the name suggests, marginal zone memory B cells have been best characterized in the marginal zone of the spleen, where they are seen to appear both immediately before and during GC reactions ( 63 – 65 ). Equivalent areas are located on the inner wall of the subcapsular sinus of LNs, and may be substantial in MLNs ( 65 ). Further studies will be necessary to characterize the B cell memory in ltβ −/− mice, including how the nature and dose of antigen might affect memory maintenance besides memory generation per se. Section title: Discussion Educational score: 4.173254013061523 Domain: biomedical Document type: Study Language: en In conclusion, this study has considered GC reactions in the MLNs of ltβ −/− mice and found that both GC and B cell memory are formed despite the complete absence of FDC networks. However, antigen-specific antibody affinity maturation is defective. This study of the MLNs of ltβ −/− mice serves as a model for the consequences of administration of soluble LTβR with respect to LNs. The spleen of ltβ −/− mice was not studied here because it is more disorganized than the MLNs ( 26 , 27 , 30 ). The fact that anti-NP memory at week 10 was much less apparent in serum than in MLNs may well be a reflection of the fact that serum antibody levels are dominated by the spleen and spleen-derived bone marrow antibody-secreting cells. Having said this, even the spleen of ltβ −/− mice generates some PNA-binding GC-like B cells ( 27 ). | Study | biomedical | en | 0.999996 |
10049950 | Section title: Cell Preparations. Educational score: 4.07581901550293 Domain: biomedical Document type: Study Language: en Ag-specific CD4 + human T cell lines were isolated as described ( 13 ). PBMCs were separated into CD4 + T cells, CD8 + T cells, CD19 + B lymphocytes, and CD14 + monocytes using immunomagnetic beads ( Dynal ). The purity of the isolated subsets was >98% as determined by flow cytometry for relevant and irrelevant subset markers (not shown). Section title: Cell Preparations. Educational score: 4.176651954650879 Domain: biomedical Document type: Study Language: en For proliferation and BDNF production assays, 5 × 10 5 freshly isolated CD4 + T cells, CD8 + T cells, and B lymphocytes were cultured in RPMI 1640 medium ( GIBCO BRL ) supplemented with 5% FCS ( GIBCO BRL ) in 96-well plates (Nunc). After a resting period of 72 h, T lymphocytes were stimulated with 5 × 10 4 irradiated allogeneic PBMCs and PHA (10 μg/ml; Sigma ). B cells were stimulated with heat-inactivated Staphylococcus aureus Cowan strain 1 (SAC; Calbiochem ) at a final dilution of 1:7,500. Monocytes were stimulated in the course of purification using anti-CD14–coated magnetic beads. Ag-specific T line cells (10 5 ) were cultured in RPMI 1640 medium supplemented with 5% FCS plus irradiated autologous or HLA-matched PBMCs and the relevant Ag (10–30 μg/ml). Proliferation was assessed by [ 3 H]thymidine uptake. Supernatants were removed at different time intervals after stimulation and analyzed for BDNF protein concentration and bioactivity. BDNF production by APCs was measured in separate wells and subtracted. The cytokine profile of CD4 + T cell lines was assessed by intracellular flow cytometry staining according to the manufacturer's protocol ( PharMingen ). Section title: Reverse-transcription PCR Analysis of BDNF Transcription. Educational score: 4.130669593811035 Domain: biomedical Document type: Study Language: en Total cellular RNA was extracted using two different RNA extraction systems (QIAGEN, and WAK Chemie). The RNA (0.5–2 μg) was transcribed with random hexamer primers ( Boehringer-Mannheim ) or oligo(dt) primers ( GIBCO BRL ), dNTP (MBI Fermentas), and Superscript™ Reverse Transcriptase ( GIBCO BRL ). All reactions were carried out in a total volume of 25 μl containing 1 U Taq polymerase (QIAGEN), 200 μM of each dNTP, cDNA, and 15 pmol of each primer for 35 or 40 PCR cycles with annealing at 60°C. The correct size of the bands was determined by comparison with a DNA mass standard (pUC MIX 8; MBI Fermentas). RNA samples transcribed in the absence of reverse transcriptase were used as negative controls to exclude genomic contamination. All PCR products were sequenced (Medigene). The primer sequences were as follows: β-actin forward: 5′-CGAGCGGGAAATCGTGCGTGA-3′ (position 622–642); β-actin reverse: 5′-CAGCGAGGCCAGGATGGAGCC-3′ ; BDNF forward: 5′-AGCGTGAATGGGCCCAAGGCA-3′ (position 208–228); BDNF reverse 5′-TGTGACCGTCCCGCCCGACA-3′ (position 570–551). Section title: Quantification of BDNF Protein Secretion. Educational score: 4.172933578491211 Domain: biomedical Document type: Study Language: en BDNF protein concentration was determined with a sensitive sandwich ELISA. In brief, 96-well flat-bottomed plates (Immulon 4; Dynatech) were coated with a chicken anti–human BDNF Ab ( Promega ). Recombinant human BDNF (used as standard; Research Diagnostics, Inc.) and cell supernatants (all made up in RPMI, 5% FCS plus 0.05% Tween 20 [ Sigma ]) were used in serial dilutions. Bound BDNF was detected by incubating the plates with a mouse anti–human BDNF Ab (Research Diagnostics, Inc.) followed by peroxidase-labeled goat anti–mouse IgG (Dianova). The plates were developed using a liquid substrate system ( Sigma ), and the OD was determined at 450 nm. For enhanced detection of BDNF in biological samples, the cell supernatants were acid-treated for 15 min before the assays. This treatment did not change detection of the standard. Specificity of the BDNF ELISA was established using recombinant BDNF and control neurotrophic factors. The sensitivity of this ELISA ranges from 20 to 40 pg/ml. Quantification of BDNF was confirmed by a second highly specific ELISA. Section title: Assay for BDNF Bioactivity. Educational score: 4.109736442565918 Domain: biomedical Document type: Study Language: en Biological assays were performed using dissociated sensory neurons prepared from nodose ganglia of 8-d-old chick embryos ( 14 ). Neurons were plated in laminin/ polyornithine-coated 48-well plastic dishes. Exogenous BDNF (1 ng/ml) and immune cell supernatants were added in the presence or absence of the anti-BDNF mAb 4.D3.3A3, which neutralizes the biological activity of BDNF. After 24 h, the surviving neurons were counted using light microscopy. For each experiment, duplicate wells were evaluated. Section title: Immunohistochemical Analysis of Brain Sections. Educational score: 4.125859260559082 Domain: biomedical Document type: Study Language: en Immunohistochemistry was performed on formalin-fixed, frozen, and paraffin-embedded human brain tissue using a mouse IgG1 mAb against BDNF (4.FL.1A1; 10 μg/ml) and a polyclonal anti-BDNF antiserum (N-20, used at 1:500 dilution; Santa Cruz Biotechnology ) ( 15 ). Two actively demyelinating cases of multiple sclerosis, one case of acute disseminated (postinfectious) leukoencephalitis, and two controls without neurological disease were examined. Control sections were incubated without primary Ab or with control IgG1 Ab. Serial sections were labeled for CD3 (1:300; Serotec), CD68 (1:100; Dako), or human IgG (1:200; Amersham Pharmacia Biotech ), using an avidin-biotin peroxidase method. Section title: T Cells, B Cells, and Monocytes Transcribe BDNF mRNA and Secrete BDNF Protein. Educational score: 4.110213756561279 Domain: biomedical Document type: Study Language: en BDNF was detected in CD4 + T cells, CD8 + T cells, B cells, and monocytes . CD4 + T cells and CD8 + T cells were stimulated with PHA and B cells with SAC. Monocytes were stimulated in the course of purification with anti-CD14–coated magnetic beads. Fig. 1 b shows the BDNF concentrations in supernatants of stimulated and nonstimulated cells after 72 h. Constitutive BDNF production was observed in T cells and B cells . All cell types produced enhanced levels of BDNF after stimulation . Reverse-transcription (RT)-PCR analysis showed BDNF transcripts in T cells, B cells, and monocytes . Section title: T Cells, B Cells, and Monocytes Transcribe BDNF mRNA and Secrete BDNF Protein. Educational score: 3.9676742553710938 Domain: biomedical Document type: Study Language: en BDNF was also produced by Ag-specific CD4 + T cell lines of different Ag specificity and cytokine profile (Table I ). Ag-induced BDNF secretion was observed both in Th1- and in Th2-type cells (Table I ). Section title: Immune Cell–derived BDNF Supports Neuronal Survival In Vitro. Educational score: 4.1630449295043945 Domain: biomedical Document type: Study Language: en Bioactivity of the BDNF produced by human immunocompetent cells was assessed by measuring the ability to rescue cultured sensory neurons from cell death ( 14 ). CD4 + T cells, CD8 + T cells, B cells, and monocytes were stimulated with PHA, SAC, or anti-CD14–coated magnetic beads, respectively. After 96 h, the supernatants were collected and added to chicken embryonic nodose ganglia neurons to determine the neuronal survival rate . Neuronal survival was supported by externally supplied recombinant BDNF and by supernatants of activated CD4 + T cells, CD8 + T cells, B cells, and monocytes . The promotion of neuronal survival by immune cell supernatant was due to biologically active BDNF as demonstrated by inhibition with anti-BDNF mAb 4.D3.3A3, which blocks the biological activity of BDNF . Section title: BDNF Is Expressed by Inflammatory Cells in Acute Disseminated Encephalitis and Multiple Sclerosis. Educational score: 4.288230895996094 Domain: biomedical Document type: Study Language: en Strong BDNF immunoreactivity was observed in inflammatory cells forming perivascular infiltrates in cases of disseminated (postinfectious) leukoencephalitis and multiple sclerosis . Using serial sections, BDNF + cells were found to correspond to infiltrating mononuclear cells . In multiple sclerosis, BDNF + lymphocytes and macrophages were not restricted to the perivascular localization but were found throughout the lesion . Lesional areas with high numbers of macrophages actively involved in demyelination showed enhanced BDNF immunoreactivity. In inflammatory central nervous system disease and healthy controls, BDNF was also detected in various types of neurons, ependymal cells, and weakly in astrocytes. No immunoreactivity was observed in oligodendrocytes or ramified microglial cells. Similar staining patterns were observed with the monoclonal and polyclonal Abs. Section title: Discussion Educational score: 4.15380859375 Domain: biomedical Document type: Study Language: en We demonstrate that human immune cells express the neurotrophic factor BDNF both in vitro and in inflammatory brain lesions. BDNF is one of the most potent factors supporting neuronal survival and regulating neurotransmitter release and dendritic growth ( 2 , 16 , 17 ). Several studies have shown that the administration of BDNF protein or the BDNF gene can rescue injured or degenerating neurons and induce axonal outgrowth and regeneration ( 6 – 10 ). Furthermore, BDNF had beneficial effects in several animal models of neurodegenerative diseases ( 11 ). Difficulties in delivering sufficient amounts of BDNF to the site of central nervous system lesions have so far hampered the successful application of BDNF for treatment of human diseases ( 12 ). Section title: Discussion Educational score: 4.186372756958008 Domain: biomedical Document type: Study Language: en One promising strategy for the delivery of neuroprotective factors relies on retroviral transduction of neurotrophic factors into T cell lines specific for Ag expressed in the nervous system ( 18 ). Our present results indicate that this experimental strategy has a natural counterpart: T cells and other immune cells homing to degenerative, infectious, or autoimmune lesions express a potent neurotrophic factor, BDNF, that may help to minimize neuronal damage. Consistent with this hypothesis, it has recently been demonstrated that the implantation of activated macrophages into experimental spinal cord lesions leads to partial functional recovery of paraplegic rats ( 19 ). In addition to its neurotrophic effect, locally produced BDNF could have an immunomodulatory function, e.g., by indirectly downregulating MHC class II expression on microglia ( 20 ). Section title: Discussion Educational score: 4.121407508850098 Domain: biomedical Document type: Study Language: en NT-mediated effects on immune cells have previously been demonstrated for nerve growth factor ( 21 ). Thus far, however, we found no evidence that BDNF acts on peripheral human immune cells. This could be explained by the fact that immune cells express only the truncated form of the trk B BDNF receptor (our unpublished observations; consistent with previous reports that the expression of the full-length, signal-transducing form gp145 trk B is restricted to neuronal cells, whereas the truncated gp95 trk B form is widely expressed in nonneuronal tissues ). Further, we did not detect significant effects of BDNF on the proliferation, cytokine production, or apoptosis of human lymphocytes (our unpublished data). Clearly, these negative observations by no means rule out that BDNF has a role in the immune system, and the question of whether BDNF can affect elements of the immune system deserves more detailed study. Section title: Discussion Educational score: 4.251530647277832 Domain: biomedical Document type: Study Language: en BDNF production by immune cells provides a novel example of the bidirectional interaction between the nervous system and the immune system ( 22 , 23 ). It is noteworthy that upon stimulation with Ag a large proportion of immune cells, including CD4 + T cell lines specific for brain autoantigens, produce BDNF. Thus, in some circumstances, the immune infiltrates commonly found in inflammatory, ischemic, degenerative, and traumatic lesions of the nervous system may have a protective rather than destructive role. This could help to explain the presence of large numbers of autoreactive T lymphocytes in the healthy immune repertoire, a phenomenon invoked by Cohen's concept of the “immune homunculus” ( 24 ). Section title: Discussion Educational score: 4.05228853225708 Domain: biomedical Document type: Study Language: en In conclusion, our demonstration that activated T cells, B cells, and monocytes produce BDNF in vitro and in inflammatory lesions raises the intriguing possibility that neuroinflammatory reactions have a neuroprotective (side) effect. This has obvious therapeutic implications for multiple sclerosis and other inflammatory diseases of the nervous system ( 25 ). Production of both neurotoxic and neuroprotective factors emphasizes the complex role of immune cells in inflammatory, degenerative, and regenerative processes of the nervous system. | Study | biomedical | en | 0.999997 |
10049951 | Section title: T Cell Epitope Prediction. Educational score: 4.134321212768555 Domain: biomedical Document type: Study Language: en TEPITOPE, a new T cell epitope prediction software, is a Windows™ application that enables the identification of (a) class II ligands binding in a promiscuous or allele-specific mode, and (b) the effects of polymorphic residues on class II ligand specificity (21, and our manuscript in preparation). 25 quantitative matrix-based HLA-DR motifs, covering the majority of class II ligand specificity, are incorporated in TEPITOPE (22, and our manuscript in preparation) and provide the basis for various algorithms included in the software package. Starting from any protein sequence, the algorithm permits the prediction and parallel display of ligands for each of the 25 HLA-DR alleles. To predict MAGE-3 CD4 + T cell epitopes, we loaded the protein sequence into the software looking for promiscuous peptide regions. We set the TEPITOPE prediction threshold at 5% ( 21 ) and picked peptide sequences predicted to bind at least 50% of the HLA-DR molecules incorporated in the software. Section title: DR–Peptide Binding Assay. Educational score: 4.1855387687683105 Domain: biomedical Document type: Study Language: en Peptide interactions with detergent-solubilized DR molecules were measured using an ELISA-based high-flux competition assay ( 23 ). HLA-DR molecules were isolated from the following human lymphoblastoid cell lines (LCL): DR1 from HOM-2, DR3 from WT49, DR4 from PREISS, DR5 from SWEIG, DR7 from EKR, and DR8 from BM9. DR2 was isolated from the L cell transfectant L466.1. The molecules were affinity purified using the mAb 1-1C4 ( 24 ) as described ( 25 ). Peptide competition assays were conducted to measure the ability of unlabeled peptides to compete with a biotinylated indicator peptide for binding to purified DR molecules. The following biotinylated indicator peptides were used: GFKA 7 for DR1 and DR7; GIRA 2 YA 4 for DR2; LAYDA 5 for DR3; UD4 for DR4 ( 26 ); TT 830–843 for DR5; and GYRA 6 L for DR8. The biotinylated indicator peptide and HLA-DR molecules were incubated with 10-fold dilutions (0.001–100 mM) of the unlabeled competitor peptides (peptides corresponding to the MAGE-3 predicted sequences). To determine relative peptide binding affinity, the promiscuous HA 307–319 peptide from influenza hemagglutinin ( 27 ) was included in each competition assay. The relative binding data of the unlabeled competitor peptides were expressed as inhibitory concentration (IC 50 ), i.e., the concentration of competitor peptide required to inhibit 50% of binding of the biotinylated indicator peptide. Section title: Peptide Synthesis. Educational score: 4.07503604888916 Domain: biomedical Document type: Study Language: en Synthetic peptides corresponding to MAGE-3 141–155 , MAGE-3 146–160 , MAGE-3 156–170 , MAGE-3 171–185 , and MAGE-3 281–295 sequences were manufactured on a 9050 Millipore synthesizer. The purity of the peptides was evaluated by reverse-phase HPLC and electron spray mass spectrometry. Synthetic peptides were lyophilized and then reconstituted in DMSO at 2 mg/ml concentration and diluted in PBS as needed. Section title: Cloning and Expression of rMAGE-3. Educational score: 4.026237487792969 Domain: biomedical Document type: Study Language: en Full-length MAGE-3 coding sequences were inserted into expression vector pET16b (Novagen), allowing the production of the NH 2 terminus 10-histidine tail as described ( 16 ). Production and purification of the recombinant fusion protein on nickel column were monitored by SDS-PAGE and Coomassie blue staining. Section title: Propagation of CD4 + T Cells. Educational score: 4.13349723815918 Domain: biomedical Document type: Study Language: en The five synthetic peptides corresponding to the MAGE-3 sequences most promiscuous for HLA-DR binding (see Table I ) were pooled (hereafter MAGE-3 pool) and used to stimulate the PBMCs of a healthy donor whose HLA type, identified by standard serologic typing, is A1, A2/ B41, B52/DR11, as described ( 28 ). In brief, 20 × 10 6 PBMCs were cultivated for 7 d in RPMI 1640 ( GIBCO BRL ) supplemented with 10% heat-inactivated human serum (Technogenetics), 2 mM l -glutamine, 100 U/ml penicillin, 50 μg/ml streptomycin (Biowhittaker) (TCM) containing the MAGE-3 pool (1 μg/ml of each peptide). The reactive lymphoblasts were isolated on a Percoll gradient ( 28 ), further expanded in T cell growth factor (Lymphocult; Biotest Diagnostic Inc.), and restimulated at weekly intervals with the same amount of antigen plus irradiated (4,000 rad) autologous PBMCs as APCs. Section title: Flow Cytometry. Educational score: 4.073018550872803 Domain: biomedical Document type: Study Language: en Cytofluorimetric analyses were performed on a FACStarPlus ® ( Becton Dickinson ). The following mAbs were used: anti-CD4–PE and anti-CD8–FITC ( Becton Dickinson ), D1.12 (purified from an anti-MHC class II hybridoma supernatant), and 57B (described in reference 16 ). FITC-rabbit anti–mouse Ig antibody (DAKO) was used as second-step reagent in indirect immunofluorescence stainings. Staining for intracytoplasmic MAGE-3 expression was performed as described ( 29 ). Intracytoplasmic staining for cytokine expression was performed using the anti–INF-γ and anti–IL-4 mAbs, following the manufacturer's instructions ( Sigma ). Section title: Proliferation Assay. Educational score: 4.12143611907959 Domain: biomedical Document type: Study Language: en CD4 + T cells and autologous irradiated PBMCs were diluted in TCM to 2 × 10 5 /ml and 2 × 10 6 /ml, respectively, and plated in triplicate in 96 round-bottomed well plates (100 μl of CD4 + T cells and 100 μl of APCs). The cells were stimulated with different concentrations of MAGE-3 pool (0.05, 0.1, 0.5, 1, and 5 μg/ml), each peptide (10 μg/ml), and different concentrations of rMAGE-3 protein (5, 10, and 20 μg/ ml). Triplicate wells with CD4 + T cells alone and APCs alone were used as controls. Three wells with CD4 + T cells plus APCs did not receive any stimulus in order to determine the basal growth rate (the blank). In inhibition experiments, different concentrations of mAb L243 or an isotype-matched irrelevant mAb (0.25 and 0.5 mg/ml) were added in triplicate wells of CD4 + cells plus APCs stimulated with MAGE-3 pool (5 μg/ml) or MAGE-3 281–295 (10 μg/ml). After 3 d, the cultures were pulsed for 16 h with [ 3 H]TdR (1 mCi/well, 6.7 Ci/mol; Amersham Pharmacia Biotech ). The cells were collected with a Titertek multiple harvester (Skatron, Inc.), and the thymidine incorporated was measured in a liquid scintillation counter. The percentage of inhibition was calculated as follows: [(cpm without mAb − cpm with mAb)/(cpm without mAb)] × 100. Section title: Cytotoxicity Assay. Educational score: 4.138515949249268 Domain: biomedical Document type: Study Language: en CD4 + T cells were tested for specific lytic activity in a standard 4-h 51 Cr-release assay as described ( 30 ). The following targets were used: melanoma cells (SK-Mel 28, HT144, OI TC described in reference 29 , and MD TC established in our laboratory from a cutaneous metastasis), and LCL. The HLA-DR type of target cells, identified by molecular or serologic typing, was SK-Mel 28 (DR*04*13), HT144 (DR*04*07), OI TC (DR*01*11), MD TC (DR*04*11), LCL (DR11). In cold target competition assays, unlabeled target cells (cold targets) were seeded in plates at serial ratios of hot-to-cold target cells. Effector CD4 + T cells and 51 Cr-labeled target cells (hot targets) were then added, and cytotoxicity was assessed as described above. Percentage inhibition was calculated as follows: [(% specific lysis without cold target − % specific lysis with cold target)/(% specific lysis without cold target)] × 100. Section title: Results and Discussion Educational score: 4.381416320800781 Domain: biomedical Document type: Study Language: en 10 synthetic peptides corresponding to sequence segments predicted by TEPITOPE to form promiscuous MAGE-3 CD4 + T cell epitopes were synthesized, and their binding to purified molecules of 7 widely diffuse HLA-DR alleles was verified. Based on the results of the competition binding assays, 5 (i.e., the sequences with the greatest degree of promiscuity) of the 10 predicted sequences were chosen for further experiments (Table I ). The five synthetic peptides were pooled (MAGE-3 pool) and used to stimulate the PBMCs of a healthy donor. T cells were 94% CD4 + after 1 wk of culture (not shown), and could be propagated in long-term culture by weekly restimulation with the MAGE-3 pool in the presence of autologous irradiated PBMCs. Reactivity of CD4 + T cells was tested in microproliferation assays : the cells responded vigorously to the MAGE-3 pool , even at low concentrations (100–500 ng/ml). Reactivity to the individual peptides forming the pool was also periodically investigated : the CD4 + T cells recognized predominantly the peptide corresponding to MAGE-3 281–295 and, although to a much lower but significant extent, the peptides corresponding to the overlapping sequences MAGE-3 141–155 and MAGE-3 146–160 . All three sequences recognized by the CD4 + T cells showed a high binding affinity to purified DR11 molecules (see Table I ). Reactivity to MAGE-3 281–295 increased during the propagation of the line . The proliferative activity of CD4 + T cells in the presence of MAGE-3 pool or MAGE-3 281–295 was inhibited by addition in culture of different concentrations of L243 mAb , demonstrating that the recognition of MAGE-3 sequences was HLA-DR restricted. We next tested the CD4 + T cells for cross-reactivity with the native protein . CD4 + T cells strongly recognized the rMAGE-3 protein after processing and presentation by autologous APCs, demonstrating that the synthetic sequences recognized by the CD4 + T cells indeed formed naturally processed epitopes. Section title: Results and Discussion Educational score: 4.083226680755615 Domain: biomedical Document type: Study Language: en Intracytoplasmic staining for IL-4 and INF-γ expression, performed after CD4 + T cell activation with PMA and ionomycin, revealed that 70% of the CD4 + T cells produced INF-γ while no cells produced IL-4 (data not shown), suggesting that they belong mostly to the Th1 type. Section title: Results and Discussion Educational score: 4.477628707885742 Domain: biomedical Document type: Study Language: en To characterize the functional activity of the MAGE-3– specific CD4 + T cells, we tested their killing potential against melanoma cells expressing the MAGE-3 protein and the HLA-DR molecules . CD4 + T cells showed cytolytic activity against OI TC and MD TC, which express the HLA-DR11 restricting allele, whereas they did not kill SK-Mel 28 and HT144, which express unrelated HLA-DR alleles . To verify whether the cytolytic CD4 + T cells recognized HLA-DR11– restricted MAGE-3 epitopes on melanoma cells, we first tested their lytic activity against HLA-DR11 + LCL unpulsed, or pulsed with the synthetic peptides recognized in microproliferation assays. LCL pulsed with MAGE-3 281–295 were strongly recognized by the CD4 + T cells, whereas no killing activity against LCL unpulsed or pulsed with MAGE-3 141–155 and MAGE-3 146–160 was detectable . Second, we performed cold target inhibition experiments which showed that the lytic activity of CD4 + T cells against OI TC was inhibited by the addition of LCL pulsed with MAGE-3 281–295 , demonstrating that this sequence is indeed presented by HLA-DR11 on the OI TC melanoma cells. These results further demonstrate that MAGE-3 281–295 is naturally processed and forms a cytotoxic CD4 + T cell epitope. Since the polyclonal CD4 + T cells proliferated in the presence of the rMAGE-3 protein, and in addition to MAGE-3 281–295 they also recognized MAGE-3 141–155 and MAGE-3 146–160 , we cannot exclude that these last two sequences may also yield natural epitopes, which are recognized by CD4 + T cells with functional activity different from killing. Moreover, although CD4 + T cells were mostly Th1 and had direct effector function upon tumor recognition, we cannot exclude that in vivo such CD4 + T cells could also exert a helper activity in the induction phase of the immune response. Section title: Results and Discussion Educational score: 4.116528511047363 Domain: biomedical Document type: Study Language: en One approach for identifying CD4 + T cell epitopes on a candidate protein is the use of overlapping synthetic peptides corresponding to the complete sequence of the protein. The major drawback of this approach is the number of peptide sequences that need to be tested, thus making this approach too expensive and time consuming. In this study, we used the TEPITOPE software package to computationally identify promiscuous HLA-DR binding sites starting from primary protein structures. We demonstrated that TEPITOPE predicted sequence segments capable of binding to multiple HLA-DR alleles. Furthermore, we showed that one or more of the predicted HLA-DR ligands were indeed naturally processed, thus confirming the validity of this approach. We expect that the application of TEPITOPE to other tumor-associated antigens will speed up identification of the antitumor CD4 + T cell epitope repertoire in humans. Section title: Results and Discussion Educational score: 4.131749629974365 Domain: biomedical Document type: Study Language: en Clinical trials based on the use of melanocyte-specific antigens (such as gp100, MART-1/Melan-A, and tyrosinase, for which CD4 + T cell epitopes were identified) are in progress in melanoma patients, and although no significant side effects were reported in a recent study that used a gp100 peptide for the treatment of HLA-A2 + patients ( 31 ), the development of autoimmune responses against normal tissue must be considered when using self-differentiation antigens as vaccines. The demonstration that MAGE-3 (i.e., an antigen not expressed in normal tissues, with the exception of testis and placenta, which are unlikely to be targets of T cells since they do not express MHC molecules), can form CD4 + T cell epitopes further supports its use for vaccination protocols in neoplastic patients using a mixture of synthetic peptides corresponding to CD8 + and CD4 + T cell epitopes. Section title: Results and Discussion Educational score: 4.121731758117676 Domain: biomedical Document type: Study Language: en Previous findings ( 13 , 32 , 33 ) reported a lytic activity of melanoma-specific CD4 + T cells. Here we give the molecular definition of an epitope able to stimulate cytolytic CD4 + T cells that can be grown in vitro with ease, raising the possibility of using those CD4 + T cells in protocols of adoptive transfer in neoplastic patients whose neoplasm expresses the MAGE-3 protein and the MHC class II molecules. Section title: Results and Discussion Educational score: 4.059020042419434 Domain: biomedical Document type: Study Language: en In conclusion, in this study we identified the first CD4 + T cell epitope on a tumor-specific antigen, and we verified that the approach used here to predict promiscuous CD4 + T cell epitopes yielded natural epitopes. It will be important to evaluate whether the identified CD4 + T cell epitopes are indeed promiscuous, making their use for peptide-based vaccines less allele dependent and more widely applicable. | Study | biomedical | en | 0.999996 |
10049952 | Section title: mAbs. Educational score: 3.3854706287384033 Domain: biomedical Document type: Study Language: en The mouse mAbs used in these studies were as follows: OX6 (anti–rat class II MHC; reference 15 ), OX7 (anti–rat Thy-1; reference 16 ), OX8 (anti–rat CD8; reference 17 ), and OX12 (anti–rat κ chain; reference 18 ). Section title: Animals and Induction of Thyroiditis and Diabetes. Educational score: 4.098909378051758 Domain: biomedical Document type: Study Language: en 3–12-wk-old female PVG and PVG.RT1 u rats, which differ only in their MHC haplotype, were obtained from the specific pathogen-free breeding facilities of the Medical Research Council Cellular Immunology Unit (Oxford, UK). Thyroiditis was induced in female PVG rats as has been previously described ( 7 ). In brief, female rats aged 3 wk were thymectomized and given four 275-rad doses of 137 Cs γ-irradiation at 2-wk intervals starting 1 wk after thymectomy. Development of thyroiditis was determined histologically and by the development of anti-Tg antibodies. Diabetes was induced in male PVG.RT1 u rats by their thymectomy at 6 wk of age followed by four 250-rad doses of 137 Cs γ-irradiation at 2-wk intervals starting 2 wk after thymectomy. Development of diabetes was determined by monitoring body weight and blood glucose levels. Section title: Ablation of Rat Thyroid Glands. Educational score: 4.054826736450195 Domain: biomedical Document type: Study Language: en Fetal PVG and PVG.RT1 u rats were exposed to 131 I ( Amersham International plc ., UK) at day 18 of gestation by intraperitoneal injection of the mother with 2 mCi of the radioactive isotope. The success of the ablation was determined both histologically and by assaying serum thyroid-stimulating hormone (TSH) levels by radio immunoassay ( Amersham International plc .) in 8-wk-old treated rats. Section title: Isolation of T Cell Subpopulations. Educational score: 4.147163391113281 Domain: biomedical Document type: Study Language: en Mature rat CD4 + T lymphocytes were negatively selected from lymph node and spleen cells using magnetic beads ( Dynal , Norway) by depletion of B cells, CD8 + T cells, and recent thymic emigrants using the mAbs OX12, OX8, OX7, and OX6. CD4 + CD8 − thymocytes were similarly purified from thymus by depletion of CD8 + cells. The purity of all isolated cells, analyzed on a FACScan ® by labeling pre- and postdepletion samples with rabbit anti–mouse Ig FITC, was >98%. Section title: Detection and Quantitation of Anti-Tg Antibodies in Sera of Rats. Educational score: 4.140382766723633 Domain: biomedical Document type: Study Language: en Sera from TxX rats were assayed by specific ELISA using 96-well microtiter plates coated overnight with purified rat Tg (20 μg/ml). After incubation of serial dilutions of sera from individual rats for 2 h at room temperature, bound rat IgG was detected using anti–rat IgG alkaline phosphatase conjugate ( Sigma Chemical Co. ) for 1 h at room temperature. The assay was developed using enzyme substrate 4-nitro-phenyl phosphate (5 mg/ml; Sigma Chemical Co. ) for 15 min at room temperature before reading the OD at 405 nm. Anti-Tg antibody titers were quantified by comparison with a standard serum pooled from TxX rats with thyroiditis and high anti-Tg antibody titers and expressed as a percentage of this standard. The level of nonspecific binding found in normal PVG serum represented a titer of ∼0.1% in the assay, and therefore only sera with titers >0.3% were considered to contain specific anti-Tg antibodies. Section title: Histological Analysis. Educational score: 3.9052188396453857 Domain: biomedical Document type: Study Language: en Whole thyroids attached to thyroid cartilage were dissected out and fresh-frozen in O.C.T. embedding medium (Sakura Finetek U.S.A. Inc.). 10-μm sections were cut from frozen blocks and stained with hematoxylin and eosin. Section title: Results and Discussion Educational score: 4.140412330627441 Domain: biomedical Document type: Study Language: en Athyroid rats were generated after a modification of a previously described protocol using 131 I ( 19 ). Rats were treated in utero with 131 I on day 18 of gestation by injecting the pregnant mother with 2 mCi of the isotope into the peritoneal cavity. Serial sections of thyroid cartilage from 8-wk-old 131 I-treated rats revealed extensive destruction of glands with little or no follicular thyroid tissue apparent . The success of the ablation was confirmed by the detection of high levels of TSH in serum of treated animals. Thyroxine normally exerts negative feedback inhibition of TSH production via the hypothalamic-pituitary axis, such that in the absence of thyroxine TSH levels are allowed to increase unchecked. Serum TSH levels in PVG or PVG.RT1 u rats treated with 131 I in utero were >10 times higher than those of normal rats (Table I ). Section title: Results and Discussion Educational score: 4.267184734344482 Domain: biomedical Document type: Study Language: en To determine whether thyroid antigen–specific T reg function had been affected by ablation of thyroid glands, CD4 + T cells from these rats were assayed for their ability to regulate thyroid-specific autoimmunity by their adoptive transfer into TxX PVG rats before development of thyroiditis. Although the protective capacity of CD4 + T cells has been shown previously to be mediated entirely by the CD45RC − subset ( 8 , 10 ), unfractionated CD4 + T cells were assayed because, at least in principle, it was possible that in the absence of specific thyroid antigen in 131 I-treated rats, T reg would still be functional but assume a naive CD4 + CD45RC + phenotype. Autoimmune thyroiditis was induced in PVG rats by their thymectomy at 3 wk of age followed by four doses of 275 rad 137 Cs γ-irradiation at 2 wk intervals starting 1 wk after thymectomy. Groups of these rats were reconstituted shortly after the final irradiation with either 5 × 10 6 CD4 + CD8 − thymocytes or 10 7 CD4 + peripheral T cells purified from either normal or athyroid 8-wk-old PVG rats. Consistent with previous studies, a highly significant proportion of rats reconstituted with CD4 + CD8 − thymocytes or CD4 + T cells from normal rats were protected from development of both serological and histological signs of disease. In contrast, TxX rats reconstituted with CD4 + T cells from athyroid rats developed high titers of anti-Tg antibody and had extensive leukocytic infiltration of thyroid glands . Significantly, CD4 + CD8 − thymocytes from the same athyroid rats were as effective as the same population from normal rats at preventing development of thyroiditis . Section title: Results and Discussion Educational score: 4.238406658172607 Domain: biomedical Document type: Study Language: en One possible explanation for the failure of CD4 + T cells from athyroid rats to control thyroiditis was that the thyroxine deficiency that resulted from 131 I treatment may have had an adverse effect on either thymopoiesis, and therefore normal repertoire selection, or on peripheral T cell function including that of T reg . Therefore, to test both the specificity of the regulatory deficit and the impact of thyroxine deficiency on T reg function, the ability of T cells from athyroid rats to prevent autoimmunity was assayed in a second autoimmune model not involving thyroid antigens. PVG.RT1 u rats were induced to develop insulin- dependent diabetes by a similar protocol of thymectomy and split dose γ-irradiation. Shortly after the final irradiation, rats were reconstituted with 10 7 CD4 + peripheral T cells from either normal or athyroid syngeneic donors. Significantly, the level of protection from diabetes afforded to TxX PVG.RT1 u rats reconstituted with 10 7 CD4 + T cells from athyroid syngeneic donors was almost identical to that observed in recipients of 10 7 CD4 + T cells from age-matched euthyroid controls. Approximately 50% of recipients were protected in both cases , a level of protection essentially the same as that observed in previous studies ( 8 ). Furthermore, that intrathymic generation of T reg was not adversely affected by thyroxine deficiency in 131 I-treated rats was evident from the observation that CD4 + CD8 − thymocytes from these rats were able to prevent thyroiditis in TxX PVG rats. Section title: Results and Discussion Educational score: 4.3823161125183105 Domain: biomedical Document type: Study Language: en The most economical interpretation of these data is that peripheral autoantigen is itself responsible for the induction of T reg , and although other interpretations are possible, T reg appear to express TCRs specific for the relevant autoantigen. Athyroid rats were deficient only in T reg that control thyroid targeted autoimmunity, since cells from these rats could still suppress diabetes . Such observations are compatible with others studies of thyroid autoimmunity that result after removal of thyroids during gestation, either surgically or by 131 I ablation. Bilateral thyroidectomy but not hemithyroidectomy of lambs at 52 d of gestation results in a loss of self-tolerance to the same thyroid glands, retransplanted after their storage in nude mice ( 20 ). Similarly, rats exposed to 131 I in utero reject syngeneic thyroid grafts as adults ( 19 ). However, when thyroid- ablated rats are surgically parabiosed with normal, syngeneic partners, self-tolerance to thyroid transplantation is restored ( 21 ). These results provide further evidence implicating a role for peripheral self-antigen in the generation of specific T reg . Experiments with T reg in mice, which prevent skin allograft rejection, are also compatible with this interpretation ( 22 ). Section title: Results and Discussion Educational score: 4.567476272583008 Domain: biomedical Document type: Study Language: en The observation of this study that CD4 + CD8 − thymocytes from athyroid rats retain the capacity to prevent thyroiditis, in stark contrast to their peripheral counterparts, has implications regarding the lineage development of these regulatory cells. Earlier studies demonstrated that CD4 + CD8 − thymocytes are a more potent source of T reg than are peripheral CD4 + T cells ( 9 ) and that this is unlikely to be attributed to differences in their mechanism of action, since protection from disease is dependent on IL-4 and TGF-β in both cases ( 10 ). One interpretation of these data is that maturation of T reg from thymic emigrants into the mature peripheral T reg population is subject to strict homeostatic regulation, such that only as many T reg undergo peripheral maturation as are required to suppress the activity of autoreactive T cells in the repertoire. Under conditions where the T reg pool has been perturbed, as occurs during the induction of autoimmunity in TxX rats, such a mechanism would stimulate the differentiation of all potential precursors among CD4 + CD8 − thymocytes. Since mature peripheral T reg have already undergone differentiation, no further recruitment is possible, and it is this difference in plasticity of the regulatory populations that results in the apparent potency of thymocytes compared with peripheral cells. That CD4 + CD8 − thymocytes from athyroid rats are able to prevent disease development shows that intrathymic generation of T reg during repertoire selection does not require peripheral thyroid tissue. Thymocytes from these animals have the potential to mature into specific T reg , but in the absence of peripheral antigen fail to do so. However, adoptive transfer of these cells into euthyroid TxX rats rescues T reg development and in doing so protects the recipients from disease development. These data suggest that the requirement for self-antigen in T reg maturation is also the point of homeostatic control, raising the possibility that it is the T reg themselves that regulate their own differentiation. With regard to the fate of the T reg precursor that emerges from the thymus of athyroid rats in the absence of autoantigen, it is not possible to conclude whether the regulatory deficit in these rats results from a failure of these cells to expand to a functionally significant frequency or a failure of the cells to survive in the absence of autoantigen. Section title: Results and Discussion Educational score: 4.340826034545898 Domain: biomedical Document type: Study Language: en The finding that the thymus is a potent source of T reg ( 9 ), together with the observation that mRNA for many “tissue-specific” autoantigens, including insulin and thyroglobulin, can be detected intrathymically ( 23 – 25 ) is suggestive of a role for positive selection of the T cell repertoire on self-antigens in the generation of T reg . There is some evidence that the self-antigens that mediate positive selection are antagonist peptides ( 26 , 27 ). Significantly, recognition of synthetic antagonistic peptide has been shown in several studies to affect cytokine production, notably that of TGF-β ( 28 ). This raises the possibility that peripheral T cell recognition of the autoantigen on which they were selected intrathymically induces generation of T reg and evokes a regulatory response from them that acts to specifically suppress autoimmune responses ( 6 ). Section title: Results and Discussion Educational score: 4.127493858337402 Domain: biomedical Document type: Study Language: en Finally, previous studies suggest that there exists in the T cell repertoire a fine balance between autoreactive T cells and the T reg that control them ( 9 ). Thus, any adverse perturbation of this balance, either congenital or environmental, could result in autoimmunity. The conclusion of this study, that peripheral autoantigen is responsible for the induction of specific T reg from precursors found in abundance amongst mature thymocytes, raises the possibility of augmenting the physiological process with a therapeutic outcome. In principle, administration of autoantigens in an appropriate form may have the effect of increasing the induction of T reg and as such represent a basis for vaccination against autoimmune disease. | Study | biomedical | en | 0.999998 |
10051512 | Section title: Spark fluorescence and the underlying time course of SR Ca 2+ release. Educational score: 4.430673599243164 Domain: biomedical Document type: Study Language: en Before discussing the number of channels responsible for generating a Ca 2+ spark, it is important to first consider what information the spark provides concerning the underlying time course of SR Ca 2+ release. The signal actually monitored in studies of Ca 2+ sparks is the change in fluorescence of a calcium indicator, generally fluo-3, within the confocal volume in a confocal line scan image. The observed time course of fluorescence at the spatial center of a spark can be interpreted using a qualitative approach that provides a general perspective on the possible time course of the underlying Ca 2+ release and serves as a starting point for the present considerations. During the rising phase of a spark, the concentration of Ca 2+ –fluo-3 must be increasing in the confocal volume. This indicates that Ca 2+ entry into the confocal volume must exceed the net effect of Ca 2+ “removal” by binding and diffusion out of the confocal volume. Thus, during the rising phase of a spark, Ca 2+ ions are being released from the channel or channels responsible for generating the spark . In contrast, during the falling phase of fluorescence in a spark, there is a net fall of Ca 2+ –fluo-3 in the confocal volume, indicating that Ca 2+ binding and Ca 2+ diffusion out of the confocal volume exceed Ca 2+ entry. Since the diffusion and Ca 2+ binding properties of the myofibril are unlikely to change significantly during the spark, the declining phase of a spark must correspond to a period during which Ca 2+ release occurs at a much lower rate than during the rising phase. In the extreme case, Ca 2+ release could occur at an approximately constant rate during the rising phase of the spark, and then stop completely during the falling phase . Section title: Ca 2+ release turns on and off abruptly at the start and peak of a spark. Educational score: 4.492668151855469 Domain: biomedical Document type: Study Language: en Our recent studies using relatively high speed (63 μs per line) line scan confocal imaging of Ca 2+ sparks in frog skeletal muscle provide a 30-fold increase in the available temporal resolution of spark time course and give results that are not inconsistent with the extreme interpretation in Fig. 1 . These studies demonstrate a very abrupt transition from rising to falling fluorescence at the peak of the spark, as shown by the theoretical time course in Fig. 1 A, which provides a good representation of most aspects of the observed sparks . The sharp peak in the observed spark time courses indicates a large and abrupt decrease in the rate of Ca 2+ release rate at the peak of the spark. From our results, it does not seem implausible that release could turn off completely at the peak of a spark. In this case, the rise time of the spark would correspond to the total time that the channel or group of channels generating the spark were open, and the declining phase would be a time during which the release rate were zero. Since the high time resolution studies also demonstrate a rather abrupt transition to a high rate of rise of fluorescence at the start of a spark, the level of Ca 2+ release activity underlying the spark also seems to achieve a near maximal rate early in the spark rising phase. In this case, the net level of overall release channel activity in a spark would rapidly jump from zero to a constant rate early in the rising phase, remain approximately constant throughout the rising phase, and then rapidly fall off to zero at the peak of the spark. As indicated diagrammatically in Fig. 1 , this interpretation could correspond to a single channel open for the entire rising phase of the spark (B), multiple channels, each of which remain open throughout the rising phase of the spark (C), or multiple channels that open and close asynchronously and repeatedly during the rising phase of the spark but close within a short interval at the time of peak of the spark (D). In a more general interpretation, the rise time of a spark provides a lower limit for the open time of channels responsible for generating the spark since some channel(s) might open or remain open during the declining phase, even though the rate of release during the declining phase of the spark must have been markedly less than during the rising phase (not illustrated). The above types of interpretation can be made quantitative through the use of detailed modelling of Ca 2+ binding and diffusion in a fiber after release from a channel or group of channels . These quantitative models indicate that release could abruptly turn on and off at the start and peak of the observed sparks. Section title: Properties of individual sparks are not obviously incompatible with Ca 2+ release from a single channel. Educational score: 4.569316864013672 Domain: biomedical Document type: Study Language: en The Ca 2+ sparks detected in a frog skeletal muscle fiber have an average rise time of 4.6 ms and an average decay time constant of 8.6 ms and an average spatial full width at half max of ∼1.4–1.5 μm . The peak change in relative fluorescence (Δ F / F ) of the larger amplitude events, which are most likely to represent events arising spatially closest to the scan line, is ∼1–2. These properties, together with the diffusion and binding properties of the fiber, provide an indication of the amount of Ca 2+ released by the channel or channels that generate the spark. It was already pointed out in early reports on cardiac Ca 2+ sparks that rough approximations of the amount of Ca 2+ released in a spark were not obviously incompatible with channel open times and Ca 2+ flux rates of single SR Ca 2+ channels in bilayers . Using detailed models of the sarcomeric distribution of myoplasmic Ca 2+ binding sites together with the diffusion properties of Ca 2+ and fluo-3, it is possible to calculate the spatio-temporal distribution of Ca 2+ –fluo-3 and the resulting Ca 2+ spark that would be produced by an assumed time course of Ca 2+ release from a point source corresponding to the channel or group of channels generating the spark. These modelling calculations, using reasonable values for the various model parameters and reasonable values for single channel current and channel open time (e.g., 1–2 pA of current for ∼10 ms) result in theoretical Ca 2+ sparks that are not obviously incompatible with the observed sparks . Thus, based on such calculations alone, it is not necessary to exclude the possibility that a Ca 2+ spark could be generated by the Ca 2+ released during the opening of a single SR Ca 2+ release channel. However, it should be noted that this finding does not imply that Ca 2+ sparks are in fact generated by the activity of a single SR Ca 2+ release channel, but only establishes that the experimentally observed sparks are not obviously quantitatively inconsistent with the possibility of spark generation by a single channel. Section title: Spark frequency increases during activation, but spark properties remain constant. Educational score: 4.608293533325195 Domain: biomedical Document type: Study Language: en The “spontaneous” sparks observed in frog fibers appear to be ligand-gated events triggered by calcium-induced calcium release (CICR) since the frequency of spontaneous events increases with increased myoplasmic [Ca 2+ ] and in the presence of caffeine , and decreases with increased myoplasmic [Mg 2+ ] , all hallmarks of CICR. The sparks initiated by fiber depolarization are voltage-activated events, presumably triggered by activation of voltage sensors , the dihydropyridine receptors in the transverse tubule (TT) membrane of the fibers. One of the salient features of Ca 2+ release by both ligand- and voltage-activated Ca 2+ sparks in frog skeletal muscle fibers is that the overall level of calcium release appears to be graded by variations of the frequency of occurrence of Ca 2+ sparks, but that the individual sparks themselves have similar average properties despite marked differences in their frequency of occurrence. For example, with protocols that use relatively brief repriming of chronically depolarized fibers, the frequency of occurrence of voltage-activated events can be modulated by both the extent of repriming and by the membrane potential of the test depolarization used to activate events after repriming, but neither of these parameters appears to affect the average amplitude or average rise time of the detected events . Lowering myoplasmic free [Mg 2+ ] increases the spontaneous frequency of ligand-gated events, but does not alter the average properties of the individual events . Thus, the average properties of the individual events appear to be quite constant despite relatively large changes in their frequency. Section title: Spark frequency increases during activation, but spark properties remain constant. Educational score: 4.409029006958008 Domain: biomedical Document type: Study Language: en An interesting implication of these observations is that the opening rates, but not the closing rates, of the channel(s) underlying the spark appear to be modulated during release activation. The opening rate of the channel(s) that initiate the spark must be increased by the voltage or ligand activation that caused the observed increases in spark frequency since spark frequency directly reflects the rate of opening of the channels that initiate the sparks. In contrast, the overall open time of the channels generating the spark, and thus the effective rate of channel closing, appears to be unchanged. The mean amplitude and rise time of the sparks should change in parallel with any changes in the net open time of the channels generating the spark , but no such changes were observed. Net channel open time could change without a parallel change in the observed spark rise time only for the case of asynchronous opening of multiple channels during the rising phase of a spark , but the spark amplitude would then still vary with changes in the net open time of the channels underlying the spark. However, no changes in mean spark amplitude were observed under the conditions in which spark frequency was markedly increased. Thus, channel closing rates do not seem to be altered under the conditions used for voltage or ligand activation of the sparks. Section title: Spark frequency increases during activation, but spark properties remain constant. Educational score: 4.1482415199279785 Domain: biomedical Document type: Study Language: en In interpreting these results, it is important to bear in mind several technical issues. In the case of both voltage- and ligand-activated sparks, only events occurring at relatively low frequency were examined in order to allow relatively unambiguous identification of individual events. Even in the case of relatively low-event frequencies, there are two other technical issues to consider. First, only events above an arbitrarily selected low-amplitude cut-off were selected for analysis. This cut-off was imposed to avoid including possible noise as events in the analysis of Ca 2+ sparks. Second, the amplitude of each observed event is very sensitive to the site of origin of the Ca 2+ release source relative to the spatial location of the confocal scan line. Since these relative locations are completely random in our studies, a variation of amplitude is introduced simply by the range of release site locations relative to the scan position. However, despite these two limitations, it would seem that if the actual average amplitude of the sparks did increase with increased frequency of occurrence, then the average amplitude of the sparks detected above the arbitrary cut-off amplitude and arising at random locations relative to the scan line should also have increased. Yet no significant changes in spark amplitudes were detected under these conditions of different spark frequencies. Furthermore, mean spark rise time, which represents the effective duration of Ca 2+ release and is less sensitive to differences in spark location relative to the scan line , was also constant under the conditions of different spark frequencies. Section title: Possible regulation of channel opening or Ca 2+ release during a spark. Educational score: 4.390407085418701 Domain: biomedical Document type: Study Language: en By analyzing sparks that occur repetitively at particular locations at much higher rates than the average frequency over the entire fiber, the limitations of variable spark origin and arbitrary cut-off of event amplitudes discussed in the preceding paragraph can to a large extent be overcome. Such higher frequency events occur spontaneously in cardiac myocytes and both spontaneously and during depolarization in frog skeletal muscle fibers and appear to represent a repetitive mode of spark activation. If these repetitive events are generated by the repeated opening of a given channel or small cluster of channels , they would all arise at the same spatial site within the fiber. Thus, variation in spark amplitude due to variation in the site of origin relative to the scan line would not be a factor in the relative amplitude of the individual events within a given repetitive train. Analysis of events in such trains indicates an unanticipated lack of smaller events, even when objective procedures are employed to evaluate possible occurrence of unidentified events between identified events . Examination of the records of repetitive events from cardiac myocytes also indicates a relative lack of smaller amplitude events. If the sparks in a repetitive train were generated by the opening of a single channel having a single or multiple exponential open time distribution and if the spark amplitude were directly related to the channel open time, then smaller events would actually be expected to occur more frequently in the train than larger events. Thus, if a single channel is responsible for generating the repetitive sparks, the observed paucity of smaller amplitude events in repetitive trains would indicate the possibility of some sort of regulation of the amount of Ca 2+ released by the single channel generating the spark. Section title: Possible regulation of channel opening or Ca 2+ release during a spark. Educational score: 4.243317604064941 Domain: biomedical Document type: Study Language: en Data from our recent high time resolution studies of nonrepetitive Ca 2+ sparks may also be consistent with regulation of the amount of Ca 2+ released in a spark. In these experiments, we have observed an inverse relationship between the mean rate of rise of fluorescence in groups of sparks having similar rise times and the mean spark rise time, resulting in a constant mean amplitude in groups of sparks having different rise times . These observations are consistent with the possibility of negative feedback between the local elevation of [Ca 2+ ] and the continuation of Ca 2+ release from the channel or channels responsible for generating the spark. Given the complexity of regulation of individual SR Ca 2+ release channels , it is not inconceivable that such feedback regulation could occur with a single channel generating a spark. Alternatively, a relatively constant amount of Ca 2+ release in different events in a repetitive train could also conceivably occur if multiple SR channels were responsible for generating a spark. Section title: Are two types of sparks activated during fiber depolarization? Educational score: 4.315967559814453 Domain: biomedical Document type: Study Language: en In our original report of the existence of ligand- and voltage-activated sparks in skeletal muscle, the observed amplitude distributions of the sparks initiated by these two types of mechanisms were different . The spontaneous (i.e., ligand-gated) sparks appeared to correspond to a single population of events, whereas the voltage-activated sparks appeared to correspond to one population of events having the same amplitude distribution as the spontaneous events together with another population of events having an amplitude distribution corresponding to approximately twice that of the spontaneous events. The smaller amplitude voltage-activated events were attributed to the opening of a single SR Ca 2+ channel directly by interaction with the voltage sensor. The larger amplitude events were attributed to opening of two channels, one activated directly by the voltage sensor and a second activated by CICR due to the locally elevated [Ca 2+ ] in the immediate neighborhood of the voltage-activated channel. In this case, at least two channels would have to be involved in generating the population of larger voltage-activated Ca 2+ sparks. However, in subsequent studies in our laboratory, we have found no obvious differences in the mean amplitudes of ligand- and voltage-activated sparks in different fibers. In our initial study , we employed levels of activation that resulted in the occurrence of relatively large numbers of sparks at relatively high frequencies since we were searching for evidence for the existence of sparks. Although this strategy may have been appropriate for an initial demonstration of the existence of these events, it was not ideal for the detailed characterization of the properties of individual events due to the possibility of events randomly overlapping in space and time. Although the possibility of chance overlap of random independent events was discounted in our original report, our estimate of the probability of random overlap did not include possibly overlapped events in the estimate of the total event rate used for the calculation. Based on our subsequent experience, it now seems possible that the event rate during the depolarizations in our initial study may have been too high to permit accurate determination of the properties of isolated individual sparks. Thus, the question of the relative amplitudes of ligand- and voltage-activated events in the same fibers should probably be reexamined using lower event rates during fiber depolarization. Section title: Are two types of sparks activated during fiber depolarization? Educational score: 4.532865047454834 Domain: biomedical Document type: Study Language: en Studies using relatively small depolarizations together with pharmacological approaches to selectively modulate a possible CICR component of voltage-activated sparks in frog skeletal muscle fibers have supported the concept of smaller events during depolarization being directly activated by the voltage sensor and larger events having an additional contribution due to local CICR . It will be important to rule out various alternative interpretations, such as possible modulation of channel open time and/or channel conductance or of SR Ca 2+ content due to the pharmacological agent in these or other studies using pharmacological interventions in which changes in average properties of individual sparks are detected. Finally, it is possible that RyR1 and RyR3, the two different mammalian skeletal muscle ryanodine receptor (RyR) isoforms that both have homologues expressed in frog skeletal muscle, may play different roles or release different amounts of Ca 2+ in the generation of Ca 2+ sparks. The failure to detect Ca 2+ sparks in adult rat skeletal muscle , which expresses only RyR1, and the observation that expression of RyR3 but not RyR1 causes the appearance of Ca 2+ sparks in myotubes from a myogenic cell line lacking any RyR expression point to different properties of these two isoforms and to the possibility that one or more RyR3 Ca 2+ channels or their frog homologue may be required for the production of a detectable Ca 2+ spark in skeletal muscle. If coordinated activity in both types of RyR channels is involved in the generation of some sparks in frog muscle fibers, then at least these sparks must require two or more channels. Section title: Could a single SR Ca 2+ channel trigger a multichannel release unit? Educational score: 4.462785720825195 Domain: biomedical Document type: Study Language: en Under conditions of low average rates of occurrence of Ca 2+ sparks as used in our recent experiments, it seems likely that each voltage-activated spark is initiated by the activation of the SR channel controlled by a single TT voltage sensor and that each ligand-activated event is initiated by the opening of a single SR Ca 2+ release channel by CICR. Thus, if a spark involves the opening of multiple SR Ca 2+ release channels, the single channel that opens to initiate the spark must activate one or more neighboring channels, presumably by CICR. These channels could in turn activate additional neighboring channels, which in principal could continue until all the SR channels along an entire region of continuous TT–SR junctional couplings were activated. For the typical relatively brief sparks having a rising phase of a few milliseconds duration, it might be imagined that the propagation of activation would have to occur rapidly at the start of the rising phase, and that the rising phase would end as the channels close in near synchrony, possibly by calcium-dependent inactivation. This general type of propagated activation scheme involving many SR Ca 2+ channels has been simulated using a model of possible local Ca 2+ signalling within the TT–SR junctional region . Thus, the possibility that activity of many SR channels coupled by CICR underlies a spark is not theoretically inconsistent with initiation of the spark by activation of a single channel. Section title: Could a single SR Ca 2+ channel trigger a multichannel release unit? Educational score: 4.353824615478516 Domain: biomedical Document type: Study Language: en In considering the possibility of propagation of activation by CICR from the SR channel to a channel along a strip of TT–SR junctional contact, it is relevant to reiterate that lowering myoplasmic [Mg 2+ ], which decreases the inhibitory influence of Mg 2+ on CICR, increases the frequency of spontaneous sparks but does not alter the mean amplitude or mean rise time of the sparks . These observations indicate that the number of SR channels contributing to a spark does not seem to be influenced by [Mg 2+ ]. Thus, if propagation of activation from the SR channel to a channel along the junctional region by local CICR is responsible for generating the spark, the number of channels activated by propagation along the TT–SR junctional strip does not seem to be significantly affected by alterations in Mg 2+ inhibition of CICR. One explanation could be that the safety factor for propagation is sufficiently high that all SR channels in the strip are activated at all levels of [Mg 2+ ] tested so that activation was essentially “all or none” at the level of an individual junctional strip. Alternatively, propagation along the junctional strip may in fact not occur, in which case each spontaneous spark would be generated by the opening of only a single SR channel by CICR. Section title: Could a single SR Ca 2+ channel maintain prolonged release in many other channels? Educational score: 4.419003009796143 Domain: biomedical Document type: Study Language: en We have observed that application of Imperatoxin A (IpTx a ) to a permeabilized frog muscle fiber causes the appearance of prolonged Ca 2+ sparks having durations of several hundred milliseconds or longer . Since IpTx a is known to produce similarly prolonged subconductance openings (about one-third conductance of normal channel opening) of individual SR Ca 2+ release channels incorporated in lipid bilayers , possibly by acting as an analogue of the 2–3 cytoplasmic loop of the TT dihydropyridine receptor/voltage sensor, it seems likely that the prolonged sparks observed in the presence of IpTx a were generated by the prolonged opening of a single SR channel to a subconductance state. The very long duration sparks initiated by IpTx a were also smaller in amplitude than normal short-duration events observed in the same fibers. These observations raise the interesting question of whether the prolonged toxin-induced opening of a single SR channel could maintain the opening of many other SR channels without the other channels inactivating. Alternatively, the prolonged toxin-induced sparks could be readily explained if toxin-induced sparks are generated by the prolonged opening of only the single channel interacting with the toxin. In this case, the observation that the amplitude of the toxin-induced event is smaller then that of a brief toxin-independent spark, together with the fact that IpTx a produces a subconductance state when applied to isolated SR channels, could indicate that the normal sparks observed in the absence of toxin could also be generated by the opening of a single SR Ca 2+ release channel, but to the full conductance state and only for a few milliseconds. Section title: Conclusion Educational score: 4.391638278961182 Domain: biomedical Document type: Study Language: en Based on the various considerations presented above, it does not appear that we can yet exclude the possibilities that either one or many channels are involved in the generation of a Ca 2+ spark. If a single channel is responsible for generating a spark, the single channel must have appropriate feedback regulation so as to account for the reproducible spark amplitude and relative lack of small events during the repetitive spark gating mode observed at occasional triads. On the other hand, the very prolonged small amplitude sparks observed in the presence of IpTx a are readily explained on the basis of prolonged subconductance opening of a single toxin-bound channel. If many channels are involved in the normal, short duration voltage- or ligand-activated spark, they would have to open and close in close synchrony, or burst over the same few milliseconds time interval to account for the abrupt start and peak of the observed spark time course. If many channels are involved in a short spark, it would also seem to be necessary for a single open channel to have the capability of maintaining long duration opening of at least some of the other channels involved in the short spark to account for the long duration events produced by IpTx a . It will be an interesting challenge to try to resolve these still viable important alternative possibilities as to the channel activity pattern underlying the Ca 2+ sparks observed in frog skeletal muscle. | Study | biomedical | en | 0.999998 |
10051514 | Section title: How Big Is a Spark? Educational score: 4.099489212036133 Domain: biomedical Document type: Study Language: en Measured spark amplitude varies widely from the lower limits of detection (defined by noise, usually ∼0.3 U F 0 ) to the highest values recorded, which in our experience with cut fiber segments under voltage clamp go routinely beyond 6 F 0 for a few sparks in every experiment. Current work with cardiac myocytes and smooth muscle also finds events of amplitude >5, much larger than in earlier reports . In our experience amplitudes are substantially lower in fibers permeabilized by saponin or by notches and immersed in internal solution . Section title: How Big Is a Spark? Educational score: 4.286579608917236 Domain: biomedical Document type: Study Language: en The reason for the differences in amplitude may be quite simple: as stated, Δ F / F 0 is close to the ratio between increase in local [Ca 2+ ] and resting [Ca 2+ ] i . The cell may be able to maintain a substantially lower internal Ca 2+ in the voltage clamp experiments, which expose it to internal solution at the cut ends only. By contrast, in permeabilized fiber segments, free [Ca 2+ ] should equilibrate rapidly within the cell at the solution value, and the dye rises rapidly, reaching values higher than the concentration in the internal solution. Therefore, the relative increase Δ F / F 0 (or Δ[Ca 2+ ]/ resting [Ca 2+ ]) should be less in the permeabilized fibers due to the buffering effect of the higher [dye] (which reduces Δ[Ca 2+ ]) and the higher resting [Ca 2+ ]. Indeed, sparks with very large values of Δ F / F 0 are only found early in voltage clamp experiments, when the resting fluorescence is very low (indicating low dye concentration, low [Ca 2+ ], or both). Section title: How Big Is a Spark? Educational score: 4.167460918426514 Domain: biomedical Document type: Study Language: en In our first study of discrete events , we evaluated event amplitude by constructing all-points histograms of the difference records between fluorescence at triadic centers and in the neighboring sarcomeres. The resulting histograms had modes at between 0.1 and 0.3 F 0 , which were interpreted as spark amplitudes. We now believe that the all-points histogram is not an adequate tool to recognize sparks because it does not take into account their multidimensional aspects. When their frequency is low, sparks will contribute negligibly to the all-points histogram, which should be dominated by very small fluctuations that may correspond to out-of-focus sparks or to continuous, eventless release. In fact, when automatic procedures are used with an amplitude selection criterion (see next section), the resulting histograms also peak at or near the lowest allowed amplitude. Therefore, modes in the amplitude distributions will always be at very small values regardless of the true amplitude of the events. We now prefer methods of recognition by multidimensional criteria (amplitude, width, duration). Such recognition was initially done by an observer , but is now entirely automatic , and yields amplitude estimates at least an order of magnitude greater. Section title: How Big Is a Spark? Educational score: 4.238024711608887 Domain: biomedical Document type: Study Language: en The width of large sparks (measured as full width at half magnitude, FWHM) varies between 1.5 and 2.2 μm in skeletal muscle. In cardiac muscle, where some sparks appear to result from multiple release sources and can be very wide, there is a well defined subset of sparks that appear to originate from single release sites. The half width of those is similar to that in skeletal muscle, ∼2 μm. Surprisingly, and as shown in Fig. 1 , in simulations of sparks resulting from a discrete Ca 2+ source the half width is only ∼1 μm, regardless of assumptions on the properties of buffers and the dye. The width of the simulated spark could be increased to reach values near 2 μm by increasing the radius of the source (to 1 μm). The discrepancy and its resolution may be taken as evidence that the source of release is extensive (rather than a single channel), or it may mean that the models of Ca 2+ release and removal are inadequate in ways we do not understand. Section title: Ca 2+ Release Flux Underlying a Spark Educational score: 4.183927059173584 Domain: biomedical Document type: Study Language: en This has been calculated in cardiac and skeletal muscle by generalizing global or whole-cell procedures of Baylor et al. and Melzer et al. to the spatially resolved images obtained by confocal microscopy. The earlier procedures first derive from the optical signals the transient increase in free [Ca 2+ ], and then calculate the increase in total calcium necessary to account for such transient by adding contributions from different Ca 2+ binding and removal processes. They are therefore subject to errors in the calculation of free [Ca 2+ ] and in the ulterior estimate of binding and removal. The generalization to sparks has additional uncertainties regarding diffusional mobility of the different Ca 2+ ligands and placement of fixed sites. If calculations are carried out assuming a homogeneous and unrestricted distribution of sites, with consensus values for concentrations, diffusion coefficients, and reaction rate constants, the values obtained are typically near 10 pA, and often >20 pA for the largest sparks in every image. Section title: Ca 2+ Release Flux Underlying a Spark Educational score: 4.3463826179504395 Domain: biomedical Document type: Study Language: en A lower bound of released Ca 2+ can be obtained without calculating local [Ca 2+ ], based on the rate of Ca 2+ binding to the dye that is needed to account for the observed fluorescence. Take as numeric example a permeabilized fiber equilibrated with a 100 nM free [Ca 2+ ], 200 μM fluo-3 solution, at a time when the internal dye concentration has risen to 300 μM. In this condition, the resting fluorescence corresponds to ∼30 μM of the dye:Ca complex . A large spark under these conditions may have an amplitude of 2.5 F 0 , requiring an additional 75 μM of dye-bound Ca 2+ . The quantity of dye-bound Ca 2+ can be found by volume integration of concentration, roughly the product of peak value by the volume of the sphere that intersects the spark at half magnitude, say 1.8 μm. Thus, 75 μM × 3.05 μm 3 or 0.23 amol Ca 2+ , equivalent to 43.4 fC, must be released within the rise time of the spark, 5 ms. Therefore, an average current close to 10 pA must flow for 5 ms just to account for the Ca 2+ bound to the dye in this example. Such values are typical of large sparks in experiments with permeabilized fibers. Section title: Ca 2+ Release Flux Underlying a Spark Educational score: 4.181893348693848 Domain: biomedical Document type: Study Language: en The free [Ca 2+ ] in cardiac SR was estimated using 19 F NMR at 1.5 mM . Mejía-Alvarez et al. used the result to estimate the physiologic current-carrying capability of cardiac release channels, measuring their Ca 2+ current in bilayers, with a luminal side [Ca 2+ ] of 2 mM and in the presence of concentrations of Cs + comparable to physiologic [K + ]. Under those conditions, the average unitary current was 0.35 pA. Extrapolated to the case of skeletal muscle, this estimate indicates that tens of channels should be open to account for large sparks. Section title: Ca 2+ Release Flux Underlying a Spark Educational score: 4.342338562011719 Domain: biomedical Document type: Study Language: en Though lower than in earlier work , these estimates of current per channel are consistent with whole-cell measures of release flux. The maximum flux density (expressed as rate of rise of total calcium concentration in the accessible myoplasmic volume) under voltage clamp in the frog is between 180 and 200 mM/s . The volume density of release channels can be gathered from morphometry. The ratio of transverse tubule length to fiber volume is 0.82 μm −2 in frog muscle . If 70% of this length is junctional and contains a double row of release channels at 30 nm spacing on each side , the number of channels per liter is 0.82 μm −2 (tubule length/fiber volume) × 0.7 (triad length/tubule length), × 2 (junctions/triad), × 2 (rows/junction), × 33 (channels/row/μm), or 1.08 × 10 17 channels per liter of fiber. At 100% activation, such channels passing 0.35 pA would generate a flux density of 180 mmol/liter of fiber per s, or 260 mM/s in terms of accessible aqueous myoplasmic volume, surpassing the highest whole-cell estimates. Section title: The Distribution of Spark Amplitudes Educational score: 1.6513818502426147 Domain: other Document type: Other Language: en The appeal of sparkology, as we stated, is to some extent esthetic—images with sparse sparks, in a dark experimental room, look very much like a starry sky. Much as stars in the sky, small sparks greatly outnumber the large ones, and the reasons for this are not very different for sparks and stars. Indeed, sparks are recorded in line scans, but may originate anywhere within the junctions of a Z disk. The smoothly decaying point spread function of the imaging system implies that the sparks originating farther from the scanned line will appear smaller in the record. And the regions of Z disk that lie “far” are greater than those that are “near,” hence the probability density function of spark amplitudes is expected to be decaying, even if sparks as objects are all of the same size. Section title: The Distribution of Spark Amplitudes Educational score: 3.929380416870117 Domain: biomedical Document type: Study Language: en The above reasoning led two groups to derive, using different formal approaches, the distribution of amplitudes expected in a line scan image for identical sparks that originate with homogeneous probability in triadic locations . This function is a simple inverse proportionality (pdf ∝ a −1 , where a is recorded amplitude). Section title: The Distribution of Spark Amplitudes Educational score: 4.146706581115723 Domain: biomedical Document type: Study Language: en In agreement with theory, the amplitude histograms of events identified by a program that locates them without human intervention are monotonically decreasing. The dependence of frequency on amplitude, however, is not the inverse function predicted for identical sparks, being consistent instead with a widely spread distribution of real spark amplitudes. The distribution of real spark amplitudes exhibits in the presence of caffeine a mode or preferred amplitude at between 2 and 3 F 0 . Section title: The Distribution of Spark Amplitudes Educational score: 4.0367231369018555 Domain: biomedical Document type: Study Language: en With this improved understanding of the meaning of recorded spark amplitude, it is natural to use large sparks, rather than those of average size, to estimate release flux, because the large ones are more likely originated near the scanned line, where recorded amplitudes are greatest. Because there is a spread of actual amplitudes, the largest sparks, whose underlying release current was estimated at >20 pA, may be outliers of unusually large amplitude as objects. For the sparks of the most common size, one third of the above estimate should apply, or ∼7 pA. Section title: Beyond Sparks Educational score: 4.334376335144043 Domain: biomedical Document type: Study Language: en The above considerations, plus the structural evidence that release channels are clustered in closely packed arrays, in skeletal as well as cardiac muscle , present a problem if single channels gate individually to produce sparks. How can a release channel pass the spark current (somewhere between 1 and 30 pA) without activating other channels that face the same junctional gap, where [Ca 2+ ] would rapidly rise beyond 100 μM ? The argument was bolstered by the demonstration in cardiac muscle of coupled fluorescence events in scans perpendicular to the fiber axis and of multiple sources of release under large sparks of cat atria . Both observations show that the activation of cardiac release channels, presumably by Ca 2+ , occasionally reaches across distances of 1 μm or more, stressing that single Ca 2+ -activatable channels within a junction can hardly operate independently. Section title: Beyond Sparks Educational score: 4.1487226486206055 Domain: biomedical Document type: Study Language: en A many-channel origin for sparks was supported by the observation in ventricular myocytes of a diffuse increase in fluorescence, devoid of sparks, when trigger Ca 2+ was delivered by photolysis of DM-nitrophen . The authors hypothesized that thus triggered SR Ca 2+ release was composed by contributions (termed quarks) that could not be individually detected. Although the terminology is intriguing, skeletal muscle soon forced consideration of another alternative, continuous release. Section title: Beyond Sparks Educational score: 4.1162109375 Domain: biomedical Document type: Study Language: en In skeletal muscle, an increase in fluorescence not composed of sparks can be readily demonstrated under a number of conditions. When slightly depolarized, some frog fibers show at triads release not constituted by discrete events, while typical sparks appear at higher voltages . In tetracaine at 200 μM, which blocks release channels in bilayers , depolarization triggers continuous release but no sparks . Section title: Beyond Sparks Educational score: 4.269600868225098 Domain: biomedical Document type: Study Language: en In mammalian muscle of adult rats, voltage-clamp depolarization caused continuous Ca 2+ release and contraction, but no sparks . The triadic origin of the release was demonstrated by the existence of a triadic gradient of fluorescence . Because the gradients of fluorescence associated with continuous release in both frogs and rats could be much lower than in sparks, it appears that continuous release may involve smaller currents, again indicating the participation of channel groups in the production of sparks. Section title: Beyond Sparks Educational score: 4.340985298156738 Domain: biomedical Document type: Study Language: en Shirokova and Ríos interpreted the continuous release observed in frogs at very low voltages or in the presence of tetracaine as flux through channels directly operated by voltage sensors. They proposed that sparks are the consequence of opening of multiple channels, caused by a local increase in [Ca 2+ ]. This increase would be started in frog skeletal muscle by release channels operated by voltage-sensing DHPrs, and reinforced by the opening of additional channels. The directly voltage-operated release component could appear alone, without triggering CICR and sparks, if the concentration of trigger Ca 2+ was insufficient (e.g., at low voltage depolarization) or the sensitivity to Ca 2+ was inhibited (in the tetracaine experiment). This hypothesis also explains why it is so difficult to demonstrate nonspark release in cardiac muscle, where presumably there is no direct activation of release by voltage. Section title: Beyond Sparks Educational score: 4.216826915740967 Domain: biomedical Document type: Study Language: en Rat muscle provides an example of eventless release that is most interesting for various reasons. The failure to observe sparks there could simply evidence difficulties in working with smaller, weaker cells, cut , and at far from physiologic temperature. We believe instead that the absence of sparks in adult rats reflects a fundamental difference in the E-C coupling mechanism, consistent with differences found in whole cell experiments . These differences include a lesser peak of Ca 2+ release flux, a lower ratio between the peak and steady levels of release under voltage clamp, the absence in the rat of the characteristic voltage dependence of this ratio observed in the frog, and the presence in the rat , but not in the frog , of the RISC phenomenon, a stop by repolarization of the release induced by caffeine. Section title: Beyond Sparks Educational score: 4.393983840942383 Domain: biomedical Document type: Study Language: en RISC is interpreted as indicating a preponderance of control by membrane voltage (via the DHPr). Its presence in the rat, and failure to appear in the frog, are consistent with our failure to demonstrate sparks (which presumably are mediated by CICR) in rats, and the ease with which continuous, voltage-operated release can be demonstrated in the mammal. In all, it appears that CICR is fundamental in the frog, where it determines sparks and the pronounced, steeply voltage-dependent peak of the release waveform, while it is much less important, perhaps absent, in the rat. Because sparks were present in myotubes from embryonic or neonatal mice, which unlike adult cells express RyR isoform 3, Shirokova et al. proposed that the production of sparks could require RyR3 (or the corresponding β isoform in frog muscle). This possibility is now bolstered by the observation of sparks in dyspedic mouse cells expressing RyR3 but not RyR1 . Section title: Beyond Sparks Educational score: 4.262981414794922 Domain: biomedical Document type: Study Language: en Finally, the molecular makeup of frog triads is usually assumed to be the same as that in mammals, but this may not be the case. The ratio of ryanodine-binding sites to DHPrs expected from the structural model of Block et al. , in which four voltage sensors face every other release channel, is 0.5. This value is found approximately in membrane fractions of rabbit or human muscle . In frogs, the value is ∼1.5 , which would suggest an excess of release channels, either outside the double row or violating the pattern in some other way. The possibility of major structural differences must be kept in mind, as the morphological alignment of mammalian and fish junctions has never been confirmed in the frog . Section title: Models of Spark Generation Educational score: 4.352523326873779 Domain: biomedical Document type: Study Language: en The “couplon” model is the latest of a class that assumes interactions within arrays of channels. The couplon, consisting of the contiguous array of RyRs associated with one side of a junctional segment of transverse tubule, includes two rows of RyRs that alternate between those associated with a DHPr tetrad, presumed to be controlled only by voltage (V channels), and those lacking contact with DHPrs, which are assumed to be activated and inactivated by Ca 2+ alone (C channels). With suitable parameters, this model reproduces many of the global features of frog skeletal muscle E-C coupling , which would be difficult to explain if release resulted from channels gating independently. Section title: Models of Spark Generation Educational score: 4.256711959838867 Domain: biomedical Document type: Study Language: en The couplon model normally generates spark-like release packets. Openings of V channels trigger, by CICR, openings of adjacent C channels, which in turn may recruit other C channels. The whole couplon, or a part of it, may thus activate to generate a phenomenon similar to a spark. Given the assumed C channel unitary current, 0.3 pA, and the size of a couplon (10–30 C channels), the simulated spark sizes are consistent with the observations. Most interestingly, the couplon sparks have a preferred amplitude , in agreement with amplitude distributions obtained by González et al. in the presence of caffeine. Section title: Models of Spark Generation Educational score: 4.338289737701416 Domain: biomedical Document type: Study Language: en Could single channel openings produce sparks with a preferred amplitude? As shown in Fig. 1 , the amplitude of a spark due to a single channel opening should be nearly proportional to the opening duration. For a single Markovian channel, gating reversibly, the distribution of open times is a sum of positively weighted decaying exponentials , so a mode in the distribution of amplitudes would not be expected. However, RyRs of striated muscle can be activated by its own permeating Ca 2+ under suitable conditions in lipid bilayers . This would couple the free energy of Ca 2+ permeating down its electrochemical gradient to the gating process, permitting irreversible gating kinetics and open time distributions with a mode . Section title: Models of Spark Generation Educational score: 4.396954536437988 Domain: biomedical Document type: Study Language: en The couplon simulations reproduce the sharp peak observed in whole-cell determinations of Ca 2+ release in the frog as essentially a sum of sparks, while the steady level that follows the peak is accounted for as a combination of sparks and continuous, directly voltage-operated release. This picture of the peak of release as composed of sparks and mediated by CICR justifies that tetracaine eliminates sparks together with the peak , and that the peak can be reconstructed by superposition of sparks whose timing is determined in fibers with partially inactivated voltage sensors . Section title: Models of Spark Generation Educational score: 4.388537406921387 Domain: biomedical Document type: Study Language: en The couplon, and related multichannel models, explain the increase of the peak relative to the steady level as voltage is increased, as a result of cooperation of multiple voltage-operated sources within the same couplon to produce a level of Ca 2+ that will elicit sparks. Such models predict that partial inactivation of voltage sensors or interference with CICR may be compensated by recruiting more voltage sensors, hence explaining that DHPr antagonists , high intracellular Mg 2+ , or BAPTA , inhibit sparks or the peak phase of release at low, but not at high voltages. Section title: Models of Spark Generation Educational score: 4.289671897888184 Domain: biomedical Document type: Study Language: en The most parsimonious model (release entirely composed of sparks reflecting opening of one or two channels operated by one voltage sensor) is ruled out by its prediction that disabling some voltage sensors would just scale down release and spark numbers. Instead, the waveform reconstructed in partially inactivated fibers showed a much smaller steady component of release flux at high voltage than in fully primed fibers , which again suggests the existence of a voltage-operated release not in discrete events, and required higher voltages to produce a peak of release , which again indicates cooperation among multiple voltage sensors to activate the same release unit. Section title: Terminating the Spark Educational score: 3.8822760581970215 Domain: biomedical Document type: Study Language: en As we stated at the outset, sparks were not expected. Their spatial discreteness could have been predicted on structural grounds , but their temporal brevity was startling. Rise times of sparks, which roughly measure release duration, are quite independent of triggering voltage, partial inactivation , or Ca 2+ channel blockers , which suggests a termination mechanism intrinsic to the release unit, rather than the trigger. Section title: Terminating the Spark Educational score: 4.50119686126709 Domain: biomedical Document type: Study Language: en The models of spark generation described above underscore the difficulties in devising a mechanism with robust termination. Local depletion of SR Ca 2+ has been ruled out in cardiac myocytes because Ca 2+ sparks may last up to a few seconds . In skeletal and cardiac muscle, images of sparks arising repetitively from the same unit also indicate that depletion does not determine spark termination. Sham et al. examined the issue in cardiac myocytes, where sparks are triggered by Ca 2+ influx through membrane channels. Observing that reopenings of membrane Ca 2+ channels, even with duration greatly prolonged by FPL64176, failed to trigger Ca 2+ release, they concluded that the termination mechanism includes desensitization to the trigger. Furthermore, they showed that this loss of sensitivity is not an adaptation because Ca 2+ release activated by the tail membrane current at the end of a pulse involved only channels that were not activated at the beginning of the pulse. Taken together, these results indicate that Ca 2+ sparks are terminated primarily by inactivation of RyRs. It remains to be established whether the inactivation is a Ca 2+ -dependent process , or is “fatal” , an obligatory coda to channel opening. Section title: Terminating the Spark Educational score: 3.877384901046753 Domain: biomedical Document type: Study Language: en In any case, the implementation of such mechanisms is more difficult if sparks are multichannel events, simply because it is more difficult to turn-off many channels synchronously. Inter-channel allosteric interactions might help, channel closing could be synchronized by negative interactions between RyRs in the quasi-crystal of the junction . Section title: Terminating the Spark Educational score: 4.645428657531738 Domain: biomedical Document type: Study Language: en In summary, sparks appear to constitute the totality of physiologic Ca 2+ release in cardiac muscle, and a major portion of it in frog skeletal muscle. The early peak of release during a voltage pulse is largely constituted by a superposition of sparks, a conclusion that can probably be extrapolated to release in response to an action potential. Release not constituted by separable events is found under various conditions in skeletal muscle; it might be the trigger of sparks in frogs, and the sole form of release in mammals. The origin of sparks is unsettled. The release flux estimated for the largest sparks seems too high to be carried by just one channel. The shift of voltage dependence of spark activation upon partial inactivation of voltage sensors and other complex properties of the whole-cell release waveform are better understood if channels engage in group interactions. Finally, if sparks were one-channel events, it would be difficult to explain why most release channels would consistently fail to activate when facing the high triadic gap [Ca 2+ ] associated with a typical spark. On the other hand, the rapid and effective termination of individual sparks can be more easily justified if sparks result from the opening of individual release channels. The distribution of spark amplitudes corrected for the distorting effects of line scanning may exhibit a modal amplitude, which is more easily explained with multichannel models of sparks, but may also be a feature of single channels that gate irreversibly. In general, it is very difficult to account for the properties of sparks, or whole-cell Ca 2+ release, by simply extrapolating the properties of individual channels in bilayers, which suggests that interactions with other triadic proteins, including release channels, and other forms of local modulation, may crucially influence physiologic gating. Section title: Terminating the Spark Educational score: 0.9288867712020874 Domain: other Document type: Other Language: en Many questions therefore remain unanswered. Going back to the stars metaphor, the trek is far from over. | Study | biomedical | en | 0.999997 |
10051516 | Section title: introduction Educational score: 4.177549362182617 Domain: biomedical Document type: Other Language: en Voltage-dependent ion channels respond to changes in the electric field across the cell membrane by undergoing conformational changes that open and close an ion-permeable pore. The voltage dependence of channel opening, or activation, is caused by rearrangement of charges within the channel protein associated with conformational changes, thereby making the rates of the conformational changes voltage dependent. The movement of charge within the channel can be detected as gating current . Section title: introduction Educational score: 4.369144439697266 Domain: biomedical Document type: Study Language: en Understanding the molecular mechanism of voltage-dependent gating is an important goal of ion channel biophysics. Voltage-dependent potassium channels are particularly attractive for structure–function studies because members of this family share considerable sequence similarity, but exhibit a wide range of gating behaviors. Also, functional voltage-dependent potassium channels can be expressed as homotetramers, and the symmetry of a homotetrameric protein is likely to be reflected in the gating process. Studies on Shaker , a voltage-dependent potassium channel cloned from Drosophila , have yielded many important insights into processes of activation. However, the ability to study the gating process in wild-type Shaker is limited by the fact that it is difficult to study individual transitions independently of one another because the rates and voltage dependences of most of the transitions in Shaker activation are too similar . The ability to perturb the energies of gating transitions with site- directed mutations provides a means to dissect out steps in the gating pathway for study using electrophysiological methods. Section title: introduction Educational score: 4.267586708068848 Domain: biomedical Document type: Study Language: en The fourth transmembrane segment (S4) of voltage-gated cation channels has been proposed to function as a voltage sensor because of its high charge density and the fact that it has been highly conserved among voltage-gated cation channels . The sequence of the S4 is unusual, consisting of repeating basic residues at every third position, separated by neutral or hydrophobic residues . Section title: introduction Educational score: 4.300406455993652 Domain: biomedical Document type: Study Language: en The results of several different lines of experimentation provide strong evidence for a role of the S4 in sensing voltage. Consistent with this hypothesis, it has been shown that mutations that neutralize S4 charged residues can decrease the amount of charge moved per channel during activation of Shaker and can decrease the voltage sensitivity of channel opening in voltage-gated sodium and potassium channels . Further, studies on skeletal muscle sodium channels and Shaker potassium channels have demonstrated that the S4 region “moves” during activation by showing that the accessibility of some S4 residues to externally and internally applied chemical modifying reagents can be manipulated by holding the channel in open or closed conformations . Section title: introduction Educational score: 4.355861663818359 Domain: biomedical Document type: Study Language: en However, the results of recent experiments suggest that the S4 is not only involved in sensing voltage during activation, but also in mediating cooperative interactions between channel subunits . Substitution of the S4 segment from the Drosophila channel Shaw into Shaker causes a dramatic decrease in the voltage dependence of channel opening and makes the time course of activation slow and single exponential . The slow, single-exponential gating kinetics suggest that the Shaw S4 mutation alters activation gating by slowing a cooperative transition in the activation pathway sufficiently to make it rate limiting. Smith-Maxwell et al. also found that the gating of heterodimers with wild-type Shaker and chimeric Shaw S4 subunits can be predicted from properties of the homotetrameric channels only if it is assumed that the mutations alter cooperative transitions in the activation pathway rather than independent transitions. Section title: introduction Educational score: 4.243534088134766 Domain: biomedical Document type: Study Language: en Further, Smith-Maxwell et al. found that the kinetic and voltage-dependent properties of the Shaw S4 ionic currents can be reproduced by introducing a subset of the substitutions present in the chimera into Shaker : V369I, I372L, S376T, to make the ILT mutant. The gating behavior of the ILT and Shaw S4 mutants can be accounted for by making the final cooperative transition rate limiting in a kinetic model of Shaker activation, without changing the rates or voltage dependences of any other transitions in the pathway . Section title: introduction Educational score: 4.278513431549072 Domain: biomedical Document type: Study Language: en Cooperativity between subunits is a recurrent feature in the various kinetic models of potassium channel gating, but it can be implemented in any of a number of ways, including: a sequential mechanism in which the movement of each voltage sensor facilitates the movement of the next one , a cooperative stabilization of the open state , and the presence of one or more highly cooperative or concerted transitions in the activation pathway . At present, little is known about the underlying conformational changes that produce the cooperativity that is observed in the activation of potassium channels. Section title: introduction Educational score: 4.232908248901367 Domain: biomedical Document type: Study Language: en In this paper, we investigate activation of the Shaw S4 chimera and ILT mutant at the level of gating currents to learn more about the role of the S4 in cooperativity and voltage sensing in the process of activation. Gating current recordings allow us to observe directly the charge movement associated with the voltage-dependent conformational changes that the channel undergoes in the activation pathway. Thus, gating current recordings from Shaw S4 and ILT channels can provide valuable insights into the nature of the cooperative conformational change and its position in the activation pathway and can reveal effects of the mutations on other transitions in the activation pathway that may be masked at the level of ionic currents by the presence of a rate-limiting transition. Section title: Molecular Biology and Terminology Educational score: 4.190155982971191 Domain: biomedical Document type: Study Language: en All experiments were performed on a mutant form of ShB , designated ShBΔ6–46, in which fast N-type inactivation was removed by deletion of amino acids 6–46 . This allowed us to study activation in isolation from the fast (N-type) inactivation process. ShBΔ6–46 still undergoes a relatively slow inactivation process (C-type inactivation), but the time constant for C-type inactivation (∼1.5 s) is sufficiently slow that it does not interfere with measurement of activation parameters . ShBΔ6-46 cDNA was further modified by the introduction of a unique “silent” StuI restriction enzyme site 3′ to the S4 coding region, at amino acid positions 380–382. This modification, which does not alter the amino acid sequence, was made using the polymerase chain reaction method to generate a cassette that was inserted between two naturally occurring unique restriction enzyme sites within the Shaker cDNA, StyI and NsiI. In this paper, the term “ Shaker ” will always refer to the ShBΔ6–46 construct. Section title: Molecular Biology and Terminology Educational score: 4.125105857849121 Domain: biomedical Document type: Study Language: en The Shaw S4 chimera and ILT mutant were constructed as described in Smith-Maxwell et al. . The Shaw S4:RRK mutant substitutes eight hydrophobic residues from the S4 of Shaw into Shaker . The Shaw S4:RRK mutation was made by annealing sense and antisense oligonucleotides spanning the unique StyI and the silent StuI restriction enzyme sites in Shaker , and ligating the annealed oligos into Shaker digested with StyI and StuI restriction enzymes. The sequence of Shaw S4:RRK was verified by dideoxy termination sequencing. Section title: Molecular Biology and Terminology Educational score: 4.16799259185791 Domain: biomedical Document type: Study Language: en For gating current experiments, expression levels were increased for ILT, Shaw S4, and Shaw S4:RRK constructs by subcloning into a high-expression Shaker vector obtained from Ligia Toro. The high-expression Shaker construct contained a pore mutation that has been reported to render the pore nonconducting . The high-expression vector was used as a background to make high-expression conducting and nonconducting versions of ILT, Shaw S4, and Shaw S4:RRK. To generate the conducting versions of ILT and Shaw S4, DNA from the S4 mutant constructs was cut with BsiWI and SpeI restriction enzymes, generating a fragment that includes the mutant S4 and the wild-type Shaker pore. These DNA fragments were substituted for the corresponding BsiWI to SpeI fragment in the high-expression W434F Shaker construct. To generate nonconducting versions of ILT, Shaw S4, and Shaw S4: RRK, DNA from the S4 mutant constructs was cut with the BsiWI and NsiI restriction enzymes to generate a fragment that includes the mutant S4 region, but not the pore region of the channel. These DNA fragments were substituted for the corresponding BsiWI-NsiI fragment in the high-expression W434F Shaker construct. Section title: Molecular Biology and Terminology Educational score: 3.7336697578430176 Domain: biomedical Document type: Study Language: en To distinguish between constructs that contain a wild-type pore sequence and the pore sequence with the W434F mutation, we refer to the channels as “conducting” and “nonconducting,” respectively. Section title: Expression System Educational score: 4.164623260498047 Domain: biomedical Document type: Study Language: en All channels were expressed in Xenopus oocytes by injection of G(5′)ppp(5′)G capped mRNA from the different channel constructs. mRNA was transcribed in vitro from linearized plasmid containing channel DNA constructs as described previously . For the original DNA constructs of Shaker and the S4 mutants, mRNA was transcribed with T7 RNA polymerase from a KpnI-linearized DNA template. For the high-expression conducting versions of ILT and Shaw S4 and for the nonconducting (W434F) version of ILT, Shaw S4, and Shaw S4:RRK, mRNA was transcribed with T7 RNA polymerase from an EcoRI-linearized DNA template. Recordings of macroscopic ionic currents typically were carried out 1–10 d after injection, whereas gating current recordings typically were carried out 14–28 d after injection. Section title: Electrophysiology Educational score: 2.1826770305633545 Domain: biomedical Document type: Study Language: en All experiments were carried out at 20 ± 0.2°C. Section title: Macroscopic ionic currents. Educational score: 4.21049165725708 Domain: biomedical Document type: Study Language: en Electrophysiological recordings of macroscopic ionic currents were carried out from excised membrane patches in the inside-out configuration . The ionic current data for Shaker , Shaw S4, and ILT presented in Figs. 2 and 3 were taken from Smith-Maxwell et al. . For all other ionic current recordings presented in this paper, currents were recorded with an Axopatch 1B ( Axon Instruments ) patch clamp amplifier and low-pass filtered using an eight-pole Bessel filter (Frequency Devices, Inc.). The standard extracellular (pipette) solution used for these experiments contained (mM): 140 NaCl, 2 KCl, 6 MgCl 2 , 5 HEPES (NaOH), pH 7.1. The standard intracellular solution (bath) contained (mM): 140 KCl, 11 EGTA, 10 HEPES ( N -methylglucamine), pH 7.2. Ionic currents were digitized at 50–200 μs/point, depending on the channel kinetics. All data were filtered at 9 kHz unless otherwise stated. Details are stated in the figure legends. A Macintosh-based system with hardware interface from Instrutech Corp . and software from HEKA Electronik was used to generate pulses and digitize and store data. Patch pipettes were constructed from VWR borosilicate glass and had initial resistances of 0.4–0.8 MΩ. No series resistance compensation was used; however, the error due to uncompensated series resistance for this series of experiments was typically <2 mV . Linear leak and capacitative currents were subtracted with a P/4 protocol from a holding potential of −110 mV. A holding potential of −80 mV, followed by a 1-s prepulse to −100 mV was used before the test pulses. Section title: Gating currents. Educational score: 3.193305015563965 Domain: biomedical Document type: Study Language: en Gating currents were measured either with a high performance cut-open oocyte voltage clamp (CA-1; Dagan Corp.) or with inside-out patches. The two different experimental procedures will be described separately. Section title: Gating currents. Educational score: 4.334772109985352 Domain: biomedical Document type: Study Language: en In the cut-open oocyte clamp configuration, the oocytes were permeabilized by addition to the lower chamber of internal solution containing 0.3% saponin. Agar bridges with platinum iridium wire were filled with 1 M sodium methanesulfonic acid (NaMES). Microelectrodes were filled with 3 M KCl and had tip resistances of <1 MΩ. No series resistance compensation was used. Gating currents were digitized at 24 μs/point and filtered at 10 kHz. The internal recording solution included (mM): 110 KOH, 2 MgCl 2 , 1 CaCl 2 , 10 EGTA, 5 HEPES, adjusted to pH 7.1 with MES. The external recording solution included (mM): 110 NaOH, 2 KOH, 2 MgCl 2 , 5 HEPES, adjusted to pH 7.2 with MES. Because Shaker , ILT, and Shaw S4:RRK gating currents activate in different voltage ranges, voltage and leak subtraction protocols had to be customized for each channel. For Shaker , ohmic capacitive and linear leak currents were substracted using either a +P/ 10 protocol from a holding voltage of +20 mV or a −P/5 protocol from a holding voltage of −120 mV. The leak-subtracted gating current records produced by the two procedures were not noticeably different. Since the gating currents of ILT and Shaw S4:RRK move over a much greater voltage range than those of Shaker , leak subtraction traces had to be taken in the voltage range of channel opening. Possible artifacts due to charge movement during leak pulses were minimized by choosing voltages for the leak subtraction where the kinetics of the channel are much slower (at least an order of magnitude) than the gating current signal of interest. For gating current recordings of nonconducting ILT, leak subtraction was performed using a +P/10 protocol from a holding voltage of +20 mV. For gating current recordings from nonconducting Shaw S4:RRK, we used a +P/5 leak subtraction protocol from a holding potential of +20 mV. For all cut-open oocyte clamp experiments, the oocyte membrane was held at −40 mV, and then stepped to −100 mV for 2 s before initiating more negative prepulse steps (prepulse voltages were −120 mV for Shaker , −140 mV for ILT, and −180 mV for Shaw S4: RRK), followed by test pulses. The properties of the gating currents did not change during the elapsed time of experiments. Details of voltage protocols, including prepulse voltages and durations, are given in the figure legends. Section title: Gating currents. Educational score: 4.247064590454102 Domain: biomedical Document type: Study Language: en The inside-out patch clamp configuration was used to measure gating currents from nonconducting and conducting versions of ILT and Shaw S4 channels. Typical pipette resistances were 0.4– 0.8 MΩ. The details of voltage protocols are given in the figure legends. Gating currents were digitized at 25 μs/point and filtered at 9 kHz. Standard patch clamp recording solutions (described above) were used, except at very positive voltages (greater than +100 mV). At voltages greater than +100 mV, patches containing nonconducting (W434F) ILT and Shaw S4 channels develop an outward ionic current that interferes with recording of gating currents. The outward current can be eliminated by perfusing the intracellular side of the inside-out patches with a K + -free solution containing (mM): 140 N -methylglucamine (NMG)–Cl, 11 EGTA, 10 HEPES, adjusted to pH 7.1 with NMG-OH. The outward current is probably caused by potassium conductance through the pores of the mutant Shaker channels, since the W434F Shaker mutant is not strictly “nonconducting,” but has a very small open probability (∼10 −5 ) , and the size of the ionic current was found to correlate with the size of the gating currents in the patches. In most inside-out patch experiments on ILT gating currents, the holding potential was 0 mV. The holding potential can be held at −80, −40, or 0 mV without introducing any detectable changes in the amplitude or time course of gating currents in a given patch, at least over the time course of our experiments. A holding potential of 0 mV was used for Shaw S4 gating current experiments. Section title: Analysis Educational score: 4.356232643127441 Domain: biomedical Document type: Study Language: en For Shaker , conductance–voltage curves were constructed by calculation of the chord conductance ( G chord ) from maximum currents ( I max ) during the test pulse at several voltages (V) assuming a reversal potential (V rev ) of −80 mV: G chord = I max /(V − V rev ). For all other channel species, conductance–voltage curves were constructed from isochronal measurements of tail currents recorded at a fixed voltage after steps to voltages that activate the channels. Isochronal measurements were made between 0.2 and 1 ms after the end of the test pulse. Conductance–voltage curves were normalized to the maximum value for comparison between patches and between channel species. Conductance–voltage ( G V) 1 curves were fit by a Boltzmann function of the form: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}\frac{G}{G_{max}}= \left( \frac{1}{1+e^{-zF(V-V_{1/2})/RT}} \right) ,\end{equation*}\end{document} Section title: Analysis Educational score: 4.123371124267578 Domain: biomedical Document type: Study Language: en where G/G max is the conductance normalized to the maximum value for each channel, V 1/2 is the voltage at which the channels are open half maximally, V is the voltage of the test pulse, z is the equivalent charge, F is the Faraday constant, R is the gas constant, and T is the absolute temperature. The slope factor is equal to RT / zF . Section title: Analysis Educational score: 4.109776496887207 Domain: biomedical Document type: Study Language: en Activation kinetics were quantified by fitting the activation time course of macroscopic ionic currents with the following exponential function: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}I(t)=A(1-e^{(-t+d)/{\tau}}).\end{equation*}\end{document} Section title: Analysis Educational score: 4.237331390380859 Domain: biomedical Document type: Study Language: en I ( t ) is the current at time t , A is the scale factor for the fit, τ is the time constant, and d is the delay or amount of time required to shift the single exponential curve along the time axis to obtain an adequate fit of the activation time course. The time course of Shaw S4, ILT, and Shaw S4:RRK currents could be fit with this function from a beginning current level of between 1 and 5% up to the maximum current level. For Shaker , single exponential fits begin at between 20 and 50% of the maximum current to allow for the large sigmoidal delays characteristic of wild-type Shaker currents. Time constants for deactivation were obtained from fits of a single exponential to tail currents measured at negative membrane potentials. Tail currents were generally well fit by a single exponential function. Section title: Analysis Educational score: 4.261784076690674 Domain: biomedical Document type: Study Language: en The voltage dependence of gating charge movement was determined by integrating the ON gating currents elicited by changes in membrane potential. Residual current at the end of test pulses was subtracted before ON gating currents were integrated. Gating charge was normalized to the maximum value to allow comparison of data across patches. To minimize artifacts caused by small drifts in the baseline, the ON gating currents were integrated over 12–30 ms, not the entire test pulse duration. Charge–voltage ( Q V) curves were fit with Boltzmann functions of the form: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}\frac{Q}{Q_{max}}= \left( \frac{1}{1+e^{-zF(V-V_{1/2})/RT}} \right) .\end{equation*}\end{document} Section title: Analysis Educational score: 4.003251075744629 Domain: biomedical Document type: Study Language: en The time constants of decay of the ON gating currents were measured by fitting a single-exponential function to the declining phase of the currents. Section title: Modeling and Simulations Educational score: 3.8404510021209717 Domain: biomedical Document type: Study Language: en Model simulations were carried out as outlined in Zagotta et al. using software developed in the Aldrich laboratory by Toshi Hoshi and Dorothy Perkins. Transitions between conformational states are assumed to obey time-homogeneous Markov processes. Voltage-dependent rate constants are assumed to be exponentially dependent on voltage. Section title: Properties of the Ionic Currents of Shaker, Shaw S4, and ILT Educational score: 4.290924072265625 Domain: biomedical Document type: Study Language: en The S4 sequences for Shaker and the mutant channels, Shaw S4 and ILT, are shown in Fig. 1 . There are 11 amino acid differences between the Shaw S4 chimera and Shaker . Three of these occur at positions occupied by basic residues in Shaker (R1, R2, and K7), decreasing the charge content of the S4 from +7 in Shaker to +3 in the Shaw S4 chimera. The ILT mutant substitutes three noncharged residues (V369I, I372L, and S376T) from the Shaw S4 sequence into Shaker , so the nominal charge of the S4 region of the ILT is +7, like Shaker . Section title: Properties of the Ionic Currents of Shaker, Shaw S4, and ILT Educational score: 3.8996641635894775 Domain: biomedical Document type: Study Language: en Despite the greater degree of similarity between the S4 regions of ILT and Shaker , the properties of the ionic currents of ILT are very similar to those of Shaw S4. The steady state, kinetic, and voltage-dependent properties of the ionic currents are summarized in Figs. 2 and 3 . A more thorough analysis of the ionic current behavior of Shaw S4 and ILT is presented in Smith-Maxwell et al. . Section title: Properties of the Ionic Currents of Shaker, Shaw S4, and ILT Educational score: 4.198606967926025 Domain: biomedical Document type: Study Language: en Fig. 2 shows ionic current traces and conductance– voltage curves of Shaker , Shaw S4, and ILT. The Shaw S4 and ILT mutations cause a large shift in the midpoint of the voltage dependence of channel opening (+120 mV) and a considerable decrease (2.7-fold) in the steepness of the conductance–voltage relation relative to Shaker . It is also apparent in Fig. 2 that the Shaw S4 and ILT mutations affect activation kinetics. The ionic current traces in Fig. 2 show that the ILT and Shaw S4 mutations greatly slow the overall rate of activation in the voltage range where channel opening probability is changing. Section title: Properties of the Ionic Currents of Shaker, Shaw S4, and ILT Educational score: 4.227400779724121 Domain: biomedical Document type: Study Language: en The effects of the Shaw S4 and ILT mutations on the time course of activation kinetics can be seen more readily in Fig. 3 . The overall rate of activation of wild-type Shaker channels is fast, but the time course of activation is clearly sigmoidal and, at most voltages, there is a considerable delay after stepping to a new voltage before the current starts to rise . This delay indicates that Shaker channels must undergo multiple transitions between closed states before opening. Further, the multiple transitions must have similar rates and voltage dependences because if one transition were rate limiting, it would dominate the time course of activation and produce a single-exponential time course. The fact that the early time course of wild-type Shaker activation cannot be fit with a single exponential function is illustrated clearly in Fig. 3 , where single exponential fits to the late phase of activation are shown superimposed on current traces. Section title: Properties of the Ionic Currents of Shaker, Shaw S4, and ILT Educational score: 4.560505390167236 Domain: biomedical Document type: Study Language: en The overall rate of activation of Shaw S4 and ILT channels is greatly slowed relative to Shaker , but, more importantly, the time course of activation of ionic currents of the mutants follows a single exponential time course over a wide voltage range. Analysis of the shape of the time course of activation of ionic currents is important because it can be used to distinguish mutations that primarily affect cooperativity from those that affect independent transitions. The slow, single-exponential gating kinetics of Shaw S4 and ILT suggest that these mutations alter activation gating by slowing a single transition in the activation pathway sufficiently to make it rate limiting . Since the mutant channels presumably assemble into tetramers of four identical subunits , the rate-limiting transition in Shaw S4 and ILT activation most likely arises from highly cooperative interactions between subunits. On the other hand, if a mutation slowed a transition that occurs independently in each of the four subunits of the channel, the time course of activation would be determined by four slow but identical transitions in the pathway to opening; as a result, the overall rate of activation would be slowed but the shape of the time course would be sigmoidal, not single-exponential . Section title: Properties of the Ionic Currents of Shaker, Shaw S4, and ILT Educational score: 4.276618957519531 Domain: biomedical Document type: Study Language: en All of the properties of the ionic currents of the Shaw S4 and ILT mutants can be accounted for by making a final cooperative step in a kinetic model of Shaker rate limiting . However, Smith-Maxwell et al. found that the rate-limiting transition is so dominant in the activation process that the properties of the ionic currents of Shaw S4 and ILT can also be described well by a simpler two-state kinetic scheme in which the rate-limiting cooperative step is the only transition in the activation pathway. This finding suggests that effects of these mutations on other transitions in the activation pathway would be difficult to resolve at the level of ionic currents, but they should be visible at the level of gating currents. Section title: Charge Movement between Closed States in ILT Educational score: 3.9348909854888916 Domain: biomedical Document type: Study Language: en The properties of the Shaker and ILT gating currents measured at negative voltages are summarized in Fig. 4 . These measurements were made from nonconducting versions of Shaker and ILT, containing the mutation W434F , in the cut-open oocyte clamp configuration. Section title: Charge Movement between Closed States in ILT Educational score: 4.292003154754639 Domain: biomedical Document type: Study Language: en The charge movement measured for ILT in the −140 to 0 mV voltage range must be associated with transitions between closed states because it moves in a voltage range where the channel does not open. The ILT mutation causes a negative shift (∼38 mV) in the voltage dependence of this component of charge movement and reduces the steepness of the Q V curve relative to wild-type Shaker . The movement of this component of gating charge saturates by −40 mV, which is 60 mV below the threshold of activation of ionic currents. There must be an additional component of charge movement in the voltage range of channel opening since the opening transition is voltage dependent. However, the gating charge that moves in the voltage range of channel opening could not be measured using the cut-open oocyte clamp technique because outward ionic currents develop at positive voltages and interfere with gating current recordings. The additional component of gating charge can be measured using inside-out patches (discussed below). Section title: Charge Movement between Closed States in ILT Educational score: 4.40866231918335 Domain: biomedical Document type: Study Language: en The ON gating currents of ILT strongly resemble those of Shaker in overall time course and relative voltage dependence, except that they activate in a more negative voltage range. Notably, both channels exhibit a rising phase in the ON gating currents, which is consistent with sequential steps in which a slower or less voltage-dependent transition is followed by a faster or more voltage-dependent transition . The time constants measured from exponential fits to the decay phase of the ON gating currents are shifted to negative potentials compared with Shaker . However, the amplitudes of the time constants of decay converge at voltages above 0 mV, consistent with the forward transitions in the activation pathways of Shaker and ILT possessing similar rates and voltage dependences. Section title: Charge Movement between Closed States in ILT Educational score: 4.333173751831055 Domain: biomedical Document type: Study Language: en The similarities observed between the ON gating currents of ILT and those of Shaker strongly suggest that Shaker and ILT channels have similar activation pathways. Considering the conservative nature of the individual amino acid substitutions introduced by the ILT mutation and the results from the analysis of ILT gating and ionic currents, it seems reasonable to propose that ILT channels undergo essentially the same conformational changes as wild-type Shaker during activation and that the ILT mutation simply alters the rates and equilibria of some transitions in the activation pathway. Section title: An Additional Component of ILT Gating Charge in the Voltage Range of Channel Opening Educational score: 4.296828269958496 Domain: biomedical Document type: Study Language: en The large separation in voltage range between the charge movement and the voltage-dependent channel opening of ILT indicates that there must be an additional component of charge movement in the voltage range of channel opening. Measurements of the charge movement in the voltage range of channel opening will allow us to assess whether or not the rate-limiting cooperative step is the only charge-moving step in this voltage range. If the rate-limiting cooperative step is the only charge-moving gating transition in this voltage range, then: (a) the amount of gating charge measured in this voltage range should be in close agreement with the amount of charge associated with the rate-limiting cooperative transition as estimated from the voltage dependence of the rates of the opening and closing transitions, and (b) the time constants of decay of the ON gating currents in the voltage range of channel opening should have the same amplitude and voltage dependence as the time constants of activation of the ILT ionic currents. Section title: An Additional Component of ILT Gating Charge in the Voltage Range of Channel Opening Educational score: 4.133686065673828 Domain: biomedical Document type: Study Language: en Gating current traces from a typical experiment in inside-out patches are depicted in Fig. 5 A. We designed voltage protocols to measure separately the charge movement at negative voltages and the charge movement in the voltage range of channel opening. First, we measured charge movement in the −140 to 0 mV range, where the channel does not open, in inside-out patches. We then changed the holding potential of the membrane to 0 mV to allow charge movement between closed states to reach equilibrium. From a holding potential of 0 mV, we stepped briefly to high voltages (+100 to +180 mV), where channels open, to elicit charge movement associated specifically with the late transitions to the open state. Section title: An Additional Component of ILT Gating Charge in the Voltage Range of Channel Opening Educational score: 4.245614528656006 Domain: biomedical Document type: Study Language: en The gating currents measured at high voltages, where channels open, are much smaller than those measured in the same patch at negative voltages. Note that the scale bars for gating currents measured in the two different voltage ranges in Fig. 5 differ by an order of magnitude. Unfortunately, over much of the voltage range of interest, the ILT gating charge movement associated with the rate-limiting transition to opening cannot be measured because the amount of charge moved is very small and the time constant of gating charge movement is prohibitively slow . Even in patches with very large numbers of channels, the gating charge signal is too small and slow to measure unless the membrane voltage is stepped to at least +130 mV. It is interesting to note that the gating currents at high voltages display a bit of a rising phase, a feature that could not be produced by a single transition. However, the presence of a rising phase should be interpreted with caution as it may be an artifact introduced by the leak subtraction procedure at these very high voltages rather than a true feature of the gating currents. Section title: An Additional Component of ILT Gating Charge in the Voltage Range of Channel Opening Educational score: 4.121088027954102 Domain: biomedical Document type: Study Language: en The voltage dependences of the total gating charge movement and of channel opening of ILT are shown plotted on the same graph in Fig. 5 B. The charge–voltage curve was constructed from the data obtained in the two different voltage ranges (−140 to 0 mV and 0 to +180 mV) by summing the amount of gating charge measured at high voltages with the amount of gating charge measured with steps to −20 mV, where the early component of gating charge movement has clearly saturated. Section title: An Additional Component of ILT Gating Charge in the Voltage Range of Channel Opening Educational score: 4.249985694885254 Domain: biomedical Document type: Study Language: en Gating current recordings detect the charge movement associated with all charge-moving transitions in the activation pathway. We can make use of this fact to determine whether or not the rate-limiting transition is the only charge-moving transition in the voltage range of channel opening. If the rate-limiting transition is the only gating transition that the ILT channel undergoes in the 0- to +180-mV voltage range, then the amount of gating charge measured in this voltage range should be in close agreement with the amount of charge estimated from the voltage dependence of the rates of the rate-limiting opening and closing transitions. On the other hand, if ILT undergoes additional charge-moving transitions that are not rate limiting in the 0- to +180-mV range, then the amount of gating charge measured in this voltage range must exceed the amount of charge estimated from the voltage dependences of the rates of the rate-limiting step. Section title: An Additional Component of ILT Gating Charge in the Voltage Range of Channel Opening Educational score: 4.3377814292907715 Domain: biomedical Document type: Study Language: en The voltage dependence of the rates of the opening and closing transitions corresponds to equivalent charge values of 0.84 and 0.90 e 0 , respectively, for a total of 1.74 e 0 predicted to move in the final opening step . Thus, 1.74 e 0 represents a lower bound estimate for the amount of gating charge that must move per channel in this voltage range to account for the voltage dependence of the rate-limiting step, and additional voltage-dependent steps will increase the amount of the gating charge measured in the 0- to +180-mV range. Assuming that the total gating charge per channel is between 12.3 and 13.6 e 0 , the proportion of gating charge moved in the rate-limiting transition corresponds to 13–14% of the total gating charge. Section title: An Additional Component of ILT Gating Charge in the Voltage Range of Channel Opening Educational score: 4.2161712646484375 Domain: biomedical Document type: Study Language: en We determined the proportions of the total gating charge moved in the two voltage ranges of interest by measuring the charge moved by steps to voltages where the two components of charge were observed to saturate. The high-voltage component was measured with steps to +180 mV from a holding potential of 0 mV, and the low-voltage component was measured with steps to −20 mV from a prepulse potential of −140 mV. The total gating charge was taken to be the sum of these two values. We calculated that 13% (SEM, 0.9%; n = 4) of the total gating charge moved in the voltage range where channels open and 87% (SEM, 1.2%; n = 4) moved between −140 and −20 mV. Given that the amount of charge moved in the activation pathway per wild-type Shaker channel has been estimated to be between 12.3 and 13.6 e 0 , these proportions correspond to gating charge movements per channel of 1.6–1.8 e 0 in the 0- to +180-mV range and 10.7–11.8 e 0 in the −140- to −20-mV range. We assumed that the amount of gating charge moved per ILT channel is the same as for wild-type Shaker based on reports in the literature that mutations at neutral residues in the S4 and at the carboxy-terminal border of the S4 do not change the total amount of gating charge moved per channel . Section title: An Additional Component of ILT Gating Charge in the Voltage Range of Channel Opening Educational score: 4.306317329406738 Domain: biomedical Document type: Study Language: en The amount of gating charge that moves in the voltage range of channel opening, 1.6–1.8 e 0 , is in very close agreement with the value for equivalent charge associated with the rate-limiting transition, 1.74 e 0 , estimated from analysis of the voltage dependence of the rates of channel opening and closing in ionic currents from Smith-Maxwell et al. . The agreement between these two independent determinations provides strong evidence that the only charge-moving step in the 0- to +180-mV range is the rate-limiting cooperative step that dominates the behavior of the ionic currents. Section title: An Additional Component of ILT Gating Charge in the Voltage Range of Channel Opening Educational score: 4.250311851501465 Domain: biomedical Document type: Study Language: en If the ILT gating charge movement at high voltages is associated with the rate-limiting cooperative transition between the final closed and open states, then the kinetics of the gating currents should be the same as the kinetics of the ionic currents. The voltage dependence of the time constants of decay of the ON gating currents at high voltages is shown plotted on the same graph as mean time constants of activation of ionic currents . The gating current time constants have the same voltage dependence (slope) as the ionic current activation time constants, but the gating currents are noticeably faster than the ionic currents. Section title: An Additional Component of ILT Gating Charge in the Voltage Range of Channel Opening Educational score: 4.579514503479004 Domain: biomedical Document type: Study Language: en The discrepancy between the kinetics of the gating and ionic currents deviates from the prediction of a singe transition. However, it can be explained in terms of a voltage shift between the gating current and ionic current data (see discussion ). If the voltage shift is corrected for, the amplitude and voltage dependence of the kinetics of the gating currents recorded in the voltage range of channel opening are consistent with those expected to accompany the gating charge movement during the rate-limiting cooperative transition that opens the ILT channel. Given that the amount of charge moved in this voltage range is in such close agreement with the equivalent charge measured for the rate-limiting cooperative transition based on the voltage dependence of the rates of ionic current activation and deactivation, it seems likely that the rate-limiting cooperative transition is the final charge-moving step in the activation pathway. Therefore, in spite of the voltage shift between the kinetics of the gating and ionic currents, it seems likely that the charge movement that we have measured corresponds to the charge moved in the rate-limiting cooperative step. The simplest interpretation of these findings is that the ILT mutation has uncovered the final voltage-dependent cooperative transition in the Shaker activation pathway by making it rate limiting. Section title: The Activation Pathway of Shaw S4 Educational score: 3.0733892917633057 Domain: biomedical Document type: Study Language: en The similar properties of the ionic currents of ILT and Shaw S4 imply that these two channels gate in the same way. However, the gating currents of Shaw S4 indicate that its activation pathway is fundamentally different from that of ILT. Section title: The Activation Pathway of Shaw S4 Educational score: 4.1662774085998535 Domain: biomedical Document type: Study Language: en The only gating charge that could be detected for nonconducting Shaw S4 channels moves at very positive voltages, in a voltage range where channels open . We could not detect any Shaw S4 charge movement in the voltage range where most of the charge moves in the activation pathways of ILT and Shaker . The Shaw S4 gating currents strongly resemble the component of ILT gating currents recorded in the voltage range of channel opening in shape and time course. As noted previously for ILT, the Shaw S4 gating currents cannot be measured over much of the voltage range of channel opening because the time constants of gating charge movement are prohibitively slow and the amount of charge moved is very small. Section title: The Activation Pathway of Shaw S4 Educational score: 4.148964881896973 Domain: biomedical Document type: Study Language: en We cannot use the method applied to ILT to determine the amount of Shaw S4 gating charge moved at high voltages because of the absence of Shaw S4 gating charge at low voltages. However, we can measure time constants from currents elicited with steps to sufficiently positive voltages. The time constants of decay of the ON gating currents are shown plotted as a function of voltage on the same graph as time constants of activation of Shaw S4 ionic currents . The kinetics of the gating currents are faster than the kinetics of the ionic currents but have a similar voltage dependence. The faster gating current kinetics correspond to a voltage shift of 20–25 mV relative to the ionic currents. This voltage shift is similar in magnitude to the voltage shift observed between the kinetics of the ionic and gating currents of ILT and probably occurs for similar reasons (see discussion). Section title: The Activation Pathway of Shaw S4 Educational score: 4.409602165222168 Domain: biomedical Document type: Study Language: en The absence of charge movement between closed states in nonconducting Shaw S4 channels indicates that the chemical or steric properties of some of the substituted S4 residues may interfere with the normal conformational changes of the channel protein during gating. If the Shaw S4 gating charge detected in the voltage range of channel opening is associated with the rate-limiting cooperative transition, then the activation pathway of Shaw S4 moves as little as ∼1.8 e 0 per channel, or 0.45 e 0 per subunit. This value is less than expected considering the charge-changing substitutions in the S4 region of Shaw S4. While there are three charge-changing substitutions between the S4 of Shaw S4 and Shaker , only two of these substitutions occur at residues that have been shown to contribute to the gating charge of the channel . With eight charge-moving residues per tetrameric channel protein remaining (i.e., two charge-moving residues per subunit), we would expect to see some charge movement between closed states in addition to the 1.8 e 0 per channel moved during the last cooperative transition. Section title: The Activation Pathway of Shaw S4 Educational score: 3.9966094493865967 Domain: biomedical Document type: Study Language: en It is also possible that the S4 mutations might interact with the pore mutation, W434F, to produce an anomalous gating phenotype. To address this possibility, we performed a series of gating current experiments with conducting ILT and Shaw S4 channels. In these experiments, we also extended the voltage range of investigation down to −240 mV in an attempt to measure Shaw S4 gating currents. Section title: The Activation Pathway of Shaw S4 Educational score: 4.108720779418945 Domain: biomedical Document type: Study Language: en We selected inside-out patches that expressed very high levels of conducting channels and measured the amount of ionic current elicited by steps to +100 mV, then we tried to detect gating currents in the same patch in the −140 to 0 mV range, where most of the charge moves in the ILT activation pathway. A representative experiment for each channel is shown in Fig. 7 . In patches expressing ILT channels, we were able to detect robust gating currents with steps to −140 to 0 mV in every patch that had at least 3.7 nA of ionic current at +100 mV. In patches expressing Shaw S4 channels, we were not able to detect any gating currents in the voltage range of −200 to 0 mV, even though we biased our experiments towards having more ionic current at +100 mV (i.e., more channels per patch) in the Shaw S4 experiments than the ILT experiments. For 6/11 patches of Shaw S4, we extended the voltage range of investigation with steps down to −240 mV (patches could not withstand more negative pulses), but we still did not detect gating currents in any patches expressing Shaw S4. Section title: The Activation Pathway of Shaw S4 Educational score: 4.133188247680664 Domain: biomedical Document type: Study Language: en The results of similar experiments on many patches are tabulated in Table I . To interpret data across patches that had different numbers of channels, we normalized the data by calculating the ratio of gating charge at 0 mV to ionic current at +100 mV. The scatter in the q/K + current data is probably due to series resistance error introduced by the very large ionic currents that were measured. The accuracy of measurements of current at +100 mV would be quite sensitive to series resistance errors because the probability of opening is still changing . However, the direction of the series resistance error would cause us to underestimate systematically the true amount of ionic current at +100 mV, which in turn would cause us to overestimate the amount of gating charge moved relative to the true amount of ionic current. Our interpretation of these experiments would not be compromised by systematically underestimating the amount of ionic current at +100 mV. Section title: The Activation Pathway of Shaw S4 Educational score: 4.299640655517578 Domain: biomedical Document type: Study Language: en As in the case of ILT, the discrepancy between the kinetics of the gating and the ionic currents of Shaw S4 can be explained readily in terms of a voltage shift, the possible causes of which will be presented in the discussion . If the voltage shift is corrected for, the amplitude and voltage dependence of the kinetics of the gating currents recorded in the voltage range of channel opening are consistent with those expected to accompany the gating charge movement during the rate-limiting cooperative transition that opens the Shaw S4 channel. Therefore, it seems likely that the charge movement that we have measured corresponds to the charge moved in the rate-limiting cooperative step. Further, it seems likely that the rate-limiting cooperative transition is the only charge-moving step in the activation pathway of Shaw S4, since we could not detect charge movement between closed states in the Shaw S4 mutant. Section title: The Activation Pathway of Shaw S4 Educational score: 3.932288408279419 Domain: biomedical Document type: Study Language: en Although we could not detect gating charge movement between closed states in the Shaw S4 mutant using the methods described above, these methods do not prove the absence of such gating charge. Gating charge from a slow transition between closed states might escape detection using these methods. However, the presence of a slow transition between closed states should manifest itself in the sigmoidicity of the time course of activation. This possibility will be addressed experimentally in the following section. Section title: Closed States in the ILT Activation Pathway Produce a “Cole-Moore Shift” that Is Absent in Shaw S4 Ionic Currents Educational score: 4.4186530113220215 Domain: biomedical Document type: Study Language: en The time course of activation of Shaker is fast, but at most voltages it is sigmoidal in shape and exhibits a large delay before the current starts to rise . This delay indicates that Shaker channels must undergo a number of voltage-dependent transitions between closed states before it can open. Depolarized holding voltages cause a decrease in the amount of delay in the time course of Shaker activation, presumably because channels now occupy states along the activation pathway that are closer to the open state of the channel . A decrease in the delay in the time course of activation in response to depolarized holding potentials is often referred to as a Cole-Moore shift because the phenomenon was first observed in squid potassium channels by Cole and Moore . Section title: Closed States in the ILT Activation Pathway Produce a “Cole-Moore Shift” that Is Absent in Shaw S4 Ionic Currents Educational score: 4.149977684020996 Domain: biomedical Document type: Study Language: en The presence of more closed states in the activation pathway of ILT than Shaw S4 can be seen on close investigation of the time course of activation of ionic currents. The properties of the ionic currents of Shaw S4 and ILT are very similar. However, at voltages above +140 mV, the time course of activation of ILT develops a very small but progressive increase in delay that is essentially absent from Shaw S4 . Section title: Closed States in the ILT Activation Pathway Produce a “Cole-Moore Shift” that Is Absent in Shaw S4 Ionic Currents Educational score: 4.27780818939209 Domain: biomedical Document type: Study Language: en From studying the voltage dependence of charge movement of ILT, we know that gating currents begin to activate at −130 mV and that charge movement between closed states saturates around −40 mV. Therefore, a prepulse to −140 mV should draw ILT channels into the closed state furthest from the open state, which will maximize the amount of delay in activation. The behavior of the ILT gating currents suggests that a prepulse to 0 mV should pool ILT channels into the final closed state adjacent to the open state, which will minimize the amount of delay in the time course of activation. Section title: Closed States in the ILT Activation Pathway Produce a “Cole-Moore Shift” that Is Absent in Shaw S4 Ionic Currents Educational score: 4.083378791809082 Domain: biomedical Document type: Study Language: en The results of prepulse experiments on the time course of activation of Shaw S4 and ILT are presented in Fig. 8 . The time course of Shaw S4 ionic currents is insensitive to the choice of prepulse voltage for all voltages tested (−180 to 0 mV). This result confirms that there are no slow charge-moving transitions between closed states that may have escaped detection in our gating current measurements. Section title: Closed States in the ILT Activation Pathway Produce a “Cole-Moore Shift” that Is Absent in Shaw S4 Ionic Currents Educational score: 4.204109191894531 Domain: biomedical Document type: Study Language: en In contrast, the ILT current traces clearly show that prepulses to −140 mV induce a small amount of delay and prepulses to 0 mV relieve this delay. When patches containing ILT channels are prepulsed to 0 mV, which effectively removes the closed state transitions from the ILT activation pathway, the time course of activation of ILT is more like that of Shaw S4 at all voltages . Thus, the small increase in the amount of delay in the ionic currents of ILT at voltages above +140 mV observed by Smith-Maxwell et al. can be accounted for by the presence of transitions between closed states in the activation pathway. Section title: Closed States in the ILT Activation Pathway Produce a “Cole-Moore Shift” that Is Absent in Shaw S4 Ionic Currents Educational score: 4.288665771484375 Domain: biomedical Document type: Study Language: en The amount of delay present in traces was quantitated by taking the x intercept of single exponential fits to current traces . The amount of delay measured is very small, even for ILT after prepulses to −140 mV. However, a simple first-order reaction (e.g., between a closed state and the open state) would produce a time course of activation with no delay. The small amount of delay observed in Shaw S4 traces that remains in ILT traces after prepulses to 0 mV is consistent with either a highly cooperative transition or with the presence of multiple charge-moving transitions. Since our gating current data indicate that there is only one charge-moving transition in this voltage range for Shaw S4 and ILT activation, the former explanation seems more likely. The small amount of delay observed for Shaw S4 and ILT after prepulses to 0 mV and the small rising phase on the gating currents are both consistent with a cooperative conformational change in which the movement of each subunit facilitates the others. Section title: Mutations at Charged Residues Affect Charge Movement between Closed States Educational score: 4.262572765350342 Domain: biomedical Document type: Study Language: en The voltage dependence of the cooperative opening transition is the same for ILT and Shaw S4, but there is no detectable charge movement between closed states in the activation pathway of Shaw S4 in the voltage range of −240 to 0 mV, where a large component of the ILT gating charge moves. The S4 sequence of Shaw S4 has charge-changing substitutions at three positions occupied by basic residues (R1, R2, and K7) in Shaker and ILT, resulting in a decrease in the net charge of the S4 from +7 to +3. To test if increasing the net S4 charge of Shaw S4 could rescue the charge movement between closed states, we constructed a mutant, Shaw S4:RRK, that contains all seven of the basic residues present in Shaker and all eight of the substitutions at noncharged residues present in the Shaw S4 chimera . Section title: Mutations at Charged Residues Affect Charge Movement between Closed States Educational score: 4.350734710693359 Domain: biomedical Document type: Study Language: en The properties of the ionic currents of Shaw S4:RRK are summarized in Fig. 9 . The voltage range of activation of Shaw S4:RRK is shifted negatively relative to ILT, but the slope of the conductance–voltage relation of Shaw S4:RRK is similar to that of ILT and considerably more shallow than that of Shaker . The time course of activation of Shaw S4:RRK shows little delay and is well described by a single-exponential function over the activation voltage range, consistent with the presence of a rate-limiting step in the activation pathway. The time constants of activation and deactivation of Shaw S4:RRK are similar to those of ILT in their amplitude and voltage dependence , but the relationship has been shifted to a more negative voltage range. Analysis of the voltage dependence of channel kinetics indicates that, for the Shaw S4:RRK mutant, 0.94 e 0 are associated with channel opening and 0.95 e 0 with channel closing, for a total of 1.89 e 0 associated with the rate-limiting cooperative transition. These equivalent charge values for the forward and backward rates of the rate-limiting transition are very similar to the charge estimates obtained for ILT of (0.84 e 0 for channel opening and 0.90 e 0 for channel closing, for a total of 1.74 e 0 ) and for Shaw S4 (0.78 e 0 for channel opening and 0.86 e 0 for channel closing, for a total of 1.64 e 0 ) . Section title: Mutations at Charged Residues Affect Charge Movement between Closed States Educational score: 4.2649688720703125 Domain: biomedical Document type: Study Language: en Overall, the properties of the ionic currents of Shaw S4:RRK are very similar to those of ILT and Shaw S4, suggesting that the properties of the rate-limiting final cooperative step in the gating pathway have not been changed by the substitutions at R1, R2, and K7, except for a shift in its voltage-dependent equilibrium. The ionic current properties of Shaw S4:RRK might be expected to resemble those of ILT and Shaw S4 because the Shaw S4:RRK mutant includes all of the substitutions (i.e., the ILT mutations V369I, I372L, and S376T) necessary to make the last cooperative gating transition rate limiting . Section title: Mutations at Charged Residues Affect Charge Movement between Closed States Educational score: 4.295810699462891 Domain: biomedical Document type: Study Language: en To investigate the effects of the Shaw S4:RRK mutation on earlier transitions in the activation pathway, we examined the properties of the gating currents . We found that Shaw S4:RRK gating currents can be recorded at voltages that are more negative than the voltage range of channel opening, demonstrating the presence of charge-moving conformational changes between closed states in the activation pathway. However, the gating currents of Shaw S4:RRK are quite different from those of Shaker and ILT . The midpoint of the charge–voltage curve of Shaw S4:RRK is shifted to an even more negative voltage range and its slope is even more shallow than that of ILT . Moreover, the behavior of the ON gating currents is not consistent with a simple voltage shift relative to Shaker and ILT. The time constants of Shaw S4:RRK are considerably slower and have a noticeably more shallow voltage dependence than the time constants for Shaker and ILT gating currents . The Shaw S4:RRK OFF gating currents are also much slower than those of Shaker and ILT. Section title: Mutations at Charged Residues Affect Charge Movement between Closed States Educational score: 4.2059431076049805 Domain: biomedical Document type: Study Language: en The relationship between gating charge movement and activation of ionic currents of Shaw S4:RRK is summarized in Fig. 11 . The charge- and conductance-voltage curves are plotted on the same graph. The threshold at which ionic currents can be detected is −30 mV. The charge–voltage curve has reached apparent saturation by –30 mV and does not increase with increasingly positive voltage steps in the voltage range considered (up to +20 mV), despite the fact that an additional component of charge movement would be expected to move during the rate-limiting transition to channel opening in this voltage range. However, the charge movement associated with the rate-limiting transition is too slow to be detected in the ON gating currents in the voltage range of −30 to +20 mV. We did not attempt to measure gating currents at still higher voltages, where the charge moved during the rate-limiting transition would be fast enough to measure, because outward currents develop at higher voltages in the cut-open oocyte configuration and interfere with measurements of gating currents. Section title: Mutations at Charged Residues Affect Charge Movement between Closed States Educational score: 4.149216651916504 Domain: biomedical Document type: Study Language: en To test whether or not these closed states are coupled to channel opening, we can look for an increase in the amount of delay in the time course of activation with appropriately negative prepulses. The traces shown in Fig. 11 B demonstrate that the amount of delay in the time course of activation of Shaw S4:RRK can be increased by a prepulse to −140 mV and decreased by a prepulse to −40 mV. Therefore, the voltage-dependent transitions between closed states that we detected as gating currents are traversed in the activation pathway. For comparison, current traces are also shown from similar experiments performed on ILT and Shaw S4. The relatively large delays in the ionic currents of Shaw S4:RRK produced by negative prepulse voltages are consistent with the slow kinetics of its gating currents compared with those of ILT. Section title: Mutations at Charged Residues Affect Charge Movement between Closed States Educational score: 4.289223670959473 Domain: biomedical Document type: Study Language: en The relationship between the gating behavior of Shaw S4:RRK, ILT, and Shaw S4 is consistent with the idea that the rate-limiting transition is late in the activation pathway and with previous work that has shown that R1 and R2 move through the membrane electric field during early gating transitions and that K7 never crosses the electric field . The rate-limiting transition in the Shaw S4:RRK activation pathway displays a voltage dependence similar to that of ILT and Shaw S4, suggesting that the charges at R1, R2, and K7 do not traverse the membrane electric field during the final cooperative transition. The gating current recordings and Cole-Moore type experiments on ILT, Shaw S4, and Shaw S4:RRK confirm that R1, R2, and K7 traverse the membrane electric field during earlier gating transitions. Section title: A Kinetic Model for ILT Educational score: 4.256759166717529 Domain: biomedical Document type: Study Language: en To further understand the changes introduced by the ILT mutation, we modified a kinetic model that was developed for Shaker . The rationale for modifying a Shaker model is that the ILT mutation should not grossly alter the gating mechanism because the amino acid substitutions introduced into the protein by this mutation are very conservative, producing small changes in the size and shape but not the chemistry of the amino acid side chains. In support of this idea, the gating currents of ILT in the −140- to 0-mV range were found to resemble those of Shaker , indicating that ILT and Shaker undergo similar numbers and types of conformational changes between closed states. Also, the individual substitution of any one of the amino acids in the ILT mutant (V369I, I372L, S376T) causes much smaller changes in gating than is observed for the ILT triple mutant, suggesting that the structure of the protein readily accommodates each of the individual substitutions . The state diagram for the Shaker model is shown in Fig. 12 A, and the rates and voltage dependences for the rate constants used are given in Table II . Section title: A Kinetic Model for ILT Educational score: 4.1983442306518555 Domain: biomedical Document type: Study Language: en In this paper, we have used a 16-state model for Shaker activation. In this model, each subunit undergoes two sequential independent transitions to reach a final closed state from which the channel opens cooperatively. A single transition has been used to represent the final cooperative transition, which may be more complex. Quantitative agreement between model predictions and Shaker data can be improved by addition of more states to this class of kinetic model . In the model of Schoppa and Sigworth , each subunit undergoes an additional independent transition, followed by two sequential cooperative transitions to reach the open state. However, we have used a 16-state model for Shaker because it is the simplest model that adequately describes most of the features of Shaker gating and ionic currents. Section title: A Kinetic Model for ILT Educational score: 4.561441898345947 Domain: biomedical Document type: Study Language: en The 16-state model is similar to the 15-state model proposed by Zagotta et al. and Smith-Maxwell et al. , except in its treatment of the last transition between the final closed state and the open state. In the 15-state model, there is no final closed state from which the channel opens cooperatively. Rather, cooperativity is implemented by slowing the first closing transition. The 15- and 16-state models describe wild-type Shaker ionic and gating currents equally well. Both the 15- and 16-state models can be readily adapted to describe the ionic currents of ILT; however, only the 16-state model can be adapted to describe the behavior of ILT gating currents. The 15-state model cannot produce the two well-separated components of gating current that were observed for ILT. This result strongly suggests that the molecular mechanism of cooperativity in the rate-limiting step in the activation of ILT is a highly cooperative step rather than a stabilization of the open state. The ability of a 16-state model to describe simultaneously the gating and ionic currents of ILT suggests that a 16-state model, in which the channel reaches a final closed state from which it opens cooperatively, represents a more reasonable approximation of the conformational changes of the protein than the 15-state model. Section title: A Kinetic Model for ILT Educational score: 4.190467834472656 Domain: biomedical Document type: Study Language: en The model predictions for wild-type Shaker and ILT gating currents are compared with data in Fig. 13 . The wild-type Shaker model does a good job of predicting many features of the Shaker gating currents, including a rising phase in the ON gating currents, a rising phase in the OFF gating currents associated with channel opening, and the position and steepness of the steady state voltage dependence of gating charge movement. The predictions for the time constants of decay of the ON gating currents agree well with the data over the −30- to +50-mV range, but less well in the −70- to −40-mV range, as was also observed in the 15-state model of Zagotta et al. . The agreement between the predictions of the time constant of decay of the ON gating currents is improved significantly by adding a third independent transition per subunit . Section title: A Kinetic Model for ILT Educational score: 3.8651180267333984 Domain: biomedical Document type: Study Language: en The gating current predictions for the ILT model are presented in two parts. In Fig. 13 , the model predictions for gating currents in the −140- to +20-mV range, where charge moves between closed states, are compared with ILT data in the same voltage range. In Fig. 14 , the model predictions for gating currents in the voltage range of channel opening are compared with ILT data. Section title: A Kinetic Model for ILT Educational score: 4.397594451904297 Domain: biomedical Document type: Study Language: en To reproduce the behavior of ILT gating currents in the −140- to 0-mV range, it was necessary to slow both of the independent backward transitions in each subunit relative to wild type Shaker , without changing the independent forward transitions. The largest change to the model was a 25-fold slowing of the rate of the β transitions. The rate of the δ transitions was slowed to a lesser degree, 3.5-fold. The fit of the model to the data was improved somewhat by slightly decreasing the amount of charge assigned to the independent backward transitions. These alterations to the backward transitions are sufficient to reproduce the features of the ILT gating currents at negative voltages, including the shift in the voltage dependence of the time constants and charge movement, the decrease in the slope of the charge movement, and the overall time course of the ON and OFF gating currents. Because the forward transitions are unchanged, the time constants of decay of the ON gating currents converge with those of the Shaker model at 0 mV , as can be observed in the data . Although the voltage dependence of the last concerted transition must be increased for the ILT model, the total amount of gating charge assigned is very similar to that of the model for wild-type Shaker : 13.8 e 0 for ILT and 13.2 e 0 for Shaker . Section title: A Kinetic Model for ILT Educational score: 4.238890647888184 Domain: biomedical Document type: Study Language: en To reproduce the gating currents of ILT in the voltage range of channel opening, the forward and backward rates of the final cooperative transition in the 16-state Shaker model must both be slowed and their voltage dependences must be increased relative to wild-type Shaker . The forward and backward cooperative transitions have been assigned equivalent charge values of 1.0 and 0.8 e 0 , respectively, for a total of 1.8 e 0 assigned to the final cooperative transition. The parameters used in this paper for the forward and backward transitions of the final cooperative step are the same as in the model for ILT gating proposed by Smith-Maxwell et al. . The ionic currents predicted by the ILT model proposed in this paper are indistinguishable from those predicted by the model of Smith-Maxwell et al. (data not shown), despite the fact that the kinetic model was modified in this paper to incorporate ILT gating current data between −140 and 0 mV. In this model, the voltage dependence of the last cooperative step must be increased significantly to reproduce ILT behavior, from 0.4 e 0 for wild-type Shaker to 1.8 e 0 for ILT. However, the true voltage dependence of the last cooperative transition in wild-type Shaker is not known for certain because the transition is too fast to be investigated thoroughly. The nature of the last cooperative step in wild-type Shaker will be discussed further in discussion. Section title: A Kinetic Model for ILT Educational score: 4.140551567077637 Domain: biomedical Document type: Study Language: en Gating current simulations were elicited from a holding voltage of 0 mV to isolate the component of charge that moves in the activation range. The model predictions are shown superimposed on representative ILT current traces in Fig. 14 A. The model does a very good job of predicting the overall time course of ILT gating charge movement over a wide voltage range, +130 to +180 mV. The ILT model predictions were compared with ILT gating currents from four different patches, and the agreement between the model and the gating currents was very good for all four patches over the range of +130 to +180 mV. Section title: A Kinetic Model for ILT Educational score: 4.290683269500732 Domain: biomedical Document type: Study Language: en However, the model does not predict the small rising phase that is observed in the gating current recordings. This discrepancy probably occurs because we have used a concerted step (e.g., a single transition in which all of the channel subunits move in unison) as an approximation for the cooperative step. A concerted step can be thought of as an extreme case of cooperativity. The presence of a small rising phase in the time course of the ILT gating currents at high voltages suggests that the channel undergoes a conformational change that is highly cooperative but not concerted. Section title: A Kinetic Model for ILT Educational score: 4.005030632019043 Domain: biomedical Document type: Study Language: en Overall, the gating currents in the voltage range of channel opening of ILT are well described by the model over a wide voltage range. The fit of the model to the data provides additional evidence that the rate-limiting cooperative transition in the gating of ILT is the final transition in the gating pathway. Section title: A Kinetic Model for Shaw S4 Educational score: 4.145597457885742 Domain: biomedical Document type: Study Language: en We used a two-state model for Shaw S4 because it is consistent with the single-exponential time course of the ionic currents and the absence of gating currents at voltages outside the activation voltage range. The state diagram for the model is shown in Fig. 12 , and the rates and voltage dependence for the rate constants used are given in Table II . The values used for the rates in the two-state model are the same as those used for the final transition in the ILT model. Section title: A Kinetic Model for Shaw S4 Educational score: 4.0989885330200195 Domain: biomedical Document type: Study Language: en For the purpose of comparison, the gating current predictions of the two-state Shaw S4 model and of the multi-state ILT model in the voltage range of channel opening have been presented together in Fig. 14 . All gating current simulations were elicited from a holding voltage of 0 mV. The gating current predictions from the ILT and Shaw S4 models are indistinguishable, except during the first 200 μs after depolarization . It is unlikely that we would detect this difference in our recordings, since the first 200 μs after a step to a new voltage is typically obscured by capacitive transients. Section title: A Kinetic Model for Shaw S4 Educational score: 4.123068809509277 Domain: biomedical Document type: Study Language: en The Shaw S4 model predictions were compared with Shaw S4 gating currents from four different patches. The model predictions agree very well with the Shaw S4 gating current recordings over the voltage range of +130 to +160 mV, but around +170 mV the predictions of the model and the recorded currents start to diverge. The model predictions are apparently too fast at the highest voltages. We observed a similar but smaller divergence between model and data for ILT at +180 mV and higher. The degree to which model and data diverged was consistently greater for Shaw S4 than ILT but varied from patch-to-patch for both channels. Section title: A Kinetic Model for Shaw S4 Educational score: 4.268764495849609 Domain: biomedical Document type: Study Language: en The divergence between model and data may indicate that the true channel behavior is more complicated than presented in the model. For example, our model does not include any transitions of the open channel to closed states that are not in the activation pathway. Open Shaker channels can close to closed states that are outside of the activation path, and the rates for some of these transitions exhibit a small voltage dependence . These transitions may make an increasingly significant contribution to the voltage-dependent and kinetic behavior of the channel as the open probability approaches its maximum. Further, the rates of transitions to closed states outside of the activation path can be affected by mutations . The difference between the gating currents of ILT and Shaw S4 at high voltages might indicate that Shaw S4 and ILT have different effects on the transitions from the open state of the channel to states outside of the activation pathway. Section title: discussion Educational score: 4.412398338317871 Domain: biomedical Document type: Study Language: en The simplest interpretation of the gating phenotype of the ILT mutant is that the ILT mutation has uncovered the final voltage-dependent cooperative transition in the Shaker activation pathway by making it rate limiting. The most compelling evidence for this interpretation is the close agreement between the amount of gating charge moved in the voltage range of channel opening and the amount of charge moved in the forward and backward transitions of the rate-limiting step, as estimated from fits to time constants of activation and deactivation. This result strongly suggests that the rate-limiting cooperative transition is the only charge-moving transition in the voltage range of channel opening, and therefore it must be the last transition in the activation pathway. This interpretation of our results is further supported by the fact that the voltage dependence of the ILT gating current kinetics at high voltages is the same as the voltage dependence of the ionic currents. Section title: discussion Educational score: 3.51324462890625 Domain: biomedical Document type: Study Language: en However, the kinetics of the ILT gating currents are noticeably faster than the ionic currents, when they should be the same if the gating charge movement is associated with the rate-limiting cooperative transition. It is important for our interpretation of the gating current data to consider the nature of this discrepancy and plausible mechanisms by which it could be produced. Section title: discussion Educational score: 4.328274250030518 Domain: biomedical Document type: Study Language: en Since the ionic and gating current kinetics have the same voltage dependence, the difference in their kinetics can be interpreted as a voltage shift of ∼20 mV. If a single charge-moving transition determines the properties of the conductance–voltage and charge–voltage curves, then a 20-mV voltage shift can be produced by a rather small energetic difference of ∼0.8 kcal mol −1 , using the relationship: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\Delta}{\Delta}G=z{\Delta}V_{1/2},\end{equation*}\end{document} Section title: discussion Educational score: 4.262732982635498 Domain: biomedical Document type: Study Language: en where z is the amount of charge moved for the final transition and ΔV 1/2 is the difference in the voltage of half-maximal activation between the gating and ionic currents. An energetic difference of ∼0.8 kcal mol −1 , and hence a 20-mV voltage shift in the gating currents, could be produced by a number of different mechanisms. We will discuss two mechanisms for which there is experimental evidence, namely, the effects on gating of the nonconducting conditions (the W434F mutation) used to measure gating currents, and variability in voltage-dependent behavior between patches. Section title: discussion Educational score: 4.3321332931518555 Domain: biomedical Document type: Study Language: en To record gating currents without interference from ionic currents, we rendered channels nonconducting by introducing the W434F mutation into the pore region . Originally, the W434F mutation was thought to block conduction of ions through the pore of the open state of the channel without interfering with activation gating transitions . However, several lines of evidence now indicate that the W434F mutation does not simply block conduction of ions and does affect gating near the open state of the channel. It has been shown that the W434F mutation decreases the probability of being open to ∼10 −5 by inactivating the channel, but does not block conduction in a normal open state conformation . Further, Chen et al. found that the W434F mutation at the homologous position in Kv1.5 slows the return of OFF gating currents after steps to voltages where the channel opens . These results clearly demonstrate that the W434F mutation alters transitions near the open state of the channel, either between the last closed state in the activation pathway and the open state or between the open state and closed states that are not traversed in the activation pathway. Section title: discussion Educational score: 4.341879367828369 Domain: biomedical Document type: Study Language: en To date, there is no direct evidence demonstrating that the kinetics of the activation pathway are affected by the W434F mutation. However, charge movement associated with slower gating transitions could mask effects of the W434F mutation on very fast transitions. Wild-type Shaker undergoes a very fast forward transition (time constant of ∼100 μs) near the open state . Changes in the kinetics of this transition would be difficult to resolve even if they were relatively large because the transition is an order of magnitude faster than the charge movement measured in our gating current records. If the W434F mutation changes the energy of the final gating transition by as little as ∼0.8 kcal mol −1 (e.g., the energy difference that is observed between the ON gating current of ILT and the ionic currents), it would not cause a measurable change in the ON gating currents of Shaker . However, the ILT mutation may unmask the effects of the W434F mutation on the kinetics of ON gating currents by isolating the last gating transition. Section title: discussion Educational score: 4.279410362243652 Domain: biomedical Document type: Study Language: en The observed voltage shift may also be due, at least in part, to variability between patches. Variability in voltage-dependent behavior between patches has been observed in both wild-type and mutant Shaker channels . The cause of this variability is not known, but it may be the result of the action of various factors (e.g., kinases, phosphatases, redox agents) that are present in oocytes. Our ILT and Shaw S4 gating current experiments may be unusually biased in this respect because of the extraordinary levels of channel expression that were required to produce a measurable signal for the small component of gating current at high voltages. As a result, all of these recordings were obtained from two batches of oocytes with exceptionally high levels of expression, and the gating current recordings will be biased towards the conditions found in these particular batches of oocytes. Further, because of the need for higher protein expression levels, gating currents were typically recorded from much older oocytes than those used to record ionic currents, and the age of the oocytes may be a factor in modification of channel by such agents as phosphatases and kinases. Meanwhile, ILT and Shaw S4 ionic currents could be measured from many batches of oocytes and, interestingly, the variability observed between patches has been considerable: the standard deviation of the midpoint of the conductance–voltage curve is ±10 mV . Such large variability in the midpoint of the conductance–voltage curve exceeds that expected based on series resistance errors, as error due to series resistance in our experiments was generally <2 mV. Section title: discussion Educational score: 4.233858108520508 Domain: biomedical Document type: Study Language: en We cannot rule out the possibility that the voltage shift (energy difference) observed between the kinetics of our ILT gating and the ionic currents is real and that the gating mechanism of ILT is actually more complicated than the mechanism presented in this paper. However, it is reasonable to suggest that the underlying small energy difference could be introduced by either the W434F mutation or the different experimental conditions required to obtain gating currents versus ionic currents. If the voltage shift is corrected for, the amplitude and voltage dependence of the kinetics of the gating currents recorded in the voltage range of channel opening are consistent with those expected to accompany the gating charge movement during the last cooperative transition that opens the channel. Therefore, in spite of the voltage shift, it seems likely that the charge movement that we have measured corresponds to the charge moved in the rate-limiting cooperative step. Section title: Insights into the Activation Pathway Educational score: 4.163218975067139 Domain: biomedical Document type: Study Language: en The ability to study the gating process in wild-type Shaker is limited by the fact that it is difficult to study individual transitions independently of one another because the rates and voltage dependences of most of the transitions in Shaker activation are too similar . The ability to perturb the energies of gating transitions with mutations provides a means to dissect out steps in the gating pathway. For this reason, we have studied the effects of mutations in the S4 region in detail and interpreted the effects of the mutations, when possible, in the context of a kinetic model for Shaker . Using a similar approach, Schoppa and Sigworth have gained insights into the gating mechanism of Shaker using the V2 mutant. Section title: Insights into the Activation Pathway Educational score: 4.171056747436523 Domain: biomedical Document type: Study Language: en For most of this discussion, we will focus on the ILT mutation, since it does not introduce any charge-changing substitutions into the S4 of Shaker and has been shown to be the least disruptive to the overall gating pathway of the three mutants in this study. Because the ILT mutation makes the last cooperative gating transition in activation rate limiting, we can study the properties of this transition in isolation from other gating transitions. Through analysis of the voltage dependence of ionic and gating currents, we have been able to determine that the equivalent charge associated with this transition is ∼1.8 e 0 , and we have shown that the rate-limiting cooperative transition is the final voltage-dependent transition in the gating pathway of ILT. Section title: Insights into the Activation Pathway Educational score: 4.204779624938965 Domain: biomedical Document type: Study Language: en Our results also provide insight into the manner in which cooperativity is implemented in the activation pathway. Activation of wild-type Shaker can be described adequately with models in which cooperativity is implemented in one of two ways: (a) as a cooperative stabilization of the open state, as in the 15-state model of Zagotta et al. , or (b) as a final cooperative transition(s) to the open state . However, our analysis of the ILT gating currents clearly supports the existence of a final cooperative transition in the activation pathway. Section title: Insights into the Activation Pathway Educational score: 4.447024345397949 Domain: biomedical Document type: Study Language: en Two lines of experimental evidence suggest that there is a similar but much faster cooperative transition to the open state in the activation pathway of wild-type Shaker . Zagotta et al. found that the first closing transition in Shaker is slower than earlier backward transitions, consistent with the presence of a strongly forward-biased cooperative transition between the final closed state of the channel and the open state (e.g., a transition with a very fast forward rate and relatively slow backward rate). But they concluded that if there is a forward cooperative transition before channel opening, it is sufficiently fast as to be “silent” in measurements of the activation kinetics of Shaker . Recently, Schoppa and Sigworth have used reactivation voltage protocols to uncover a strongly forward-biased cooperative transition near the open state. They found that the forward transition is very fast, at least as fast as 9,100 s −1 (which corresponds to a time constant on the order of 100 μs). However, their estimate of the rate and voltage dependence of this transition should be considered as a lower bound because the time course of such a fast transition may not be fully resolved from the contribution of other slower transitions to the time course of reactivation. Cooperative interactions in proteins can occur on time scales much faster than 100 μs. For example, haemoglobin undergoes its oxy–deoxyhaemoglobin conformational change in 20 μs . Even if such a fast transition could be isolated experimentally from slower transitions in the activation pathway of wild-type Shaker , it would be difficult to measure using standard electrophysiological techniques. Section title: Insights into the Activation Pathway Educational score: 4.251092910766602 Domain: biomedical Document type: Study Language: en The amount of charge assigned to the cooperative final transition in ILT gating, ∼1.8 e 0 , is greater than the amount of charge assigned to the final opening transition of Shaker in all kinetic models of Shaker , except for an early model presented by Schoppa et al. . There is no consensus between the various models on the voltage dependence of the final opening transition in wild-type Shaker , which reflects the fact that it is difficult to measure such a fast transition as a separate component in the kinetic measurements. The ILT mutation makes the final cooperative transition rate limiting, making it possible to measure the voltage dependence of this transition in the ILT mutant. Section title: Insights into the Activation Pathway Educational score: 4.548901081085205 Domain: biomedical Document type: Study Language: en Is the voltage dependence of the final cooperative transition in wild-type Shaker similar to that of ILT? The results of studies on the V2 mutant, which substitutes a valine for L382 at the carboxy-terminal border of the S4, may provide insight into this matter . The V2 mutation causes a large positive shift in the voltage dependence of a component of gating charge of ∼1.8 e 0 and shifts the probability of channel opening to a much more positive voltage range than Shaker . In these respects, the effect of the V2 mutation is strikingly similar to that of the ILT mutation. However, in the kinetic model for Shaker and V2 gating developed by Schoppa and Sigworth , the voltage-shifted component of gating charge (1.75 e 0 ) is moved in not one but two sequential concerted transitions preceeding channel opening, each of which moves ∼1 e 0 . The primary reason cited for introducing two final cooperative transitions is to explain the features of Shaker and V2 OFF gating currents, which show a rising phase followed by a slow decay. Schoppa and Sigworth found that one cooperative transition must be introduced to account for the rising phase of the OFF gating currents, and a second cooperative transition must be introduced to account for the slow decay, which is slower than the independent intermediate gating transitions. However, the kinetics of the OFF gating currents, specifically the presence of a rising phase, may be affected by channels opening under nonconducting conditions . The question of the validity of the rising phase in the OFF gating kinetics of wild-type Shaker could be addressed using the experimental approach of Chen et al. , whereby OFF gating currents are measured in conducting solutions by stepping to the reversal potential of the solutions. Section title: Insights into the Activation Pathway Educational score: 4.247397422790527 Domain: biomedical Document type: Study Language: en It is not clear how to reconcile our ILT results with the model of Schoppa and Sigworth . Their model is well constrained by measurements of gating currents, ionic currents, and single-channel currents. However, the time course of activation of the V2 channel is fast and sigmoidal like Shaker and, accordingly, the final two cooperative transitions for Shaker and V2 in the model are very fast. As in the case of wild-type Shaker , the last cooperative transition in the gating of V2 may be too fast to be studied independently of other gating transitions. Thus, it seems plausible that the last cooperative transition has a similar voltage dependence (∼1.8 e 0 ) for Shaker , ILT, and V2, and that the disagreement between the models arises because of the difficulties inherent in characterizing the last cooperative transition in Shaker and V2. Section title: Insights into the Activation Pathway Educational score: 4.154575824737549 Domain: biomedical Document type: Study Language: en Interestingly, Smith-Maxwell et al. found that the I372L substitution, which is one of the three substitutions made in ILT, is responsible for increasing the equivalent charge movement associated with the final opening transition. The I372L substitution also greatly slows the final transition, which allows the properties of the final transition to be determined. Again, it is possible that the final opening transition in Shaker has the same voltage dependence as in the I372L and ILT mutants but that the transition is simply too fast to measure in Shaker . Section title: Insights into the Activation Pathway Educational score: 4.281566619873047 Domain: biomedical Document type: Study Language: en Alternatively, the I372L mutation may have altered the amount of equivalent charge moved during the last cooperative gating transition. It is conceivable that charge-conserving mutations could alter the amount of charge moved during a transition by changing the movement of the voltage sensor, thereby increasing the fraction of the electric field through which the charged residues move or increasing the number of charged residues that interact with the electric field during the transition between the final closed state and the open state. Section title: Insights into the Activation Pathway Educational score: 4.092594146728516 Domain: biomedical Document type: Study Language: en However, we have found that the amount of charge moved in this step is not readily altered by mutations elsewhere in the S4 sequence. The cooperative transition has the same voltage dependence for all three mutant channels studied in this paper, namely ILT, Shaw S4, and Shaw S4:RRK, despite the fact that these three mutants have different S4 sequences and produce different effects on earlier steps in the gating pathway. Section title: Channel Structure and Conformational Changes during Activation Educational score: 4.256178379058838 Domain: biomedical Document type: Study Language: en As yet, we do not understand the molecular basis for structural transitions or cooperativity in Shaker . But several aspects of the molecular basis for structural transitions and cooperative interactions are well understood for a handful of allosteric proteins that have been crystallized in both active and inactive conformations . Flexibility of helix packing seems to be essential for the structural transitions of many allosteric proteins. α-Helices can shift relative to each other by up to 1.6 Å and turn by several degrees with only minor adjustments to their side chain packing. When coupled in series, the small shifts of α-helices can be amplified to produce large structural transitions (e.g., for citrates synthase, haemoglobin, and aspartate transcarbamylase). Section title: Channel Structure and Conformational Changes during Activation Educational score: 4.631374359130859 Domain: biomedical Document type: Study Language: en The final cooperative transition in Shaker gating may have a similar design, in which a series of small shifts and turns of many α-helical regions of the protein produce the structural transition that opens or closes the channel. Consistent with this hypothesis, cysteine-modifying experiments have shown that the process of activation changes the exposure of residues to solution in many regions of the protein, including the S4 region , the S4–S5 linker region , and the S6 region . This hypothesis is also consistent with the collective findings of a number of mutagenesis studies. In addition to the ILT and V2 mutations, a number of mutations have been identified in the S4, S4–S5 linker, and S5 that affect cooperativity by increasing the backward rate of the cooperative transition, which manifests itself as an increase in the first closing rate from the open channel in single channel recordings. These mutations include R3Q in the carboxy-terminal half of the S4 helix , E395C in the S4-S5 linker region, and F401I in the amino terminal of the S5 helix (Kanevsky and Aldrich, manuscript submitted for publication). In the Shaker literature, there are many additional reports of Shaker mutations in the S4– S6 regions that produce strong effects on gating that are consistent with changes in cooperativity but for which the reported data are incomplete or inconclusive . Section title: Channel Structure and Conformational Changes during Activation Educational score: 4.2663679122924805 Domain: biomedical Document type: Study Language: en Our knowledge of the structure of Shaker , albeit limited, is also consistent with this hypothesis. All of the transmembrane regions of Shaker are predicted to be α-helical, and experimental evidence is emerging in support of this prediction. In the three-dimensional structure of the bacterial channel, the sequences corresponding to the S5 and S6 regions of Shaker are α-helical . Also, evidence from cysteine-scanning mutagenesis suggests that at least part of the S4–S5 linker is α-helical . Section title: Channel Structure and Conformational Changes during Activation Educational score: 4.430809020996094 Domain: biomedical Document type: Study Language: en This physical model of gating, in which a series of small shifts of many α-helices produce a larger structural transition that opens the channel, can explain why even conservative substitutions like ILT and V2 can produce such strong effects on gating: they may be involved in close-packed contacts between helices that shift during gating. It can also explain why mutations in different regions of the protein affect a single conformational change: extensive regions of the protein move during the conformational change that accompanies channel opening. Section title: Channel Structure and Conformational Changes during Activation Educational score: 4.243600845336914 Domain: biomedical Document type: Study Language: en The ILT mutant, which isolates the last cooperative gating transition from earlier gating transitions, may provide a valuable experimental tool for dissecting out the molecular basis of structural transitions in the activation of Shaker . The ILT channel could be used as a background for cysteine-scanning mutagenesis experiments in regions that have been shown to change their exposure during channel opening, like the S4 region or the S6 region . With this approach, we could correlate the conformational changes of the protein with either the final cooperative step of the gating pathway or the early charge moving steps. Section title: Role of the S4 in Cooperative Interactions between Subunits Educational score: 4.4614763259887695 Domain: biomedical Document type: Study Language: en The molecular basis for cooperativity between subunits is not understood for Shaker , but it is well understood for a number of allosteric proteins that have been crystallized in active and inactive forms . Subunit contacts are of critical importance for stabilizing the alternative structures of the active and inactive forms of haemoglobin, phopsphofructokinase, and aspartate transcarbamylase. The contacts between subunits strongly stabilize only the alternative quaternary structures of the protein and do not stabilize intermediate forms of the protein (e.g., where some subunits are in a different conformation from the others). Thus, concerted transitions between alternative quaternary structures of the protein are strongly favored energetically. The contacts between subunits in alternative structures are stabilized chiefly by electrostatic interactions and hydrogen bonds between side chains of opposite charge. Section title: Role of the S4 in Cooperative Interactions between Subunits Educational score: 4.301651477813721 Domain: biomedical Document type: Study Language: en The molecular basis of cooperativity in Shaker may have a similar design. The results presented in this paper and Smith-Maxwell et al. clearly demonstrate that the S4 region is involved in cooperative interactions between subunits, not just in voltage sensing. Although the involvement of the S4 in cooperativity may be mediated indirectly, our results point to the intriguing possibility that the S4 helix may form part of the interface between subunits. If the S4 forms part of the interface between subunits, then it is possible that some of the charged residues in the S4 region participate in salt bridges across subunits that stabilize the alternative quaternary structures of the closed and open Shaker channel. Section title: Role of the S4 in Cooperative Interactions between Subunits Educational score: 4.306891441345215 Domain: biomedical Document type: Study Language: en The evidence in favor of the charges in the S4 region participating in salt bridges is compelling. Neutralizations at K5 and R6 are “lethal” (prevent proper folding and expression of channels), but can be rescued by making complementary neutralizations at E293Q in the S2 region and D316N in the S3 region, suggesting that the S4 basic residues K5 and R6 participate in electrostatic interactions (salt bridges) with acidic residues in the S3 . Tiwari-Woodruff et al. have shown that a lethal charge reversal mutation at K5 can be rescued by a second charge reversal mutation at E293 and that a lethal charge reversal mutation at E283 in the S2 can be rescued by making a second charge reversal mutation at either R3 or R4. Section title: Role of the S4 in Cooperative Interactions between Subunits Educational score: 4.215595722198486 Domain: biomedical Document type: Study Language: en Although there is strong evidence to suggest that several of the charges in the S4 region participate in salt bridges, the question of whether these bridges form within or across subunits has not been as thoroughly investigated. Studies on heteromultimeric channels suggest that the interaction between K5 and E293 is within, not across, subunits , but this result alone does not preclude the possibility that some of the charges in the S4 region can participate in salt bridges across subunits. | Other | biomedical | en | 0.999997 |
10051517 | Section title: introduction Educational score: 4.442716121673584 Domain: biomedical Document type: Study Language: en With the determination of the high-resolution structure of the KcsA K + channel from Streptomyces lividans , structure–function studies of ion channel proteins can henceforth include structure. This advance has revealed in unprecedented detail the molecular makeup of a K + -selective permeation pathway. Moreover, homology considerations argue that the pores of all K + channels are built along the basic outlines observed in KcsA, a member of the family of K + channels constructed from two membrane-spanning sequences. Until structures of other K + channels become available, however, indirect means must be used to attack questions of molecular architecture of protein domains not directly forming the pore. In contrast to KcsA, K v -type channels are built from six membrane-spanning sequences, the first four of which, S1–S4, are associated with the voltage-dependent conformational changes that culminate in pore opening. Section title: introduction Educational score: 4.434162139892578 Domain: biomedical Document type: Study Language: en Functional K + channels are formed by the assembly of four identical or similar subunits . The canonical topology-model for the individual K v channel subunit, shown in Fig. 1 A, is supported by ample biochemical and electrophysiological evidence . Comparison to KcsA firmly establishes the pore-forming sequences S5 and S6 as α helical, but no experimental results directly bearing on the secondary structures of the S1–S4 sequences have yet been advanced; likewise, S1 and S4 are definitively known to span the membrane, but this cannot be asserted for S2 or S3, since the S2–S3 “loop” has not yet been nailed down as cytoplasmic. Because of the proposed importance of S2 in cooperating with the charge-bearing S4 sequence in the early steps of gating , we chose S2 as the focus of a tryptophan-perturbation mutagenesis study designed to achieve two goals: (a) to ascertain the secondary structural character of this putative transmembrane sequence, and (b) to locate the lipid-facing residues of S2. The experimental premise is that bulky Trp substitutions will wreak functional havoc if made at positions that interact intimately with other parts of the channel protein, while they will often be accommodated at lipid-exposed positions. We find that S2 accommodates point mutations with a periodicity that strongly implicates an α-helical structure and identifies a lipid-exposed face of the helix. Section title: Recombinant DNA Methods Educational score: 4.3055620193481445 Domain: biomedical Document type: Study Language: en The channel used here is inactivation-removed (Δ6-46) Shaker B carrying two point mutations: L338R in the S3–S4 loop to create a MluI site, and F425G in the external vestibule. This Shaker variant, which we denote “wild-type,” is functionally distinguishable from traditional inactivation-removed Shaker B in three respects. Our construct binds charybdotoxin 2,000-fold more strongly than Shaker B , its voltage activation curve is right-shifted ∼15 mV, and C-type inactivation is ∼10-fold slower. Mutations were generated on this background in pBluescript KS(+) using PCR-based mutagenesis incorporating 3′ XbaI and 5′ MluI or BamHI restriction sites. PCR products were purified by agarose gel electrophoresis, digested, and reintroduced into the Shaker cDNA. All constructs were confirmed by sequencing through the cloning cassette. cRNA was transcribed in vitro from a FspI-linearized plasmid using T7 RNA polymerase ( Promega Corp. ) or the T7 mMessage mMachine (Ambion Inc.). Section title: Electrophysiology Educational score: 4.177486419677734 Domain: biomedical Document type: Study Language: en Defolliculated Xenopus oocytes were injected with 1–5 ng cRNA (enough to produce ∼5 μA of current 1–5 d after injection) and stored at 17°C in ND96 solution containing (mM): 96 NaCl, 2 KCl, 1.8 CaCl 2 , 1 MgCl 2 , and 10 HEPES, pH 7.6, and also containing gentamicin (10 mg/liter). Oocytes were examined 2–5 d after injection using two-electrode voltage clamp (Warner Instruments, New Haven, CT) in ND96 solution (containing 0.3 mM CaCl 2 ) to check expression levels and K + selectivity. Electrodes were filled with 3 M KCl, 5 mM EGTA, and 10 mM HEPES or 10 mM Tris, pH 7.6. Tail currents were recorded in KD98 solution containing (mM): 98 KCl, 0.3 CaCl 2 , 1 MgCl 2 , and 10 HEPES, pH 7.6. Standard pulse protocols used a holding potential of −90 mV, a test pulse between −60 and +50 mV in 5- or 10-mV increments (50-ms duration), followed by a tail pulse to −70 mV (30-ms duration). Values of test and tail voltages and pulse duration were modified according to the kinetic properties of individual mutants. Section title: Data Analysis Educational score: 4.207278251647949 Domain: biomedical Document type: Study Language: en Voltage-activation curves were calculated using standard tail-current analysis , with tail amplitudes measured 2–3 ms into the pulse. The activation curve was fit using a Boltzmann function to produce the usual parameters V o (half-activation voltage), z (slope factor), and Δ G o , the free energy of channel opening at zero voltage (a nearly model-free measure of the intrinsic stability of the open conformation with respect to the closed), according to Δ G o = zF V o . Section title: Data Analysis Educational score: 4.089470863342285 Domain: biomedical Document type: Study Language: en Activation kinetics were compared among the various mutants at V o by the time required for half-opening, t o , and the time constant for deactivation, τ d , determined by fitting the falling exponential component of the tail. Section title: results Educational score: 4.332226753234863 Domain: biomedical Document type: Study Language: en The S2 segment in Shaker spans residues 278–300 . In integral membrane proteins of known structure, lipid-exposed residues in transmembrane helices tend to be more hydrophobic and less well conserved than residues facing the interior of the protein . Comparison of S2 sequences in K + channels highlights the variable residues , which when mapped onto a helical projection lie predominantly on one face . The present experiments seek to test this sequence-based suggestion for an α-helical disposition of S2. We proceed from the idea that a bulky, hydrophobic tryptophan substitution is more likely to disrupt channel structure (and hence ablate function) when placed at positions packed against other parts of the protein than when introduced at lipid-exposed areas. We therefore mutated each S2 residue individually to tryptophan (or the single tryptophan to alanine), anticipating that positions functionally forgiving of mutation would be interspersed, possibly in helical periodicity, with positions marked by failure to express K + current. Though not rigorously justified, this expectation is plausible since such substitution-toleration patterns have been observed with several membrane proteins, originally with randomly selected residues , and more recently with specifically inserted tryptophans . Section title: results Educational score: 4.2983717918396 Domain: biomedical Document type: Study Language: en Our results contradict this expectation. Of the 23 substitutions made, only one (R297W) failed to express current in Xenopus oocytes. Representative currents in response to depolarizing voltage pulses are shown in Fig. 2 for the wild-type channel and two mutants. These particular mutant channels display altered voltage dependencies; the activation curve for I287W is right-shifted 27 mV, while that for E293W is left-shifted 22 mV. These two mutants are altered in other ways; the slope of the activation curve is decreased in I287W, and both activation and deactivation kinetics of E293W are slowed substantially. Similar recordings were collected for the remaining Trp-substituted channels. The effects of the substitutions were analyzed using several empirical parameters: Δ G o (the zero-voltage free energy of opening), t o (the activation half-time measured at the half-point on the activation curve), and τ d (the deactivation time constant at −70 mV). These parameters are shown in Table I for all mutant channels. Fig. 3 A plots the open-state stabilization energy ΔΔ G o ; i.e., the difference in Δ G o between mutant and wild type. In all, 13 mutant channels have electrophysiological properties similar to wild type; we define these residues as “low- impact” or “tolerant,” with |ΔΔ G o | < 1 kcal/mol. The remaining 9 “high-impact” positions (E283, T284, C286, I287, W289, F290, E293, L294, and A300) have properties substantially different from wild type. This binary cut between the two types of residues is of course arbitrary, but it naturally falls out of the results; our overall conclusions do not change if we double or halve this cutoff value. In most channels with altered equilibrium activation properties, the kinetic parameters were also changed greater than twofold . We emphasize that the intention of this gating analysis is empirical, not mechanistic: to identify positions at which mutations produce obvious and gross changes in gating, not to understand the steps in the activation pathway that are altered by the mutations. Section title: results Educational score: 4.132120609283447 Domain: biomedical Document type: Study Language: en Most of the tolerant positions bear large, hydrophobic residues , and so their mutation to Trp causes only moderate changes in side chain volume. We therefore challenged these positions with more radical changes of side chain chemistry. We constructed a set of single Asn substitutions that would alter both side chain volume and polarity of Trp-tolerant positions: L285N, I288N, F292N, V296N, F298N, and L299N. Remarkably, all six mutants expressed channels exhibiting electrophysiological properties similar to those of the wild-type channel . Section title: discussion Educational score: 4.487220764160156 Domain: biomedical Document type: Study Language: en The S2 transmembrane segment has been proposed to participate in two ways in the charge movements underlying voltage-dependent gating in K v -type K + channels: first by providing a helical foundation from which two negative residues, E283 and E293, form salt bridges with positive residues in the charge-carrying S4 sequence , and second by moving at least one of these negative residues inward during gating; i.e., in the direction opposite to S4 movement . These proposals are intriguing, but neither can be considered established. Indeed, there has been no evidence demonstrating that S2 (or any of the first four transmembrane sequences) is helical, and even the accepted topology identifying S2 and S3 as crossing the membrane is unsupported by any experimental results on K + , Na + , or Ca 2+ channels. Section title: A Pattern of Tryptophan Toleration Educational score: 4.2801055908203125 Domain: biomedical Document type: Study Language: en In this study, we used the tryptophan-perturbation strategy previously applied to three other membrane proteins: MotA, a component of the E . coli flagellar motor complex , and two inward rectifier-type K + channels, RomK1 and K ir 2.1 . This approach exploits the Janus-like nature of transmembrane α-helices in integral membrane proteins. Transmembrane helices often have two distinct faces: one exposed to bilayer lipid, which is expected to accommodate tryptophan, the other packed at more structurally stringent protein– protein interfaces. Not only does the Trp-scanning strategy make intuitive sense, but it now may be placed on a firm empirical foundation : comparison to the structure of KcsA . Here, we map the Trp-tolerant residues of the first transmembrane helix found previously in RomK1 onto the equivalent positions of KcsA. The figure demonstrates an impressive correspondence between the willingness of positions to accommodate Trp and their disposition on the outer, lipid-facing surface of the channel protein. This agreement enhances our confidence in a Trp-perturbation scan as a probe of helical orientation in membrane proteins. Section title: A Pattern of Tryptophan Toleration Educational score: 4.235780715942383 Domain: biomedical Document type: Study Language: en We carried out an initial scan of 23 point mutations along the S2 segment of a Shaker K + channel. Had we assayed only the expression of functionally active protein, we would have failed to discern any pattern of Trp toleration whatsoever: all mutants save one expressed voltage-dependent K + channels. The fact that all these mutants were properly folded and faithfully delivered to the plasma membrane is in itself a notable result for several reasons. First, it corroborates the well-known flexibility of proteins in adjusting local structure to accept mutations even in closely-packed regions . Second, two of the functionally competent mutants are at the absolutely conserved glutamate residues (E283, E293) thought to be critical for proper folding and for salt-bridging to positive charges in S4 . We were surprised that alterations as outrageous as Glu → Trp simultaneously in all four subunits would be tolerated, but our results are in harmony with other studies showing that these residues are not strictly essential for channel function. Section title: A Pattern of Tryptophan Toleration Educational score: 4.416504383087158 Domain: biomedical Document type: Study Language: en A clear pattern of response to Trp substitution emerges upon examination of empirical gating characteristics of the mutant channels , such as the intrinsic free energy of opening, ΔG o . Between E283 and A300, the tolerant and high-impact residues follow an α-helical pattern, with the two types of positions segregating on opposite sides of a helical wheel diagram , the single exception being T284. The two absolutely conserved glutamates (E283, E293), which have been proposed to pack against the S4 segment, are located squarely within the high-impact face. Moreover, the tolerant residues coincide with the region of highest natural sequence variability , a hallmark of lipid exposure. These considerations argue that S2 is in fact α-helical from position 283 to 300, that the tolerant face of the helix projects side chains into membrane lipid, and that the high-impact face packs against other membrane-spanning parts of the Shaker protein. Section title: Side Chain Chemistry and the Lipid-exposed Face of S2 Educational score: 4.276187896728516 Domain: biomedical Document type: Study Language: en It is both notable and satisfying that a clean helical pattern emerges from a technique such as Trp substitution scanning, which is at best a diagnostic bludgeon. However, we worried that this pattern might be an artifact arising from the nature of the S2 sequence itself. The tolerant face of the proposed helix tends towards larger and more hydrophobic residues than the high-impact face, and so it could be argued that Trp substitution would be inherently less perturbing on the former side of the helix than on the latter. This possible objection is weak, however, since the differences are slight in both side chain volume (98 vs. 84 Å 3 mean volume per residue) and polarity (six hydrophobic/three uncharged-polar on the tolerant side, six hydrophobic/ three charged-polar on the high-impact side), and since the channel responds differently to the same mutations (I, L, F → W) made on the different sides of the putative helix. Nevertheless, the pattern of Trp toleration is so fundamental to our interpretation that we sought to test further whether the tolerant face remains tolerant if subjected to a wider range of substitutions. Section title: Side Chain Chemistry and the Lipid-exposed Face of S2 Educational score: 4.1988325119018555 Domain: biomedical Document type: Study Language: en For this reason, we attempted to abuse the channel by introducing residues that would simultaneously alter side chain polarity and volume. Six hydrophobic residues on the tolerant face, L285, L288, F292, V296, F298, and L299, were substituted singly by asparagine, a small, polar side chain that is not found in any of the S2 sequences we surveyed and is almost never observed in lipid-exposed domains of membrane proteins . We might therefore expect that these mutations would be disruptive, but this was not the case; all six Asn mutants expressed K + channels with properties close to wild type, a result that further illustrates the permissive character of the Trp-accepting positions. (We note, however, that double, triple, and quadruple alanine mutations on the tolerant face did lead to channels with significantly left-shifted voltage-activation curves.) Section title: Conformational Stabilization by S2 Substitutions Educational score: 4.364651203155518 Domain: biomedical Document type: Study Language: en The Shaker channel's toleration of Trp substitutions is in harmony with the proposal that the tolerant face of the S2 helix is lipid exposed. However, its similar compliance towards Asn residues appears to contradict this idea. It seems alarming that the channel structure readily admits these polar groups into the hydrocarbon-rich bilayer interior. This fact would be disquieting if our assay for the impact of a substituted residue had been the ability of the protein to fold properly in the membrane; i.e., if we had measured the standard-state free energy of Shaker folding from the denatured state. But no such measurement exists; instead, our assay quantifies a process entirely different from folding and assembly: the relative thermodynamic stability of the conducting vs. nonconducting conformations. Section title: Conformational Stabilization by S2 Substitutions Educational score: 4.251859664916992 Domain: biomedical Document type: Study Language: en Nearly all our mutants assemble well enough to produce immediately recognizable K + channels. It may be unexpected that Shaker polypeptides bearing lipid-exposed Asn residues behave in so forgiving a manner, but that is the experimental fact; our assay has nothing to say about the channel's thermodynamic comfort once it is in the membrane. Instead, the assay identifies mutations having strong differential effects on the stability of the open compared with the closed conformations; this circumstance is expected to occur where close-packed parts of the protein move upon gating. In contrast, residues that remain lipid-exposed in both open and closed conformations should be functionally unresponsive to substitution regardless of side chain chemistry, as long as the protein achieves a transmembrane insertion in the first place. This is the situation that we observe on the tolerant face of the S2 helix. Section title: Conformational Stabilization by S2 Substitutions Educational score: 4.327164649963379 Domain: biomedical Document type: Study Language: en Closer inspection of the high-impact residues hints at a picture of S2 movement during gating. We notice that the high-impact residues in the first half of the Trp-scan (positions 278–287) tend to destabilize, while those in the latter half (289–300) tend to stabilize the open state. Assuming that most Trp substitutions destabilize a protein interface, this pattern suggests that upon opening, S2 moves so that the external part of the helix packs against the rest of the protein more tightly, while the cytoplasmic end of the helix loosens its interaction with other protein regions. These could be subtle motions, since the free energies involved are small. Section title: Conclusions Educational score: 4.379150390625 Domain: biomedical Document type: Study Language: en The helical periodicity observed in this mutational perturbation scan suggests a structural picture of Shaker S2, a picture that most likely applies to all K v -type K + channels. We conclude that S2 is indeed an α helix, as envisioned in most models of voltage-gated ion channels. Our results are also consistent with an orientation of S2 that places the variable residues in the sequence projecting into the membrane lipid. Finally, the present results position the two conserved glutamates in the core of a protein–protein interface, a picture consistent with earlier proposals of a salt bridge between S2 and S4. Section title: Conclusions Educational score: 4.300334930419922 Domain: biomedical Document type: Study Language: en The impressive correspondence between sequence variability and Trp-toleration in S2 prompts more confidence in a sequence-based analysis of the other “outer” helices S1 and S3. A BLAST search on S1 and S3 identified 120 and 135 sequences, respectively, and the conserved and variable positions were identified. These residues display a clear segregation on helical wheel projections . In analogy to the S2 results, we therefore consider it likely that S1 and S3 are indeed helical, with their variable sides facing membrane bilayer lipid. We note that the putative lipid-exposed face of S1, like that of S2, covers approximately half the helical surface, while that of S3 is much smaller. This leads to the speculation that S1 and S2 are positioned towards the periphery of the tetrameric channel, while S3 is more buried within the protein complex. | Study | biomedical | en | 0.999996 |
10051518 | Section title: introduction Educational score: 4.308370113372803 Domain: biomedical Document type: Study Language: en Large conductance Ca 2+ -activated K + channels (BK channels) 1 play an important role in regulating the excitability of nerve, muscle, and other cells by stabilizing the cell membrane at negative potentials . BK channels are activated by both intracellular calcium (Ca 2+ i ) and membrane depolarization, and hence can serve as an important link to couple the effects of Ca 2+ i and membrane potential, two common forms of signaling in cells. To facilitate this linkage, the Ca 2+ i sensitivity of BK channels, defined as the Ca 2+ i required for half-maximal activation at a given voltage, is set to match the specific needs of the various cells. In some tissues, such as smooth muscle, BK channels are highly Ca 2+ i sensitive, while in other tissues, such as skeletal muscle, the channels are much less Ca 2+ sensitive . Section title: introduction Educational score: 4.706063270568848 Domain: biomedical Document type: Study Language: en Recent studies have given some insight into the molecular basis for differences in Ca 2+ sensitivity. BK channels can be formed of either α subunits alone or of α together with β subunits . The larger pore-forming α subunits are encoded by the gene at the slo locus, mutations of which underlie the Drosophila slowpoke phenotype . The α ( slo ) subunit shows homology with the pore-forming subunits of the voltage-dependent superfamily of K + channels, which have at least six putative transmembrane domains, a pore-forming region between S5 and S6, and an S4 voltage-sensor region . However, the NH 2 - and COOH-terminal ends of the α subunits differ from those of the superfamily. The NH 2 terminus of mammalian α subunits displays an additional transmembrane domain, S0, that places the amino terminal into the extracellular space and is required for the action of the β subunit . The COOH-terminal tail is greatly extended, displays four hydrophobic domains, and appears to provide the Ca 2+ -sensing domain of the channel . The β subunit, with two putative transmembrane domains, shows no homology with other ion channel subunits . Section title: introduction Educational score: 4.491808891296387 Domain: biomedical Document type: Study Language: en While α subunits assemble as tetramers to form functional channels by themselves , β subunits expressed alone do not . Rather, β subunits can associate with α subunits in a 1:1 stoichiometry , increasing the apparent Ca 2+ sensitivity of the α subunits ∼10-fold . It is the presence of the β subunit that confers the greatly increased Ca 2+ sensitivity to BK channels in smooth muscle . Although it is known that the β subunit slows activation and deactivation kinetics , while having little effect on channel open probability in the absence of Ca 2+ i , the mechanism by which the β subunit increases the apparent Ca 2+ sensitivity of BK channels is not known. The β subunit could increase apparent Ca 2+ sensitivity through fundamental changes in the gating mechanism, such as by generating additional conformational states or Ca 2+ -binding sites. Alternatively, the β subunit might act by modulating the gating of the α subunit to increase the rate of Ca 2+ binding or to change the rates of selected transitions among the various conformational states. Section title: introduction Educational score: 4.379117965698242 Domain: biomedical Document type: Study Language: en We now use the resolving power of the single-channel recording technique to differentiate among these possible types of action by studying the kinetics of single BK channels comprised of α subunits alone, or of both α and β subunits. Our data suggest that the β subunit does not act by changing the fundamental gating mechanism, as neither the Hill coefficients for Ca 2+ binding nor the numbers of detected kinetic states entered during gating were changed by the β subunit. The data also suggest that the β subunit had little effect on the initial Ca 2+ -binding steps involved in activation of the channel, as the durations of the gaps (the long closed intervals) between bursts of activity were little changed. Instead, the β subunit increased Ca 2+ sensitivity through selected modulation of transition rates to retain the channel in the open and closed states that generate the bursts of activity (bursting states), increasing burst duration 20–100-fold. We also found that the β subunit inhibited transitions to subconductance states, and that the gating of native BK channels from cultured rat skeletal muscle was similar to the gating of BK channels expressed from α subunits alone. Section title: Heterologous Expression of BK Channels in Human Embryonic Kidney 293 Cells Educational score: 4.162913799285889 Domain: biomedical Document type: Study Language: en Human embryonic kidney (HEK) 293 cells were transiently transfected with expression vectors (pcDNA3) encoding the α subunit and the β subunit of BK channels, kindly provided by Merck Research Laboratories, and also with an expression vector encoding the green fluorescent protein (GFP, Plasmid pGreen Lantern-1; GIBCO BRL ). Cells were transfected transiently using the Lipofectamine Reagent (Life Technologies, Inc.). The GFP was used to monitor successfully transfected cells. For transfection, cells at 30–40% confluency in 30 mm recording Falcon dishes were incubated with a mixture of the plasmids (total of 1 μg DNA), Lipofectamine Reagent (optimal results at 7 μl), and Opti-MEM I Reduced Serum Medium ( GIBCO BRL ). The mixture was left on the cells for 1 h, after which it was replaced with standard tissue culture media: DMEM with 5% fetal bovine serum ( GIBCO BRL ) and 1% penicillin-streptomycin solution ( Sigma Chemical Co. ). The cells were patch-clamped 2–3 d after transfection. Section title: Heterologous Expression of BK Channels in Human Embryonic Kidney 293 Cells Educational score: 4.123671531677246 Domain: biomedical Document type: Study Language: en In the coexpression experiments, a fourfold molar excess of plasmid encoding the β subunit was used to drive coassembly with the α subunits in the expressed channels . Using the same promoter (cytomegalovirus) for the α and β subunits and the GFP increased the probability that if the GFP was expressed, the included subunits would also be expressed. While we did not prove directly that all BK channels studied from cells cotransfected with plasmids encoding for α and β subunits were indeed composed of both α and β subunits, the markedly different bursting kinetics of BK channels from such cells (see results ) indicated that the coexpression of the β with the α subunit altered the gating of the channels. Section title: Solutions Educational score: 4.270883083343506 Domain: biomedical Document type: Study Language: en The intracellular solution contained 175 mM KCl, 5 mM TES [ N -tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid] pH buffer, and 10 mM EGTA and 10 mM HEDTA to buffer the Ca 2+ (see below). The extracellular solution contained either 150 or 175 mM KCl and 5 mM TES and had no added Ca 2+ or Ca 2+ buffers. Both the intracellular and extracellular solutions were brought to pH 7. The amount of Ca 2+ added to the intracellular solution to obtain approximate free Ca 2+ concentrations of 0.1– 100 μM was calculated using stability constants for EGTA from Smith and Miller and for HEDTA from Martell and Smith . These solutions were then calibrated using a Ca 2+ electrode (Ionplus from Orion Research, Inc.) standardized against solutions with KCl and TES (as in the experimental solutions) in which a known amount of Ca 2+ was added. Before adding Ca 2+ , any contaminating divalent cations were removed from the solution by treatment with Chelex 100 (Bio-Rad Laboratories). The solutions bathing the intracellular side of the patch were changed by means of a valve-controlled, gravity-fed perfusion system using a microchamber . Section title: Single-Channel Recording and Analysis Educational score: 4.174707889556885 Domain: biomedical Document type: Study Language: en Currents flowing through single BK channels in patches of surface membrane excised from HEK 293 cells transfected with clones for either α or α and β subunits were recorded using the patch-clamp technique . All recordings were made using the excised inside-out configuration in which the intracellular surface of the patch was exposed to the bathing solution. BK channels were identified by their large conductance and characteristic voltage and Ca 2+ dependence . Endogenous BK channels in nontransfected HEK 293 cells were not seen, but we cannot exclude that they might exist at a low density. Currents were recorded with an Axopatch 200A amplifier ( Axon Instruments ) and stored on VCR tapes using a VR-10B digital data recorder. The currents were then analyzed using custom programs written in the laboratory. Single-channel patches were identified by observing openings to only a single open-channel conductance level during several minutes of recording in which the open probability was >0.4. Except for two experiments in which patches containing two BK channels were used to measure the effect of Ca 2+ on open probability ( P o ), all data were from patches containing a single BK channel. Experiments were performed at room temperature (20–25°C). Section title: Single-Channel Recording and Analysis Educational score: 4.13999080657959 Domain: biomedical Document type: Study Language: en Single-channel current records were low-pass filtered with a four-pole Bessel filter to give a final effective filtering of typically 4.5–10 kHz (−3 dB) and were sampled by computer at a rate of 125–250 kHz. The methods used to select the level of filtering to exclude false events that could arise from noise, measure interval durations with half-amplitude threshold analysis, and use stability plots to test for stability and identify activity in different modes have been described previously, including the precautions taken to prevent artifacts in the analysis . The kinetic analysis in this study was restricted to channel activity in the normal mode, which typically involves ∼96% of the detected intervals . Activity in modes other than normal, including the low activity mode , was removed before analysis, as were transitions to subconductance levels, except when the subconductance levels were being studied specifically. The numbers of intervals during normal activity analyzed for each experimental condition ranged from ∼1,500 to ∼200,000, with the greater numbers of intervals being obtained for higher Ca 2+ i where the channel activity was higher. Section title: Single-Channel Recording and Analysis Educational score: 4.114556312561035 Domain: biomedical Document type: Study Language: en The methods used to log-bin the intervals into dwell-time distributions, fit the distributions with sums of exponentials using maximum likelihood fitting techniques (intervals less than two dead times were excluded from the fitting), and determine the number of significant exponential components with the likelihood ratio test have been described previously . Dwell-time distributions are plotted with the Sigworth and Sine transformation, which plots the square root of the number of intervals per bin without correcting for the logarithmic increase in bin width with time. With this transform, the peaks in the plots fall at the time constants of the major exponential components. Section title: Single-Channel Recording and Analysis Educational score: 4.147980690002441 Domain: biomedical Document type: Study Language: en The method of defining a critical gap (closed interval) in order to identify bursts is detailed in Magleby and Pallotta . In brief, the distributions of closed-interval durations were first fitted with, typically, the sum of five exponential components. The closed intervals from the one to two exponential components with the longest time constants were then defined as gaps between bursts, as there was typically a difference of one to three orders of magnitude in the time constants separating the components generating gaps between bursts from those generating closed intervals within bursts. A critical time was then defined to separate closed intervals that were gaps between bursts from those that were gaps within bursts, so that the numbers of misclassified closed intervals would cancel out. The critical time was found to be relatively insensitive to the numbers of exponentials used to fit the dwell-time distribution. Burst analysis was performed on data sets from single channels in which P o was typically less than ∼0.8, since it became increasingly difficult to define gaps between bursts as the P o approached its maximum value of ∼0.96 during activity in the normal mode. Section title: Native BK Channels from Cultured Rat Skeletal Muscle Educational score: 3.6092824935913086 Domain: biomedical Document type: Study Language: en The parameters describing bursting kinetics for native BK channels from cultured rat skeletal muscle were obtained by analyzing data from previous experiments by McManus and Magleby and Rothberg and Magleby , which can be consulted for the experimental details. Section title: The β Subunit Alters the Gating of mslo as Revealed by Single-Channel Kinetics Educational score: 4.143200874328613 Domain: biomedical Document type: Study Language: en The patch-clamp technique was used to record currents from single BK channels in patches of membrane excised from HEK 293 cells after transfection with either the α subunit (referred to as α channels) or with both α and β subunits (referred to as α + β channels). The effects of the β subunit on the gating are illustrated in Fig. 1 , which shows representative single-channel currents recorded with 1.8, 3.6, or 5.4 μM calcium at the inner membrane surface (Ca 2+ i ). The activity of both α and α + β channels increased with increasing Ca 2+ i , and for each Ca 2+ i , the presence of the β subunit further increased the activity. These observations are fully consistent with earlier studies, using mainly currents through multiple channels, that established that the β subunit increases the open probability ( P o ) . Section title: The β Subunit Alters the Gating of mslo as Revealed by Single-Channel Kinetics Educational score: 4.114405632019043 Domain: biomedical Document type: Study Language: en The first clues towards the mechanism by which the β subunit increases P o are readily apparent in Fig. 1 . The β subunit had marked effects on the single-channel gating kinetics: at a fixed Ca 2+ i , the β subunit greatly increased the durations of the bursts of activity while appearing to have little effect on the durations of the gaps (closed intervals) between bursts. This characteristic effect of the β subunit on the bursting kinetics of the single-channel current records was consistently observed in comparisons of data from 19 α channels and 10 α + β channels. 10 of these channels (5 α and 5 α + β) were then analyzed in detail to obtain the results in the rest of the paper. Section title: The β Subunit Increases P o while Having Little Effect on the Hill Coefficient Educational score: 4.16262149810791 Domain: biomedical Document type: Study Language: en As evident in Fig. 1 , α + β channels are open a greater fraction of the time at a given Ca 2+ i than are α channels. To further examine this difference in Ca 2+ sensitivity, we plotted P o vs. Ca 2+ i for α and for α + β channels. Typical results are shown in Fig. 2 A, where the Ca 2+ i for a P o of 0.5 ( K d ) was 9.2 ± 2.3 μM (mean ± SD) for the α channels, shifting to 2.6 ± 0.52 μM for the α + β channels (+30 mV). In a series of similar experiments, the K d was 14.2 ± 7.2 μM (range: 6.9–22.86 μM, n = 5) for a channels and 3.5 ± 1.3 μM (range: 2.2–4.9 μM, n = 5) for α + β channels. Thus, the effect of the β subunit on P o was equivalent to increasing Ca 2+ i approximately fourfold. Section title: The β Subunit Increases P o while Having Little Effect on the Hill Coefficient Educational score: 4.1682658195495605 Domain: biomedical Document type: Study Language: en The Hill coefficients derived from the P o vs. Ca 2+ i plots like those in Fig. 2 A were 4.6 ± 1.8 for α channels and 4.5 ± 1.6 (mean ± SD) for α + β channels (the slopes were not significantly different: P > 0.9), suggesting that four to five Ca 2+ ions (range: 2–6) typically bound to activate the BK channels in our experiments. These values are within the ranges of 2–5 typically observed for both native and cloned BK channels . Section title: The β Subunit Increases Mean Open Time and Decreases Mean Closed Time Educational score: 4.136660099029541 Domain: biomedical Document type: Study Language: en To investigate the basis for the β subunit–induced increase in P o , we measured the observed durations of the open and closed intervals for data obtained from patches containing either a single α or an α + β channel over a range of Ca 2+ i . Since determinations of observed mean open- and closed-interval duration are highly dependent on the time resolution, comparisons between specific α and α + β channels were made only for data obtained at the same level of filtering. Results are shown in Fig. 2 , B and C, for a representative comparison, where the β subunit increased mean open times 3–7-fold and decreased mean closed times ∼10-fold over the examined ranges of Ca 2+ i . Similar results were found for comparisons between four additional α and four additional α + β channels, each channel from a different experiment, paired for the same level of filtering, over a range of filtering (4.5–10 KHz). Section title: The β Subunit Increases Mean Open Time and Decreases Mean Closed Time Educational score: 4.144859313964844 Domain: biomedical Document type: Study Language: en Thus, the β subunit increases P o through a dual effect of increasing observed mean open times and decreasing observed mean closed times. (It will be shown in a later section that the decrease in mean closed times with the β subunit results in large part from a decrease in the frequency, rather than the duration, of the longer closed intervals.) At high levels of Ca 2+ i , and consequently high P o , the mean durations of the closed intervals were brief, and the β subunit had less of an effect on the durations of these already brief closed intervals. We did not explore the effects of nominally zero Ca 2+ i , where the β subunit has been reported to have little effect on P o . Section title: The β Subunit Does Not Change the Number of Detected Kinetic States Entered during Gating Educational score: 4.1413254737854 Domain: biomedical Document type: Study Language: en The gating of BK channels has been described by kinetic schemes in which the channel makes transformations among a number of different kinetic states . To examine whether the β subunit changes the number of kinetic states entered during gating, we fitted sums of exponential components to dwell-time distributions (frequency histograms) of open and closed interval durations for four single α and three single α + β channels. The numbers of significant exponential components required to fit the distributions gives an estimate of the minimum number of states entered during gating . (Examples of dwell-time distributions will be presented in a later section.) Section title: The β Subunit Does Not Change the Number of Detected Kinetic States Entered during Gating Educational score: 4.177182197570801 Domain: biomedical Document type: Study Language: en Fig. 3 plots the number of significant exponential components required to describe the open (A) and closed (B) dwell-time distributions for α channels and α + β channels. The estimates are plotted against the numbers of analyzed intervals, as the ability to detect exponential components is dependent on the numbers of intervals analyzed . Estimates of the minimal numbers of open states (the number of significant exponential components) ranged from two to four for both types of channels, with the lower estimates of two open states associated with the smaller data sets. The mean number of detected open states for α channels (3.0 ± 0.5; mean ± SD) was not significantly different from the mean number of detected open states for α + β channels (3.1 ± 0.6). Estimates of the numbers of detected closed states ranged from three to seven for α channels and from four to seven for α + β channels, with the estimate of three associated with a small data set. The mean number of detected closed states for α channels (5.4 ± 0.9) was not significantly different ( P > 0.37, Mann-Whitney test) from the mean number of detected closed states for α + β channels (5.6 ± 1.0). Section title: The β Subunit Does Not Change the Number of Detected Kinetic States Entered during Gating Educational score: 4.167675971984863 Domain: biomedical Document type: Study Language: en While it cannot be ruled out that changes in the numbers of kinetic states did occur with the β subunit but were not detected due to overlapping time constants and/or small areas of some of the exponential components, the data in Fig. 3 do indicate that the pronounced effect of the β subunit on channel activity did not arise from an obvious change in the numbers of detected kinetic states entered during gating. This observation, that the β subunit did not change the numbers of detected kinetic states, and the observation in a previous section that the β subunit did not change the Hill coefficients, suggests that the β subunit may exert its effects by changing transition rates among states rather than through fundamental changes in the gating mechanism, such as changes in the numbers of states or in the number of Ca 2+ -binding sites. Section title: The β Subunit Greatly Increases Burst Duration Educational score: 4.1135406494140625 Domain: biomedical Document type: Study Language: en As a first step towards determining which transition rates may be affected, we examined the effect of the β subunit on bursting kinetics, since the single-channel records in Fig. 1 suggest that the β subunit greatly increases the durations of the bursts. A critical gap (closed interval between bursts of openings) was used to identify bursts (see methods ). Over the examined range of Ca 2+ i , the β subunit increased mean burst duration 20–100-fold , while having little effect on the mean durations of the gaps (closed intervals) between or within bursts . Section title: The β Subunit Greatly Increases Burst Duration Educational score: 4.328653335571289 Domain: biomedical Document type: Study Language: en This marked increase in mean burst duration by the β subunit was associated with pronounced increases in both the mean open times and in the mean number of openings per burst . Since the addition of each opening to a burst requires an intervening (brief) closed interval, the numbers of closings per burst also increased dramatically. The β subunit– induced lengthening of bursts decreased the fraction of the total closed intervals that were gaps between bursts . For example, with 3.6 μM Ca 2+ i , 21.7% of the observed closed intervals were gaps between bursts for α channels, and this decreased to 1.8% for α + β channels. Since the gaps between bursts are the longer closed intervals, this fractional reduction in the numbers of such intervals by the β subunit contributes greatly towards the increase in P o by the β subunit. Section title: The β Subunit Greatly Increases Burst Duration Educational score: 4.134299278259277 Domain: biomedical Document type: Study Language: en Since estimates of both mean burst duration and the mean duration of gaps between bursts were relatively insensitive to the level of filtering, these parameters were compared directly for five α channels and five α + β channels, each obtained from a patch containing a single channel, in Fig. 4 , E and F. The data from the 10 channels support the representative data shown in Fig. 4 , A and B, for one channel of each type: the β subunit greatly increased mean burst duration while having little effect on the duration of gaps between bursts. While there was considerable variability in estimates of mean burst duration among channels of the same type, all of the individual estimates of mean burst duration for α channels when compared with α + β channels were clearly separated at each examined Ca 2+ i , differing by at least an order of magnitude . Hence, the magnitude of the effect of the β subunit on mean burst duration was greater than the variability among channels of the same type. Section title: The β Subunit Greatly Increases Burst Duration Educational score: 4.1605916023254395 Domain: biomedical Document type: Study Language: en Since the mean open time, the mean number of openings per burst, and the mean duration of the gaps within bursts were all highly sensitive to differences in filtering, we only compared estimates of these parameters for channels that were filtered the same. Results similar to those in Figs. 2 , B and C, and 4, A–D, were found for four such additional detailed comparisons between α and α + β channels, paired for the same level of filtering. In each case, the β subunit increased P o by prolonging the bursts through increases in both the numbers of openings per burst and in the mean open time. Prolonging the bursts also decreased the fraction of shut intervals that were gaps between bursts by preventing the channel from entering the longer closed intervals that separate bursts. Section title: Increasing P o with the β Subunit Was Not Equivalent to Increasing Ca 2+ Educational score: 4.1901350021362305 Domain: biomedical Document type: Study Language: en Similar to the effects of the β subunit on increasing mean open time, increasing Ca 2+ i also increases mean open times for BK channels and the numbers of openings per burst . Thus, a potential mechanism for the action of the β subunit is that it may increase the rates at which the channel binds the activating Ca 2+ ions. If the addition of the β subunit increases all the Ca 2+ -binding rates proportionally, then α and α + β channels should display identical kinetics when the Ca 2+ i is adjusted to give the same P o for both types of channels. To examine this possibility, we compared the dwell-time distributions of α and α + β channels at the same P o . Section title: Increasing P o with the β Subunit Was Not Equivalent to Increasing Ca 2+ Educational score: 4.184001922607422 Domain: biomedical Document type: Study Language: en Results are shown in Fig. 5 , which presents open dwell-time distributions on the left and closed dwell-time distributions on the right for both α and α + β channels, each at two different Ca 2+ i . At 1.8 μM Ca 2+ i , the P o for the α channel was 0.004 , while the P o for the α + β channel was 0.15 . The increase in P o induced by the β subunit was due to both a pronounced shift in the open intervals to longer durations and a marked decrease in the number of longer closed intervals (gaps between bursts), as indicated by a decrease in the amplitude of the component marked gaps. Section title: Increasing P o with the β Subunit Was Not Equivalent to Increasing Ca 2+ Educational score: 4.241535186767578 Domain: biomedical Document type: Study Language: en By increasing Ca 2+ i from 1.8 to 5.4 μM, the P o of the α channel was increased from 0.004 to 0.16 to approximate the P o of 0.15 for the α + β channel at 1.8 μM Ca 2+ i . A comparison of the dwell-time distributions for the α and α + β channels at the same P o showed marked differences in the kinetics: both the mean open times and the mean durations of the gaps between bursts were approximately an order of magnitude less for the α channel than for the α + β channel , while the relative numbers of gaps between bursts were greater for the α channel than for the α + β channel. These marked differences in the kinetics of α and α + β channels at the same P o exclude the possibility that the β subunit acts by the same proportional increases in all the rate constants for Ca 2+ binding. Section title: The β Subunit Has Little Effect on the Durations of the Gaps between Bursts Educational score: 4.115137577056885 Domain: biomedical Document type: Study Language: en One reason why increasing P o with the β subunit was not equivalent to increasing Ca 2+ i in the absence of the β subunit was the differential effects of the β subunit and Ca 2+ i on the gaps between bursts. Fig. 4 , B and D, shows that the β subunit had little effect on the durations of the gaps between bursts, while decreasing their relative numbers. This can also be seen in Fig. 5 , where the addition of the β subunit at a fixed Ca 2+ i had little effect on the mean durations of the gaps between bursts (positions of the peaks labeled gaps) while it decreased the relative numbers of the gaps, as indicated by the decrease in amplitude of the peaks in the presence of the β subunit . Section title: Ca 2+ i Decreases the Durations of the Gaps between Bursts Educational score: 4.159010887145996 Domain: biomedical Document type: Study Language: en In contrast to the little effect of the β subunit on the durations of the gaps between bursts, increasing P o by raising Ca 2+ i decreased the durations of gaps between bursts for both α and α + β channels. This decrease is shown in Fig. 4 B, where increasing Ca 2+ i reduced the durations of gaps between bursts for both types of channels. This effect of Ca 2+ i in reducing the durations of gaps between bursts for both the α and α + β channels can also be seen in the dwell-time distributions in Fig. 5 , where increasing Ca 2+ i from 1.8 to 5.4 μM shifted the peaks labeled gaps to briefer durations . Thus, a major means by which Ca 2+ i increases P o for both α and α + β channels is to drive the channels from the gaps between bursts into the bursting states, decreasing the durations of the gaps between bursts. Section title: The β Subunit Acts Specifically to Stabilize Bursting Activity Educational score: 4.11030912399292 Domain: biomedical Document type: Study Language: en The results in Figs. 1 and 4 A showed that burst duration was markedly greater for α + β channels than for α channels for data obtained at the same Ca 2+ i . The results also showed that increasing P o by increasing Ca 2+ i increased burst duration for both α and α + β channels. Since the β subunit increases P o , the greater burst duration for α + β channels could have been a consequence of the increased P o , rather than a specific effect of the β subunit on lengthening the bursts. Section title: The β Subunit Acts Specifically to Stabilize Bursting Activity Educational score: 4.149831295013428 Domain: biomedical Document type: Study Language: en To distinguish between these two possibilities, the effects of the β subunit on the bursting kinetics were studied at the same P o for α and α + β channels over a wide range of P o , obtained by changing Ca 2+ i . The results are shown in Fig. 6 , where the parameters describing bursting kinetics are plotted against P o . When α and α + β channels were compared at the same P o (the Ca 2+ i was higher for the α channels to obtain the same P o ), mean burst duration was still greatly increased for α + β channels when compared with α channels , due mainly to increases in both mean open times and the mean number of openings per burst . Thus, the β subunit directly facilitates bursting, as its effects on bursts are greater than if the P o were elevated to the same level in the absence of the β subunit by increasing Ca 2+ i . Section title: The β Subunit Acts Specifically to Stabilize Bursting Activity Educational score: 4.127613067626953 Domain: biomedical Document type: Study Language: en In contrast to the β subunit–induced increases in the burst parameters, the mean durations of the gaps (closed intervals) between bursts were about an order of magnitude less for α channels than for α + β channels at the same P o . Since the β subunit has little effect on the durations of the gaps between bursts , this difference reflects the fact that the data from α channels were obtained at a higher Ca 2+ i to obtain the same P o . The higher Ca 2+ i for the α channels reduced the durations of the gaps between bursts. Section title: Consistency of Bursting Kinetics as a Function of P o Educational score: 4.119722366333008 Domain: biomedical Document type: Study Language: en Fig. 7 , A and B, plots the mean burst duration and the mean duration of the gaps between bursts against P o for five α and five α + β channels. P o ranged from ∼0.0003 to ∼0.85. The points cluster around the lines (linear least squares fits to the log of the points), indicating a relative lack of variability when these bursting parameters are plotted against P o . This can be compared with the data in Fig. 4 , E and F, where the variability is greater when the same bursting parameters are plotted against Ca 2+ i . Nevertheless, for both types of plots, the variability among channels of the same type was less than the effect of the β subunit on the indicated bursting parameters. Section title: Consistency of Bursting Kinetics as a Function of P o Educational score: 3.921022653579712 Domain: biomedical Document type: Study Language: en A variability in the Ca 2+ dependence of BK channels has been described previously for native, purified, and cloned channels . Plotting the bursting parameters against P o rather than Ca 2+ i may provide a means to remove much of this variability when studying the detailed single-channel kinetics. Section title: Native BK Channels from Cultured Rat Skeletal Muscle Have Bursting Kinetics Like α Channels Educational score: 4.153964996337891 Domain: biomedical Document type: Study Language: en The significant separation of the bursting parameters between α and α + β channels, together with the relative lack of variability in the parameters for channels of the same type , makes it possible to functionally identify whether native BK channels are composed of α subunits alone, of both α and β subunits, or of a mixture of the two. Bursting parameters for data from six patches from cultured rat skeletal muscle, each containing a single BK channel, are plotted in Fig. 7 , C and D. The dotted lines replot the continuous lines from Fig. 7 , A and B, defining the bursting parameters for the cloned α and α + β channels. Section title: Native BK Channels from Cultured Rat Skeletal Muscle Have Bursting Kinetics Like α Channels Educational score: 4.1794281005859375 Domain: biomedical Document type: Study Language: en The symbols for the native channels are in the immediate vicinity of the line for the bursting parameters of α channels. The simplest explanation of these observations is that native BK channels from cultured rat skeletal muscle are composed of α subunits alone. This conclusion is consistent with the studies of Tseng-Crank et al. and Chang et al. who found low or no β mRNA expression in human, canine, or rat skeletal muscle. We cannot exclude, however, that the native channels may have one or more β subunits per channel, but appear to gate like α channels because of other factors. For example, the alternative splice structure of BK channels can alter gating . Since the structure of the studied native BK channels is not known, they may be of a different splice variant than the cloned channels. Section title: The β Subunit Inhibits Entry into Subconductance States Educational score: 4.121829986572266 Domain: biomedical Document type: Study Language: en Ion channels can enter subconductance levels during gating, reflecting the entry of the channel into conformations that are not fully open or are perhaps partially blocked . Fig. 8 shows a typical example of gating to a subconductance level that was observed in α channels, but was seldom observed in α + β channels. To examine the subconductance gating, 210 min of current records from 24 α channels and 230 min from 15 α + β channels were visually inspected for transitions to subconductance levels with durations longer than 50 ms. There were 382 transitions to such subconductance levels with a mean duration of 0.4 s for the α channels, 9 transitions with a mean duration of 0.3 s, and 1 transition with a duration of 9 s for the α + β channels. The total time spent in subconductance levels for each channel was divided by the total time of the recording for that channel and converted to percentage for the plots in Fig. 8 C. Section title: The β Subunit Inhibits Entry into Subconductance States Educational score: 4.161802768707275 Domain: biomedical Document type: Study Language: en The mean percentage of time that α channels spent gating to subconductance levels (1.1 ± 1.2%, mean ± SD) was decreased 32-fold in the α + β channels (0.034 ± 0.097%). While 20 of 24 α channels spent >0.1% of their time gating to subconductance levels, only 1 of 15 α + β channels did. Thus, the β subunit inhibits entry into partially conducting states that give subconductance levels of the type shown in Fig. 8 . Section title: discussion Educational score: 4.197632312774658 Domain: biomedical Document type: Study Language: en The accessory β subunit of mammalian BK channels greatly increases Ca 2+ sensitivity . The single-channel analysis in our study provides insight into the mechanism for this apparent increase in Ca 2+ sensitivity. We found that the β subunit increased burst duration 20–100-fold by increasing both the number of openings per burst and the mean open times, while having little effect on the mean durations of the gaps (closed intervals) between bursts. Section title: discussion Educational score: 4.349795341491699 Domain: biomedical Document type: Study Language: en The bursting kinetics of channels can be described by the highly simplified Scheme I, where k 1 is the rate constant for entering bursts and k −1 is the rate constant for leaving bursts. For simple models that describe the basic single-channel properties of the gating of BK channels, the gaps between bursts in Scheme SI are generated by potential transitions among three to eight closed states, and the bursts in Scheme SI are generated by potential transitions among three to four open states and three to six brief closed states . Because the data in our study were recorded from patches containing a single BK channel, each gap between bursts represents the sum of the dwell times in the closed states entered between bursts for that single channel. Some of the closed states contributing to the gaps between bursts would be expected to bind Ca 2+ , with the binding driving the channel through one or more closed states towards the first open state that terminates the gap between bursts. It is this Ca 2+ dependence of the closed states entered between bursts that produces long gaps between bursts at low Ca 2+ and brief gaps at high Ca 2+ . Section title: discussion Educational score: 4.262058258056641 Domain: biomedical Document type: Study Language: en Inspection of Scheme SI suggests that the β subunit could promote bursting by facilitating the entry of the channel into bursts (by increasing k 1 ) or by preventing the channel from leaving the bursts once entered (by decreasing k −1 ), or by both actions. If the β subunit acts to facilitate entry into bursts, then the durations of the gaps (closed intervals) between bursts should be decreased by the β subunit. Alternatively, if the β subunit acts by retaining the channel in bursts once entered, then the β subunit should have little effect on the durations of the gaps between bursts, but should decrease their relative numbers since the channel would enter the gaps between bursts less often. Section title: discussion Educational score: 4.053552150726318 Domain: biomedical Document type: Study Language: en Our observations that the β subunit increased burst duration and decreased the numbers of gaps between bursts while having little effect on the durations of the gaps between bursts suggest that the β subunit had little effect on k 1 . Thus, the β subunit increases P o mainly by slowing k −1 to retain the channel in the bursting states. Section title: discussion Educational score: 4.180962562561035 Domain: biomedical Document type: Study Language: en In contrast to the relative lack of effect of the β subunit on the durations of the gaps between bursts, increasing Ca 2+ i decreased the durations of gaps between bursts for both α and α + β channels . Thus, a major means by which Ca 2+ i increased P o for both α and α + β channels was to increase k 1 to drive the channels from the gaps between bursts into the bursting states. Increasing Ca 2+ i also increased the durations of the bursts, but not as much as the increase induced by the β subunit for the same increase in P o . Section title: discussion Educational score: 4.334908485412598 Domain: biomedical Document type: Study Language: en How might the β subunit effectively slow k −1 to retain the channel in the bursting states? One possibility would be for the β subunit to add additional states that are entered during the bursting. Gating in these additional states could then retain the channel in the bursts. Another possibility would be for the β subunit to add additional Ca 2+ binding sites that would act to retain the channel in bursts. Our observations that the β subunit did not change the numbers of exponential components in the dwell-time distributions or the Hill coefficients suggest that the β subunit does not act by changing either the numbers of kinetic states entered during gating or the effective number of Ca 2+ binding sites. (Our observations that the number of subunits per channel could be doubled, from four for α channels to eight for α + β channels, without changing the numbers of detected kinetic states, indicate that the number of states entered during gating is not necessarily related to the total number of subunits comprising the α channel.) Section title: discussion Educational score: 4.139481544494629 Domain: biomedical Document type: Study Language: en The above findings, when coupled with previous observations that the β subunit does not appear to change the effective gating charge , suggest that the β subunit acts not by fundamental changes in the gating mechanism, such as alterations in either the number of Ca 2+ -binding sites or the number of major conformational changes, but rather through modulation of the gating of the α subunits. Section title: discussion Educational score: 4.2393646240234375 Domain: biomedical Document type: Study Language: en One possible way the β subunit might modulate the gating of the α subunits would be through changes in the Ca 2+ binding rates. If the β subunit increased all the Ca 2+ -binding rates to the α subunits proportionally, then increasing Ca 2+ i sufficiently to obtain the same P o for α channels as for α + β channels should give the same single-channel kinetics for both types of channels. This was found not to be the case, as the durations of the bursts, the mean open times, the mean numbers of openings per burst, and the durations of the gaps between bursts were all considerably less for α channels than for α + β channels at the same P o . Section title: discussion Educational score: 4.187192916870117 Domain: biomedical Document type: Study Language: en Since the β subunit does not increase all of the Ca 2+ -binding rates proportionally, could it act by increasing a subset of the Ca 2+ -binding rates? Our observation that the β subunit had little effect on the durations of the gaps between bursts suggests that the β subunit has little effect on the Ca 2+ -binding rates to the closed states that dominate the gaps between bursts. This observation does not exclude the possibility that the β subunit may increase some of the Ca 2+ -binding rates in the bursting states, but such an effect would require a differential effect of the β subunit on the bindings of successive Ca 2+ . Section title: discussion Educational score: 4.252943515777588 Domain: biomedical Document type: Study Language: en If the β subunit does act by retaining the channel in the bursting states, then this is functionally equivalent to imposing a barrier to prevent the channel from leaving the bursting states. If this is the case, then the deactivation from the bursting states that occurs in the presence of Ca 2+ after a step to negative membrane potentials might be expected to be slowed by the β subunit. Consistent with this possibility, the β subunit does slow deactivation after steps to negative membrane potentials . Section title: discussion Educational score: 4.49908971786499 Domain: biomedical Document type: Study Language: en The β subunit of the BK channel bears no sequence homology with accessory subunits from other channels , suggesting that modulatory subunits may have evolved separately as needed to modulate specific channels. It also appears that the β subunit of the BK channel works differently from the modulatory subunits of other channels that increase expression levels and speed activation and inactivation rates . However, the actions of the β subunit for the BK channel seem to have some features in common with the actions of the accessory Ca 2+ channel β 2A subunit on Ca 2+ channels in the presence of a dihydropyridine derivative; both subunits increase burst duration, although in the case of the Ca 2+ channel this increase occurs only when the Ca 2+ channel is in a high P o mode. Interestingly, these increases in burst duration by the different subunits on different channels occur even though the β subunit of BK channels has two putative transmembrane segments , while the β 2A subunit of Ca 2+ channels is cytoplasmic . Section title: discussion Educational score: 4.247118949890137 Domain: biomedical Document type: Study Language: en A comparison of the bursting kinetics of BK channels from cultured rat skeletal muscle to the bursting kinetics of α channels and α + β channels indicated that BK channels in cultured rat skeletal muscle have bursting kinetics similar to α channels . Thus, BK channels in cultured rat skeletal muscle gate as if they are composed of α subunits alone. This conclusion is consistent with the studies of Tseng-Crank et al. and Chang et al. , who found low or no β mRNA expression in human, canine, and rat skeletal muscle. In contrast to skeletal muscle, BK channels in tracheal smooth muscle are composed of α + β subunits, and most BK channels in human coronary artery smooth muscle function as if they are composed of α + β subunits . The β subunit would confer a greater Ca 2+ sensitivity to BK channels in smooth muscle. Section title: discussion Educational score: 4.150602340698242 Domain: biomedical Document type: Study Language: en Our observation that the β subunit of BK channels decreases the percentage of time spent in gating to subconductance levels suggests that the β subunit of BK channels stabilizes the full conductance level of the open states. Similar to our observation for BK channels, the presence of an auxiliary subunit for the ryanodine receptor also decreases the percentage of time spent in gating to subconductance levels . Section title: Conclusion Educational score: 4.420631408691406 Domain: biomedical Document type: Study Language: en From a functional viewpoint, it is the retention of the BK channel in the bursting states by the β subunit that increases the apparent Ca 2+ sensitivity of the channel. In the presence of the β subunit, each burst of openings is greatly amplified in duration through increases in both the numbers of openings per burst and in the mean open times. The physical mechanism by which the β subunit retains the channel in the bursting states is not known, but one possibility is that selective allosteric effects of the β subunits on the α subunits facilitate some conformational changes and/or inhibit others. This selective facilitation and/or inhibition would work to increase the effective energy barrier for leaving the bursting states, through increases in both mean open time and the numbers of openings per bursts. | Study | biomedical | en | 0.999998 |
10051519 | Section title: introduction Educational score: 3.9957354068756104 Domain: biomedical Document type: Other Language: en Maintenance and regulation of cell volume are fundamental physiological processes. In response to swelling, cells activate anion and cation channels that allow the passive loss of inorganic ions and organic solutes. Net solute and osmotically obliged water efflux functions to return cell volume towards its original value, a process termed regulatory volume decrease. Section title: introduction Educational score: 4.294467926025391 Domain: biomedical Document type: Study Language: en Swelling of vertebrate cells activates an outwardly rectifying anion current termed I Cl, swell . The general characteristics of I Cl, swell include an Eisenman type I anion permeability sequence (I − > Br − > Cl − > F − ), modest outward rectification, voltage-dependent inactivation at potentials above E Cl , inhibition by a wide variety of compounds, including conventional anion transport inhibitors, and block by extracellular nucleotides . The degree of rectification, voltage sensitivity, and pharmacology of I Cl, swell can vary somewhat between different cell types. It is not clear whether the differences observed reflect the existence of distinct channels or whether they are due to experimental and/or physiological variables. Section title: introduction Educational score: 4.058167457580566 Domain: biomedical Document type: Study Language: en The mechanism by which swelling activates I Cl, swell is unknown . However, two important physiological parameters have been shown to modulate channel activation. Changes in cytoplasmic ionic strength alter both the rate of channel activation and channel volume sensitivity , and swelling-induced activation requires the presence of intracellular ATP . Section title: introduction Educational score: 4.09185266494751 Domain: biomedical Document type: Review Language: en Intracellular ATP plays a crucial role in regulating many ion channels and transporters . In most cases, the underlying cellular and molecular mechanisms of ATP-dependent regulation are incompletely understood. Hilgemann recently reviewed the mechanisms by which ATP can regulate channel/transporter function. These mechanisms include protein and lipid phosphorylation, ATP binding, ATP hydrolysis, modulation of cytoskeletal function, chelation of polyvalent cations, and modulation of membrane trafficking. Section title: introduction Educational score: 4.248978614807129 Domain: biomedical Document type: Study Language: en The mechanisms of ATP-dependent regulation of the I Cl, swell channel are largely unknown. Most studies of I Cl, swell have shown that nonhydrolyzable ATP analogues readily substitute for intracellular ATP, suggesting that the requirement for ATP involves binding rather than phosphorylation or hydrolysis reactions . However, studies in cultured mouse cortical collecting duct and carotid body type I cells indicate that phosphorylation of the I Cl, swell channel and/or accessory proteins is required for swelling-induced activation. Section title: introduction Educational score: 4.270689487457275 Domain: biomedical Document type: Study Language: en In the present investigation, we demonstrate that I Cl, swell activation in N1E115 neuroblastoma cells does not involve ATP hydrolysis or phosphorylation reactions. Furthermore, we demonstrate that the ATP dependence of the I Cl, swell channel is modulated by the rate of cell volume change. At high rates of swelling, I Cl, swell activates rapidly and activation is insensitive to intracellular ATP concentration. Our results indicate that there are ATP-dependent and -independent mechanisms by which I Cl, swell can be activated. In addition, they demonstrate that cells sense not only the magnitude of volume change, but also the rate at which volume is perturbed. These findings have important physiological implications for control of both cell volume and metabolic homeostasis, and they provide new insights into regulation of the I Cl, swell channel. Section title: Cell Culture Educational score: 4.142851829528809 Domain: biomedical Document type: Study Language: en N1E115 mouse neuroblastoma cells were cultured in high glucose Dulbecco's modified eagle's medium (DMEM; GIBCO BRL ) with 10% fetal bovine serum, 50 U/ml penicillin, 50 μg/ml streptomycin, and 25 mM HEPES in the presence of 5% CO 2 / 95% air. Cells were used between passages 30 and 45. The osmolality of the growth medium was measured by vapor pressure osmometry and was 295–305 mOsm. Section title: Patch Clamp Recordings Educational score: 4.13291072845459 Domain: biomedical Document type: Study Language: en N1E115 cells were grown in 35-mm culture dishes and dissociated by brief treatment with Ca 2+ - and Mg 2+ -free modified Hank's solution. Dissociated cells were allowed to reattach to the poly- l -lysine–coated cover slip bottom of a bath chamber (R-26G; Warner Instrument Corp.) that was mounted onto the stage of an inverted microscope (TE 300; Nikon Inc. ). Patch electrodes were pulled from 1.5-mm outer diameter borosilicate glass microhematocrit tubes ( Fisher Scientific Co. ) that had been salinized with dimethyl-dichloro silane ( Sigma Chemical Co. ). Electrodes were not fire polished before use. Section title: Patch Clamp Recordings Educational score: 3.8682949542999268 Domain: biomedical Document type: Study Language: en The standard bath solution contained (mM): 70 N -methyl- d -glucamine (NMDG) 1 -Cl, 5 MgSO 4 , 1.3 CaCl 2 , 12 HEPES, 8 Tris, 5 glucose, 2 glutamine, 120 sucrose (pH 7.4; 300 mOsm). Section title: Patch Clamp Recordings Educational score: 3.7847847938537598 Domain: biomedical Document type: Study Language: en Bath was reduced to 150–250 mOsm by reducing sucrose concentration. In one set of experiments, bath was reduced to 100 mOsm. For these studies, the NMDG-Cl concentration in the control (i.e., 300 mOsm) bath solution was reduced to 40 mM and sucrose concentration was increased to 200 mM. Section title: Patch Clamp Recordings Educational score: 4.180596351623535 Domain: biomedical Document type: Study Language: en Patch clamping was carried out using a pH 7.2 pipette solution that contained 125 mM CsCl, 20 mM HEPES, 6 mM CsOH, 1 mM EDTA, 40 μM oligomycin, 5 μM iodoacetate, and 20 μM rotenone. Sodium and K + -free bath and pipette solutions were used to minimize the contribution of cation currents to the whole cell conductance. To prevent spontaneous cell swelling, the osmolality of the pipette solution was hypotonic (280 mOsm) with respect to the bath. Metabolic inhibitors were added from concentrated stock solutions dissolved in DMSO. Final DMSO concentration in the pipette solutions was 0.15%. ATP and ATP analogues were added as sodium and lithium salts, respectively. Electrodes had DC resistances of 3–5 mΩ. Cells were used only if the series resistance was no greater than ∼150% of the pipette resistance and the reversal potential (E rev ) was within ±3 mV of the calculated value. Section title: Patch Clamp Recordings Educational score: 4.133987903594971 Domain: biomedical Document type: Study Language: en An Axopatch 200A ( Axon Instruments ) patch clamp amplifier was used to voltage clamp N1E115 cells after gigaseal formation and attainment of whole cell access. Command voltage generation, data digitization, and data analysis were carried out on a 200 mHz Pentium computer (Dimension XPS M200s; Dell Computer Corp.) using a DigiData 1200 AD/DA interface with pClamp 6 software ( Axon Instruments ). Data were digitized at 5 kHz and filtered at 0.5 kHz using an eight pole Bessel filter (902; Frequency Devices). Electrical connections to the amplifier were made using Ag/AgCl pellets and 3 M KCl/agar bridges. Whole cell currents were measured by varying membrane potential from −80 to +80 mV at 80 mV/s every 5–15 s. Section title: Patch Clamp Recordings Educational score: 4.0596208572387695 Domain: biomedical Document type: Study Language: en Cells were swollen by reduction of bath osmolality. At the end of a patch clamp recording, bath osmolality was returned to 300 mOsm and another cell was selected for study. Cells were exposed to hypotonicity approximately every 15–25 min and to at most four to five hypotonic shocks before they were removed and replaced with fresh cells. This number of repetitive hypotonic shocks had no effect on the rate or extent of current activation (data not shown). Section title: Patch Clamp Recordings Educational score: 4.05426025390625 Domain: biomedical Document type: Study Language: en Whole cell current measured before induction of cell swelling is defined as “baseline current.” This current was recorded every 5 s for 60 s before the bath osmolality was reduced. These 13 data points were averaged to yield a mean baseline current. This current was subsequently subtracted from all data points within a given current record to correct for variability in resting current levels between different cells. Section title: Measurement of Relative Cell Volume Changes Educational score: 4.202826976776123 Domain: biomedical Document type: Study Language: en Whole cell currents and volume changes were measured simultaneously in single, patch clamped cells. Cells attached to the cover slip bottom of the patch clamp bath chamber were visualized by video-enhanced differential interference contrast microscopy using a 63× oil-immersion objective lens (1.25 numerical aperture; Carl Zeiss, Inc. ) and a 32× condenser lens (0.4 numerical aperture; Leitz). Optical sectioning demonstrated that the cells maintained a spherical morphology for at least 60 min after attachment to the cover slip. Cells were routinely removed from the bath chamber and replaced with fresh ones every 30–45 min. Given that the cells have a spherical morphology, relative cell volume change can be determined as: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}relative\;cell\;volume=(experimental\;CSA/control\;CSA)^{3/2},\;\end{equation*}\end{document} Section title: Measurement of Relative Cell Volume Changes Educational score: 3.8356130123138428 Domain: biomedical Document type: Study Language: en where CSA is the cell cross-sectional area (CSA) measured at a single focal plane. In all CSA measurements described in this paper, we used focal planes located at the point of maximum cell diameter. Section title: Measurement of Relative Cell Volume Changes Educational score: 4.113949775695801 Domain: biomedical Document type: Study Language: en Cell images were recorded continuously throughout a patch clamp experiment using a super VHS video cassette recorder and a CCD camera . Cross-sectional areas of single cells were quantified by digitizing recorded video images with an image processing computer board with 512 × 480 × 8 bit resolution and a 300 mHz Pentium II computer (Dimension XPS D300; Dell Computer Corp.). Digitized images were displayed on the computer monitor and cell borders were traced using a mouse and a computer-generated cursor. The CSA of a traced region was determined by image analysis software (Optimas; Bioscan, Inc.). This image acquisition and analysis system allows detection of changes in CSA with an accuracy of ±2–3%. Optical sectioning methods have demonstrated that this approach reliably tracks relative cell volume increases up to 250% of control values (our unpublished observations). Section title: Measurement of Relative Cell Volume Changes Educational score: 2.8678767681121826 Domain: biomedical Document type: Study Language: en During swelling, a small percentage of cells exhibited bleb formation. These cells were excluded from the analysis of both volume changes and current activation. Section title: Measurement of Relative Cell Volume Changes Educational score: 4.032529830932617 Domain: biomedical Document type: Study Language: en Cell volume typically increased in a linear fashion when bath osmolality was reduced. The rate of cell swelling was therefore determined by performing linear regression analysis on volume changes measured for 30–60 s after swelling was initiated. Section title: Data Analysis Educational score: 4.048883438110352 Domain: biomedical Document type: Study Language: en The effect of intracellular ATP concentration on I Cl, swell was quantified using two parameters, the rate of current activation initiated by cell swelling and the cell volume set-point of the channel. Cell volume set-point is defined as the volume of the cell at which current activation is initiated. Section title: Data Analysis Educational score: 4.185640811920166 Domain: biomedical Document type: Study Language: en Rate of current activation and cell volume set-point were measured simultaneously in single, patch clamped cells. Fig. 1 shows volume and current measurements in a single cell and illustrates how the set-point and rate of current activation were quantified. Current activation is defined as the point at which there is a significant and continuous increase in current amplitude above the baseline current. Rate of current activation was quantified by performing linear regression analysis on whole cell currents measured for 30–60 s after activation was detected. The threshold for current activation is the intercept between the line defining rate of current activation and the current immediately before activation begins . The relative volume of the cell at the current activation threshold is defined as the cell volume set-point of the channel. Section title: Statistical Analysis Educational score: 3.0187604427337646 Domain: biomedical Document type: Study Language: en Data are presented as means ± SEM. Statistical significance was determined using Student's two-tailed t test for unpaired, independent means. When comparing three or more groups, statistical significance was determined by one-way analysis of variance. P ≤ 0.05 indicated statistical significance. Section title: Enzymes Educational score: 1.2143441438674927 Domain: biomedical Document type: Other Language: en Grade VI apyrase was purchased from Sigma Chemical Co. Alkaline phosphatase and creatine kinase were purchased from Boehringer Mannheim Biochemicals . Section title: The ATP Requirement of I Cl, swell Activation Does Not Involve Hydrolysis and/or Phosphorylation Reactions Educational score: 3.5693061351776123 Domain: biomedical Document type: Study Language: en In most cell types, I Cl, swell activation is supported normally by nonhydrolyzable ATP analogues . However, in carotid body type 1 and mouse cortical collecting duct cells, phosphorylation reactions appear to be needed for channel activation . The reason for these contradictory findings is unclear and we therefore carried out a series of experiments to look more closely at the role of phosphorylation. Section title: The ATP Requirement of I Cl, swell Activation Does Not Involve Hydrolysis and/or Phosphorylation Reactions Educational score: 4.129541873931885 Domain: biomedical Document type: Study Language: en In their studies on cortical collecting duct cells, Meyer and Korbmacher exposed cells to extracellular metabolic inhibitors for 20–45 min before they were patch clamped. Such prolonged metabolic inhibition may result in dephosphorylation of the channel and/or other proteins. If the putative phosphorylation of these proteins is crucial to channel function, then intracellular ATP rather than nonhydrolyzable analogues would be required for swelling-induced activation. We therefore pretreated N1E115 neuroblastoma cells with 5 mM 2-deoxyglucose and 100 nM rotenone for 25–35 min, and then patch clamped them with the standard pipette solution containing (μM) 40 oligomycin, 5 iodoacetate, and 20 rotenone. Both 2-deoxyglucose and rotenone were present in the bath throughout the entire experiment. Cells were dialyzed for 4–5 min with the metabolic inhibitors before swelling was induced. Preliminary studies demonstrated that a dialysis period of 2–3 min was sufficient to maximally inhibit swelling-induced current activation, presumably by maximally reducing intracellular ATP levels. Section title: The ATP Requirement of I Cl, swell Activation Does Not Involve Hydrolysis and/or Phosphorylation Reactions Educational score: 4.197310924530029 Domain: biomedical Document type: Study Language: en In the presence of intracellular metabolic inhibitors and ATP or ATP analogues, an outwardly rectifying (data not shown) current was activated when cells were swollen by reduction of bath osmolality. With 73 and 125 mM Cl − in the bath and pipette solutions, respectively, the mean ± SEM E rev observed was 13.1 ± 0.3 mV ( n = 109). This value is close to the value of 12.8 mV predicted from the Goldman-Hodgkin-Katz equation and the previously measured relative cation conductance ( P cation / P Cl ) of 0.03 . These results indicate that the current activated by swelling is a Cl − current (i.e., I Cl, swell ). Section title: The ATP Requirement of I Cl, swell Activation Does Not Involve Hydrolysis and/or Phosphorylation Reactions Educational score: 4.108909606933594 Domain: biomedical Document type: Study Language: en Results of the metabolic inhibition studies are shown in Fig. 2 A. When exposed to intracellular metabolic inhibitors alone, and a Mg 2+ -free pipette solution containing 2 mM ATP, the mean ± SEM rate of chloride current activation measured at +60 mV was 3.95 ± 0.26 pA/pF per min ( n = 28). This value was not significantly ( P > 0.1) different from those obtained in cells pretreated for 25–35 min with 2-deoxyglucose and rotenone, and then dialyzed with metabolic inhibitors in the presence of 2 mM ATP and 0 mM Mg 2+ or 2 mM ATP and 2 mM Mg 2+ . Section title: The ATP Requirement of I Cl, swell Activation Does Not Involve Hydrolysis and/or Phosphorylation Reactions Educational score: 4.113866806030273 Domain: biomedical Document type: Study Language: en As a further test for the involvement of phosphorylation in regulating I Cl, swell , we patch clamped cells with a pipette solution containing alkaline phosphatase. As shown in Fig. 2 B, there was no significant ( P > 0.8) difference in the rate of swelling-induced current activation in the presence of 2 mM ATP and 0 mM Mg 2+ , 2 mM AMP-PNP, and 0 mM Mg 2+ , or 2 mM AMP-PNP, 0 mM Mg 2+ , and 30 U/ml of alkaline phosphatase. We conclude from the results shown in Fig. 2 that swelling- induced activation of I Cl, swell in N1E115 neuroblastoma cells requires only ATP binding, and not phosphorylation events and/or ATP hydrolysis. Section title: The Rate of I Cl, swell Activation Is a Saturable Function of Intracellular ATP Concentration Educational score: 4.202001571655273 Domain: biomedical Document type: Study Language: en To further characterize the dependence of I Cl, swell activation on ATP binding, we quantified the effect of ATP concentration on the rate of current activation, the rate of cell swelling, and the cell volume set-point of the channel simultaneously in single patch-clamped cells. We define cell volume set-point as the relative cell volume at which current activation is initiated . In initial experiments, cells were swollen at a rate of 35–40%/min via reduction of bath osmolality by 100 mOsm . As shown in Fig. 3 (right), intracellular ATP concentration had no effect on the rate of cell swelling. The rate of current activation, however, was increased by increasing ATP levels in the patch pipette solution . The rate of current activation was a saturable function of intracellular ATP concentration . The maximal rate of current activation ( R max ) was 4.11 pA/pF per min and the EC 50 for ATP was 0.32 mM. The cell volume set-point of the channel was 1.09–1.15 and was unaffected by ATP concentration . Section title: The Rate of I Cl, swell Activation Is a Saturable Function of Intracellular ATP Concentration Educational score: 4.237910270690918 Domain: biomedical Document type: Study Language: en With prolonged exposure to hypotonic medium, N1E115 cells underwent dramatic volume increases . The ability to withstand such large degrees of swelling most likely reflects the presence of extensive membrane microvilli and possibly membrane infolding. Similar large volume increases have been observed in patch-clamped lymphocytes and have been attributed to the presence of numerous microvilli that amplify the total membrane surface area. Section title: The Rate of I Cl, swell Activation Is a Saturable Function of Intracellular ATP Concentration Educational score: 4.23946475982666 Domain: biomedical Document type: Study Language: en It is likely that ATP-consuming reactions are active in patch-clamped cells. These reactions may occur at a rate that approaches the rate of ATP diffusion from the patch pipette solution. Thus, it was conceivable that ATP-consuming reactions close to the channel and/or its activation machinery may have reduced local ATP concentrations, resulting in an overestimation of the concentration of the nucleotide required for any given rate of current activation. To address this important issue, we dialyzed cells with an ATP regeneration system consisting of 10 mM creatine phosphate and 80 U/ml of creatine kinase. The solution also contained 0.25 mM ATP. This concentration was close to the EC 50 , and was chosen so that small increases in ATP concentration potentially brought about by the regeneration system would be detected as relatively large increases in the rate of current activation. The rate of current activation in the presence of the ATP regeneration system was not significantly ( P > 0.15) different from that observed with 0.25 mM ATP alone. We conclude that the ATP concentration sensed by the channel and/or its regulatory machinery is similar to that in the bulk pipette solution. Section title: The Rate of I Cl, swell Activation Is a Saturable Function of Intracellular ATP Concentration Educational score: 4.130408763885498 Domain: biomedical Document type: Study Language: en Surprisingly, swelling-induced activation of an outwardly rectifying anion current was also detected in metabolically poisoned cells dialyzed with an ATP-free pipette solution, albeit at a greatly reduced rate . The mean ± SEM rate of current activation observed was 0.44 ± 0.09 pA/pF per min ( n = 10). This suggested that the cells may contain trace amounts of ATP. To test for this possibility, we dialyzed cells for 5 min with an ATP-free pipette solution containing metabolic inhibitors and the ATP-consuming enzyme apyrase. Apyrase was present at a concentration of 2 U/ ml. Concentrations of apyrase of 0.5–1 U/ml have been shown previously to be effective in depleting both intracellular and extracellular ATP. The presence of apyrase had no significant ( P > 0.25) effect on the rate of current activation . Section title: The Rate of I Cl, swell Activation Is a Saturable Function of Intracellular ATP Concentration Educational score: 4.153097152709961 Domain: biomedical Document type: Study Language: en The observation that a small amount of current is activated in the absence of intracellular ATP is consistent with findings of Volk et al. . These investigators noted that swelling-induced anion current activation required intracellular ATP when inner medullary collecting duct cells were swollen by a 50-mOsm reduction in bath. In contrast, when swelling was induced by 100-mOsm reduction in bath osmolality, an ATP-independent anion current was activated. Section title: The Rate of I Cl, swell Activation Is a Saturable Function of Intracellular ATP Concentration Educational score: 4.192873001098633 Domain: biomedical Document type: Study Language: en Given these results, we examined the effect of increasing the rate of swelling on the ATP dependence of I Cl, swell . N1E115 cells were dialyzed for 4–5 min with an ATP-free pipette solution containing metabolic inhibitors, and then exposed to a 150-mOsm reduction in bath. Under these conditions, the cells swelled at a rate of 65–70%/min . As shown in Figs. 3 and 5 , cell swelling induced a rapid increase in whole cell current. The mean ± SEM E rev for the current was 12.8 ± 1.2 mV ( n = 17), indicating that it is carried by Cl − . The mean ± SEM rate of current activation was 5.0 ± 0.8 pA/pF per min ( n = 17). Addition of 2 U/ml of apyrase to the patch pipette solution had no significant ( P > 0.4) effect on the rate of current activation . Activation of the ATP-independent current was reversed by cell shrinkage. Exposure of swollen cells to a 300-mOsm bath solution induced cell shrinkage and a rapid reduction in whole cell current . Section title: The Rate of I Cl, swell Activation Is a Saturable Function of Intracellular ATP Concentration Educational score: 4.178239345550537 Domain: biomedical Document type: Study Language: en To determine whether the I Cl, swell channel was responsible for this ATP-independent current, we compared its biophysical and pharmacological characteristics to the current activated by a 100-mOsm reduction in bath in metabolically poisoned cells dialyzed with 2 mM ATP. As shown in Table III , both currents had similar reversal potentials, rectification and voltage sensitivity, and were inhibited in a voltage-dependent manner and to the same extent by 100 μM DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonate). In addition, the channels responsible for the currents had similar relative anion permeabilities. We conclude from these results that the I Cl, swell channel gives rise to both currents. Section title: The Rate of I Cl, swell Activation Is a Saturable Function of Intracellular ATP Concentration Educational score: 4.132968902587891 Domain: biomedical Document type: Study Language: en The effect of ATP concentration on I Cl, swell activation in cells swollen by a 150-mOsm hypotonic shock is shown in Fig. 7 . There was no significant difference ( P > 0.2) between rates of current activation measured in the presence of different intracellular concentrations of ATP. Therefore, at high rates of cell swelling, current activation is insensitive to cytoplasmic ATP levels. The cell volume set-point of the channel was also unaffected by ATP concentration and was similar to that observed in cells swollen more slowly with smaller hypotonic shocks (Table II ). 3 Section title: The Rate of I Cl, swell Activation Is a Saturable Function of Intracellular ATP Concentration Educational score: 4.16580867767334 Domain: biomedical Document type: Study Language: en The effect of high rates of swelling on the ATP sensitivity of current activation suggested that it would also be altered by slower rates of cell swelling. Fig. 3 shows the effects of intracellular ATP concentrations and different osmotic perturbations on whole cell current activation and the rate of cell swelling. When cells were swollen at a rate of 13–17%/min by a 50-mOsm hypotonic shock, current activation was again a saturable function of ATP concentration , but both the EC 50 (0.8 mM) and R max (1.37 pA/pF per min) were dramatically altered compared with cells swollen by a 100-mOsm reduction in bath . The cell volume set-point of the channel was unaffected by ATP concentration and was similar to that observed at higher rates of swelling (Table II ). Section title: The Rate of I Cl, swell Activation Is a Saturable Function of Intracellular ATP Concentration Educational score: 4.133785247802734 Domain: biomedical Document type: Study Language: en The effects of varying the rate of cell swelling on ATP dependence are shown in Figs. 3 and 7 and summarized in Fig. 8 . Fig. 8 A shows the rate of current activation in metabolically poisoned cells dialyzed with an ATP-free pipette solution (i.e., ATP-independent current activation). I Cl, swell activation was relatively slow (0.26–0.44 pA/pF per min) when the rate of cell swelling was 15–40%/min (50–100-mOsm reductions in bath). The rates of ATP-independent current activation observed under these conditions were not significantly different ( P > 0.3), indicating that they were relatively insensitive to the rate of cell volume increase. In only 1 (swollen by a 50-mOsm reduction in bath) of the 20 cells used in these experiments, were we unable to detect swelling-induced, ATP-independent current activation. Section title: The Rate of I Cl, swell Activation Is a Saturable Function of Intracellular ATP Concentration Educational score: 4.146740913391113 Domain: biomedical Document type: Study Language: en When cells were swollen at 55%/min (120-mOsm reduction in bath), the rate of ATP-independent current activation increased significantly ( P < 0.05) to 0.85 pA/pF per min . Importantly, current activation under these conditions was still a saturable function of intracellular ATP concentration . Section title: The Rate of I Cl, swell Activation Is a Saturable Function of Intracellular ATP Concentration Educational score: 4.1626482009887695 Domain: biomedical Document type: Study Language: en ATP-independent current activation increased dramatically to ∼5 pA/pF per min when the rate of cell swelling was increased to 65–70%/min (150-mOsm reduction in bath). Increasing the rate of cell swelling to 100%/min (200-mOsm reduction in bath) 4 had no further effect on the rate of ATP-independent current activation . Section title: The Rate of I Cl, swell Activation Is a Saturable Function of Intracellular ATP Concentration Educational score: 4.1078267097473145 Domain: biomedical Document type: Study Language: en The EC 50 for ATP varied inversely with the rate of cell swelling . At low rates of swelling, half maximal rates of current activation required higher concentrations of ATP compared with that observed in cells swollen at higher rates. R max was a direct function of the rate of cell swelling . Section title: Current Rundown Educational score: 4.18320369720459 Domain: biomedical Document type: Study Language: en Transient activation of I Cl, swell has been observed in several cell types when they are patch clamped with ATP-free solutions and activated shortly after obtaining the whole cell configuration . This “rundown” of the current is prevented when the pipette solution contains ATP or ATP analogues. The simplest interpretation of these findings is that current activation is initially supported by the presence of endogenous ATP. As this ATP is dialyzed away, the I Cl, swell channels spontaneously inactivate . Section title: Current Rundown Educational score: 4.128730773925781 Domain: biomedical Document type: Study Language: en To assess whether N1E115 neuroblastoma cells also exhibited such rundown behavior, we patch clamped cells with an ATP-free pipette solution containing metabolic inhibitors. Cells were swollen by exposure to a 50-mOsm reduction in bath within 60 s after obtaining the whole cell configuration. As shown in Fig. 9 A, I Cl, swell activated in response to cell swelling. The activation was transient, however, and the current slowly inactivated despite continued volume increase. Section title: Current Rundown Educational score: 4.174099922180176 Domain: biomedical Document type: Study Language: en Fig. 9 B shows the effect of increasing the rate of swelling on current rundown. The cell shown in the figure was initially swollen at a rate of 12%/min by exposure to a 50-mOsm reduction in bath. Current activation was initiated at a relative cell volume of 1.04 and spontaneous rundown was observed when the relative cell volume was 1.38. Approximately 5 min after rundown began, relative cell volume was 1.8, and the cell was then swollen at a rate of 63%/min by exposure to a 150-mOsm reduction in bath. Rapid current reactivation occurred immediately upon switching the solution. Similar experiments were carried out in five additional cells. In all cases, exposure to a 150-mOsm reduction in bath induced a rapid reactivation of current (data not shown). The amount of additional swelling that occurred before current reactivation was initiated was between 6 and 12% in three cells. In three other cells, current reactivation was initiated immediately after the solution switch and it was not possible to accurately measure the cell volume change at which it occurred. Section title: Role of Phosphorylation in I Cl, swell Activation Educational score: 4.277532577514648 Domain: biomedical Document type: Study Language: en Most studies of I Cl, swell activation that have examined channel ATP dependence have concluded that nonhydrolyzable ATP analogues substitute normally for ATP . However, studies in cultured mouse cortical collecting duct and carotid body type I cells have shown that activation only occurs with ATP or ATPγS , that it requires the presence of intracellular Mg 2+ , which is needed for kinase activity, and that it can be inhibited by the protein kinase inhibitor staurosporine . These results have been interpreted as indicating that phosphorylation of the I Cl, swell channel and/or accessory proteins is required for swelling-induced activation. Section title: Role of Phosphorylation in I Cl, swell Activation Educational score: 4.421638011932373 Domain: biomedical Document type: Study Language: en The reasons for the contradictory findings on the role of intracellular ATP are unclear. One possibility is that methodological differences may unmask constitutive phosphorylation of the channel and/or accessory proteins. Meyer and Korbmacher exposed collecting duct cells to metabolic inhibitors for 20–45 min before they were patch clamped. Such prolonged metabolic inhibition is likely to lead to dephosphorylation of the channel and/or important regulatory proteins. If phosphorylation were crucial to the function of these proteins, rephosphorylation by ATP would have to occur before the channel could be activated by swelling and ATP binding. Our findings do not support this hypothesis, however. Channel activation is normal in cells exposed to prolonged metabolic inhibition after they were dialyzed with Mg-free pipette solutions containing 2 mM ATP . Furthermore, channel activation is unaffected by dialyzing metabolically poisoned cells with Mg-free solutions containing 2 mM AMP-PNP and 30 U/ml of alkaline phosphatase to dephosphorylate proteins. We conclude therefore that in N1E115 neuroblastoma cells, activation of I Cl, swell requires ATP binding rather than hydrolysis and/or phosphorylation reactions. The requirement for phosphorylation observed in other cell types may reflect the existence of distinct channel types. Alternatively, it may reflect the existence of multiple signaling/regulatory pathways involved in swelling-induced channel activation. These pathways could be cell-specific, they may reflect the physiological status of the cell, and/or they may be sensitive to experimental parameters such as solution composition 3 and the mechanism or rate of cell swelling. Section title: Characteristics of I Cl, swell Activation Educational score: 4.192854881286621 Domain: biomedical Document type: Study Language: en There are several aspects of I Cl, swell channel activation that bear on the discussion of the modulatory role of intracellular ATP. First, it is important to note that graded increases in cell volume do not activate I Cl, swell by inducing graded changes in the P o of a single, homogeneous population of channels. Instead, channels exist in either an inactive state where P o = 0, or an active state where P o ∼ 1 . Swelling increases the number, n , of active channels in the cell membrane , and n is therefore a direct function of the magnitude of the volume increase. Section title: Characteristics of I Cl, swell Activation Educational score: 4.330197334289551 Domain: biomedical Document type: Study Language: en I Cl, swell can be activated by forcing fluid into a cell via the patch pipette . This indicates that activation does not require transmembrane water flow or the presence of an osmotic gradient. Spontaneous channel activation does not occur with prolonged dialysis of the cell, suggesting that dilution of a freely diffusible factor is not involved. Activation only requires expansion of the cell volume. Thus, activation must ultimately involve mechanical events. These mechanical events may be detected directly by the channel and/or associated regulatory proteins. Putative regulatory proteins may include components of the cytoskeleton that are altered by cell swelling. Alternatively, cell swelling may alter the activity of signal transduction pathways that ultimately are responsible for activating the channel. Section title: Characteristics of I Cl, swell Activation Educational score: 4.231610298156738 Domain: biomedical Document type: Study Language: en When using whole cell patch clamp to study I Cl, swell , current activation can be characterized in terms of a cell volume set-point of the channel and a rate of current activation. Cell volume set-point is defined as the relative cell volume at which current activation is detected and is a measure of the sensitivity of the channels to swelling. The set-point is regulated by intracellular ionic strength and G-proteins . The fact that n increases as a direct function of the amount of cell swelling suggests that individual channels or groups of channels have different cell volume set-points. Alternatively, macroscopic cell swelling may have different effects on the mechanical properties of microdomains within the plasma membrane or in submembrane structures. Section title: Modulation of I Cl, swell Activation by Intracellular ATP Educational score: 4.323298454284668 Domain: biomedical Document type: Study Language: en It has been concluded previously that ATP is an essential cofactor for swelling-induced activation of I Cl, swell . However, as shown in Figs. 3 – 9 , Table III , and by Volk et al. , current activation can be observed under ATP-free conditions. With rates of swelling between 15 and 55%/min, increases in intracellular ATP increase the rate of I Cl, swell activation without changing the cell volume set-point of the channel . The rate of I Cl, swell activation is a saturable function of ATP concentration, which can be defined as an EC 50 for ATP and a maximal rate of current activation ( R max ) at saturating ATP levels . Two conclusions can be drawn from these observations. First, ATP interacts with a saturable binding site(s) that selectively recognizes the nucleotide and certain analogues. Second, ATP binding induces a conformational change(s) in the channel and/or regulatory proteins that is required for channel activation. The rate at which this conformational change, and hence current activation, occurs is a saturable function of ATP concentration. Section title: Modulation of I Cl, swell Activation by Intracellular ATP Educational score: 3.7604382038116455 Domain: biomedical Document type: Study Language: en Rundown of I Cl, swell occurs when endogenous ATP is dialyzed out of the cytoplasm . This indicates that ATP is required to maintain the channel and/or regulatory proteins in the activated conformation. Thus, ATP must be bound to the activated channel and/or regulatory proteins. Section title: Modulation of I Cl, swell Activation by Intracellular ATP Educational score: 4.069479465484619 Domain: biomedical Document type: Study Language: en One of the more novel findings of our studies is that the rate of cell swelling alters channel ATP sensitivity . As the rate of cell swelling is increased, the EC 50 for ATP falls . At high rates of volume increase, I Cl, swell activation is insensitive to intracellular ATP concentration . Section title: Modulation of I Cl, swell Activation by Intracellular ATP Educational score: 4.014826774597168 Domain: biomedical Document type: Study Language: en How does the rate of cell swelling modulate the ATP dependence of I Cl, swell activation? There are two obvious models to consider. First, the rate of cell swelling could somehow change the affinity of the ATP binding site(s). Alternatively, channel activation may occur via distinct ATP-dependent and -independent pathways. Section title: Modulation of I Cl, swell Activation by Intracellular ATP Educational score: 4.182236671447754 Domain: biomedical Document type: Study Language: en Changes in ATP affinity cannot explain all of the experimental observations adequately. As shown in Figs. 4 , 5 , 7 , and 8 , ATP-independent current activation occurs, albeit slowly, at rates of cell swelling (15–55%/ min) where there is a strong dependence of current activation on intracellular ATP concentration. Furthermore, if ATP affinity were a function of the rate of volume expansion, the K d of the binding site will fall as the rate of swelling is increased. However, this effect would have to be reversed at high rates of swelling where current activation is independent of intracellular ATP concentration . Indeed, it would be necessary to postulate that the K d for ATP rises to infinity (i.e., the site is no longer capable of binding ATP) when cells are swollen at ≥65%/min. Section title: Modulation of I Cl, swell Activation by Intracellular ATP Educational score: 4.260629653930664 Domain: biomedical Document type: Study Language: en The alternative and, in our opinion, simpler explanation for the observed changes in ATP sensitivity is that the I Cl, swell channel can be activated via distinct ATP- dependent and -independent mechanisms. Swelling- induced changes in the EC 50 for ATP can be explained if increases in the rate of cell swelling increase the proportion of channels activating in the ATP-independent mode. As the rate of swelling is increased, less ATP would be needed (i.e., the EC 50 for ATP would fall) to drive a given rate of current activation. Section title: Modulation of I Cl, swell Activation by Intracellular ATP Educational score: 4.004492282867432 Domain: biomedical Document type: Study Language: en The results of our studies have important implications for understanding the regulation of I Cl, swell . It is entirely possible that channel activation is modulated by multiple factors and signal transduction pathways. The physiological status of the cell, as well as experimental variables such as the rate and mechanism of swelling, and the magnitude of the volume increase may determine which factors and signaling pathways control channel activation. This possibility needs to be taken into account in the design and interpretation of future studies of I Cl, swell regulation. Section title: Modulation of I Cl, swell Activation by Intracellular ATP Educational score: 4.040921211242676 Domain: biomedical Document type: Study Language: en Clearly, extensive additional biophysical and cellular studies are required to test the models described above. A complete understanding of the ATP dependence of the channel ultimately requires molecular identification of the pore-forming and associated regulatory proteins. Section title: How Is the Rate of Cell Swelling Sensed? Educational score: 4.239816665649414 Domain: biomedical Document type: Study Language: en An important finding of these studies is the demonstration that the rate of cell swelling alters the ATP requirement of the I Cl, swell channel . How does a cell sense the rate at which its volume is changing? One possibility is that rate per se is not sensed, but instead there is some critical volume that is reached more rapidly at higher rates of swelling. This critical volume could in turn determine whether the channel activates via the ATP-independent pathway. The data in Table II argue strongly against this possibility. When cells were swollen at 65–70%/min (150-mOsm reduction in bath) in the absence of intracellular ATP, the cell volume set-point of the channel was 1.14. This value is not significantly different from those observed under all other experimental conditions (Table II ). Furthermore, in cells that are exhibiting current rundown, current can be reactivated rapidly at various times after rundown begins simply by increasing the rate of swelling to 65–70%/min . Taken together, these results indicate that the cells are sensing the rate of volume increase and not some absolute volume change. Section title: How Is the Rate of Cell Swelling Sensed? Educational score: 3.9953572750091553 Domain: biomedical Document type: Study Language: en The molecular mechanisms responsible for sensing the rate of cell swelling are completely unknown. However, it is interesting to speculate that the cytoskeleton may play an important role. Numerous studies have demonstrated that the cytoskeleton undergoes structural reorganization in response to mechanical forces. These changes can occur on a time scale of seconds to minutes and appear to play important roles in fundamental processes such as maintenance of cell shape, cell locomotion, mechanosensitivity, and signal transduction . Section title: How Is the Rate of Cell Swelling Sensed? Educational score: 4.297729015350342 Domain: biomedical Document type: Study Language: en It is reasonable to postulate that the mechanical stress associated with cell swelling may also induce changes in cytoskeletal organization. These putative changes could be important for maintaining plasma membrane–cytoskeleton interactions, the spatial organization of membrane and submembrane proteins, and the overall mechanical properties of the cell. If the rate of volume perturbation was more rapid than the rate at which cytoskeletal reorganization could take place, it is easy to envision that protein–protein and protein–membrane interactions might be altered. Changes in protein–protein interactions could in turn modulate the ATP dependence, as well as other aspects of I Cl, swell activation. Assessment of the role of the cytoskeleton in regulation of I Cl, swell will require extensive additional investigation using electrophysiological, pharmacological, molecular, and biophysical approaches. Section title: Physiological Significance of ATP-dependent Channel Regulation Educational score: 4.396942138671875 Domain: biomedical Document type: Study Language: en Volume regulatory efflux of structurally diverse organic osmolytes appears to occur primarily via the I Cl, swell channel . In addition, the channel is highly permeable to important metabolic intermediates such as pyruvate, the short-chain fatty acids acetate, and butyrate, the ketone body β-hydroxybutyrate, and several amino acids . These metabolites are major inputs into the tricarboxylic acid cycle. Because of its high organic solute permeability, we have suggested previously that modulation of I Cl, swell channel activation by ATP levels may function as an important feedback regulatory mechanism . Unregulated loss of cellular metabolites would likely disrupt cellular energy production. Regulation of I Cl, swell by cellular ATP levels could therefore prevent depletion of energy-producing carbon sources when cellular energy production is reduced. Section title: Physiological Significance of ATP-dependent Channel Regulation Educational score: 3.8390047550201416 Domain: biomedical Document type: Study Language: en I Cl, swell activation has been invoked as an important component of cellular volume regulation when cells are swollen by anoxic and ischemic insults . We have argued previously that the requirement for intracellular ATP would rule out or diminish such a role . However, as we have demonstrated here, it is clearly possible to activate the channel in the absence of ATP. Section title: Physiological Significance of ATP-dependent Channel Regulation Educational score: 4.355465412139893 Domain: biomedical Document type: Study Language: en In patch-clamped cells, channel activation via the ATP-independent pathway is controlled by the rate of volume increase. Other factors may control activation by this pathway as well. Whether the ATP dependence of I Cl, swell is modulated in intact cells is uncertain and deserves further study. If such modulation occurs, it is interesting to speculate on its physiological significance. As noted above, activation of I Cl, swell in a metabolically compromised cell may further compromise metabolism and lead to additional disruption of cellular functions. Thus, if swelling is not too severe, it may be beneficial to the cell to protect its metabolism at the expense of volume homeostasis. On the other hand, if swelling is extreme and membrane lysis is imminent, volume control may take precedence over energy metabolism. We suggest that the modulation of channel ATP dependence may be an adaptation that allows cells to cope with the potentially competing demands of preservation of energy metabolism and volume regulation. | Other | biomedical | en | 0.999994 |
10051520 | Section title: introduction Educational score: 4.249289035797119 Domain: biomedical Document type: Study Language: en ClC proteins represent a class of voltage-dependent Cl − channels with several members involved in hereditary human diseases. The Cl − channel prototype ClC-0 from Torpedo is a dimeric protein behaving as if comprising two protopores that can gate independently from each other and a common gate that acts on both protopores . Such behavior, suggestive of a “double-barreled” structure, has not been demonstrated for other ClC proteins at the single channel level. Indeed, a double-barreled structure of ClC-1 has recently been challenged . Section title: introduction Educational score: 4.452826023101807 Domain: biomedical Document type: Study Language: en Using single channel recording, we show that also the muscle Cl − channel, ClC-1, has two equidistant open conductance levels of ≈ 1.2 and 2.4 pS whose open probability and kinetics are consistent with the presence of two independently gated conductance states modulated in parallel by a common gate, although the relatively fast kinetics of the common gate render their separation less obvious than for ClC-0. We verified that the most simple scheme implementing a double-gate two-protopores model fits well the single-channel data and predicts, for the same single-channel parameters, current fluctuations consistent with macroscopic measurements. Several mutations of ClC-1 causing dominant myotonia lead to a positive shift of the voltage dependence of the conductance that is only partially reversed in mutant/wild-type (WT) 1 heterodimers . We have used macroscopic fluctuation analysis to characterize changes in the double-gate behavior of two mutations of ClC-1 causing dominant or recessive myotonia. Mutation I290M, causing a positive shift of the voltage dependence of the conductance that is only partially reversed in mutant/WT heterodimers , shows a strong reduction of the open probability of the common gate. In contrast, we found that for mutation I556N, which causes a recessive or benign form of dominant myotonia and does not impose its “shift” on WT/ mutant heterodimers , the open probability of the common gate is reduced less dramatically. Section title: introduction Educational score: 4.12291145324707 Domain: biomedical Document type: Study Language: en Our results suggest that the mammalian ClC homologues have the same double-barreled structure as the Torpedo channel ClC-0. In addition, our findings for recessive and dominant mutations raise the possibility that, for other mutations also, the pattern of inheritance may derive from differential effects on the double-gate mechanism of ClC-1 activation. Section title: Electrophysiology Educational score: 4.123246192932129 Domain: biomedical Document type: Study Language: en Channels were expressed in Xenopus oocytes and currents were measured at 18°C 2–5 d after injection using the inside-out configuration of the patch clamp technique . Bath solution contained (mM): 120 N -methyl- d -glucamine (NMDG)-Cl, 2 MgCl 2 , 5 HEPES, 2 EGTA, pH 7.3 or 6.5. Extracellular (pipette) solution contained (mM): 100 NMDG-Cl, 5 MgCl 2 , 5 HEPES, pH 7.3. Data were low-pass filtered at one third of the sample frequency. Mutant I290M is described in Pusch et al. , and mutant I556N in Kubisch et al. . RNA synthesis and oocyte injection were performed as described . Section title: Data Analysis Educational score: 4.120640277862549 Domain: biomedical Document type: Study Language: en Single channels were analyzed as described . Amplitude histograms were fitted with the sum of three gaussian distributions with means i 0 , i 0 + i 1 and i 0 + 2 i 1 and with variances σ 0 2 , σ 0 2 + σ 2 , σ 0 2 + 2σ 2 , where i 0 and σ 0 2 are the mean and variance of the leakage current in the patch; i 1 and σ 2 are the mean and variance of the current flowing through a single pore. Section title: Data Analysis Educational score: 4.235263824462891 Domain: biomedical Document type: Study Language: en Dwell-times of the three conductance levels were fitted on the basis of Scheme B with the four rate constants (α, β, λ, μ) as free parameters using maximum likelihood techniques : the two open conductance levels are associated with a single kinetic state; thus, the dwell-time distributions are given by \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}f_{i}(t)=\frac{1}{{\tau}_{i}}exp(-t/{\tau}_{i})\end{equation*}\end{document} Section title: Data Analysis Educational score: 2.0419323444366455 Domain: biomedical Document type: Other Language: el i = 1, 2, with τ 1 = 1/(α + β + μ); τ 2 = 1/(2β + μ). Section title: Data Analysis Educational score: 2.785583019256592 Domain: biomedical Document type: Study Language: en The closed level is associated with four kinetic states, and therefore the distribution is given by \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}f_{0}(t)={ \,\substack{ ^{4} \\ {\sum} \\ _{j=1} }\, }a_{j}{\lambda}_{j}exp\hspace{.167em}(-{\lambda}_{j}t);{ \,\substack{ ^{4} \\ {\sum} \\ _{j=1} }\, }a_{j}=1,\end{equation*}\end{document} Section title: Data Analysis Educational score: 4.071135520935059 Domain: biomedical Document type: Study Language: en where the eigenvalues λ i and the coefficients a i are functions of all rate constants. The overall log-likelihood for the observed dwell-times is given by \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}\hspace{.5em}{\mathrm{ln}}[L({\theta})]={ \,\substack{ ^{2} \\ {\sum} \\ _{i=0} }\, }{ \,\substack{ ^{n_{i}} \\ {\sum} \\ _{j=1} }\, }\frac{f_{i}( \left t_{ij} \right{\mid} {\theta})}{prob(t_{min}<=t_{ij}{\theta})},\end{equation*}\end{document} Section title: Data Analysis Educational score: 4.124255180358887 Domain: biomedical Document type: Study Language: en where θ denotes the rate constants, n i is the number of events in level i , t ij is the j th dwell time in level i , and the denominator is the probability that the observed events will fall in the experimental time range. t min was set to 5 ms. The log-likelihood was calculated numerically and maximized by varying the rate constants using the simplex algorithm. Section title: Data Analysis Educational score: 4.164961814880371 Domain: biomedical Document type: Study Language: en The noise analysis was performed as follows. From repeated voltage stimulations, from an activating positive voltage to a negative “test” voltage, the mean current, < I ( t )>, and the variance, <σ 2 ( t )>, were calculated . The capacity transient was measured from the response to a step to 0 mV; i.e., close to the reversal potential, and subtracted off line. Leakage currents were estimated exploiting the strong rectification of ClC-1 [| I (−100 mV)|/ I (+100 mV)] ≈ 8.5, measured from patches with large expression where the contribution of leakage currents was negligible; when a weaker rectification was measured in a given patch, the leakage current was estimated assuming a linear leakage conductance with a reversal potential of 0 mV. Section title: Data Analysis Educational score: 4.033226013183594 Domain: biomedical Document type: Study Language: en Assuming their independence, the “fast” and “slow” gates of Fig. 3 were modeled as having time-dependent open probabilities given by: 1 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}P_{f}(t)=P_{f}[1+a_{f}exp(-t/{\tau}_{f})];\;P_{s}(t)=P_{s}[1+a_{s}exp(-t/{\tau}_{s})],\end{equation*}\end{document} Section title: Data Analysis Educational score: 4.201749324798584 Domain: biomedical Document type: Study Language: en with initial values P f (0) = P f (1 + a f ) and P s (0)= P s (1 + a s ), steady state values P f and P s , and time constants τ f and τ s . Accordingly, the mean current was fitted by 2 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}<I(t){\backslash}>=2niP_{f}(t)P_{s}(t)=I_{{\infty}}[1+a_{f}exp(-t/{\tau}_{f})][1+a_{s}exp(-t/{\tau}_{s})],\end{equation*}\end{document} Section title: Data Analysis Educational score: 4.001684665679932 Domain: biomedical Document type: Study Language: en where n is the number of channels, i is the single channel current, and I ∞ = 2 niP f P s . The expected time course of the variance according to the scheme of Fig. 3 is given by : 3 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}<{\sigma}^{2}(t){\backslash}>=i<I(t){\backslash}>\{1+P_{f}[1+a_{f}exp(-t/{\tau}_{f})]-<I(t){\backslash}>/(ni)\}.\end{equation*}\end{document} Section title: Data Analysis Educational score: 4.09681510925293 Domain: biomedical Document type: Study Language: en Eq. 3 was fitted to the measured variance using i , n , and P f as free parameters. The fit was constrained by imposing that all the resulting estimates of the open probabilities, \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}P_{f};\;P_{f}(0)=P_{f}(1+a_{f});\;P_{s}=I_{{\infty}}/(2niP_{f});\;P_{s}(0)=P_{s}(1+a_{s}),\end{equation*}\end{document} Section title: Data Analysis Educational score: 2.5490126609802246 Domain: biomedical Document type: Study Language: en had to be in the range [0...1]. In most cases, this constraint led to a best fit for P s (0) = 1. The best fit for P f (0) was always independent of constraints and was >0.4. P f (0) was generally smaller for the two mutations. Section title: Single Channel Recordings Educational score: 4.208148956298828 Domain: biomedical Document type: Study Language: en Single channel recordings of the Cl − channel ClC-0 from Torpedo are characterized by an apparent “double-pore” behavior in which relatively long closures separate bursts of activity during which the channel opens stochastically to two nonzero, equidistant conductance levels . Single-channel studies of ClC-1 are more difficult due to ClC-1's low conductance and fast gating . Low intracellular pH (pH i ) leads to a slowing of gating kinetics and to an increase of the residual open probability at negative voltages . This effect of intracellular pH is illustrated in Fig. 1 . We have exploited both effects to resolve single channel events of ClC-1 at low pH i . Section title: Single Channel Recordings Educational score: 4.289295196533203 Domain: biomedical Document type: Study Language: en To this end, ClC-1 was expressed in Xenopus oocytes and we recorded currents from membrane micropatches using the inside-out configuration of the patch-clamp technique and pH 6.5 in the bath (internal) solution. To search for single-channel events, we first used relatively large-tipped patch- pipettes (resistance of 1–3 MΩ) to identify a region of the oocyte with significant but not too high expression. We then patched the same region of the oocyte membrane using pipettes with smaller tip openings (resistance of 4–8 MΩ). In a few patches ( n = 9), we succeeded in obtaining recording conditions (high seal resistance, low background noise, and relatively long stability) allowing fairly clear resolution of single-channel events . These records showed invariably two approximately equidistant open conductance levels easily distinguishable above the background noise at voltages between −100 and −140 mV. From Fig. 2 A, another important qualitative feature can be observed; i.e., fairly common closing periods separate bursts of openings that almost invariably contain both conductance levels. This behavior is similar to that of ClC-0 channels and has been modeled as arising from the gating transitions of a double-pore channel with an independent parallel modulation by a common gate. Section title: Single Channel Recordings Educational score: 4.299438953399658 Domain: biomedical Document type: Study Language: en Amplitude histograms of long recordings confirm these qualitative observations. The histograms are very well fitted by the sum of three gaussian distributions with equidistant peaks ( i 0 ≡ 0, i 1 , 2 i 1 ) and additive variances (σ 0 2 , σ 0 2 + σ 2 , σ 0 2 + 2σ 2 ) as explained in methods . Accordingly, the relative areas covered by the three gaussian components yield the probabilities P 0 , P 1 , and P 2 of the closed and of the two open levels, respectively . A simple analysis shows that the values of the probabilities are grossly departing from the expectations from a simple binomial superposition of two independently gating channels each with open probability P . Therefore, it can be excluded that the two open conductance levels are due to the presence of two identical and independent channels, and the invariable presence of both levels in all patches forces the conclusion that our recordings of the type shown in Fig. 2 A represent a single channel that has two open states, one with twice the conductance of the other. Based on the stationary probabilities obtained from the amplitude histograms, a model with two independently gated and equally sized conductances with possibly different open probabilities P A and P B , respectively , can be excluded. Best fits of such a model to experimental amplitude histograms invariably yielded P A = P B , and thus the same bad prediction as the binomial superposition of two identical channels . Section title: Single Channel Recordings Educational score: 4.070598602294922 Domain: biomedical Document type: Study Language: en One long registration at −140 mV from a patch containing a single channel allowed us to perform a fair statistical analysis of dwell times . This showed that histograms of single- and double-opening times were well fitted by single exponential distributions, whereas a single exponential was inadequate to fit the closed time histogram . Section title: Single Channel Recordings Educational score: 4.437413215637207 Domain: biomedical Document type: Study Language: en A model that can account for our observations must include a common gate that modulates the access to a conduction pathway that can have three different conductances (0, γ, 2γ). The simplest kinetic scheme (with the smallest number of parameters) is shown in Fig. 3 B. The three conductance levels of the “open” common gate are modeled by a binomial superposition of two equal and independent processes. The kinetic scheme can be mechanistically interpreted according to either one of the models shown in Fig. 3 (C or D). Fig. 3 C assumes the classical double-barreled structure where Cl − has equal access to two parallel, equal and, independent two-state protopores . In Fig. 3 D, Cl − is allowed by the common gate to enter a single permeation pathway that, however, can assume three different conformations. As discussed later, there are several arguments in favor of either interpretation, but it is important to stress that both lead to the very same kinetic scheme. For analogy with ClC-0, we adopt in the following the interpretation and terminology of the double-pore model for describing the data in terms of the kinetic scheme of Fig. 3 B. Section title: Single Channel Recordings Educational score: 4.285689353942871 Domain: biomedical Document type: Study Language: en The simple double-barreled model is characterized by the single-protopore open probability P f , a probability P s for the opening of the common slow gate, and the independence of the two gating processes. It can reproduce exactly the stationary probabilities of the three conductance levels obtained from the amplitude histograms using the relationships: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}P_{0}=1-P_{s}+P_{s}*(1-P_{f})^{2};\;P_{1}=2*P_{s}*P_{f}*(1-P_{f});\;P_{2}=P_{s}*P^{2}_{f};\;P_{0}+P_{1}+P_{2}=1\end{equation*}\end{document} Section title: Single Channel Recordings Educational score: 4.1881208419799805 Domain: biomedical Document type: Study Language: en . The exact fit of the data does not demonstrate the model because we have two parameters and two independent data points. However, the consistency of the values of P f , P s , and γ with other measurements can be used as a more stringent test of its validity. Firstly, the single channel currents give a conductance γ of 1.2–1.3 pS for each protopore , which is in good agreement with previous and present noise analysis (see below) of macroscopic currents clearly attributable to ClC-1 channels. This also supports the assumption that the events indeed represent openings of ClC-1 and not endogenous channels. Furthermore, both P s and P f are only a little voltage dependent in the small voltage range investigated and have values well above 0.5, that are consistent with the macroscopic activation curve of ClC-1 at pH i 6.5 . Section title: Single Channel Recordings Educational score: 4.349194049835205 Domain: biomedical Document type: Study Language: en The double-barreled model is also consistent with our measurements of dwell-time distributions, since it predicts in particular single exponential dwell-time distributions for both open levels and a multiexponential distribution for the closed times (the theory predicts four exponentials, but major contributions are only from two components). To obtain best estimates for the four rate constants in Fig. 3 B, the dwell-time histograms were fitted simultaneously according to the double-barreled model using maximum likelihood techniques (see methods ). The histograms are well fitted with the rate constants given in the legend to Fig. 4 . From the rate constants, the stationary probabilities can be calculated as P f = α/(α + β) = 0.59 and P s = λ/(λ + μ) = 0.68. These values are close to those obtained independently from the amplitude histogram of the same patch ( P f = 0.48, P s = 0.74), supporting further the validity of the double-barreled model. We notice that, while the relaxation time constant of the fast gate, τ f [defined as 1/(α + β)], is similar to that of ClC-0 at −100 mV, that of the slow gate, τ s [defined as 1/(λ + μ)], is less than three times larger and more than two orders of magnitude smaller than for ClC-0 making the dissection of the two gating processes much more difficult. These two time constants of gating probably correspond to the two macroscopic time constants of current deactivation described by Fahlke et al. and Rychkov et al. that also differ by a factor of 3–5. Section title: Single Channel Recordings Educational score: 4.34506368637085 Domain: biomedical Document type: Study Language: en In contrast to their qualitative similarities in showing two equally spaced conductance levels, a major difference in the gating properties of ClC-1 and ClC-0 is the different voltage dependence of the slow gating mechanism. In ClC-0, the slow gate closes at positive voltages leading to a small steady state macroscopic conductance in the positive voltage range. In contrast, in ClC-1, the overall steady state conductance increases monotonically with increasing voltage , implying that the slow gate remains open at positive voltages. From the noise analysis described below, it appears that the open probability of the slow gate approaches a value of 1 at positive voltages, indicating that the slow gate has a reversed voltage dependence in ClC-1 compared with ClC-0. However, several point mutations of ClC-0 also lead to a loss of voltage sensitivity or even a reversed voltage dependence of the slow gate (our unpublished results). Section title: Macroscopic Current Fluctuations Educational score: 4.126330375671387 Domain: biomedical Document type: Study Language: en To extend our studies to physiological pH conditions in which the faster kinetics prevent the recordings of single channel events, we developed a method to estimate P f and P s from macroscopic current fluctuations. The method is illustrated in Fig. 5 for measurements at pH i 6.5, and the analysis procedure is described in detail in the methods . From repeated stimulations , the time course of the mean current and of its variance were calculated and fitted with Eqs. 2 and 3 . The best fitting values of P f , P s , and i thus obtained were consistent with those measured from the single-channel amplitude histograms . As already mentioned, this good accordance of the single channel analysis and the fluctuation analysis supports the assumption that the single-channel events described above are indeed due to ClC-1 channels and not to endogenous oocyte channels. Vice versa, it justifies the use of macroscopic fluctuation analysis for estimating the single channel parameters of our model. Section title: Myotonic Mutations Educational score: 4.399335861206055 Domain: biomedical Document type: Study Language: en Mutations of ClC-1 can cause either dominant or recessive myotonia . Recessive myotonia is frequently associated with mutations leading to a partial or total loss of function . This is consistent with the in vitro result that a reduction of the macroscopic muscle Cl − conductance by 50% is not sufficient to cause myotonia . Several dominant mutations (as for example mutation I290M) lead to a large shift of the conductance–voltage curve to positive voltages of homomeric channels that is only partially reduced in wild-type (WT)/mutant heteromers, leaving a strong reduction of the chloride conductance at the relevant physiological voltages . Section title: Myotonic Mutations Educational score: 4.305668830871582 Domain: biomedical Document type: Study Language: en Recently, mutations that cause either recessive myotonia or a benign form of dominant myotonia with incomplete penetrance have been identified that show a positive shift of the macroscopic conductance–voltage relationship in homodimeric mutant channels, but form mutant/WT heterodimers that behave almost like homomeric WT channels . An example of these mutations that do not impose the shift on WT/mutant heteromers is the mutation I556N . The different behavior of mutant/WT heteromeric channels indicates that the mechanism underlying the shift of the conductance–voltage curve of mutants I290M and I556N is different. Section title: Myotonic Mutations Educational score: 4.245243549346924 Domain: biomedical Document type: Study Language: en To investigate whether these two mutations produce different effects on the two gates postulated by Fig. 3 B, we used macroscopic fluctuation analysis at the physiological pH i 7.3 to estimate and compare the values of γ and of the stationary probabilities for WT and mutants I290M and I556N . The WT value of γ at pH i 7.3 was not significantly different from that at pH i 6.5, and γ was also not significantly affected by the mutations. The WT estimates of P f and P s at −100 mV were smaller at pH i 7.3 than at pH i 6.5, consistent with the effect of pH i on the macroscopic conductance . The relative macroscopic open probability compares well with the calculated product P f * P s from the noise analysis at both pH i values (not shown), indicating that P f as well as P s approach 1 at large positive voltages. Also, the initial values [ P f (0), P s (0)] obtained from the noise analysis were close to 1 in most cases (see methods ). For the same pH i 7.3 conditions, the estimated stationary open probabilities were both significantly reduced by the mutations . In particular, mutant I290M shows a significantly larger reduction of P s than mutant I556N, whereas both mutations cause a similar reduction of P f . Section title: discussion Educational score: 4.353165626525879 Domain: biomedical Document type: Study Language: en We presented in this work some new information that is relevant for the modeling of the gating mechanism of the muscle Cl − channel ClC-1. Our single-channel recordings show that the gating of this channel has strong similarities with that of the “classical” ClC prototype ClC-0, although the slow common gate of ClC-1 has faster kinetics and a different voltage dependence. Therefore, we have assumed that many of the arguments suggesting that ClC-0 channels have a double-barreled structure apply also to ClC-1, and we have used a simple kinetic scheme involving a common gate on top of two parallel and independently gated pores to fit our single-channel data. We have also shown that the analysis of macroscopic current fluctuations on the basis of that scheme gives consistent estimates of the single-channel parameters. Macroscopic fluctuation measurements were then used to characterize two ClC-1 mutants linked to hereditary myotonias. Such analysis supports the simple mechanistic hypothesis of Kubisch et al. for the different inheritance pattern of the two mutations in terms of the two-gate double-barreled model: monomers of the recessive mutant I556N may associate with WT subunits to form fairly functional dimers because only the protopore provided by the mutant subunit has strongly modified properties, whereas monomers of the dominant mutant I290M modify both homodimers and mutant/WT heterodimers because they affect the common gate. Section title: discussion Educational score: 4.294920921325684 Domain: biomedical Document type: Study Language: en To what extent do our results support the hypothesis of a double-barreled structure of ClC-1? The fact that our single-channel recordings are consistent with the simple kinetic scheme of Fig. 3 B does not exclude the possibility that more complicated gating schemes are needed for a quantitative description of ClC-1 gating in a wider range of voltage, in different ionic conditions, and at a higher time resolution. Due to their small conductance and relatively fast kinetics, single ClC-1 channels can be observed with much less resolution than ClC-0 , and the argument in favor of an apparent double-barreled structure is correspondingly weaker. In particular, the relative kinetic overlap of the fast and slow gates makes their distinction more difficult. Since ClC-0 and ClC-1 are structurally very similar and have several common permeation and gating properties our finding that they also show the same basic subconductance behavior further supports the idea that these two channels share similar structure–function relationships. Section title: discussion Educational score: 4.239302635192871 Domain: biomedical Document type: Study Language: en Recently, Fahlke et al. suggested that ClC-1 has only one pore and supported this hypothesis with measurements of the effect on macroscopic currents of various channel-modifying reagents in homo- and heteromeric ClC-1 mutant constructs. With the assumption that the reagents act by a pore-blocking mechanism, they interpret their results as incompatible with a channel structure with two physically distinct conduction pathways. This conclusion would be hard to extend to ClC-0 channels, for which the evidence for the presence of two physically distinct conduction pathways, based on mutagenesis and single-channel recordings, is very strong . Section title: discussion Educational score: 4.411706447601318 Domain: biomedical Document type: Study Language: en Almost symmetric subconductance states have also been observed in other channel types that are definitively single-barreled channels . Root and MacKinnon observed independent protonation of two identical sites that led to the appearance of binomially distributed subconductance states in a cyclic nucleotide–gated cation (CNG) channel. Such a mechanism is similar to the interpretation of ClC-1 gating as shown in Fig. 3 D, even though the gating of the CNG channel is different from ClC-0/ ClC-1 gating in at least two aspects: (a) the subconductance states in the CNG channel are not equidistant and (b) the lowest conductance state with an open “common” gate is not zero in the CNG channel. Most importantly, however, the results of Middleton et al. and Ludewig et al. for ClC-0 are extremely difficult to explain on the basis of a channel with a single pore: in these studies, the conductance, ion selectivity, and gating properties of single protopores could be altered independently from the other protopore, resulting in heteromeric asymmetric double-barreled channels in which the properties of the two protopores were identical to those of the respective homomeric parent channels. Section title: discussion Educational score: 4.454170227050781 Domain: biomedical Document type: Study Language: en On the other hand, we argue that the results of Fahlke et al. for ClC-1 can also be easily interpreted in the framework of a double-barreled channel. First, it has not been ruled out by Fahlke et al. that the reagents used in their study decrease the macroscopic current amplitude not by a pore-blocking mechanism, but by acting on the gating of the channel. In the voltage-dependent sodium channel, for example, amino acids of the “S4 segment” that probably do not contribute to the pore are accessible to cysteine-modifying reagents, and their modification leads to persistent changes in gating properties . The fact that some of the cysteine substitutions, used by Fahlke et al. to make the channels sensitive to the modifying reagents, do per se modify drastically the macroscopic currents mediated by ClC-1, suggests that this could indeed be the case. If, for example, the effect of the cysteine substitution is on the common gate, it is plausible that the ulterior modification produced by the binding of MTSES [sodium (2-sulfonatoethyl)methanethiosulfonate] (or other reagents) forces this gate into a closed conformation. This would explain the results of Fahlke et al. , which would then be fully compatible with a double-barreled model. In addition, even accepting the pore-blockage assumption, it could be that the two pores of the channel have common intracellular and/or extracellular vestibules and that the modifying reagents act by blocking in these regions the entrance to both pores simultaneously. Section title: discussion Educational score: 4.180719375610352 Domain: biomedical Document type: Study Language: en In conclusion, our present data demonstrate that single ClC-1 channels behave like ClC-0 channels. Therefore, considering that the basic molecular structure of both channels is similar and, in the absence of more compelling evidence to the contrary, we maintain our preference for the idea that both channels are characterized by a double-barreled structure with two identical and physically distinct conduction pathways. In fact, we think that future observations will likely establish the notion of the double barrel as a distinctive common motif of the structure of all ClC channels, some of which have functional properties that are very poorly understood except for their involvement in hereditary diseases like kidney stone diseases and Bartter's syndrome . | Study | biomedical | en | 0.999997 |
10051521 | Section title: introduction Educational score: 4.791410446166992 Domain: biomedical Document type: Study Language: en The intracellular signal that triggers the contraction of cardiac muscle is a transient rise in intracellular free calcium. The majority of this calcium (50–95%, depending upon species and conditions) is released from the sarcoplasmic reticulum (SR). 1 This calcium is released by a process of calcium-induced calcium release (CICR) via release channels, which have been shown to be type-2 ryanodine receptors (RyR2). It is generally believed that the major stimulus for CICR is calcium entering the cell via sarcolemmal dihydropyridine–sensitive L-type calcium channels. The rate and amount of calcium release from the SR is tightly controlled by the magnitude and duration of the L-type calcium current . This graded control is paradoxical because the released calcium, which is roughly 10× larger in amount than the trigger, would be expected to stimulate further CICR, leading to a regenerative, nearly all-or-none release. Several years ago , we proposed that this paradox of control might be explained if the stimulus for release of calcium by RyRs were actually the local nanodomains of [Ca 2+ ] generated by nearby L-type channels, rather than the global cytosolic [Ca 2+ ]. According to this local control hypothesis, the graded control of macroscopic SR calcium release would actually be achieved by graded statistical recruitment of individual, autonomous, stochastic release events. Section title: introduction Educational score: 4.453918933868408 Domain: biomedical Document type: Study Language: en Recent ultrastructural studies (Franzini-Armstrong, C., F. Protasi, and V. Ramesh, manuscript in preparation) show that, depending on species, from a few tens to ∼200 RyRs are clustered in a two dimensional crystal-lattice array on the surface of SR release terminals, apposed, across the 15-nm cleft of the diad junction, to clusters of sarcolemmal dihydropyridine receptors (DHPRs). RyR2 is a homotetramer of a polypeptide of roughly 5,000 amino acids. The transmembrane calcium-sensitive channel is formed by the COOH-terminal ∼600 amino acids , while the remainder of the molecule forms a 30-nm quatrefoil “foot process” that spans the diadic cleft, and is required for interaction of the channel with a variety of modulators . The DHPRs in cardiac muscle, whose number varies in different studies from 10–100% of the number of RyRs , are localized at the junctions, but are randomly positioned relative to the ryanodine receptor lattice (Franzini-Armstrong, C., F. Protasi, and V. Ramesh, manuscript in preparation). This contrasts with the regular arrangement of DHPR tetrads found in skeletal muscle. Section title: introduction Educational score: 4.433066368103027 Domain: biomedical Document type: Study Language: en We carried out numerical simulations of this system of channels, interacting stochastically via calcium diffusing in the diadic cleft. These simulations have revealed a new paradox. Local control succeeds if the gating of the RyR is represented by a simple, phenomenological, four-state scheme. However, published schemes derived from actual gating statistics of single RyR2 channels incorporated into lipid bilayers give rise to unacceptable instabilities when used in the local control model. These instabilities are traceable to two deficiencies in bilayer-derived gating schemes. The absence of strong inactivation prevents termination of locally regenerative release by clustered RyRs. Activation by binding of a single Ca 2+ ion, in some bilayer-derived schemes, does not provide adequate discrimination against activation by global (rather than microdomain) Ca 2+ . This suggests that the gating of RyR2 in situ may differ significantly from its behavior in bilayers. One possible explanation of this difference would be the existence of allosteric interactions between the large foot processes of adjacent RyRs, which appear anatomically to be in contact. We show by simulation that RyR–RyR allosteric interaction energies can be chosen in such a way as to remedy both the inactivation and the cooperativity deficiencies of RyR2 gating schemes. Such interactions may be one of the important functions of the foot process, which has been highly conserved in evolution. Section title: Simulation Educational score: 4.298552513122559 Domain: biomedical Document type: Study Language: en Monte Carlo simulations were carried out using a modification of an algorithm previously reported . In brief, the set of channels at each diad was treated as a single stochastic system whose state (“macrostate”) is defined by specifying the Markov state of each of the individual channels. State transitions of individual channels are considered to take place instantaneously so that a macrostate transition corresponds to a transition of exactly one channel. The transition rates of the individual channels were determined from their gating schemes as a function of the local [Ca 2+ ] at the position of each channel. The dwell time and destination of each transition were selected by use of appropriately distributed random numbers, while the local [Ca 2+ ] was determined concomitantly by solving the partial differential equations governing diffusion and binding reactions in the diadic cleft. The aggregate calcium fluxes produced by 1,000–10,000 such diads were taken as input to a conventional lumped-compartment model of global cytosolic and SR calcium dynamics. This model (a set of ordinary differential equations) was solved to update the values of [Ca 2+ ] cytosolic and [Ca 2+ ] SR . These were used, in turn, to determine unitary currents of the channels and to set boundary conditions at the edges of the diadic clefts. The global model consisted of seven differential equations governing the following dynamical variables: [Ca 2+ ] cyto , SR lumenal calcium (in rapid equilibrium with calsequestrin), and calcium bound to five types of buffer sites, representing calcium-sensing dye (fluo-3), troponin, calmodulin, SR membrane sites, and low affinity sarcolemmal sites. The SR calcium pump was modeled as a steady uptake, represented by a Hill function of [Ca 2+ ] cyto , balanced (in the resting state) by a leak proportional to lumenal [Ca 2+ ]. Compartmental volumes and the kinetic parameters of all buffers and pumps were taken from the literature (Table I ) and were not treated as adjustable parameters of the model. The details of the cytosolic calcium removal model have very little effect on the short term simulations presented in this paper. Section title: Simulation Educational score: 4.2416157722473145 Domain: biomedical Document type: Study Language: en The extreme computational demands of such a simulation required the use of approximations to the reaction–diffusion equations in the diadic cleft. Because of the large aspect ratio of the cleft (diameter 100–400 nm, height 15 nm), diffusion was treated in two dimensions only, and the equations were discretized on a 10-nm mesh. This coarse approximation can be justified because the locations of the exit pore(s) and calcium sensing site(s) of the ryanodine receptor, as well as the diffusion paths of Ca 2+ through the space occupied by the RyR foot processes, are not known with any greater precision. L-type channels were located at random on the mesh, while RyRs were located regularly at 30-nm center-to-center spacing . Effective values of the cleft height and calcium diffusion coefficient were used to approximate the effects of surface charge, viscosity and tortuosity, as estimated by Soeller and Cannell . Diads containing up to 121 RyRs were simulated; most of the results shown here were computed for 25-RyR diads containing an average of five randomly located DHPRs that display the qualitative features and save computation time. Section title: Simulation Educational score: 4.255377292633057 Domain: biomedical Document type: Study Language: en Even with these approximations, computation times of hours were required for the full simulation. For this reason, many of the results shown were computed using the approximation that calcium diffusion in the cleft is at steady-state during the dwell time in any given macrostate, which accelerates the computation by about two orders of magnitude. The accuracy of this approximation depends on the density of fixed calcium binding sites in the diadic cleft. In the absence of such sites, it agrees with the full simulation to within a few percent. In the presence of low affinity fixed Ca 2+ binding sites at the density estimated by Post and Langer , errors of 30–50% may occur with the steady state approximation, but the qualitative features of the results appear to be correct, and most of the quantitative results can be recovered by use of effective values of channel parameters that are well within the large experimental uncertainties . The fixed buffer sites in the cleft do not appear explicitly in the steady state diffusion approximation since these sites are nonmobile and therefore have no effect on the steady state distribution of [Ca 2+ ], although they control the rate at which the steady state profile is established after a change in the sources. Section title: L-Type Channel Educational score: 4.1558074951171875 Domain: biomedical Document type: Study Language: en There is no consensus on the gating scheme of the L-type sarcolemmal calcium channel. In these simulations, it was modeled using a novel 24-state gating scheme that we developed as an improvement of the scheme recently proposed by Jafri et al. . The new scheme combines the scheme of Imredy and Yue for calcium-dependent inactivation with that proposed by Shirokov et al. to explain gating charge movement and voltage-dependent inactivation. This scheme consists of four-gating modes (normal, calcium-inactivated, voltage inactivated, and both calcium and voltage inactivated). This model is in the process of being validated and parametrized using measured whole cell and single channel calcium currents. For the purposes of this paper, it may be thought of as an empirical source of DHPR openings that act as input to the CICR process. Fig. 3 , B and C, shows representative L-type calcium currents measured from a rat cardiac myocyte in the presence of 10 μM ryanodine. The parameters of the L-type gating model were adjusted manually to fit these data, as shown, and these parameter values were used for most of the simulations shown in the rest of the paper without any further adjustment. They should not be considered necessarily optimal or unique. Section title: RyR Gating Schemes Educational score: 4.122317314147949 Domain: biomedical Document type: Study Language: en Simulations were carried out using six different gating schemes for RyR2, shown in Fig. 4 . Two of these are phenomenological schemes representing, in simplest form, the combination of activation by binding of two calcium ions and either calcium-dependent or fateful inactivation. The other four are published gating schemes based on single-channel observations of purified RyRs in lipid bilayers. Section title: Measurement of SR Release Flux Educational score: 4.145599842071533 Domain: biomedical Document type: Study Language: en SR release flux was measured by the recently developed Oregon green/EGTA method, which has been described in detail by Song et al. . In brief, adult rat ventricular cardiac myocytes were enzymatically isolated and studied under whole cell patch-pipette voltage clamp. The fast, low affinity fluorescent calcium indicator Oregon green 488 BAPTA 5N (1 mM) and EGTA (4 mM) were dialyzed into the cell from the pipette. As we have shown previously, this combination gives a fluorescence signal dominated by a term directly proportional to SR release flux. Myocytes were imaged in the line scan mode of an inverted confocal microscope (LSM-410; Carl Zeiss, Inc. ), with the scan line oriented longitudinally. This method makes it possible to see the localized SR release at the Z line of each sarcomere. Whole cell calcium release rate was estimated by averaging over the length of the scan line. Section title: Local Control Produces Graded Release of SR Calcium Educational score: 4.3358378410339355 Domain: biomedical Document type: Study Language: en Fig. 5 shows representative measurements of calcium release flux from the SR of a rat cardiac myocyte, compared with simulations using the phenomenological RyR gating Scheme 5 in Fig. 4 . Fig. 5 (left) shows the time course of SR release flux during a single voltage clamp depolarization to 0 mV from a holding potential of −70 mV. The figure demonstrates that the local control model can simulate the observed time course of release, particularly the fact that release declines during sustained depolarization. The fact that release in this model is dependent on local geometry is illustrated by the much smaller dotted curve, which shows the release flux that would be computed assuming a diadic cleft height of 30 nm instead of the correct 15 nm. Fig. 5 (right) shows the peak release flux as a function of the voltage of the clamp pulse. I Ca , the macroscopic current of the L-type channels, shows the bell-shaped voltage dependence typical of voltage-controlled ion channels, while the SR flux recapitulates (approximately, see below) the voltage dependence of I Ca , but at an absolute magnitude 10-fold larger. The simulation reproduces these features well. This demonstrates the central point of the local control hypothesis: the complicated stochastic interactions of calcium-coupled channels can produce high amplification with stable, graded control by trigger calcium. Section title: Local Control Produces Graded Release of SR Calcium Educational score: 4.350250244140625 Domain: biomedical Document type: Study Language: en As shown in Fig. 6 , if the bell shaped I CaL vs. V and I SR vs. V curves are normalized to their respective peaks, there is a small difference between their positions. When these curves are then divided to give a plot of the gain of excitation–contraction (EC) coupling (peak I SR divided by peak I CaL ), the resulting gain diminishes with voltage. This was one of the original predictions of the local control theory, which has been confirmed in several laboratories . It stems from the fact that local activation of the RyR depends on the unitary current of the triggering L-type channel(s) rather than on the macroscopic ensemble-average current. As pointed out previously , the decline of gain at positive voltages is a manifestation of the cooperativity of RyR activation by calcium, because the diminution of L-type unitary current as the calcium reversal potential is approached reduces I SR disproportionately in comparison to its linear effect on macroscopic I CaL . As argued by those authors, a quadratic dependence of RyR activation on local calcium should result in a macroscopic gain that is roughly proportional to the L-type unitary current. The decline of gain with voltage in the simulations is steeper than that of the L-type current over the midrange of voltages , even though activation of the RyR was assumed to require two Ca 2+ ions. This “elbow” in the voltage-gain relation results from the depletion of SR calcium (by release to the cytosol) during the depolarization, as indicated by the fact that it is prevented if SR depletion is minimized by reducing the number of diads per unit cytosolic volume, or reducing the number of available L-type channels (not shown). Such an elbow has been observed experimentally in intact cardiac myocytes . It was not seen when I CaL (and therefore I SR and SR depletion) was reduced by a factor of 20 with nifedipine in order to study calcium sparks . Section title: SR Calcium Release Is Locally Regenerative Educational score: 4.446274280548096 Domain: biomedical Document type: Study Language: en The number of RyRs that contribute to SR calcium release at a single diad is not known. As an index of that number, we computed the mean number of RyR openings per diad during a clamp pulse to 0 mV for 50 ms, averaged over those diads at which at least one RyR opening occurred. Neither the unitary current i RyR of the RyR nor its calcium sensitivity in situ is well known. We therefore varied the RyR permeability over two orders of magnitude, adjusting the sensitivity k o in each case to give the observed macroscopic gain of 10 at 0 mV and standard SR loading, to determine whether there could be a regime in which an L-type channel communicates with a single RyR without regenerative local recruitment of other RyRs. As shown in Fig. 7 , the mean number of RyR openings per diad has a U-shaped dependence on i RyR , with a nadir of ∼11 . This shows that, at least in the context of this model, RyR activation is always locally regenerative. This result can be understood qualitatively as follows. If i RyR were small, the RyR sensitivity would have to be high, since many RyRs would need to be recruited to achieve the macroscopic gain of 10. If i RyR were high, on the other hand, the sensitivity must be low, as a result of which the coupling from DHPR to RyR frequently fails. The release in this case is made up of rare diadic release events, each of which is highly regenerative. The fact that the DHPR-RyR communication fails before RyR–RyR recruitment is basically a consequence of geometry: if the DHPRs are located randomly at the junction, they cannot, on average, be much closer to RyR sensing sites than the latter are to the release pores of neighboring RyRs. If DHPRs were located in registry with RyRs, and if the calcium-sensing site of the latter were located on the “top” of the foot process, within a few nanometers of the DHPR, then nonregenerative 1:1 communication would be possible, as originally envisioned in the model of Györke and Palade . Section title: Local and Global Stability Educational score: 4.310222148895264 Domain: biomedical Document type: Study Language: en Before discussing simulations using empirically determined RyR gating schemes, we define two different concepts of stability. Local stability refers to the fact that there must be some mechanism to terminate the regenerative release at an individual diad. Global stability refers to the fact that the resting state of the myocyte must be stable against regenerative build up of global calcium, and the cell must eventually relax to this resting state after stimulation. The average diadic release rate triggered by global cytosolic calcium must, therefore, be less than the removal rate (SR pump + Na/Ca exchange) in the vicinity of the resting state. These two concepts of stability are distinct, though related. If there were no calcium removal, [Ca 2+ ] cyto would eventually build up to cause global regenerative release, no matter how well behaved the RyRs. On the other hand, it is evident that a cell cannot return to the resting state unless local release terminates. Both forms of stability are adversely affected by the clustering of RyRs. Clustered RyRs will have a more regenerative local release, which will be more difficult to terminate. Release triggered by background calcium will also be enhanced by clustering, since if a single RyR in the cluster opens in response to background calcium, this will trigger opening of most of the other RyRs in the cluster. Section title: Local and Global Stability Educational score: 4.5069475173950195 Domain: biomedical Document type: Study Language: en There are three mechanisms that contribute to the termination of local release: (a) depletion of SR calcium, (b) inactivation of RyRs, and (c) stochastic attrition. The last refers to the fact that for a finite-size cluster of RyRs, gating stochastically, there is always a nonzero probability that all RyRs will be closed at the same time, and this event will interrupt the positive feedback and extinguish the activity of the cluster, as long as there are no further DHPR openings to re-ignite it. SR depletion cannot be the principal mechanism of local release termination, since release terminates after voltage steps to very negative or positive voltage, which release very little of the SR calcium stores, and release in calcium sparks also terminates despite the fact that there is no global SR depletion. Conversely, greatly prolonged sparks occur in the presence of ryanodine , indicating the absence of local SR depletion. Inactivation and stochastic attrition must, together, be capable of terminating release at a single diad. Section title: Local and Global Stability Educational score: 4.3329877853393555 Domain: biomedical Document type: Study Language: en The contribution of stochastic attrition may be roughly estimated from a simple, analytical model. Consider a “cluster” of n identical channels, each of which has only two states, open and closed. Start with each channel in equilibrium, with an open probability P o and an opening duration τ o . The channels are assumed to gate independently, except that, if all close at once, the cluster is considered to be extinct. So long as the cluster is “alive,” the number of open channels, n o , will be binomially distributed, except that n o = 0 is excluded. The probability that a cluster becomes extinct during a time interval dt is given by \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}\frac{dt}{{\tau}_{attrit}}=\begin{matrix}(probability\;that\;only\;one\;channel\;is\;open) \enskip & \\ {\times}(probability\;that\;last\;channel\;closes), \enskip & \end{matrix}\end{equation*}\end{document} Section title: Local and Global Stability Educational score: 3.9789083003997803 Domain: biomedical Document type: Study Language: en where τ attrit is defined to be the time constant for extinction of clusters by stochastic attrition. Using this relationship together with the binomial distribution, it is straightforward to determine the attrition time constant: 1 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\tau}_{attrit}=\frac{{\tau}_{o}[1-(1-P_{o})^{n}]}{n(1-P_{o})^{(n-1)}P_{o}}.\end{equation*}\end{document} Section title: Local and Global Stability Educational score: 4.472432613372803 Domain: biomedical Document type: Study Language: en This time constant varies roughly exponentially with the product nP o , the expected number of open channels. Some numerical values are instructive. For a cluster of 25 channels with a mean opening duration of 10 ms and P o = 10%, τ attrit is ∼46 ms. For 75 channels with a 50% open probability, it becomes 160 billion years, ∼10× the age of the universe! This makes it clear that stochastic attrition alone cannot be relied upon to terminate local release robustly if the number of RyRs in a diad is as large as present ultrastructural data indicate. There must also be an inactivation process that is capable of substantially reducing P o . For an opening duration of 10 ms, one finds numerically that to extinguish clusters with a time constant of 50 ms requires nP o < 2.6. Therefore, for RyR clusters of the size found in cardiac muscle, local stability requires an inactivation process that, while it need not be completely absorbing, can reduce the RyR open probability to a few percent or less. It needs to be kept in mind, however, that stochastic attrition is not an alternative to inactivation, but an intrinsic feature of multichannel local control models, which always contributes to the cluster extinction process regardless of what other mechanisms are present. Section title: Fateful Inactivation of RyR Gives Stable EC Coupling Educational score: 4.412230491638184 Domain: biomedical Document type: Study Language: en Since the stability of local control EC coupling depends critically on the inactivation process of the RyR, it is reasonable to ask whether the calcium-dependent inactivation mechanism employed in the phenomenological gating of Fig. 4 , Scheme 5, is essential to the success of the model. The answer is no; as shown in Fig. 8 , qualitatively comparable results can be obtained with Fig. 4 , Scheme 6, in which inactivation is fatefully linked to activation, but not explicitly dependent on calcium . In this scheme, unlike Scheme 5, the activation and inactivation “gates” are not independent. The inactivation and repriming rate constants (vertical transitions) are different in the calcium-free and -activated states of the channel (subject to the constraint of microscopic reversibility, which requires that the products around the loop of forward and reverse rates be equal). By proper choice of these rate constants, it can be arranged that, on exposure to Ca 2+ the channel first activates and then inactivates, while on removal of Ca 2+ , channels predominantly deactivate before repriming, avoiding another passage through the open state. This arrangement requires some tuning to secure sufficiently rapid decay of release both on depolarization and repolarization. Section title: Bilayer-derived RyR Gating Schemes Give Unstable EC Coupling Educational score: 4.237214088439941 Domain: biomedical Document type: Study Language: en We now turn to consideration of models based on the empirically derived RyR gating Schemes 1–4 in Fig. 4 . Each of these schemes proves to have one or more deficiencies that preclude local and global stability. We can consider, first, Scheme 3 proposed by Keizer and Levine . This scheme was developed to explain the “adaptation” of the RyR observed by Györke and Fill . When used in an ensemble-average sense, where [Ca 2+ ] is taken to be a global value imposed by the researcher, this scheme gives a reasonable description of RyR adaptation as seen in bilayers with Cs + as current carrier. It is clear, however, that it cannot be considered as a description of microscopic events in a local control model, because it has a calcium-dependent transition between two open states. As shown in Fig. 4 , once the channel reaches state O1, it will be exposed to the microdomain of high [Ca 2+ ] created by its own permeating calcium. This will immediately induce a transition to O2, precluding any opportunity for inactivation or adaptation. The rate and extent of inactivation will be minimal in a local control setting, preventing termination of local release, as was confirmed by carrying out the simulation with this gating scheme (not shown). Section title: Bilayer-derived RyR Gating Schemes Give Unstable EC Coupling Educational score: 4.394636631011963 Domain: biomedical Document type: Study Language: en The remaining three schemes of Fig. 4 share certain features. All have a slow inactivation process, which is calcium dependent only in Scheme 4. Each also has the possibility of opening the channel after binding only a single calcium ion, although only Scheme 2 of Zahradnikova and Zahradnik has only one calcium binding site. Each of these schemes was put forward with a set of rate constants determined by measurements in lipid bilayers, in the absence of Mg 2+ and ATP. To incorporate these schemes into the local control simulation, it is necessary to decide how (if) these constants should be modified to describe the channel in the intracellular milieu. Fig. 4 , Scheme 1, for example, has an open state reachable after binding two Ca 2+ , as well as one reachable with only a single calcium. Clearly, by completely rearranging the rate constants, it would be possible to make this scheme behave somewhat similarly to the phenomenological Scheme 6. But Scheme 1 was determined by very careful fitting to a series of data on transient activation after flash photolysis of caged calcium, taking into account the transient overshoot of [Ca 2+ ] after the flash. It makes little sense to discard this information. We therefore explored parameter values according to the following strategy. Since all lipid bilayer schemes studied in the absence of Mg 2+ and ATP gave activation rates that were much too high for local control, we reduced the rates of calcium binding by about two orders of magnitude to account for the effect of Mg 2+ competition; the exact factor used was determined by requiring the gain (ratio of peak release current to peak I CaL ) to be 10 for a depolarization to 0 mV. This criterion was relatively independent of the values of rate constants related to adaptation/inactivation. When the resulting models failed to show adequate stability, we examined the effect of increasing the kinetic rates of adaptation/inactivation steps by a factor of ∼20, consistent with the observations of Valdivia et al. . This also failed, whereupon we examined the effect of increasing only the forward rates of inactivating steps, thereby stabilizing the inactivated/adapted states. This strategy was limited by the onset of an unacceptable degree of inactivation in the resting ([Ca 2+ ] = 100 nM) condition, and moved the steady state activation curve rightward so that the steady state P o approached unity only at [Ca 2+ ] values over 100 mM. Even with these adjustments, all three schemes gave models that manifested local instability (failure of release termination after activation) or global instability (spontaneous activation by background [Ca 2+ ]). Because of these instabilities, [Ca 2+ ] cyto fails to relax to the resting value following repolarization, as shown in Fig. 9 A (Schemes 1 and 2). The process of parameter exploration is illustrated for Scheme 2 in B–D. Initially, the bilayer-derived rate constants were kept, except that the rate of calcium binding to the activating site ( K RC1 ) was decreased from 1,000 to 16 mM −1 ms −1 , consistent with a competitive effect of Mg 2+ , giving an apparent gain of 10 for a depolarization to 0 mV, starting with all channels in the resting state . The apparent success of this parameter set is illusory, as shown in Fig. 9 C, where the depolarization is preceded by a 1-s conditioning run-in at the holding potential. Exposure to background calcium triggers a spontaneous SR release, which continues indefinitely, partially inactivating the channels so that, in the true resting state, the gain at 0 mV is reduced to only ∼1. As shown in Fig. 9 D, variation of the activating rate constant ( K RC1 ) over a fourfold range and of the inactivating rate constants over a 100-fold range was powerless to recover the normal gain in the true resting state of the model. Extensive parameter variations of this kind failed to identify a satisfactory parameter set for Schemes 1 or 2. The authors of Fig. 4 , Scheme 4, did not specify a complete set of kinetic constants for the scheme, but it suffers from the same generic problems as the first two: slow and incomplete inactivation (73% at pCa 3) and activation by only a single Ca 2+ ion. Section title: Bilayer-derived RyR Gating Schemes Give Unstable EC Coupling Educational score: 4.312808036804199 Domain: biomedical Document type: Study Language: en The global stability problem that arises if the RyR can be activated by a single calcium ion can be demonstrated in a relatively model-independent way. The time-averaged local [Ca 2+ ] at a distance r from an L-type channel with an open probability P o , passing a unitary calcium current i CaL , considered as a point source, is given by 2 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}[Ca^{2+}]_{av}=\frac{i_{CaL}P_{o}}{8{\pi}D_{Ca}Fr}.\end{equation*}\end{document} Section title: Bilayer-derived RyR Gating Schemes Give Unstable EC Coupling Educational score: 4.479275703430176 Domain: biomedical Document type: Study Language: en The openings of the L-type channel are brief and sparse, with P o probably not in excess of 5% . Using this value, and assuming a unitary current of 0.1 pA and an (effective) calcium diffusion coefficient of 0.15 × 10 −5 cm 2 s −1 , the time-average local [Ca 2+ ] at a distance of 10 nm from the L-type channel is only 1.4 μM, which is comparable to the global [Ca 2+ ] cyto reached during a forceful beat, and only 14× resting [Ca 2+ ] cyto . A RyR that opens as the first power of local [Ca 2+ ] cannot, therefore, discriminate against activation by global background calcium, because the average release flux will be proportional to the average [Ca 2+ ], which has a major contribution from nonlocal background calcium. This implies that globally stable local EC coupling cannot be achieved by any RyR that can be activated by a single Ca 2+ ion, assuming that the position of DHPRs is random in relation to the RyR lattice so that the average distance from DHPR pore to RyR sensing site is ∼10 nm. Section title: Bilayer-derived RyR Gating Schemes Give Unstable EC Coupling Educational score: 4.617624282836914 Domain: biomedical Document type: Study Language: en The previous argument tacitly assumed that an RyR with only a single calcium binding site would have a time-averaged open probability roughly proportional to the time-averaged local [Ca 2+ ]. It might be argued that this is not necessarily so: there could be schemes in which the occupancy of the single binding site is proportional to the local [Ca 2+ ], but the mean RyR P o during repeated exposure to brief pulses of high [Ca 2+ ] is larger, as a result of nonlinear steps downstream from the calcium binding reaction. The difficulty with this argument becomes apparent if one tries to construct such a model. For a single RyR in isolation, the binding site occupancy translates into the probability that the Markovian channel is found in certain states. But how is one to make the open probability depend nonlinearly on this probability when the master equations governing Markov transitions are linear? For the case of a channel with a microreversible (i.e., thermodynamically passive) gating scheme exposed to a steady calcium level, we can be quite rigorous about this. The equilibrium law of mass action requires that the P o be a rational function of degree 1; i.e., a Michaelis-Menten function. The question, then, is whether there could be a gating scheme in which the non–steady state effects of exposure to a train of brief, very high pulses of [Ca 2+ ] could “pump” the channel into a higher P o than predicted by linear extrapolation from the resting state. The experimental constraints on this problem are actually very strong. In a resting cardiac myocyte, calcium sparks occur at a rate of 100 s −1 . If the spark corresponds to a release of 4 pA for 10 ms, and if the RyR unitary current is 0.4 pA, then resting spark release amounts to an average of only ∼10 open RyRs per cell. If there are, conservatively, 10 5 RyRs per myocyte, the resting P o at [Ca 2+ ] = 100 nM is 10 −4 . Is it possible that a one-calcium gating scheme with this resting P o could, in response to a 200-μs pulse of [Ca 2+ ] = 100 μM, open with a probability of, say, 0.1? This is a special case of a problem that has been solved previously in connection with thermodynamic constraints on RyR adaptation . Using the methods in that paper, we found that it is impossible, assuming that the Markov scheme follows the kinetic law of mass action. We can therefore be fairly confident that the difficulty in obtaining global stability in simulations with single-calcium RyR gating schemes is generic. Section title: Local Control Can Be Stabilized by RyR–RyR Allosteric Interactions Educational score: 4.530867576599121 Domain: biomedical Document type: Study Language: en The fact that many RyR gating schemes derived from lipid bilayer data fail to support stable EC coupling in simulations suggests that RyR gating in situ may differ from that in bilayers. Two features are required to achieve local and global stability: “strong” inactivation and cooperative activation by more than one Ca 2+ ion. A possible clue as to how these features might arise comes from the ultrastructure and molecular biology of the RyR. In all striated muscles from crustaceans to man, including those that are activated purely by CICR (e.g., crayfish skeletal, mammalian cardiac) RyRs are found in dense two-dimensional crystalline arrays, although the details of the crystal lattice have varied somewhat . Moreover, the amino acid sequence of the foot process has been strongly conserved. Tunwell et al. found 98.6% amino acid identity between rabbit and human RyR2. We have identified a sequence of 200 amino acids located in the foot region that is 100% identical between mouse and human RyR2, despite the presence of 82 synonymous mutations at the DNA level (data not shown). Since only ∼29% of random mutations are synonymous , the probability that all would be synonymous in the absence of selection is only 0.29 82 (8.2 × 10 −45 ). This implies that the foot has an important (though unknown) function even though the COOH-terminal region of the molecule suffices to form a calcium-sensitive channel . Could the difference between in situ and in vitro gating of the RyR be due to allosteric interactions between the foot processes of nearest-neighbor RyRs, which appear ultrastructurally to be in contact? To test this possibility, we modified the simulation algorithm to make the transition rates of each RyR dependent on the states of its (up to) four nearest neighbors. To satisfy the constraint of microscopic reversibility, the interactions were specified in the form of free energies of interaction between neighboring RyRs, depending (symmetrically) on the states of each. To determine the transition rate K ij of a given RyR from state i to state j, the sum of the allosteric contact energies with its neighbors (in their present states) were added up in the initial and final states of the transition, and the exponential of the difference, divided by kT was multiplied into the affinity k ji / k ij of the transition. This still leaves one kinetic degree of freedom to determine how the effects of the free energy change are to be partitioned between the forward and backward rate constants. The transition rate therefore has the form 3 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}K_{ij}=k_{ij}exp\hspace{.167em} \left[ {\eta}_{ij}{ \,\substack{ \\ {\sum} \\ }\, }\begin{matrix}4 \enskip & \\ m=1 \enskip & \end{matrix}(E_{js_{m}}-E_{is_{m}})/(kT) \right] ,\end{equation*}\end{document} Section title: Local Control Can Be Stabilized by RyR–RyR Allosteric Interactions Educational score: 4.37904167175293 Domain: biomedical Document type: Study Language: en where k ij is the transition rate of the RyR in isolation, E js = E sj is the allosteric interaction energy between a RyR in state j and a neighbor in state s (zero if the neighbor is absent from the array), and η ij = 1 − η ji is a “splitting coefficient.” From the point of view of reaction rate theory, η may be considered to be the weighting factor that would be required to express the allosteric interaction energy of the transition state as a weighted average of the allosteric energies of the initial (“reactant”) and final (“product”) states. This makes it reasonable that η should lie between 0 and 1, but this is not required. In principle, each source of allosteric energy could have its own value of η for each transition, but, to avoid excessive proliferation of parameters, we assumed that a single value applies to all allosteric contributions affecting the rate of a given transition. By default, η = 0.5 was assumed, except for the case of a diffusion-limited calcium binding reaction, for which η = 0 is logical, since a change in the free energies cannot increase the on-rate and would decrease it only if it engendered a sufficient conformational change in the molecule to restrict access to the binding site, or a sufficient reduction in the magnitude of the binding energy to prevent capture of the ion. Section title: Local Control Can Be Stabilized by RyR–RyR Allosteric Interactions Educational score: 4.588857650756836 Domain: biomedical Document type: Study Language: en For the allosteric model, we used gating Scheme 2 of Zahradnikova and Zahradnikov , since it can be considered a “worst case,” having slow inactivation and only a single calcium binding site. In this model, RyRs interact both allosterically and by CICR— a process difficult to analyze intuitively. The strategy we used to choose the allosteric interaction energies had two parts. To produce local stability, the forward rates of inactivating transitions need to be increased, which requires stabilizing the inactivated state(s) relative to the noninactivated. A simple counting argument shows that adding an allosteric energy E ij that is equal to − E /4 if one of the states i and j is an inactivated state and − E /2 if both are, will contribute − E to the free energy change associated with any inactivating transition of a channel that has four nearest neighbors. To produce global stability is somewhat more subtle, because it requires creating, de novo , cooperativity of RyR activation by calcium. Since, in Scheme 2, each RyR has only a single Ca 2+ binding site, it is necessary to create positive cooperativity between binding sites on different RyRs. The strategy here is to add a large, positive allosteric contact energy between a RyR in an open state and one in the resting state. Initially, all RyRs in the diad are in the resting state R. Binding of calcium to one of these RyRs will move it to state C 1 , from which it could open if the channel were in isolation. But, to move this RyR to states O 1 or O 2 when it is surrounded by resting channels would cause a large increase in allosteric energy, so this transition is effectively prevented by an energy barrier. If more of its neighbors acquire bound calcium, moving them out of state R, the energy barrier is decreased so that the RyR can open, effectively responding to the binding of several Ca 2+ ions by the array as a whole. The important insight here is that, for this strategy to work, the underlying single-RyR gating scheme must have a closed state with bound calcium, so that binding of multiple Ca 2+ ions by the array can occur before any channel opens. If binding of the first Ca 2+ is tantamount to opening of the channel, then this event, however rare, will supply the local calcium to permit opening of nearby channels so that the whole array is effectively triggered by a single Ca 2+ . Section title: Local Control Can Be Stabilized by RyR–RyR Allosteric Interactions Educational score: 4.448300361633301 Domain: biomedical Document type: Study Language: en Once one RyR opens, a complicated interplay of allosteric and CICR interactions ensues, including some unexpected effects. For example, the energy barrier that prevented opening of a RyR surrounded by resting neighbors will, during the relaxation phase, prevent calcium from dissociating from RyRs that are in contact with one that is still open. We started with allosteric energies chosen by the two strategies described in the preceding paragraph, which succeeded in creating cooperativity of activation and strong inactivation. We then made adjustments, guided by admittedly incomplete intuition, until the performance of the model was satisfactory. The final matrix of allosteric interaction energies is shown in Fig. 10 A; they are not necessarily either unique or optimal. It should be noted that in this model we made no changes in the bilayer-derived parameters other than the introduction of the allosteric interactions. In Fig. 10 B, we show typical time courses of SR release in response to 50-ms depolarizations to the voltages shown. While the responses are not exactly the same as those of the phenomenological model, they serve to demonstrate that Fig. 4 , Scheme 2, when augmented by the allosteric couplings, produces locally stable release. Fig. 10 C shows the rate of release stimulated by global cytosolic Ca 2+ , for Scheme 2 with and without allosteric couplings. The presence of the allosteric couplings converts the first order dependence of release flux on global [Ca 2+ ] cyto into a cooperative response. This markedly reduces the rate of release triggered by global [Ca 2+ ] cyto in the physiologic range, as required for global stability. Fig. 11 , A and B, shows explicitly that the dynamics of [Ca 2+ ] cyto after repolarization, and the spontaneous release rate when exposed to resting [Ca 2+ ] cyto have been stabilized by the allosteric interactions. These allosteric interactions are basically inhibitory in character, as demonstrated in Fig. 11 , C and D, which shows the effect of reducing the number of RyRs by leaving random vacancies in the RyR lattice. When the lattice is more than ∼60% filled, increasing the number of RyRs actually decreases the absolute release rate, because the effect of allosteric inhibitions more than compensates for the increased number of RyRs carrying the release flux. It is striking that even a few vacancies per diad markedly increase background calcium release, impairing global stability . Section title: discussion Educational score: 4.232623100280762 Domain: biomedical Document type: Study Language: en In contrast to the situation in skeletal muscle, it is well established that mammalian cardiac excitation–contraction coupling is mediated by calcium-induced calcium release. This creates the paradox of control: why does the calcium released from the SR not feed back to trigger more release, causing an explosive chain reaction that would release all of the calcium in the SR? Previously , we analyzed some highly simplified and idealized models of stochastic CICR, with the conclusion that local control might be able to explain graded coupling. Since that time, a considerable amount of evidence has arisen in support of local control , but we have yet to see a single local calcium release event triggered by an identified L-type channel opening. However, considerably more information has become available about the ultrastructure of the diad junction and the gating of L-type channels and RyRs, and computer power has increased to the point where it is now practical to make use of this information in simulations. Section title: discussion Educational score: 4.308520317077637 Domain: biomedical Document type: Study Language: en Ideally, such simulations should be used to rule out erroneous mechanisms, prove the uniqueness of correct models, and identify the numerical values of their parameters. In practice, it is impossible to explore completely the multidimensional parameter space of these models, so the goal must be approached in stages. First, we ask whether local control, in a realistic setting of diad geometry, number, and location of channels, could explain the paradoxical gradedness of cardiac EC coupling. This question is answered in the affirmative by the demonstration that a phenomenological local control model with numerous, regularly spaced RyRs and multiple randomly located DHPRs at each diad junction can display graded control of SR calcium release flux by a sarcolemmal calcium current only one tenth as large. In the second stage, we ask whether one or more of the proposed RyR gating schemes based on lipid bilayer data are compatible with local control. It is difficult to answer this question with complete rigor, because of the large parameter space of these schemes. The approach that we have taken is to begin with the parameters obtained in lipid bilayers and make a trial series of parameter modifications consistent with known effects of the intracellular milieu; e.g., competition of Mg 2+ at Ca 2+ binding sites and acceleration of adaptation/inactivation kinetics. Based on these explorations, we have drawn inferences about how and why RyR gating schemes fail in the local control model, and what properties a successful RyR must possess. We have tried to back these inferences with relatively model-independent arguments. The third stage would involve detailed fitting of models to large amounts of whole-cell EC coupling data, identifying parameter values and establishing what experiments would discriminate between competing models. This stage goes beyond the scope of this paper, although the results obtained so far offer some tantalizing hints. Section title: discussion Educational score: 4.467318058013916 Domain: biomedical Document type: Study Language: en The somewhat surprising result of our stage-2 explorations is that none of the published bilayer-derived gating schemes gives a successful local control model, even when plausible modifications are made to adjust for the effects of the intracellular milieu. While it is difficult to exclude rigorously the possibility that some alternative set of parameters might solve the problem, it appears from our explorations that this could not be done without sacrificing crucial features of the gating behavior seen in bilayers; e.g., turning an adaptive inactivation into an irreversible one. All the schemes failed by lack of sufficient stability. Based on our explorations, this failure appears to result from the absence of two critical features: cooperative activation by more than one calcium ion and a “sufficiently” rapid and complete inactivation mechanism. The isolated RyR in bilayer is a very artificial system, and there are many reasons why it might not demonstrate physiological gating behavior, including the absence of other proteins associated with the RyR at the junction (e.g., triadin, junctin, calsequestrin). However, the most intriguing possibility is that the missing factor is the densely packed regular array of other RyRs that has been so well conserved through hundreds of millions of years of evolution. Recently, it has been reported that pairs (and, on occasion, groups of three, four, or even five) of skeletal muscle RyRs in lipid bilayers can gate synchronously when the associated protein FKBP-12 is present . Similar behavior is sometimes observed with RyR2 (A.R. Marks, private communication). This positive cooperativity was seen even when barium, rather than calcium, was the permeant ion, indicating that it is probably not mediated by the CICR mechanism. Our simulations show that cooperative allosteric interactions between nearest neighbor RyRs, interacting with CICR, can lead to stable local control of SR calcium release by a RyR whose gating scheme in isolation lacks the properties required for stability. Section title: Limitations of the Allosteric Gating Model Educational score: 4.436428070068359 Domain: biomedical Document type: Study Language: en Our construction of a stable gating scheme using allosteric interaction energies should be considered only as a proof of principle. In particular, our use of gating Scheme 2 from Fig. 4 as a starting point should not be construed as an endorsement of that gating scheme— we chose it only because it displays the pathologies of both inactivation and cooperativity in a simple form, and because it has a calcium-bound closed state, needed for our construction of RyR–RyR cooperativity. Our particular choice of allosteric energies should also not be considered either unique or optimal. They were obtained by starting with a certain strategy and then “tinkering” to get improved activation and inactivation behavior. The resulting model may deviate significantly from observations in many areas that we did not examine carefully, such as repriming dynamics. Our choice of allosteric interactions, while it produces positive cooperativity between RyRs, does not give rise to the perfect synchronization of opening and closing of coupled RyRs observed by Marx et al. . Bers and Fill have suggested that such synchronization among all the RyRs in a cardiac diad might be a mechanism to generate calcium sparks, as an alternative to stochastic CICR, which also produces spontaneous multi-channel release events consistent with observed sparks (results not shown). It is not likely that allosteric interactions are required for the production of sparks since removal of FKBP12, the immunophyllin that is associated with the native RyR and is required for synchronized gating , does not abolish sparks , but may actually increase their frequency and duration and induce global calcium oscillations . This observation finds a natural place in our allosteric coupling scheme, which is fundamentally inhibitory in character, as shown in Fig. 11 . In fact, for the model based on RyR gating Scheme 2, even partial relief of the allosteric inhibitions unmasks a much greater degree of instability than is observed in intact cardiac myocytes deprived of FKBP12 by either knockout or the drug FK506. In interpreting such findings, it is important to bear in mind that FKBP has major effects on the gating properties of RyRs even in isolation, perhaps by mediating coordination between monomers in the tetramer . Section title: Limitations of the Allosteric Gating Model Educational score: 4.576557159423828 Domain: biomedical Document type: Study Language: en There are, moreover, fundamental reasons to doubt that allosteric coupling alone, unaided by CICR, could produce acceptable synchronous gating of all the RyRs at a diad. Marx et al. reported that all the components of the dwell time distribution of synchronously gating pairs of RyR1 (two open times and two closed times) were unchanged from those of single channels. As pointed out by Bers and Fill , this is quite surprising on energetic grounds. One way to model such an array is to assume that the coupling gangs together the molecular degrees of freedom involved in gating so that the only available states of the array are those in which all channels are in the same state. If that were the case, then an array of n channels would have all the state and, presumably, transition-state, energies n -fold larger than a single channel. For the large n found in the cardiac diad, these increased energies, which appear as negative exponentials, would produce an overwhelming slowing of the gating process, as well as reduce the occupancy of the less probable states in the gating scheme to negligible values. Another way to model a synchronized array, more in keeping with our approach in the simulations, is to assume that the gating of individual channels in the array remains well defined, but is modulated by allosteric interaction energies that are additive over all the points of RyR–RyR contact in the array. The fact that Marx et al. did not observe intermediate states (one channel open, one closed) in coupled channel pairs implies that occupancy of such states is suppressed by a positive allosteric energy, large compared with kT , at a contact between open and closed channels. In order for all the RyRs at a diad to go from closed to open, it would be necessary to pass, however briefly, through such intermediate states, which would have an allosteric energy proportional to the perimeter of contact between open and closed channels. This perimeter must, at some point in the process, be as large as the minimum diameter of the diad, 10 or more RyRs for large diads (Franzini-Armstrong, C., F. Protasi, and V. Ramesh, manuscript in preparation). This would represent an enormous energy barrier, which would, again, greatly slow the synchronous gating. Section title: Limitations of the Allosteric Gating Model Educational score: 4.422982692718506 Domain: biomedical Document type: Study Language: en We are led to the conclusion that coupled gating, unaided by CICR or other exogenous sources of energy, should be greatly slowed compared with single channel gating. These arguments fall short of rigorous proof, because we have assumed that the states and transitions states of individual channels remain well defined in the array, and that allosteric interactions can be treated as an additive perturbation generated at the interface between nearest-neighbor RyRs. One could, instead, achieve coupled gating without change in the dwell times by assuming that ganging together n channels has a catalytic effect that reduces the transition state energies of individual channels by a factor of 1/ n . Such a long range mechanical interaction in a lattice of huge macromolecules would be, at the least, ad hoc, although it might not be physically impossible. This argument shows that synchronous gating of many channels, with unchanged dwell times, is not a natural consequence of the interaction of multiple RyRs. It is therefore likely, as indicated by Bers and Fill , that allosteric interactions collaborate with local CICR to produce the observed macroscopic and microscopic phenomena of cardiac EC coupling. Whether this collaboration is of the form modeled here is a matter presently beyond the resolution of direct measurement. In light of the observations by Marx et al. , it is also likely that allosteric interactions would contribute substantially to the quantitative behavior and robustness of gating schemes that are not intrinsically unstable, but require “tuning” to give acceptable behavior. Section title: Limitations of the Allosteric Gating Model Educational score: 4.35972785949707 Domain: biomedical Document type: Study Language: en Although the possible role of allosteric interactions in cardiac EC coupling is the most novel phenomenon to emerge from the modeling, it should be put in perspective. To achieve stable local control, two characteristics are required: “strong” RyR inactivation and cooperative RyR activation by more than one Ca 2+ ion. Inactivation could be produced by any interaction of the RyR with its native environment that stabilizes the inactivated state—it is not necessary to invoke state-dependent allosteric interactions with neighboring RyRs for this purpose. In contrast, the creation of cooperative activation, starting from a gating scheme that permits RyR opening in response to a single Ca 2+ , does require the exchange of information between RyRs. However, it is not entirely certain that the cooperativity problem is a real one. While published gating schemes based on bilayer data all permit single-Ca 2+ activation, some studies have shown the steady state open probability of a single RyR2 depending on [Ca 2+ ] with a Hill coefficient of >2 , suggesting that intramolecular cooperativity might be adequate to achieve global stability. Section title: Limitations of the Allosteric Gating Model Educational score: 4.34488582611084 Domain: biomedical Document type: Study Language: en In any case, it is unlikely that any of the currently proposed RyR gating schemes captures all of the features of importance for physiologic EC coupling. One complicating factor that we have not considered here is the possibility that SR lumenal calcium may directly influence gating kinetics. Another (possibly related to the first) is that increasing the SR calcium load of a rat myocyte modestly above normal provokes periodic calcium oscillations, whereas “latch up” of the cell in a stable state of high [Ca 2+ ] cyto and high RyR open probability is never observed, even when egress of calcium from the cell is prevented. None of the models described above reproduces this global behavior accurately. It is possible that “adaptation” or other modulation of RyR gating by sustained elevation of [Ca 2+ ] in the submicromolar range plays an important role in determining global stability. Further simulation and experimentation will be required to determine how this and other physiologic constraints translate into required properties of the RyR at the molecular level. | Study | biomedical | en | 0.999996 |
10051522 | Section title: introduction Educational score: 4.829248905181885 Domain: biomedical Document type: Study Language: en The visual cycle is the series of reactions occurring in the eye through which visual pigment proceeds from photoactivation to regeneration. Absorption of a photon by the pigment of a photoreceptor triggers within milliseconds a series of reactions leading to a cell response. This sequence of reactions, collectively termed the phototransduction cascade, has been extensively studied and is now well characterized . According to this scheme, photoisomerization of the chromophore from the 11-cis to the all-trans conformation ultimately results in the closure of cation channels and the hyperpolarization of the cell. Another consequence of the photon absorption is the eventual dissociation of the chromophore from the protein moiety of the visual pigment to form all-trans retinol and free opsin. The visual pigment is then regenerated through a series of reactions requiring less than a minute in cones and tens of minutes in rods . In the vertebrate retina, this is a complex process that involves both photoreceptors and the pigment epithelium . Reduction of all-trans retinal to all-trans retinol must occur in the bleached outer segment of photoreceptors before the opsin binding site becomes accessible for regeneration. All-trans retinol is then removed from the outer segment and translocated to the pigment epithelium. There it is converted back to 11-cis retinal, and then returned to the receptor outer segment where it combines with opsin to form visual pigment. Section title: introduction Educational score: 4.85314416885376 Domain: biomedical Document type: Study Language: en Aside from the ultimate recovery of visual sensitivity , very little is known about the physiological consequences of the reactions involved in the reconstitution of the visual pigment within photoreceptors. An important initial step in the recovery process is likely to be the binding of 11-cis retinal, supplied from the pigment epithelium, into the chromophore pocket of opsin . The effects on phototransduction and regeneration of a whole host of retinal analogues have been studied with biochemical as well as with physiological methods . Treating bleached pigment from rods with derivatives of retinal having shortened polyene chains produces a catalytically active complex that promotes phosphorylation of opsin by rhodopsin kinase . Competition studies with 11-cis retinal suggest that these compounds exert their effect by binding in the chromophore pocket of opsin . These retinoids, including β-ionone , all-trans C17 aldehyde, and 9-cis C17 aldehyde do not form a covalent link with opsin via a Schiff base because the shorter length of their side chain apparently does not allow them to span from the ring-binding site to the corresponding lysine of opsin . The fact that these compounds do not form a covalent bond with the protein suggests that occupancy of the chromophore binding site of rod opsin alone is sufficient for catalytic activation of the complex. Thus, these biochemical experiments indicate that, in rods, retinoids with shortened polyene chains act as agonists, activating the photoreceptor. On the other hand, physiological studies on intact isolated cone photoreceptor cells show that in these cells short-chain retinal analogues produce an opposite effect: treatment of bleached cones with β-ionone or with 9-cis C17 aldehyde results in downregulation of transduction, characterized by slowed flash responses, increased sensitivity, and increased dark current. Thus, in cones, short-chain retinal analogues act as reverse agonists, inactivating the photoreceptor. These latter studies were interpreted as demonstrating that the noncovalent binding of retinal in the chromophore pocket of cone opsin might be an important initial step in dark adaptation. Section title: introduction Educational score: 4.483108043670654 Domain: biomedical Document type: Study Language: en The present study was undertaken to resolve the contradictory findings regarding the role retinoids play in regulating the activity of rod and cone photoreceptors. The experiments described here also address the question of whether the occupancy of the chromophore pocket of rod opsin by a retinoid produces physiological consequences that are separate from those due to the consequent formation of the Schiff base linkage. The retinal analogue chosen to address these questions was β-ionone because of its capability of binding in the chromophore pocket of opsin without forming a covalent bond. Furthermore, as we demonstrate here, its binding is rapidly and totally reversible. The use of β-ionone enabled us to study specifically the physiological effect of the noncovalent binding of retinal in the chromophore pocket of opsin without the interfering effects of the covalent bond between retinal and opsin. In addition, treating bleach-adapted rods with β-ionone allowed us to compare directly the effect of its binding in the chromophore pocket of rod opsin with its previously described effect in cones . We present evidence that, in bleach-adapted rods, occupancy of the chromophore binding site of opsin by β-ionone causes activation of the transduction cascade and desensitization of the cell. This observation is opposite to the expected downregulation of transduction during dark adaptation, when 11-cis retinal occupies the chromophore pocket. It is also opposite to the effect of β-ionone observed in bleach-adapted cone photoreceptors. This newly discovered distinction between rods and cones reconciles the conflict between biochemical and physiological results, and implies a fundamental difference in the interaction of rod and cone opsins with retinal. Section title: materials and methods Educational score: 4.214090347290039 Domain: biomedical Document type: Study Language: en Single rod photoreceptors from the retina of the larval tiger salamander (Ambystoma tigrinum ) were isolated in physiological solution as previously described . In brief, animals were dark-adapted overnight. They were decapitated and pithed in dim red light, following which the eyes were removed, hemisected, and placed in ∼1.5 ml saline solution. The retina was torn free of the pigment epithelium and chopped into small pieces with a razor blade. A fraction of the resulting suspension was transferred to a recording chamber located on the stage of an inverted microscope (Invertascope D; Carl Zeiss, Inc. ). The remaining portion of the suspension was stored at 4°C in the dark and remained viable for several hours. The preparation was viewed via an infrared television camera fitted to the inverted microscope and connected to a video monitor. Membrane currents were recorded from solitary rods with a suction microelectrode using methods similar to those reported previously . The inner segment of the rod was drawn into the pipette so that the outer segment could be rapidly superfused with test solutions. The tip of the electrode was heat-polished to a diameter of ∼10 μm. The current recorded from the cell was converted to voltage and amplified using a patch clamp amplifier (EPC-7; List Electronic), and low-pass filtered with an active eight-pole filter at 20 Hz cutoff frequency (902LPF; Frequency Devices Inc.). The data were digitized at 250 Hz, stored on a computer, and subsequently analyzed using the pCLAMP 6 data acquisition and analysis software ( Axon Instruments ) and the Origin 4 graphics and data analysis software (Microcal Software, Inc.). Section title: Light Stimulation Educational score: 4.271381378173828 Domain: biomedical Document type: Study Language: en A dual-beam optical stimulator provided test flashes as well as bleaching and background lights . The light source was calibrated at the beginning of each experiment with a photometer (80×; United Detector Technology). The absolute intensity of the test flash/bleaching beam was 1.36 × 10 7 photons μm −2 s −1 (500 nm); the absolute intensity for the background beam was 1.28 × 10 7 photons μm −2 s −1 (520 nm). Light intensity for each beam was attenuated with a series of calibrated neutral density filters. The wavelength was set with narrow band interference filters (10 nm bandwidth at 1/2 transmission; Corion Optics). A stimulus spot 1 mm in diameter was focused at the plane of the preparation by a 0.25 NA × 10 achromat objective located above the stage of the inverted microscope. Test flash duration was 20 ms. The fraction of bleached pigment was calculated according to the relation: 1 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}F=1-exp\hspace{.167em}(-IPt),\end{equation*}\end{document} Section title: Light Stimulation Educational score: 4.080883026123047 Domain: biomedical Document type: Study Language: en where F is the fraction of bleached pigment, I is the light intensity in photons μm −2 s −1 , and t is the duration of light exposure in seconds. The value used for the photosensitivity of the cell P was 6.2 × 10 −9 μm 2 . Section title: Solutions Educational score: 4.163321495056152 Domain: biomedical Document type: Study Language: en The cell was perfused with saline solution that contained 110 mM NaCl, 2.5 mM KCl, 1.6 mM MgCl 2 , 1.0 mM CaCl 2 , 10 mM dextrose, 10 mM HEPES, pH 7.8, and bovine serum albumin (100 mg/liter −1 ). The solution in which the guanylyl cyclase activity was measured was made by adding 3-isobutyl-1-methylxanthine (IBMX) 1 to normal saline solution to a final concentration of 500 μM (IBMX solution). The solution in which phosphodiesterase activity was measured (Li + solution) was made by replacing NaCl in the saline solution with equimolar LiCl. β-Ionone was purchased from Sigma Chemical Co. and was repurified by double distillation. It was dissolved to 20 mM in EtOH and then to its final concentration with saline solution (final EtOH concentration <0.1%). β-Ionone concentration was measured with a spectrophotometer (ε = 8,700 M −1 cm −1 ). Despite all attempts to minimize the binding of β-ionone to the plastic tubing by replacing most of it with glass, the concentration of β-ionone at the outflow of the microperfusion system in the recording bath was significantly reduced. For this reason, the concentration of β-ionone to which the cell was exposed was determined from samples collected from the outflow of the microperfusion tubing after each experiment. Section title: Solutions Educational score: 4.2218170166015625 Domain: biomedical Document type: Study Language: en The outer segment of the cell was exposed to β-ionone and test solutions using a double-barrel microperfusion system driven by a stepping motor . This system allowed for quick change of the solution perfusing the outer segment. The time required for a complete solution change, estimated from the junction current recorded while the cell was exposed to bright light, was ∼25 ms. Test solutions for estimation of rate constants of guanylyl cyclase and phosphodiesterase (to be referred to as rate of guanylyl cyclase and rate of phosphodiesterase) were delivered through one of the barrels of the microperfusion system using a multiple-way valve. The time for removing the dead volume in the system was ∼45 s. This allowed for effective estimation of both cyclase and phosphodiesterase rates within 1 min. β-Ionone was delivered to the cell through the other barrel of the microperfusion system. Initially, β-ionone flow was turned off so that jumps could be performed from saline solution into test solution (Li + and IBMX). Subsequently, β-ionone flow was turned on and its effect on cyclase and phosphodiesterase rates was estimated by jumping the cell from β-ionone into the test solution. The lack of β-ionone in the test solutions had only a negligible effect on the measurements of cyclase and phosphodiesterase rates. Section title: Solutions Educational score: 3.9885895252227783 Domain: biomedical Document type: Study Language: en The flow rates of the β-ionone and test solutions were regulated by injecting phenol red solution into the experimental chamber before each experiment. The sharp interface between the bath solution and the solutions flowing through the solution changer allowed us to observe and adjust the flow of both β-ionone and test solutions. The cells were not exposed to phenol red because in control experiments we observed that phenol red activates phototransduction in bleach-adapted rods. Section title: Data Analysis Educational score: 4.265142917633057 Domain: biomedical Document type: Study Language: en To estimate the rates of cyclase and phosphodiesterase in the cell, we have used a modification of the method initially described by Hodgkin and Nunn and subsequently used by Cornwall and Fain . The light-sensitive current in photoreceptors, i , is controlled by the concentration of cyclic GMP according to the relation : 2 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}\frac{i}{i_{m}}=\frac{[cGMP]^{N}}{[cGMP]^{N}+K^{N}_{1/2}},\end{equation*}\end{document} Section title: Data Analysis Educational score: 4.380439758300781 Domain: biomedical Document type: Study Language: en where i m is the maximum possible membrane current, K 1/2 is the Michaelis constant for binding of cGMP to the light-sensitive channels, and N is the Hill coefficient, reflecting the cooperative binding of cGMP to the channels. The level of cGMP in the cell is regulated by the relative rates of guanylyl cyclase, responsible for cGMP synthesis, and cGMP phosphodiesterase, responsible for its hydrolysis. The change in free cGMP concentration in the cell is given by: 3 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\eta}\frac{d}{dt}[cGMP]={\alpha}[GTP]-{\beta}[cGMP],\end{equation*}\end{document} Section title: Data Analysis Educational score: 4.321455478668213 Domain: biomedical Document type: Study Language: en where η is the buffering capacity of the cell for cGMP, α is the rate constant of cyclase, and β is the rate constant of phosphodiesterase. Following Hodgkin and Nunn , we assumed that the cGMP binding sites are of high affinity and saturated (η = 1). Combining Eqs. 2 and 3 , one obtains a relation between the current and the rates of cyclase and phosphodiesterase: 4 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}\frac{d}{dt}[K_{1/2} \left( \frac{i}{i_{m}-i} \right) ^{1/N}]={\alpha}[GTP]-{\beta}K_{1/2} \left( \frac{i}{i_{m}-i} \right) ^{1/N}.\end{equation*}\end{document} Section title: Data Analysis Educational score: 4.368486404418945 Domain: biomedical Document type: Study Language: en One can estimate the rate of cyclase or phosphodiesterase in the intact photoreceptor in different conditions of adaptation or in the presence of drugs by suddenly blocking one of these two enzymes. The rate at which the current changes immediately after the block provides an estimate of the velocity at which cGMP is being synthesized or hydrolyzed by the unaffected enzyme. Using this scheme, the rate of cyclase can be estimated by treating the cell with the phosphodiesterase inhibitor, IBMX. Sudden exposure of the cell to saline solution containing 500 μM IBMX substantially inhibits phosphodiesterase and the continued production of cGMP by cyclase results in current increase. The rate of increase of the current provides an estimate of the rate of cGMP synthesis by cyclase. Alternatively, saline solution in which Li + is substituted for Na + can be used to estimate the rate of phosphodiesterase. Li + permeates the light-sensitive channels but the presence of Li + inside the cell blocks the Na + /Ca 2+ –K + exchange mechanism . As a result, [Ca 2+ ] i increases rapidly and inhibits cyclase. The rate of the resulting decrease in current provides an estimate of the rate of hydrolysis of cGMP by phosphodiesterase. Section title: Data Analysis Educational score: 4.469204902648926 Domain: biomedical Document type: Study Language: en Derivation of a useful expression of the current as a function of the enzymatic rates of cyclase or phosphodiesterase with the help of test solutions requires several important assumptions that were implicitly made in the original work of Hodgkin and Nunn . However, new information makes necessary the explicit reexamination of these assumptions. The simpler case of the Li + jump requires the assumption that the cGMP channel characteristics ( K 1/2 and N ), as well as i m , do not change during or immediately after the solution change. Recent evidence has revealed that of these three parameters only K 1/2 is not constant but rather varies as a function of the concentration of calcium . As pointed out above, stepping into Li + results in an increase of [Ca 2+ ] i . A decrease in [Ca 2+ ] i within the physiological range can cause a significant decrease of K 1/2 . However, an increase in [Ca 2+ ] i beyond the physiological range, as in the case of Li + step, causes very little or no change of K 1/2 . Thus, the proposition that K 1/2 , N , and i m do not change during and immediately after the step in Li + solution is valid. A second assumption is that the current i , recorded with the suction electrode, is much smaller than i m . Thus, the ratio i /( i m − i ) in Eq. 4 can be replaced by the more simple i / i m . Using these assumptions for the case of blocked cyclase (α = 0), Eq. 4 simplifies to a form that relates the rate of phosphodiesterase to the normalized current: 5 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}\hspace{.5em}{\mathrm{ln}}(J)=-N{\beta}t.\end{equation*}\end{document} Section title: Data Analysis Educational score: 3.969026565551758 Domain: biomedical Document type: Study Language: en From Eq. 5 , the relative change in the rate of phosphodiesterase compared with that of the dark-adapted cell will be given by: 6 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}\frac{\hspace{.5em}{\mathrm{ln}}(J)}{\hspace{.5em}{\mathrm{ln}}(J_{D})}=\frac{{\beta}}{{\beta}_{D}}.\end{equation*}\end{document} Section title: Data Analysis Educational score: 4.009087085723877 Domain: biomedical Document type: Study Language: en After subtracting the junction current resulting from the solution change, the normalized current was plotted on a semilogarithmic graph. β was estimated from the slope of the straight line fitted to the current trace, and then the ratio β/β D was calculated. Section title: Data Analysis Educational score: 4.377346515655518 Domain: biomedical Document type: Study Language: en In the more complicated case of the IBMX jump, one again has to assume that the cGMP channel characteristics ( K 1/2 , N ) and i m do not change during the solution jump. Since the step into IBMX causes an increase in the inward current, the level of [Ca 2+ ] i should either stay the same or increase slightly. Thus, in addition to being valid for N and i m , the above assumption still applies for K 1/2 . One potential problem, however, derives from the current increase during the step into IBMX solution. The assumption i << i m , which is again required, may no longer hold. To test the validity of this approximation, we calculated the rates of cyclase from a set of data with and without this assumption and compared the results. The relative velocities of cyclase in the two cases differed by <2%, leading us to ignore this factor in further analysis. Using the same assumptions as made in the case of Li + , Eq. 4 can be simplified for the case of blocked phosphodiesterase (β = 0). Then, dividing by Eq. 2 written for the dark-adapted condition, one gets: 7 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}\frac{K_{1/2}\frac{d}{dt}[(i)^{1/N}]}{K^{D}_{1/2}[(i^{D})^{1/N}]}=\frac{[GTP]}{[cGMP]_{D}}={\alpha}^{\prime},\end{equation*}\end{document} Section title: Data Analysis Educational score: 4.220890998840332 Domain: biomedical Document type: Study Language: en where D indicates the parameters of the cell in its dark-adapted state. Following Hodgkin and Nunn , we have used α′ as a measure of the relative changes in the rate of cyclase . The expression for the relative rate of cyclase derived from Eq. 7 is: 8 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}\frac{K_{1/2}\frac{d}{dt}[(i)^{1/(3)}]}{K^{D}_{1/2}[(i^{D})^{1/3}]}=\frac{{\alpha}^{\prime}}{{\alpha}^{\prime}_{D}}=\frac{{\alpha}}{{\alpha}_{D}},\end{equation*}\end{document} Section title: Data Analysis Educational score: 4.268185615539551 Domain: biomedical Document type: Study Language: en where we assumed N = 3 . Another potential complication is that K 1/2 is likely to be different in rods that are dark-adapted, bleached, or chemically treated. As can be seen from Eq. 8 , in contrast to the Li + method, calculation of the relative rate of cyclase requires direct comparison of K 1/2 in the dark-adapted and in the bleached or otherwise manipulated cell. As pointed out above, K 1/2 of the cGMP-gated channel is not constant, but rather varies as a function of the concentration of calcium. The decrease in [Ca 2+ ] i produced by the bleach and probably by β-ionone can cause a significant decrease of K 1/2 . To improve the accuracy of the calculation of the rate of cyclase, we have incorporated an estimate of the change of K 1/2 caused by the bleaching of pigment in our analysis. This was done in the following way. The time derivative of the current di/dt was calculated using the Savitzki-Golay method . Since the data were acquired at 250 Hz (4 ms/point) and filtered at 20 Hz (50 ms/point), the linear regression was performed over 13 points (13 × 4 ms = 52 ms, corresponding to 19 Hz). The change in K 1/2 after the bleach was calculated from the corresponding drop in the dark current using the relation between current and [Ca 2+ ] i in Nakatani et al. . Using Eq. 6 , α′ was obtained in each experimental condition, and then the ratio α/α D (equal to α′/α′ D ) was calculated. Section title: Data Analysis Educational score: 4.189187049865723 Domain: biomedical Document type: Study Language: en The measurements of the rates of cyclase and phosphodiesterase in this study were done pairwise in the same cell, under the same conditions, and within 1 min of each other. To minimize possible side effects, the order of measurements was varied from cell to cell. Thus, direct comparison between the two enzymatic rates should be much more accurate than when two different cells from two different animals are considered. Since all of the measurements of the velocities of cyclase and phosphodiesterase were done in steady state, it would be expected that the two relative velocities (see Eqs. 6 and 8 ) will be equal. Accounting for the change in K 1/2 and modifying the analysis of IBMX jumps originally devised by Hodgkin and Nunn and later used by Cornwall and Fain compensated for the mismatch between the rates of cyclase and phosphodiesterase observed in these studies. In our experience, the two rates were very similar for activation of the phototransduction cascade by bleaches of up to 40%. For even higher transduction levels (higher bleaches), the Li + method saturates due to the overlap of the initial rapid influx of Li + ions, caused by the higher permeability of the light-dependent channels to Li + than to Na + , and the decrease in cGMP-activated current, caused by the block of cyclase. In addition, after higher bleaches, the dark current is significantly reduced, which makes the rate of its decrease in Li + solution more difficult to measure. Section title: Photocurrent and Light Sensitivity Educational score: 4.389056205749512 Domain: biomedical Document type: Study Language: en The experiment illustrated in Fig. 2 shows the effect that β-ionone has on the dark current and sensitivity of a bleach-adapted rod. The cell was stimulated with a series of test flashes of increasing intensity to monitor changes in the amplitude of the current and in sensitivity. Fig. 2 A illustrates a series of superimposed flash responses recorded from a dark-adapted rod. After a brief exposure to bright light that bleached 20% of the visual pigment, the cell was allowed to recover for 35 min until its dark (light-suppressible) current reached a new steady state level from which no further recovery occurred. The bleach caused a steady reduction in the dark current, as evidenced by the decreased amplitude of the saturating flash response . When the rod was then exposed to β-ionone, the dark current declined further within several seconds to a new, lower level , which persisted for as long as the cell remained in β-ionone solution. After returning the rod to normal saline solution, the dark current recovered to its bleach-adapted level within 5 min . The amplitudes of the flash responses in each condition were plotted against flash intensity to create the corresponding intensity–response curves . Bleaching increased the test flash intensity required to elicit a threshold response compared with the dark-adapted state (▪). Thus, the sensitivity of the cell after the bleach was lower than that of its dark-adapted state. Treatment of the bleached cell with β-ionone reduced further its sensitivity , at the same time decreasing the maximal response amplitude. After returning the rod to normal saline solution, both sensitivity and maximal response amplitude recovered to their bleach-adapted levels . Thus, β-ionone caused a reversible decrease in dark current and sensitivity in bleach-adapted rods. When cells were exposed to a sufficiently bright light to totally suppress the dark current and treated with β-ionone, only a small junction current was observed. Thus, the decreased amplitude of the responses elicited in β-ionone solution appeared to result from a decrease in the light-sensitive conductance. Section title: Photocurrent and Light Sensitivity Educational score: 4.1528496742248535 Domain: biomedical Document type: Study Language: en The effect of β-ionone on the dark current and the sensitivity was tested in a total of 36 bleach-adapted cells. All of them exhibited a decrease in both dark current and sensitivity in the presence of β-ionone. The magnitude of pigment bleaching ranged from 3 to 80%. β-Ionone concentrations used varied from 1 to 20 μM. For low concentrations of β-ionone (up to 5 μM), the decrease in the dark current was greater for larger bleaches until it saturated at ∼20% pigment bleach. For higher concentrations of β-ionone, the decrease in the dark current was also larger for larger bleaches, but the effect did not saturate in the range of bleaches that were tested. Owing to their variable nature, it was not possible to determine the exact relationship between the decrease in the dark current and the magnitude of the pigment bleach or the concentration of β-ionone. For this reason, we chose to restrict our analysis to the effect of 10 μM β-ionone in rods in which 20% of the pigment had been bleached. In a total of 21 cells tested under these conditions, the mean decrease in the dark current caused by β-ionone was 51 ± 8% (SEM, n = 21). The corresponding decrease in sensitivity was 0.91 ± 0.08 log units (SEM, n = 10). Section title: Dim Flash Kinetics Educational score: 4.317389488220215 Domain: biomedical Document type: Study Language: en The response amplitude is a linear function of light intensity at the lower end of the intensity–response curve for both rods and cones . This implies that for dim flashes, the photoresponse is equal to the linear sum of the responses to single photons. Thus, analysis of the kinetics of dim flash responses allows for investigation of the kinetics of the phototransduction cascade. Activation of transduction by a bleach or by background light accelerates the dim flash response . To investigate whether the effect of β-ionone on the dark current and photosensitivity is a result of activation of phototransduction as opposed to a nonspecific effect, we looked at its impact on the kinetics of dim flash responses. Fig. 3 shows normalized dim flash responses elicited from a cell, first in the dark-adapted state, 40 min after 20% of the pigment had been bleached, and then during and after exposure to β-ionone. As expected, bleaching accelerated significantly the photoresponse. After bleaching, the time to peak of the dim flash response was reduced by >25%. Treatment of the bleached cell with β-ionone resulted in further acceleration of the kinetics of the response. The effect of β-ionone was completely reversible and several minutes after returning the cell to normal saline solution, the response slowed to its pretreatment, bleach-adapted level. Thus, β-ionone caused a reversible acceleration of the dim flash response in a bleach-adapted rod. Section title: Rate of Guanylyl Cyclase Educational score: 4.335272789001465 Domain: biomedical Document type: Study Language: en To investigate the effect of β-ionone on phototransduction in detail, we performed a series of experiments designed to measure directly the rates of cyclase and phosphodiesterase and to evaluate their change when the cell was exposed to β-ionone (see materials and methods ). Fig. 4 shows typical results from one experiment in which cyclase was studied. Using the microperfusion system, the rod was quickly exposed to saline test solution containing 500 μM IBMX in order to block phosphodiesterase. Fig. 4 (left) shows current recordings during the solution change. Fig. 4 (right) plots d ( J 1/3 )/ dt as a function of time, where J = i / i D . The time course of the solution change in both cases is shown on the top of the figure. Since the derivative of J 1/3 is proportional to the rate of change in cGMP concentration (see materials and methods , Eq. 2 ), its maximum represents a measure of the rate of synthesis of cGMP by guanylyl cyclase. The rate of cyclase was first measured in the dark-adapted cell . Here, cyclase had only a low basal activity. After a 20% bleach followed by a recovery period, the rate of cyclase was measured once again . The rate of current increase upon jumping into IBMX solution was higher than in the dark-adapted state . As shown previously , the rate of cyclase increases as a result of a bleach, as can be seen here from the greater than threefold increase in the peak of the derivative as compared with that measured before bleaching . Exposure of the bleached cell to β-ionone produced a twofold further acceleration of cyclase . The effect of β-ionone was completely reversed a few minutes after the cell was returned to normal saline solution . Thus, β-ionone caused a reversible acceleration of guanylyl cyclase in a bleach-adapted rod. Section title: Rate of Guanylyl Cyclase Educational score: 4.169776916503906 Domain: biomedical Document type: Study Language: en The effect of β-ionone on the rate of cyclase was studied in a total of 30 bleach-adapted rods. In all of them, β-ionone caused acceleration of cyclase. The range of pigment bleaches used was from 3 to 80%, and the range of β-ionone concentrations was from 3 to 20 μM. No clear quantitative correlation between the fraction of pigment bleached, or the concentration of β-ionone, and the corresponding acceleration of cyclase could be observed. However, in general, for a given bleached fraction of visual pigment, the relative rate of cyclase, compared with the bleach-adapted state, was higher for higher concentrations of β-ionone. On the other hand, for a given concentration of β-ionone, the relative acceleration of cyclase was higher for smaller bleaches. As in the experiments described in the previous section, we studied in detail the effect of 10 μM β-ionone after a 20% pigment bleach. A total of 18 rods were studied under these conditions. The rate of cyclase in the presence of β-ionone was 2.5 ± 0.3 (SEM, n = 18) times higher than in the corresponding bleach-adapted state. Section title: Rate of Phosphodiesterase Educational score: 4.161959171295166 Domain: biomedical Document type: Study Language: en To substantiate further the conclusion that β-ionone activates phototransduction, the rate of phosphodiesterase was measured within 2 min of the cyclase rate measurement in these same rods. The minimum time between the two measurements was limited by the time required for the new test solution to fill the dead space in the microperfusion tubing. Since the current was at steady state while the cell was exposed to β-ionone, the concentration of cGMP should be constant, and the rates of its synthesis by cyclase and its hydrolysis by phosphodiesterase should be equal. The activation of cyclase as a result of treatment of the bleached cell with β-ionone then should be accompanied by a corresponding acceleration of phosphodiesterase. Section title: Rate of Phosphodiesterase Educational score: 4.210229873657227 Domain: biomedical Document type: Study Language: en Fig. 5 shows one example of an experiment in which the rate of phosphodiesterase was measured. In this case, cyclase was blocked by suddenly exposing the cell to a test solution in which Li + was substituted for Na + on an equimolar basis. The time course of the jump into Li + solution is shown at the top of the figure. Fig. 5 (left) shows the current recordings from the cell; Fig. 5 (right) shows semi-logarithmic plots of the normalized current as a function of time. Here, the slope is proportional to the rate of the phosphodiesterase (Eq. 5 ). As with cyclase, bleaching produced an acceleration of phosphodiesterase , and subsequent treatment with β-ionone resulted in an even higher enzymatic rate . Again, the effect of β-ionone was completely reversible upon return of the cell to normal saline solution . Thus, β-ionone caused a reversible acceleration of cGMP phosphodiesterase in a bleach-adapted rod. Section title: Rate of Phosphodiesterase Educational score: 4.18336296081543 Domain: biomedical Document type: Study Language: en The effect of β-ionone on phosphodiesterase was measured under the same bleaching conditions and the same β-ionone concentrations as cyclase (see the previous section) in a total of 30 cells. In 25 cells, β-ionone caused acceleration of phosphodiesterase and in the remaining 5 either no effect or a small deceleration was observed. However, in 4 of these 5 cells the fraction of the bleached pigment was 40% or higher. We interpret the inconsistency in these cells as being due to the inability of the Li + jump method to resolve the high rate of phosphodiesterase after large bleaches (see materials and methods ). This speculation is supported by the fact that in contrast to phosphodiesterase, cyclase was accelerated in these cells and there was a decrease in both the dark current and the sensitivity as a result of the treatment with β-ionone. In the remaining 25 cells, the acceleration of phosphodiesterase was higher after smaller bleaches for each β-ionone concentration. In 17 experiments similar to those presented in Fig. 5 , we examined the effect of 10 μM β-ionone after a 20% pigment bleach. In these cells, the rate of phosphodiesterase in the presence of β-ionone was 1.7 ± 0.2 (SEM, n = 17) times higher than in the corresponding bleach-adapted state. Section title: Phototransduction in a Dark-adapted Rod in the Presence of β-Ionone Educational score: 4.260398864746094 Domain: biomedical Document type: Study Language: en Experiments were designed to investigate the possible site of action of β-ionone on phototransduction. Specifically, we were interested in testing the hypothesis that β-ionone activates transduction by binding directly in the chromophore pocket of opsin. To test this idea, we compared the effect of β-ionone in a dark-adapted and background light-adapted rod with its effect after a bleach. As a first step, we examined the effect of β-ionone in dark-adapted cells. A total of 16 cells were tested. In a small fraction of these, treatment with β-ionone resulted in a slight activation of phototransduction. The decrease in the dark current, the loss of sensitivity, and the acceleration of cyclase and phosphodiesterase produced by β-ionone in these dark-adapted rods, although significant, was small compared with the effect of β-ionone seen after 20% or higher pigment bleaches. The observed acceleration of phototransduction in some of the control experiments could be due to the fact that some of the cells were not completely dark adapted and contained some small fraction of bleached pigment. In accord with this notion, we observed that β-ionone activates phototransduction even after bleaches as low as 3%. Because the cells were exposed to dim red and infrared light during preparation, it is possible that in some cases a small but significant fraction of pigment was bleached. This idea is supported by the observation that the sensitivity of the cells slightly increased during the first 30–60 min after the dissection (data not shown), despite the fact that the animals were dark-adapted overnight. The interaction of the pigment bleached during the dissection with β-ionone could produce the observed activation of phototransduction. Section title: Phototransduction in a Dark-adapted Rod in the Presence of β-Ionone Educational score: 4.2630205154418945 Domain: biomedical Document type: Study Language: en To test this hypothesis directly, two dark-adapted cells that showed some acceleration of the transduction cascade during exposure to β-ionone were treated with 11-cis retinal in an effort to regenerate any residual bleached pigment. In both cells, about 1 h after addition of 11-cis retinal, the effect of β-ionone on sensitivity, dim flash kinetics, and cyclase and phosphodiesterase velocities was either significantly reduced or absent. Thus, complete regeneration of the pigment abolished the effect of β-ionone. Another argument, consistent with this notion, is based on the following observation: a hemisected eyecup was dark adapted for 24 h at 4°C after the dissection. Treatment of five cells from this preparation with β-ionone produced no effect, presumably because the pigment bleached during the dissection was regenerated in the intact retina during this additional dark period. Taken together, these observations indicate that treatment with β-ionone produces a small effect on transduction in freshly dissected dark-adapted cells and no effect in fully dark-adapted cells where all of the pigment has been regenerated. Section title: Rate of Guanylyl Cyclase and Phosphodiesterase in Bleached vs. Light-adapted Cell Educational score: 4.2170562744140625 Domain: biomedical Document type: Study Language: en As a second step in investigating the possible site of action of β-ionone, we compared its effect in a background light-adapted rod with its effect after a bleach. In both cases, the level of phototransduction activity is higher than in the dark-adapted state. However, the background light intensity required to produce activation comparable with that of a significant pigment bleach is so low that it only photoactivates a negligible fraction of the pigment. For instance, a steady background producing activation of cyclase (phosphodiesterase) equivalent to a 20% bleach would bleach <0.03% of the pigment in 1 h. Thus, even though both bleaching and background light produce elevation of the rate of phototransduction, the retinal binding pocket on opsin may be free and available for binding only in the bleached state. Section title: Rate of Guanylyl Cyclase and Phosphodiesterase in Bleached vs. Light-adapted Cell Educational score: 4.202095031738281 Domain: biomedical Document type: Study Language: en In four rods, the effect of β-ionone on cyclase in background light was directly compared with its effect on cyclase after a bleach. Each cell was first exposed to a series of backgrounds of increasing intensity. For each background, the rate of cyclase was measured in both saline solution and in β-ionone solution. An example at one background is shown in Fig. 6 , A (saline solution) and B (β-ionone). The cell was then bleached and allowed to recover to a steady state. The rate of cyclase was measured once again in saline solution , and then in β-ionone (D). Comparison between Fig. 6 , A and C, shows that one of the backgrounds induced exactly the same activation of cyclase as the subsequent bleaching. β-Ionone failed to produce any effect on the rate of cyclase in the background light-adapted state , but in contrast increased significantly the rate of cyclase after the cell was bleached (D). This result indicates that the activation of phototransduction is not sufficient to induce acceleration of cyclase by β-ionone. Section title: Rate of Guanylyl Cyclase and Phosphodiesterase in Bleached vs. Light-adapted Cell Educational score: 4.144697189331055 Domain: biomedical Document type: Study Language: en In an experiment similar to the one just described, the effects of β-ionone on the rate of phosphodiesterase in the light- and bleach-adapted states were also compared. In the two tested rods, β-ionone only accelerated phosphodiesterase in the bleach-adapted state, but did not affect the background light-adapted state. This result complements the result for cyclase described in the previous paragraph. Taken together, they indicate that β-ionone activates phototransduction only in the presence of bleached pigment. This observation supports the hypothesis that β-ionone affects phototransduction by interacting selectively with bleached pigment. Section title: Time Course of the Effect of β-Ionone After a Bleach Educational score: 4.279671669006348 Domain: biomedical Document type: Study Language: en The results illustrated in Figs. 7 and 8 are from an experiment designed to determine the time course of onset of transduction activation triggered by β-ionone. Since for the first few minutes after bleaching the rates of both cyclase and phosphodiesterase are too high to be measured reliably with a solution step, we chose the amplitude of the dark current as an index of the level of transduction. The cell was exposed to saturating test flashes to measure the dark current first in saline solution and then 25 s after exposure to β-ionone solution. The fact that the effect of β-ionone is completely reversible allowed us to do repetitive measurements in the course of recovery of the cell after the bleach. Current recordings from one such experiment are presented in Fig. 7 . Fig. 7 A shows recordings from one rod in its dark-adapted state and at different times after the bleach. In the dark-adapted cell, a jump into β-ionone caused only a small junction current shift, but not a change in the amplitude of the saturating photoresponse. Thus, as described above, β-ionone did not affect the light-sensitive current in the dark-adapted cell. After bleaching 20% of the pigment, the current was initially completely suppressed, and then started to rapidly recover. During this period when the dark current was increasing quickly, estimation of the effect of β-ionone was rendered unreliable by the fact that measurements of the current in saline solution and in β-ionone were made 45 s apart. Several minutes after the bleach, the recovery of the cell slowed down sufficiently to allow reliable comparison of the dark current in saline solution and in β-ionone solution without any significant drift in the current during the 45 s between test flashes. Jumping into β-ionone solution produced a drop in the current whose total amplitude increased with time after the bleach. The kinetics of the decrease in the current caused by β-ionone were carefully examined. The effect of β-ionone on the current at different times after the bleach can be seen in Fig. 7 B. These recordings are from the same cell as Fig. 7 A, but are superimposed and plotted on a different time scale. For each of the traces recorded after the bleach, the current decrease caused by β-ionone could be described by a single exponential decay function. For the cell shown on Fig. 7 , the time constant of these exponential functions changed from 12 s, measured 13 min after the bleach, to 3.7 s 1-h later. The time constant of the exponential current decay decreased with time after the bleach in all nine rods tested. On average, the decrease of the current caused by β-ionone shortly after the bleach was two times slower than the decrease in the current recorded 1-h later. Section title: Time Course of the Effect of β-Ionone After a Bleach Educational score: 4.335844993591309 Domain: biomedical Document type: Study Language: en Fig. 8 shows the time course of the effect of β-ionone on the dark current amplitude for the same cell as a function of time after the bleach. Fig. 8 A presents the amplitude of the current in saline solution and in β-ionone in the dark-adapted cell and at different time points after the bleach. As pointed out above, the current immediately after the bleach was completely suppressed. The amplitude of the current in β-ionone 7 min after the bleach was slightly higher than the corresponding amplitude in saline solution measured 45 s earlier. As discussed above, we interpret this observation not to be a result of the treatment with β-ionone, but rather to follow from the fast recovery of the cell during that period. 13 min after the bleach, the amplitude of the current in β-ionone was already clearly smaller than the corresponding current in saline solution and with time that difference became more pronounced. To estimate the rate of the increase of this effect, we calculated the relative decrease in the current caused by β-ionone as a function of time after the bleach. Fig. 8 B shows the ratio of the amplitudes of the current in β-ionone and in saline solution from A, plotted against the time after the bleach. The decrease of that ratio could be fit by a single exponential decay function with a time constant of 13 ± 1.1 (SEM) min. The average time constant for all cells tested in this way was 13 ± 2.1 (SEM, n = 8) min. This rate of increase of the effect of β-ionone as a function of time after a bleach is of the same order as the rate of decay of photoactivated pigment to free opsin and all-trans retinol derived from spectrophotometric experiments or with early receptor potential measurements . The fact that the amplitude of the effect of β-ionone correlates with the concentration of free opsin in the cell provides another argument in support of the notion that β-ionone exerts its effect by interacting exclusively with free opsin. Section title: Effect of β-Ionone on Phototransduction in a Bleach-adapted Rod Educational score: 4.479435920715332 Domain: biomedical Document type: Study Language: en Our experiments show that in bleach-adapted rods β-ionone causes (a) a decrease in the dark current, (b) a decrease in the sensitivity, (c) an acceleration of the dim flash photoresponse, and (d) an increase in the rates of guanylyl cyclase and cGMP phosphodiesterase. Together, these results indicate that in bleach-adapted rods β-ionone acts as an agonist and activates phototransduction in the dark. β-Ionone produces no effect in fully dark-adapted cells, where the basal level of transduction is low and the chromophore pockets of the opsin molecules are still occupied by native 11-cis retinal. Furthermore, β-ionone produces no effect in background light-adapted cells, where the level of transduction is high but still most of the chromophore pockets are occupied by 11-cis retinal. The rate at which the amplitude of the observed effect increases in time after the bleach is of the same order as the rate of decay of photoactivated rod pigment to free opsin. Based on these observations, we conclude that the effect of β-ionone is the result of its direct interaction with the free opsin molecules produced by the bleaching light. Section title: Effect of β-Ionone on Phototransduction in a Bleach-adapted Rod Educational score: 4.665852069854736 Domain: biomedical Document type: Study Language: en We suggest that β-ionone exerts its physiological effect by binding directly in the chromophore pocket of free opsin. Fig. 9 presents a model of the proposed mechanism of activation of phototransduction by β-ionone in rods. The chromophore pocket of opsin is shown schematically and the points of interaction of opsin with retinal or with β-ionone are indicated by a star. For each intermediate state, the relative enzymatic activity is indicated by the amplitude of the vertical bar to the right. In the dark-adapted state, 11-cis retinal is bound in the chromophore pocket of opsin and is covalently attached to it via a Schiff base linkage . The dark-adapted pigment is not capable of activating phototransduction beyond its basal level, as indicated by the absence of a vertical bar on the right. Absorption of a photon by the pigment molecule results in the photoisomerization of retinal and the full activation of the complex . Decay of this photoactivated rhodopsin produces all-trans retinol and free opsin. The bleached pigment exhibits low catalytic activity , resulting in a level of transduction in the bleach-adapted state higher than in the dark-adapted state. Binding of β-ionone in the free chromophore pocket of opsin produces a complex with catalytic activity that is higher than that of free opsin alone . The upregulation of phototransduction caused by this increase in the catalytic activity of the bleached pigment can be observed physiologically and is reported here. As expected for the noncovalent type of interaction between β-ionone and opsin, the binding of β-ionone is reversible and its removal from the pocket results in restoration of the bleach-adapted catalytic activity of free opsin . Section title: Contrast between the Effect of β-Ionone in Rods and Cones Educational score: 4.419572830200195 Domain: biomedical Document type: Study Language: en The effect of β-ionone in bleach-adapted salamander cones has been studied previously . In cones, β-ionone partially reverses the effect of bleaching and downregulates phototransduction. In other words, administration of β-ionone to a bleach-adapted cone restores some of the large loss in sensitivity produced by the bleach and reverses the acceleration of the dim flash response. The dark current recovers partially. Treatment of bleach-adapted cones with β-ionone also causes a decrease in the rate of guanylyl cyclase. These results on cones are in direct contrast with the results reported here for rods. Thus, the interaction of β-ionone with the chromophore-binding pocket of rod and cone opsins leads to opposite physiological effects. This observation resolves the current discrepancy between biochemical and physiological data and suggests that the biochemical observation that opsin is activated by the binding of retinoid in the chromophore pocket is correct but applies only to rod opsin rather than to both rod and cone opsin. Section title: Contrast between the Effect of β-Ionone in Rods and Cones Educational score: 4.141580581665039 Domain: biomedical Document type: Study Language: en The contrast between the chromophore pockets of rods and cones is not limited to the interaction with β-ionone. Jones et al. observed a significant decrease in the sensitivity of bleach-adapted rods treated with 11-cis retinol. In the same study, 11-cis retinol caused recovery of sensitivity in bleach-adapted cones. Together, these observations indicate that the contrast between rods and cones outlined here may be a fundamental feature of the interaction of the two types of opsin with their native chromophore. Section title: Nature of Bleaching Adaptation Educational score: 4.48913049697876 Domain: biomedical Document type: Study Language: en The state of opsin that is responsible for bleaching adaptation is a matter of controversy. According to one model, the free opsin produced after bleaching exhibits a low catalytic activity that sustains an elevated level of transduction in the bleached cell . An alternative view is that interaction of all-trans retinal with free opsin is the factor causing bleaching adaptation . A strong argument for a free opsin mechanism of bleaching adaptation deriving from our findings is that the occupancy of the chromophore pocket by β-ionone produces opposite effects in rods and cones. The noncovalent binding of all-trans retinal in the pocket would also be expected to produce opposite effects in rods and cones and therefore could not account for the bleaching adaptation in both photoreceptor types. In contrast, the sustained catalytic activity of free opsin could explain the elevated rate of phototransduction observed in both rods and cones after a bleach. Thus, the results from our experiments make it unlikely that bleaching adaptation is the result of the interaction of all-trans retinal with the chromophore pocket of free opsin. Section title: Nature of Bleaching Adaptation Educational score: 4.371402740478516 Domain: biomedical Document type: Study Language: en Finally, our observations of the effect of β-ionone in bleach-adapted rods may have important implications for the physiology of regeneration of rhodopsin in the intact retina. By analogy with the results reported here, binding of the native 11-cis retinal in the chromophore pocket of rod opsin might be expected to form an active noncovalent complex that results in transient upregulation of phototransduction and transient reduction in the sensitivity of the rod. In contrast, binding of retinal in the chromophore pocket of cone opsin might be expected to downregulate transduction, and thus contribute to the partial recovery of the sensitivity of the cone even before the formation of the Schiff base linkage and the regeneration of the pigment. Verification of this model will require further investigation. | Study | biomedical | en | 0.999998 |
10063307 | Section title: INTRODUCTION Educational score: 3.1745996475219727 Domain: biomedical Document type: Study Language: en Although the development mechanism of chronic viral hepatitis B(CVH-B), which is the most important etiologic factor in liver cirrhosis(LC) and hepatocellular carcinoma (HCC) in Korea, has not been fully understood until now, immune mechanism of the host(especially cellular immunity) seems to play a major role in this area 1 ) . Section title: INTRODUCTION Educational score: 4.025213718414307 Domain: biomedical Document type: Study Language: en Hepatitis B virus (HBV) by itself does not appear to induce hepatic lesion directly, and lysis of infected hepatocytes depends on the immune response of the host 2 ) . In these circumstance, IFN induces the recognition of cytotoxic T cells to infected hepatocytes and lysis of the infected hepatocyte 1 – 5 ) . Section title: INTRODUCTION Educational score: 4.0212297439575195 Domain: biomedical Document type: Study Language: en Immune system abnormality of the host accompanies incomplete removal of the infected hepatocytes and persistent replication of HBV introduces the development of CVH-B 6 , 7 ) . Several studies 8 – 10 ) reported that decrease of the IFN concentration, CD4/CD8 ratio and value of CD4, and increase of the value of CD8 were discovered in patients with CVH-B. These results suggested that the defective immune system of the host participated in the pathogenesis of CVH-B 11 – 15 ) . Section title: INTRODUCTION Educational score: 3.858980655670166 Domain: biomedical Document type: Study Language: en According to the above results, one hypothesis 18 , 19 ) was that therapeutic agents (which improve the host immunity and prevent the proliferation of HBV) prevent the progression of CVH-B to LC or HCC and, therefore, many antiviral and immunosuppressive agents were tried and variable therapeutic effects were observed 18 – 22 ) Among these, IFN may be the most valuable and effective treatment modality 15 – 22 ) . Section title: INTRODUCTION Educational score: 3.953298330307007 Domain: biomedical Document type: Study Language: en Therefore, the authors have undertaken a controlled study to determine the effectiveness of combination treatment with prednisolone withdrawal and IFN α-2b to find out the abnormalities of cellular immunity on patients with CAH-B, and compared the effectiveness of combination treatment with IFN α-2b with prednisolone withdrawal to IFN alone . Section title: 1. Patients Educational score: 3.738924503326416 Domain: biomedical Document type: Study Language: en Sixty two patients with CAH-B and 12 normal controls were examined. All patients had HBsAg, HBeAg and positive results for HBV DNA. Among these patients with CAH-B, 32 patients were treated with prednisolone withdrawal followed by IFN α -2b for a total of 6 months, and 30 patients were treated with conventional hepatotonics for the same duration as the control patient group. Section title: 1. Patients Educational score: 2.403346538543701 Domain: biomedical Document type: Study Language: en All cases of the normal control group had negative results for serum HBsAg and anti-HCV, and the results of LFT were within normal limits. Section title: 2. Treatment protocol Educational score: 2.0700979232788086 Domain: biomedical Document type: Other Language: en Patients had been taking 45 mg prednisolone per day for the initial 2 weeks and then 30 mg per day and 15 mg per day for the subsequent 2 weeks and, thereafter, they had not been taking any drug for 2 weeks. Section title: 2. Treatment protocol Educational score: 2.7211978435516357 Domain: biomedical Document type: Other Language: en IFN α -2b(Intron A®, Schering-Plough, Kenilworth, New Jersey, USA) was injected in doses of 3×10 6 units subcutaneously. The same dose was given every day during the first week and, thereafter, three times a week for the subsequent 15 weeks. Section title: 3. Serological assays, T cell subsets determinations and LFTs Educational score: 4.072591304779053 Domain: biomedical Document type: Study Language: en HBsAg, anti-HBs, HBeAg and anti-HBe were tested with radioimmunoassay using CIS® Kit(Abbott Laboratories, North Chicago, USA) and HBV-DNAs were quantitated by the spot hybridization 36 ) (EXPIDITE® Persepiv Biosystem INC, USA) 1 month before administration of prednisolone and 1 month after termination of IFN α-2b treatment in patient with CAH-B, and 1 month before the study in normal controls. Section title: 3. Serological assays, T cell subsets determinations and LFTs Educational score: 2.684673547744751 Domain: biomedical Document type: Study Language: en Anti-HCV was checked by second generation of enzyme immmunoassay(IMX®, Abbott GmbH Diagnostica, Max-Planck-Ring 2, Wiesbaden-Delkenhein, Germany) 1 month before the study in all patient and normal controls. Section title: 3. Serological assays, T cell subsets determinations and LFTs Educational score: 4.061993598937988 Domain: biomedical Document type: Study Language: en The subsets of lymphocytes were examined by flow cytometry(COULTER EPICS XL®, Coulter Corporation, Miami, Florida, USA) with mouse monoclonal antibody(OKT3, OKT4 and OKT8) 23 ) in all participants in this study at 1 month before administration of prednisolone. Thereby, peripheral total T cell fraction and T cell subsets were assessed in IFN α -2b treated patients with CAH-B at 1 month before administration of prednisolone and after IFN α-2b treatment. Section title: 3. Serological assays, T cell subsets determinations and LFTs Educational score: 4.074771881103516 Domain: biomedical Document type: Study Language: en Serum levels of aspartate aminotransferase(AST), alanine aminotransferase(ALT), alkaline phosphatase(AP), total bilirubin(TB), total protein(TP), albumin(Alb), blood urea nitrogen(BUN) and creatinine(Cre) were examined by 17 Hi-cell autoanalyzer before prednisolone treatment and 1, 3 and 6 months after the beginning of treatment with IFN α-2b . Section title: 3. Serological assays, T cell subsets determinations and LFTs Educational score: 2.6305222511291504 Domain: biomedical Document type: Study Language: en Blood cell counts were examined every 2 weeks during this study for the evaluation of leukopenia and thrombocytopenia. Section title: 3. Serological assays, T cell subsets determinations and LFTs Educational score: 4.006919860839844 Domain: biomedical Document type: Study Language: en The patients who were treated with prednisolone withdrawal and IFN α-2b were divided in to two groups, responder and non-responder, according to the response to treatment. The response was defined as one of these was achieved: normalization, disappearance of HBV DNA and seroconversion of HBeAg. Assessment was carried out at 1 month after IFN a-2b treatment and based on seroconversion of HBeAg and HBV DNA at 1 month after IFN α-2b treatment, and LFTs after 1, 3, and 6 months. Section title: 4. Statistical Analysis Educational score: 2.5700018405914307 Domain: biomedical Document type: Study Language: en All data were analysed by using a paired t-test and given as mean±SEM. Section title: 1. Clinical characteristics of the patients Educational score: 2.2810895442962646 Domain: biomedical Document type: Study Language: en Numbers, mean age(years old) and sex ratios of IFN α-2b treated patients with CAH-B, control patient group treated with conventional hepatotonics and normal control group were 32, 35.7, 28:4, 30, 31.6, 21:9 and (12, 33.1, 9:3. Section title: 1. Clinical characteristics of the patients Educational score: 2.037813901901245 Domain: biomedical Document type: Study Language: en Among these groups, age and sex ratios were not significantly different from each other( Table 1 ). All the clinical profiles and laboratory findings were not significantly different in both groups of patients at the beginning of this study(p>0.05). Section title: 2. Changes of the levels of liver function test and adverse effects according to IFN α -2b treatment in patients with CAH-B Educational score: 4.028957366943359 Domain: biomedical Document type: Study Language: en Both serum levels of AST and ALT were significantly decreased after IFN α-2b treatment(p<0.001) in patients with CAH-B. Section title: 2. Changes of the levels of liver function test and adverse effects according to IFN α -2b treatment in patients with CAH-B Educational score: 4.091884613037109 Domain: biomedical Document type: Study Language: en In 26(81.3%) of 32 patients with IFN α -2b treatment, levels of AST and ALT significantly decreased to normal or one and a half of normal level. On the other hand, levels of these enzymes in the control patient group decreased to normal range or one and a half of normal level in only 8 (26.7%)of 30 patients,(p<0.05). Section title: 2. Changes of the levels of liver function test and adverse effects according to IFN α -2b treatment in patients with CAH-B Educational score: 3.406852960586548 Domain: biomedical Document type: Study Language: en The levels of AP, TP, BUN, creatinine(Cre) and hemoglobin(Hb) had no significant difference statistically between pre- and post-treatment periods(p<0.05, Table 2 ). Section title: 2. Changes of the levels of liver function test and adverse effects according to IFN α -2b treatment in patients with CAH-B Educational score: 2.3171656131744385 Domain: biomedical Document type: Study Language: en Flu-like syndromes were observed in all patients, but the syndromes disappeared with sulindac preparation in all cases within 3 to 14 days. Section title: 2. Changes of the levels of liver function test and adverse effects according to IFN α -2b treatment in patients with CAH-B Educational score: 3.6816492080688477 Domain: biomedical Document type: Study Language: en Thrombocytopenia and leukopenia were noted in 7(21.9%) and 6 cases(18.8%) of the 32 patients respectively, and mostly prominent in the second week, but almost all of the patients had improved during IFN α -2b treatment. Section title: 3. Changes of HBeAg, Anti HBe and HBV DNA in patient group with IFN α -2b treatment and control patient group Educational score: 4.135086536407471 Domain: biomedical Document type: Study Language: en A marked reduction of serum HBV DNA was noted in the IFN α-2b group as compared with the patient control group. In the IFN α -2b group, loss of HBV DNA occurred in 21 of 32 cases(65.6%) and was significantly more frequent than in control patient group(7 of 30 cases, 23.3%, p<0.05) ( Table 3 ). Section title: 3. Changes of HBeAg, Anti HBe and HBV DNA in patient group with IFN α -2b treatment and control patient group Educational score: 4.111354827880859 Domain: biomedical Document type: Study Language: en A seroconversion rate of HBeAg was significantly higher in the IFN α -2b treatment group(12 of 32 cases, 37.5%) than in patient control group(7 of 30 cases, 23.3%, p<0.05). None of both the IFN α -2b treatment group and patient control group had resulted in loss of HBsAg( Table 3 ). Section title: 4. Comparison of the peripheral total lymphocytes and T cell subsets between IFN α -2b treatment group and normal control group Educational score: 4.128462791442871 Domain: biomedical Document type: Study Language: en Before administration of prednisolone, the numbers of CD3 cells of the IFN α -2b group were similiar to those of the control group(p>0.05), CD4 cells were significantly reduced in the IFN α -2b group than in the control patient group(36.3±7.7% vs 42.1±5.7%, p<0.05) and CD8 cells also were significantly increased in the IFN α -2b group than in that of the control group(30.6±10.3% vs 24.3±5.2%, p<0.05). The ratio of CD4 to CD8 cells was more decreased in the IFN α -2b group than in the control group, but was statistically insignificant (p>0.05) ( Table 4 ). Section title: 5. Comparison of T cell subsets between the responder group and non-responder group for IFN α -2b treatment with prednisolone withdrawal Educational score: 4.12587833404541 Domain: biomedical Document type: Study Language: en Before administration of prednisolone, CD3 cells in the responder group were slightly higher than those of the non-responder group, but insignificant(p>0.05). CD4 cells in the responder group were significantly lower than in the normal control group(35.2±6.8% vs 42.1±5.7%, p<0.05). CD8 cells in both responder and non-responder group were higher than in the normal control, but insignificant(31.6±9.6%, 31.0±8.9% vs 24.3±5.2%, p>0.05)( Table 5 ). Section title: 6. Comparison of changes of T cell subsets according to IFN α -2b treatment between responder and non-responder group Educational score: 4.117156028747559 Domain: biomedical Document type: Study Language: en In the responder group, numbers of CD4 cells were increased after IFN α -2b treatment, but statistically insignificant(35.2±6.8% vs 40.0±9.3%, p>0.05) and numbers of CD8 cells were decreased but insignificant also( Table 6 ). In contrast to the responder group, numbers of CD4 cells were decreased from 36.9±8.3% to 35.4±4.0% and numbers of CD8 cells were increased from 31.0±8.9% to 33.9±9.7% after IFN α -2b treatment in the non-responder group, but these results were not statistically significant, too(p>0.05, Table 6 ). Section title: DISCUSSION Educational score: 2.4669911861419678 Domain: biomedical Document type: Other Language: en CAH-B is one of the most important causes of the development of LC and HCC. Its clear pathogenesis was not proven until now, but defective cellular immunity of the host has been suggested as playing an important role in the development of CVH-B 1 – 5 ) . Section title: DISCUSSION Educational score: 4.174231052398682 Domain: biomedical Document type: Study Language: en With HBV infection to the hepatocytes, HBeAg and HBeAg were expressed on the surface of the hepatocyte 24 ) , and major histocompatability antigen complex-I(MHC-I) was expressed concomittently 4 ) . These MHC-I and HBeAg or HBeAg interact synergically and induce cytolysis of the infected liver cells by the cytotoxic T cell 5 ) . Section title: DISCUSSION Educational score: 4.220802307128906 Domain: biomedical Document type: Study Language: en IFN is released by activated lymphocytes and activates the intracellular enzyme(2,5-oligoadenylate synthetase) and this enzyme induces the release of ribonuclease which destroys viral mRNA, and eventually prevents viral translation and propagation to the adjacent tissue 5 ) . IFN increases the expression of MHC-I on the hepatocytes also, and induces the cytolysis of the infected hepatocytes by cytotoxic T cell 5 ) . Section title: DISCUSSION Educational score: 3.326385259628296 Domain: biomedical Document type: Study Language: en Several studies reported that reduced serum IFN level and qualitative or quantitative abnormalities of cellular immunity of the host were discovered in patients with CVH-B 8 – 10 ) . Section title: DISCUSSION Educational score: 3.9794845581054688 Domain: biomedical Document type: Study Language: en Many reports for T cell subsets in CVH-B showed variable results, but usually many authors 10 , 11 , 24 , 25 ) agree that decreased ratio of CD4 to CD8 cells is largely dependent on significantly decreased CD4 cells in patients with CVH-B. Barnaba et al 26 ) had demonstrated that the ratio of CD4 to CD8 cells was decreased during acute viral hepatitis B and that it resulted from increased CD8 cells or decreased CD4 cells or both. Thomas et al 12 ) also reported similiar results in chronic liver disease and that it resulted from decreased CD4 cells mostly. Section title: DISCUSSION Educational score: 2.8345859050750732 Domain: biomedical Document type: Study Language: en In this report, increase of CD8 cells appeared but was statistically insignificant. In our study, decreased CD4 cells and increased CD8 cells were also significantly noted( Table 4 ). Section title: DISCUSSION Educational score: 4.04905891418457 Domain: biomedical Document type: Study Language: en Similiarly as in previous reports 11 , 12 , 24 – 26 ) , the ratio of CD4 to CD8 cells was decreased in patients with CAH-B as compared with normal control in our study too, but was statistically insignificant(p>0.05, Table 4 ). These results may be due to both an decrease of CD4 cells and an increase of CD8 cells. In our study, CD4 cells of patients were lower significantly in the responder group compared with that of the normal control group, but insignificant in the non-responder group( Table 5 ). It is suggested that decrease of CD4 cells may be used as a potential prognostic factor to predict the course of CVH-B, but further studies can be assessed to confirm this suggestion. Section title: DISCUSSION Educational score: 4.087085723876953 Domain: biomedical Document type: Study Language: en In our study, a larger decrease of CD4 cells and a larger increase of CD8 cells also appeared in the responder group than in the non-responder group at the pre-treatment period. It is interesting that CD4 cells have increased significantly from 35.2±6.8% to 40.0±9.3% (P<0.05) and that CD8 cells have decreased from 31.6±9.6% to 30.2±8.8%(P>0.05) in the responder group, but were insignificant in the non-responder group in our study( Table 6 ). Section title: DISCUSSION Educational score: 4.03333044052124 Domain: biomedical Document type: Study Language: en Many authors agree that persistent proliferation of HBV may play an essential role in the progression of CVH to LC or HCC 6 , 7 ) . In our study, all of the patients with CVH-B had HBeAg and HBV DNA which were serologic markers for proliferation of HBV( Table 3 ). Loss of these markers was accompanied by improvement of symptoms and laboratory data 27 – 29 ) . These findings propose that various antiviral therapies will prevent the progression of CVH-B to LC or HCC. Section title: DISCUSSION Educational score: 3.1605112552642822 Domain: biomedical Document type: Study Language: en In various reports, Immunosuppressants(prednisolone, azathioprine etc) and several antiviral or immunomodulating agents(adenosine arabinoside, adenosine arabinoside monophosphate, IFN, and interleukin-2 etc) had been studied and many authors agree that IFN is the most effective agent among these 15 – 22 , 30 ) . Section title: DISCUSSION Educational score: 3.9881374835968018 Domain: biomedical Document type: Study Language: en Yasushi et al 30 ) reported that loss of HBV DNA occurred in 42% with treatment of IFN α for 12 to 24 months, and 54% in Lok et al 17 ) and 52% in Thomas et al 18 ) . In Korea, Choi et al 15 ) and Choi et al 16 ) reported that seroconversion rates of HBeAg were 25% and 17.6% apart. These results were superior to the spontaneous conversion rate of HBeAg in Korea reported by Yoon et al 31 ) , but inferior to those in western countries 16 – 18 ) . Section title: DISCUSSION Educational score: 3.0523741245269775 Domain: biomedical Document type: Study Language: en However, the reason why these results were worse than western countries is suspected as follow; first, transmaternal transmission of HBV was more common in Korea and patients with CVH-B occurred in this manner were more resistent to IFN treatment and, second, we commonly used a lower dose than western countries 16 – 18 ) . Therefore, if the dose of IFN is increased, a better response will be acquired. Section title: DISCUSSION Educational score: 4.063802719116211 Domain: biomedical Document type: Study Language: en Recently, several studies 32 , 33 ) reported that IFN therapy with prednisolone withdrawal may be more effective for the seroconversion of HBeAg than in IFN therapy alone. In Korea, Kim et al 34 ) reported that combined therapy with IFN and short-term prednisolone withdrawal was more effective(50% rate of seroconversion) than single IFN therapy. It has been demonstrated that seroconversion of HBeAg occurred in 12 cases(37.5%) of 32 patients with interferon treatment, and it is significantly higher than in the control patient group(7 Of 30 cases, 23.3%, p<0.05) in our study( Table 3 ) and this result was inferior to Kim et al 34 ) , but superior to Choi et al 15 ) (17.6%) and Choi et al 16 ) (25.0%). Therefore it may be suggested that combination therapy of IFN with prednisolone withdrawal is more effective than IFN alone. Section title: DISCUSSION Educational score: 3.8731133937835693 Domain: biomedical Document type: Study Language: en Disappearance of HBV DNA occurred in 21 cases(65.6%) of 32 patients and these results were similiar to those of other previous reports. The above results were significantly better than the control patients. Loss of HBV DNA occurred faster than seroconversion of HBeAg and similiar results were reported by other authors 37 – 39 ) . Section title: DISCUSSION Educational score: 3.8177132606506348 Domain: biomedical Document type: Study Language: en The good responder group for the IFN treatment includes the patients with recently acquired infection, higher levels of serum aminotransferases, lower serum level of HBV DNA and active hepatitis on liver biopsy. Heterosexuals and women respond better than homosexuals and men 35 ) . However, in our study, the levels of aminotransferase and sex difference did not influence the prognosis of IFN treatment(p>0.05). | Study | biomedical | en | 0.999997 |
10063308 | Section title: INTRODUCTION Educational score: 4.26658821105957 Domain: biomedical Document type: Study Language: en Helicobacter pylori infection is now recognized as the major cause of chronic gastritis throughout the world 1 – 3 ) . A fraction of infected persons developed peptic ulcer disease 4 ) or gastric cancer 2 , 5 , 6 ) , accounting for its clinical significance. The pathophysiology of this infection can be better understood by several concepts, such as heterogeneity of strains 7 ) , persistence of infection 8 ) , immunological down regulation 9 ) , physiological consequences and variability in outcome 10 ) . Microbial, host and environmental factors must contribute to the outcome variation, respectively. Especially, the relapse of H. pylori has been reported after antimicrobial therapy 11 – 13 ) , and its transmission is still unknown. The fact that H. pylori can convert under unfavorable conditions into a metabolically active but non-culturable coccoid state has stimulated speculation about its role in transmission and reinfection 14 , 15 ) . Some investigators 16 – 18 ) suggested that the coccoid forms are degenerative and have no potentiality of infection, like Campylobacter jejuni , and others 14 , 19 – 21 ) supposed that they are dormant and one stage of the biological cycle of H. pylori , like Vibrio vulnificus . Some reported some studies 22 , 23 ) have suggested that the coccoid forms of H. pylori could be potentially viable. However, regrowth of H. pylori from the coccoid forms could not be possible. In the present study, to elucidate the viability of coccoid forms, we induced conversion from bacillary to coccoid forms and studied the morphological changes and antigenic evolutions during coccoid conversion. Section title: 1. Strain and culture conditions Educational score: 4.1880998611450195 Domain: biomedical Document type: Study Language: en The H. pylori strain used (C001) was isolated in the Research Institute for Gastroenterology of Dankook University (Chunan, Korea) from a gastric biopsy of a patient with gastric cancer. Cells were grown at Brucella blood agar with 5% horse serum and antimicrobial agents, at 37°C under microaerophillic conditions with 10% CO 2 and 5% O 2 . Added antimicrobial agents were vancomycin (10 mg/L), colistin (5 mg/L), trimethoprim (5 mg/L) and amphotericin B (5 mg/L). The cells of a 2day culture of H. pylori C001 were harvested from one plate, suspended in phosphate-bufferd saline (PBS, pH 7.4), and adjusted to a turbidity of 1 MacFarland unit. After Gram staining, it was microscopically observed that this suspension contained only bacillary forms. The suspension was then used to innoculate 10 plates of Brucella blood agar (0.1 ml/plate). The plates were incubated at 37°C under microaerophillic conditions. The cells of one plate were harvested daily and suspended in 1 ml of PBS (pH 7.4). Enumeration of colony forming unit were performed by standard serial dilution and plate count procedures. Total cell (bacillary or coccoid forms) counts were assessed by turbidimetry at 540 nm. To each suspension, Gram-stained smears were used to assess the relative percentages of coccoid and bacillary forms. These measurements were made blindly by 30 different persons, and the averages of their determinations were considered as the final results. Also, the suspensions of day 2, 7, 9 and 15 were used for electron microscopic examinations. Section title: 2. Preparation for electron microscopic examination Educational score: 4.036088943481445 Domain: biomedical Document type: Study Language: en Bacteria were harvested from plates, fixed for microscopy with 2.5% glutaraldehyde in 100mM sodium cacodylate buffer. Cells were concentrated by centrifugation and samples were removed for examination by differential interference contrast light microscopy. To assist in identification, selected samples were also negatively stained with 1% uranyl acetate for transmission electron microscopy (TEM). Section title: 3. Western blotting Educational score: 4.225968837738037 Domain: biomedical Document type: Study Language: en The evolution of the antigenic profiles during coccoid conversion of H. pylori C001 was studied by Western blotting, using sera from thirty patients known to be colonized by H. pylori . Total antigens of H. pylori C001 were obtained by sonification of bacterial cells harvested at different stages of rod and coccoid, respectively, and suspended in PBS (pH 7.4). After sonification, the unlyzed bacteria were eliminated by centrifugation , and the supernatants were saved, adjusted to 1 ml of protein per ml, and frozen at −70°C until use. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the method of Laemmeli 24 ) with a 4% stacking gel and a 10% separating gel. Prior to electrophoresis, the protein solutions were heated at 100°C for 5 min in a sample buffer containing 5% (wt/vol) SDS and 0.42% (wt/vol) 2-mercaptoethanol. Electrophoresis was conducted for 1 hour under a constant voltage (15 v/um) using the minigel system (Bio-Rad). Proteins were blotted onto a pre-wetted nitrocellulose membrane (Bio-Rad) by using a Mini Trans-Blot electrophoretic transfer cell (Bio-Rad) under a constant current of 200 mA for 1 hour. The blots were incubated for 1 h with human sera diluted at 1/100; they were then rinsed and incubated in goat serum anti-human IgG conjugated with alkaline phosphatase (Dakopatts, Copenhagen, Denmark). After a final wash, the nitrocellulose filters were developed with 5-bromo-4-chloro-3-indolylphosphosphate (BCIP) as a substrate, and nitroblue tetrazolium as a chromogenic indicator. The reactions were stopped after 20 min by washing the filters extensively with distilled water. Section title: 1. Examination of morphological changes Educational score: 4.226076126098633 Domain: biomedical Document type: Study Language: en As assessed by turbidimetry, the bacterial mass increased from day 1 to day 3. The maximal active growth was observed on day 2–3 (36 hours). The stationary phase was day 4–6. The proportion of coccoid forms increased from 0 to 100% from day 2 to day 15 . Between day 5 and day 9, the fraction of coccoid forms on the plates was increased. Transmission electron micrographs at day 2 showed spiral rod forms. At day 7, the relative number of bacillary forms decreased and U-shaped forms became predominant, and they looked like invaginated bacilli. At day 9, U-shaped forms were converted to doughnut-shaped forms. At day 15, only globular full coccoid forms were observed . The ultrastructure of the inner cell side was not observed. Section title: 2. Evolution of antigens of H. pylori during coccoid conversion Educational score: 4.249167442321777 Domain: biomedical Document type: Study Language: en The evolution of the antigenic profiles during coccoid conversion of H. pylori was studied by Western blotting, using different sera from culture positive patients. These sera were used to reveal the total antigens of the strain cultured for 2 day (0% coccoids) and 15 days (>99% coccoids). SDS-PAGE analysis of whole cell preparations of H. pylori showed numerous bands between 30 and 125 kDa. These proteins included CagA at 125 kDa, VacA (vacuolating cytotoxin) at 88 kDa, an adhesin and porin at 35 kDa and urease subunit at 30 kDa 25 , 26 ) . The antigenic profiles were not changed in 46.7% (14/30 cases) and were changed in 53.3% (16/30 cases) during coccoid conversion. Antigenic fractions changed during coccoid conversion were protein band at 125 kDa and band at 35 kDa, which were intensively detected in bacillary forms. Those proteins which disappeared included CagA and porin, outer membrane . Disappearance of CagA protein (125 kDa) during coccoid conversion was observed in 68.8% (11/16 cases) and disappearance of porin (35 kDa) in 62.5% (10/16 cases). The rest of the profiles were identical between rod and coccoid forms. Section title: DISCUSSION Educational score: 4.672504901885986 Domain: biomedical Document type: Study Language: en Helicobacter pylori , a gram negative spiral bacterium, can survive in such diverse environments as the human stomach of low pH 23 ) , gut of high osmolarity 27 ) and water 28 ) . In order to adapt to such crucial conditions, the bacteria should carry systems that respond to changes in nutrient, osmolarity, temperature and other external factors. The organisms exist in two forms, an actively dividing spiral forms and a coccoid form of arrested growth under various stress, including the starvation for nutrients 29 ) , extended incubation 28 ) , accumulation of metabolic products 14 ) , pH alteration 14 ) and exposure to antimicrobial agents 30 ) . The viability of the coccoid form of H. pylori , the possible role of this form in transmission and as a cause of reinfection is controversial 14 , 15 ) . Although the mode of transmission still remains unclear, oral-oral and oral-fecal transmission have been suggested 31 , 32 ) . If H. pylori follows the latter route, they must pass through the anaerobic atmosphere of the alimentary tract which is an adverse situation for H. pylori . Under these stressed conditions, infected bacillary forms might be changed to coccoid form. Shirai et al. 20 ) have reported that almost 100% cells changed to coccoid-like bodies within 24 hr of anaerobic incubation, and 0.1–1% produced colonies on Brucella agar. Also, they revealed that the colonies appeared from coccoid bodies which remained viable under anaerobic conditions, although some of them appeared from a few spiral bodies, thereby they assisted H. pylori in passing the adverse anaerobic route of the human alimentary tract by changing their morphology. However, it is still unknown whether coccoid forms of H. pylori can revert to vital organism in vivo or if they are of any patho-physiological significance at all. According to some reports 16 – 18 ) , the coccoid forms are degenerative and incapable to form complex adhesions and, hence, are of low pathogenic potential. But others 14 , 19 – 21 ) suggested that coccoid forms are dormant cells and viable cells. Vijayakumari et al. 19 ) revealed that specialized attachment sites were seen in the interaction between coccoids and epithelial cells of KATO III cell, and these adherence patterns were similar to those observed with spiral forms in vivo, suggesting a possible pathogenic role for the coccoids of H. pylori . Also, with antigens prepared from both coccoid and spiral forms, immunoreactive protein bands of 128, 116, 110, 95, 91, 66, 60, 54, 50 and 33 kDa were conserved in both the coccoid and spiral forms by the results of Western blotting. Vijayakumari et al. 19 ) suggested that the coccoids could be differentiated infective form of H. pylori , and that they could evoke an immune response from the host after attachment to gastric epthelial cells. We found the morphological changes during coccoid conversion by the transmission electron microscopy. Spiral bacilli forms at day 2 converted to full coccoid forms at day 15, through the U-shaped forms at day 7, and doughnut-shaped forms at day 9. Although we could not detail the change of ultrastructure during coccoid conversion under our transmission electron microscopy, coccoid forms were considered via U-shaped and doughnut-shaped forms. Benaïssa et al. 21 ) revealed that initiation of conversion from bacillary was the formation of dense periplasmic material, followed by an inwardly curved formation of bacilli as an intermediate step between the bacillary and coccoid forms and, then, a change in the protoplasmic cylinder was evoked to full coccoid forms, strongly suggesting a transition of bacillary to full coccoid forms via U-shaped forms. The extracytoplasmic side of the invaginated membrane might serve as a site for the oxidation of toxic material, which has been described for Ecthiorhodospira mobilis and other spirilloid or vibroid gram negative organism 29 , 33 ) , just like H. pylori . Therfore, similar invagination of the membrane could consolidate the potential viability of H. pylori coccoids. On the antigenic evolution, we observed the identical antigenic profile between the bacillary and coccoid forms in 46.7%, and changed antigenic profile in 53.3%. Interestingly, disappeared protein bands during coccoid conversion were CagA and porin or adhesin, which were more intensively detected in bacillary forms. Those findings could suggest that virulent factors of H. pylon might vanish in some coccoid forms. Vijayakumari et al. 19 ) and Benaïssa et al. 21 ) have reported that the protein bands were the same pattern in both the coccoid and bacillary forms, which were a little bit different from our results. We think the same protein bands between the bacillary and coccoids highlight the significance of these antigens. However, we cannot clarify the role of the antigenic components absent in the coccoid forms. In conclusion, these results showed that coccoid forms of H. pylori retain antigenic characteristics similar to bacillary forms, and some of the antigens disappeared in coccoid forms. Therefore, coccoid forms might be viable, and represent one of the stages of the H. pylori biological cycle. However, it is still unclear whether the coccoid forms can revert to infective bacillary forms in vivo, whether they represent a temporary adaptation to a particular environment, and whether they are actually involved in the transmission of the bacterium. | Study | biomedical | en | 0.999997 |
10063309 | Section title: INTRODUCTION Educational score: 4.557106018066406 Domain: biomedical Document type: Study Language: en Helicobacter pylori (H. pylori) infection is now recognized as the cause of type B gastritis, as a critical factor in the development and the recurrence of duodenal ulcer disease, and as an essential co-factor in the development of gastric carcinoma and gastric MALT-lymphoma 1 – 5 ) . Although estimation of the lifetime risk of developing an ulcer in people with H. pylori infection is difficult, it is believed that approximatley 10 – 15 % of individuals with H. pylori infection may develop an ulcer. 6 , 7 ) The link between H. pylori and the development of peptic ulcer disease may be related to the inappropriate release of gastrin observed in H. pylori -positive patients. One of the most notable H. pylori -associated changes in gastric secretion is an increased gastrin release after meals and after bombesin stimulation. This abnormality is alleviated following eradication of the infection 8 – 14 ) . It has also been shown that H. pylori infection can induce reversible increased basal and gastrin mediated acid secretion 15 , 16 ) . Section title: INTRODUCTION Educational score: 4.1363725662231445 Domain: biomedical Document type: Study Language: en The mechanism by which H. pylori enhances gastrin release is not yet known but there have been increasing numbers of studies which show that changes in the numbers of antral G-cells and D-cells are responsible for the physiologic regulation of gastrin and gastric acid secretion. Many of the studies have suggested that H. pylori infection results in reduction of the number of antral D-cells and in the soamatostatin level resulting in a lack of inhibition of G-cells which leads to an increased amount of gastrin in the antrum and the serum. 17 – 19 The purpose of this study was to evaluate the influence of H. pylori infection on the behaviour of the G-cell and D-cell populations and on the relationship between the serum gastrin concentration and the G-cell to D-cell ratio. Section title: Study Population Educational score: 4.018710136413574 Domain: biomedical Document type: Study Language: en The study population consisted of 37 patients with infection and 33 patients without H. pylori infection. All of the 70 patients were either endoscopically and histologically confirmed benign gastric(GU) or duodenal ulcers (DU). Among H. pylori -positive patients, 29 patients had DU and 8 had GU. H. pylori -negative patients were divided as 22 DU and 11 GU patients. None of them had received H 2 -receptor antagonists, antibiotics, proton pump inhibitors or NSAIDs for at least 30 days prior to biopsy. Patients with any other chronic illness were also excluded. Section title: Methods Educational score: 4.152525424957275 Domain: biomedical Document type: Study Language: en Four gastric mucosal specimens were obtained from the antrum within 3 cm proximal to the pylorus. Two of the specimens were examined for identification of H. pylori and the other two were evaluated for the numbers of G-cells and D-cells. Patient selection criteria included subjects with two good histologic specimens which contained the entire section from the surface epithelium to the muscularis mucosae. The presence of H. pylori infection was confirmed by H & E staining, culture, and histologic examination of biopsy specimens. For measurement of the serum gastrin concentration, a blood sample was collected after overnight fast from each patient. Fasting gastrin concentrations were measured by the radioimmunoassay technique using a Gamm Dab[ 125 I] Gastrin RIA kit (INCSTAR Co. UK) which specifically measures both G17 and G34. Results were expressed as ng/ml G17 equivalents. Each examination was duplicated. Section title: Evaluation of Antral G-Cells and D-Cells Educational score: 4.077020168304443 Domain: biomedical Document type: Study Language: en Gastric mucosal biopsy specimens were fixed in 10% buffered formalin and embedded in paraffin after routine dehydration and cleansing. Sections 5μ m in thickness perpendicular to the surface of the mucosa, including a complete glandular portion and intact muscularis mucosae, were subjected to immunoperoxidase staining. Section title: Evaluation of Antral G-Cells and D-Cells Educational score: 4.118151664733887 Domain: biomedical Document type: Study Language: en A complete glandular profile was defined as a gland totally within the microscopic field with a clearly visible lumen. For gastrin immunocytochemistry, the tissues were incubated overnight with rabbit antibiodies against the non-sulfated form of gastrin-17 (DAKO Corp., Copenhagen, Denmark). For somatostatin immunocytochemistry, the tissues were incubated with a polyclonal antibody raised against synthetic somatostatin (DAKO Corp., Copenhagen, Denmark). The secondary system in both cases consisted of an anti-rabbit ABC (avidin-biotin complex) kit (Biomeda Co., Foster). In both cases the reaction was developed with diaminobenzidine as the chromogen. Finally, the tissues were counterstained with H&E, then mounted. Section title: Evaluation of Antral G-Cells and D-Cells Educational score: 4.106655120849609 Domain: biomedical Document type: Study Language: en Complete glandular profiles confined in the 7×7 grids of an eyepiece micrometer (Eyepiece Micrometer 20,4 OCM 7/7 SQ, Olympus Co., Japan) were selected and examined under a 40x objective for observation and quantitation of G-cells and D-cells. The total numbers of G-cells and D-cells in an entire grid square, including the nucleus in the plane section, were counted and the mean number of cells per millimeter of muscularis mucosae was calculated. All microscopic examinations were performed blindly by a pathologist unaware of either the individual serum gastrin level or the status of H. pylori infection. Section title: Statistical Analysis Educational score: 3.9769487380981445 Domain: biomedical Document type: Study Language: en Results were expressed as the means±SD. A two-tailed, unpaired t-test and a Wilcoxon rank sum test were used to determine the significance of difference between means, with differences giving a p value less than 0.05 being considered significant. The number of each peptide-producing cell per gastric gland was calculated by dividing the total number of each cell type by the number of complete gland profiles counted from the same subject. The G-cell to D-cell ratio was calculated by dividing the numbers of G-cells by D-cells from the same subject, then averaging for the group. Section title: RESULTS Educational score: 3.9224307537078857 Domain: biomedical Document type: Study Language: en Results are summarized in Table 1 . There was no significant difference in the number of complete gastric gland profiles per field between patients with and without H. pylori infection. (9.8±2.7 vs 9.0±2.9, respectively, p>0.5). Section title: Serum Gastrin Concentration Educational score: 4.104611396789551 Domain: biomedical Document type: Study Language: en The fasting serum gastrin concentration was significantly higher in patients with H. pylori infection compared to patients without infection (80.3±23.5 vs 47.6±14.1 pg/ml, respectively, p<0.001) . There were no differences in serum gastrin concentration between GU and DU patients within both the H. pylori -infected group (81.6±18.8 vs 80.0±24.9, respectively, p>0.5) and the H. pylori -uninfected group (44.7±10.5 vs 49.1±15.6, respectively, p>0.5). Section title: G Cells Educational score: 4.096458911895752 Domain: biomedical Document type: Study Language: en The mean number of G-cells per gastric gland was similar in patients with and without H. pylori infection. (7.1±3.1 vs 7.3±3.9, respectively, p>0.5) . There were no differences in the number of G-cells between GU and DU patients within both the infected group (8.6±4.3 vs 6.7±2.6, respectively, p>0.5) and the uninfected group (6.6±2.3 vs 7.7±4.5, p>0.5). Section title: D Cells Educational score: 4.094610691070557 Domain: biomedical Document type: Study Language: en The mean number of D-cells per gastric gland was significantly lower in both GU and DU patients with H. pylori infection compared to patients without H. pylori infection (1.3±0.4 vs 2.5±1.7, respectively, p<0.001) . There were no differences in the number of D-cells between GU and DU patients within both the H. pylori -infected group (1.3±0.4 vs 13.±0.4, respectively, p>0.5), and H. pylori -uninfected gruop (2.1±1.0 vs 2.6±2.0, respectively, p>0.5). Section title: The Ratio of G-cells to D-Cells Educational score: 4.088212013244629 Domain: biomedical Document type: Study Language: en The ratio of G-cells to D-cells was significantly higher in patients with H. pylori infection compared to patients without H. pylori infection (5.7±2.7 vs 3.5±1.9, p<0.001, respectively) . There were no differences in the ratio between GU and DU patients within both the H. pylori -infected group (7.0±4.2 vs 5.3±2.0, respectively, p>0.5), and the H. pylori -uninfected group (3.5±1.6 vs 3.5±2.1, respectively, p>0.5). Section title: DISCUSSION Educational score: 4.249841690063477 Domain: biomedical Document type: Study Language: en The pathogenetic mechanism of H. pyori -associated peptic ulcer diseases have not been proved yet. However it is well known that a direct cytopathic effect of the organism on either the gastric or the duodenal epithelium is not involved in the development of peptic ulcer diseases because H. pylori is a non-invasive microorganism. One of the most characteristic abnormalities in gastric secretion induced by H. pylori infection is increased gastrin release after meals or after bombesin stimulation; this abnormality restored to its original state following eradication of the organism 8 – 14 ) . It has also been shown that H. pylori infection can induce reversible increased both basal and gastrin-mediated acid secretion 15 , 16 ) . However, the mechanism by which the microorganism alters gastrin metabolism is still unclear. Since synthesis and secretion of somatostatin, which is a physiologic paracrine inhibitor of antral G-cell function, are directly regulated by the intragastric pH, there is substantial evidence that the increased gastrin secretion of H. pylori -positive patients is related to an interplay between antral G-cells and D-cells. Therefore, we measured the numbers of antral G-cells and D-cells and correlated these numbers with serum gastrin concentrations in both H. pylori- infected and H. pylori -uninfected patients. Section title: DISCUSSION Educational score: 4.091869354248047 Domain: biomedical Document type: Study Language: en Our study showed that the number of complete gastric gland profiles was not different between patients with and without H. pylori infection. The number of G-cells per complete gastric gland was also not different between H. pylori -positive patients and -negative patients. Similar observations were made by Queiroz et al 18 ) , Sankey et al 20 ) and Moss et al 21 ) , who demonstrated that the number of antral G-cells is apparently not affected by the presence of H. pylori . With regard to the serum gastrin concentration our results are in agreement with earlier studies which show that H. pylori -associated abnormalities in gastrin secretion are reversible with alleviation of the bacterial infection 8 –, 11 , 13 , 22 – 25 ) . Section title: DISCUSSION Educational score: 4.05339241027832 Domain: biomedical Document type: Study Language: en There were also no differences in serum gastrin concentration between GU and DU patients within both the H. pylori- infected group and the H. pylori -uninfected group. In this study, the number of D-cells per complete gastric gland was significantly lower in both GU and DU patients with H. pylori infection compared to patients without infection. Consequently, the ratio of G-cells to D-cells was also significantly higher in H. pylori -positive patients than in H. pylori -negative patients. There were no differences in the ratio between GU and DU patients within both the H. pylori -infected group and H. pylori -uninfected group. Similar results have also been reported 17 , 18 , 26 , 27 ) . Section title: DISCUSSION Educational score: 4.677500247955322 Domain: biomedical Document type: Study Language: en The mechanism by which H. pylori decreases the number of antral D-cells should be considered. One possible explanation is an inflammatory change in the region of the D-cells. Recently, Kaneko H. et al 17 ) and Moss et al 19 ) have reported that H. pylori infection is associated with a decrease in somatostatin-mRNA and is associated with the somatostatin-immunoreactive cell density of the antral mucosa. These changes were reversed after eradication of H. pylori . The degree of reversal was correlated with the grade of chronic inflammation. These findings are consistent with the reports of Domschke et al 28 ) and Sumii et al 26 ) . Ito et al 29 ) also reported that the number of D-cells decreased in proportion to the extent and degree of chronic atrophic gastritis, and that D-cells disappeared earlier and more diffusely than G-cells. Another possibilty may be a local alkaline environment induced by ammonia which is produced by bacterial urease. This possibility is supported by previous studies 30 – 34 ) . These reports indicate that changes in intragastric acidity exert an influence on the antral D-cell density, on the tissue content of soamtostatin and on both the plasma and the antral gastrin concentration. Recently, there have been new efforts to explain the mechanism of increased gastrin release which is observed in patients with H. pylori infection. Graham et al 35 ) observed that the number of G-cells was significantly lower in patients with DU than in either infected or uninfected controls, and that the ratio of G-cells to D-cells was similar in duodenal ulcer patients and in uninfected controls. They also found that, although eradication of the H. pylori infection results in a dramatic reduction in stimulated gastrin secretion, infection was not associated with a change in the number of either antral G-cells or D-cells in patients with DU. Based on these results they concluded that an H. pylori -associated increase in gastrin secretion appears to be related to local factors which regulate G-cell function. There are several reports regarding the role of cytokines, which are released by the inflammatory cells activated by H. pylori , in the regulation of antral G-cell function. These reports have suggested that interleukins, TNF- α or interferons stimulate gastrin secretion via receptors potentially residing on antral G-cells 36 – 38 ) . Section title: DISCUSSION Educational score: 4.115068435668945 Domain: biomedical Document type: Study Language: en In conclusion, our results strongly suggest that the exaggerated response of gastrin secretion observed in H. pylori -positive patients is due to a reduction of the antral D-cell mass because these cells normally inhibit the synthesis and release of gastrin. | Study | biomedical | en | 0.999995 |
10063310 | Section title: INTRODUCTION Educational score: 4.471651554107666 Domain: biomedical Document type: Study Language: en Nitric oxide (NO), produced by the conversion of the terminal guanidino nitrogen atoms from L-arginine, accounts for many of the biological properties of endothelium-derived relaxing factor (EDRF) 1 ) . NO synthesis is catalyzed by three distinct forms of nitric oxide synthases (NOS), i.e., brain (bNOS), inducible (iNOS) and endothelial constitutive (ecNOS) isozymes 2 ) . The importance of NO in the physiological control of blood pressure is now well established. It causes vascular relaxation by activation of soluble guanylate cyclase and, hence, increasing cyclic guanosine 3′, 5′-monophosphate (cGMP) levels in the smooth muscle 3 ) . Therefore, an alteration in NO synthesis may be a significant factor in the pathogenesis of hypertension. A decreased responsiveness to endothelium-dependent vasodilators is characteristically seen in isolated arteries from various models of experimental hypertension 4 – 7 ) . Section title: INTRODUCTION Educational score: 4.380224227905273 Domain: biomedical Document type: Study Language: en A large number of studies evidence the capacity of the kidney to produce NO and its relevant role in renal function 8 – 12 ) . It has been demonstrated that the kidney is very sensitive to the reduction of NO, as low doses of NOS inhibitors reduce sodium and water excretion without affecting renal hemodynamics or systemic arterial pressure 8 ) . The kidney has an intrinsic mechanism, in which an increased renal perfusion pressure promotes sodium excretion, a phenomenon called pressure natriuresis 13 ) . It has been shown that chronic inhibition of NO synthesis impairs sodium excretion, resulting in right shift of the pressure-natriuresis curve and hypertension 14 ) . In spontaneously hypertensive rats (SHR), pressure-natriuresis is also blunted 15 ) and papillary blood flow is reduced 16 ) . Recently, Larson et al. 17 ) have shown that administration of L-arginine to SHR restores the pressure-dependent increase in renal medullary hemodynamics in association with restoration of pressure-natriuresis. These results suggest that a defective NO pathway may be involved in the blunted pressure-natriuresis. However, the precise status and role of the NO-cGMP pathway in the kidney in SHR is not clear. Section title: INTRODUCTION Educational score: 4.067678451538086 Domain: biomedical Document type: Study Language: en The present study was aimed at investigating whether the hypertension is related with an altered activity of NO system in the kidney of SHR. The expression of NOS isozymes and tissue NO levels were determined in the kidney isolated from SHR and their normotensive control Wistar-Kyoto rats (WKY) by Western blot analysis. Section title: 1. Animals Educational score: 4.063571929931641 Domain: biomedical Document type: Study Language: en Experiments were carried out using adult (12-week old) male SHR and WKY. Systolic blood pressure was measured in a conscious state by the tail-cuff method. Rats were killed by decapitation. Blood was collected from the trunk, and centrifuged (3,000 rpm for 30 min) for the determination of plasma NO contents. Kidneys were rapidly removed, immediately frozen in liquid nitrogen and stored at −80°C until extraction. Section title: 2. Colorimetric Assay of Nitrite/Nitrate Educational score: 4.1369309425354 Domain: biomedical Document type: Study Language: en NO x (nitrite/nitrate) contents in the kidney and plasma were measured with a colorimetric nitric oxide assay kit (Oxford). A microplate was used to perform enzyme reactions in vitro. For spectrophotometric assay of nitrite with Griess reagent, 80 ml MOPS (50 mmol/L)/EDTA (1 mmol/L) buffer and 5 μl tissue samples were added to wells. Nitrate reductase (0.01 U) and 10 μl NADH (2 mmol/L) were added to the reaction mixture, and the plate was shaken for 20 minutes at room temperature. Color reagents, sulfanilamide and N-(1-Naphthayl) ehylenediamine dihydrochloride were added, and absorbance values at 540 nm were read in a microtiter plate reader . The concentration of nitrite/nitrate was estimated from a standard curve, which was constructed with the use of standard reagents included in the assay kit. Section title: 3. Protein preparation Educational score: 4.1331892013549805 Domain: biomedical Document type: Study Language: en The cortex, outer medulla and inner medulla from frozen kidney tissues were dissected and were homogenized with Polytron homogenizer at 3,000 rpm in a solution containing 250 mmol/L sucrose, 1 mmol/L EDTA, 0.1 mmol/L phenylmethylsulfonyl fluoride and 50 mmol/L potassium phosphate buffer at pH 7.6. Large tissue debris and nuclear fragments were removed by two consecutive low speed centrifuge spins (3,000g, 5 min; 10,000g, 10 min). The protein concentration of the homogenate was determined by the method of Bradford 18 ) , with bovine serum albumin as a standard. In the case of cortex, the membrane-bound protein was further centrifuged at 100,000g for 60 min. The pellet was resuspended for protein blotting of ecNOS and the supernatant was used for blotting of bNOS and iNOS. Section title: 4. Western blot analysis Educational score: 4.164218425750732 Domain: biomedical Document type: Study Language: en Protein samples were electrophoretically size-separated with a discontinuous system consisting of a 7.5% polyacrylamide resolving gel and 5% polyacrylamide stacking gel. High-range molecular weight markers (Biorad; Hercules, CA, USA) were loaded as size standard. An equivalent amount of total tissue protein (100 μg) was loaded on each lane. After separation, the proteins were electrophoretically transferred to a nitrocellulose membrane at 20 V overnight. The membranes were washed in Tris-based saline buffer (pH 7.4) containing 1% Tween-20 (TBST), blocked with 5% nonfat milk in TBST for one hour and incubated with a 1:2,000 dilution of monoclonal mouse anti-bNOS, anti-ecNOS and anti-iNOS antibodies (Transduction Laboratories; Lexington, KY, USA) in 2% nonfat milk/TBST for one hour at room temperature. The membranes were then incubated with a horseradish peroxidase-labeled goat anti-mouse IgG (1:1,000) or goat anti-rabbit IgG in 2% nonfat milk in TBST for 2 hours. The bound antibody was detected by enhanced chemiluminescence on X-ray film or hyperfilm (Amersham, Little Chalfont, Buckinghamshire, England). The membranes were stripped between incubations with different antibodies in a Tris-buffered solution containing 2% sodium dodecyl sulfate and 100 mmol/L β-mercaptoethanol at 50°C. Section title: 5. Statistical analysis Educational score: 2.834812879562378 Domain: biomedical Document type: Study Language: en The results are presented as means±SEM. The significance of the differences was analyzed by the Student’s t -test of unpaired data. Section title: 1. Blood pressure and plasma NO concentration Educational score: 3.819302797317505 Domain: biomedical Document type: Study Language: en Body weight, systolic blood pressure and heart rate in SHR and WKY were summarized in Table 1 . Systolic blood pressure and heart rate were higher in SHR compared with those in WKY. The plasma NO x (nitrite/nitrate) concentration was significantly higher in SHR compared with that in WKY . Section title: 2. NOx contents and NOS expression in the kidney Educational score: 3.913455009460449 Domain: biomedical Document type: Study Language: en Fig. 2 shows NO x contents in the inner medulla, outer medulla and cortex of the kidney in SHR and WKY. The NO x contents were higher in the outer medulla and cortex of the kidney by about 50% and 60%, respectively, in SHR compared with those in WKY. Section title: 2. NOx contents and NOS expression in the kidney Educational score: 4.131820201873779 Domain: biomedical Document type: Study Language: en Three isoforms of NOS (bNOS, iNOS, ecNOS) were determined in the renal cortex and outer and inner medulla of SHR and WKY by Western blot analysis . Anti-bNOS, anti-iNOS and anti-ecNOS antibodies recognized protein bands with molecular sizes of 155, 130 and 140 kDa, respectively. Fig. 4 shows densitometric analysis of NOS in the kidney. ecNOS protein expression did not significantly differ between SHR and WKY. However, bNOS proteins were expressed higher In the outer medulla and cortex by 80% and 70%, respectively, and iNOS proteins were higher in the inner medulla, outer medulla and cortex by 95%, 135% and 50%, respectively, in SHR. Section title: DISCUSSION Educational score: 4.1872782707214355 Domain: biomedical Document type: Study Language: en It is well known that acetylcholine-induced vasodilation is impaired in SHR 19 – 21 ) , however, of which precise mechanisms are not fully understood. A growing number of studies 22 – 25 ) have demonstrated that NO synthesis may be increased in SHR. It was reported that an inhibition of NOS produced a greater reduction of vasorelaxation to acetylcholine 26 ) and an exaggerated hypertension in SHR 27 ) . Furthermore, the present study showed that plasma NO x (nitrite/nitrate) concentration and NO x contents in the kidney were significantly higher in SHR compared with those in WKY. It has been known that the release of NO by endothelial cells can be altered by changes in blood flow 28 ) , and their mRNA and protein for ecNOS can be induced by mechanical forces 29 ) . The augmented expression of ecNOS may play an important role in the compensatory mechanisms to counteract the hypertension. Section title: DISCUSSION Educational score: 4.130202293395996 Domain: biomedical Document type: Study Language: en The present study showed that renal NO x contents and NOS protein expression in the medulla were higher than in the cortex. These findings are consistent with previous studies revealing the largest signal for constitutive NOS protein expression in the inner medullary collecting duct 30 ) , and with the report showing the highest amount of cGMP in the inner medulla, when stimulated with acetylcholine 31 ) . These results suggest that the medullary NO system plays a more important role in renal function, such as control of sodium and water excretion. Section title: DISCUSSION Educational score: 4.421009540557861 Domain: biomedical Document type: Study Language: en Tubuloglomerular feedback (TGF) is the critical component of a negative feedback control system that effectively buffers the hemodynamic influences on GFR and stabilizes the amount of NaCl entering the distal nephron 32 ) . In the kidney, bNOS protein has been demonstrated exclusively in the macula densa cells, and NO produced in these cells counteract the TGF-mediated vasoconstriction 33 , 34 ) . This finding indicates that, under normal conditions, NO is one of major determinants of TGF sensitivity. SHR are known to have a sensitive TGF response during development of hypertension 35 , 36 ) . The exaggerated response could contribute to a lower glomerular filtration and, perhaps, to the development of hypertension 37 ) . Thorup and Persson 38 ) compared the effects of intratubular inhibition of NO by [N ω -nitro-L-arginine (L-NNA)] on the TGF between SHR and WKY rats, and showed that L-NNA did not change either sensitivity or the magnitude of the TGF response in SHR, whereas their normotensive control strains responded with a very strong resetting toward higher sensitivity. This finding indicates that NO-mediated vasodilation that counteracts TGF-induced vasoconstriction of the afferent arteriole is less pronounced in SHR than in their normotensive controls. Our study demonstrated that the expression of bNOS protein in renal cortex and outer medulla in SHR was higher than in WKY. Since bNOS in macula densa seems to attenuate the afferent arteriolar constriction induced by the TGF 33 , 34 ) , enhanced bNOS expression would be expected to increase GFR and sodium excretion which act as a compensatory function opposing the hypertension. Section title: DISCUSSION Educational score: 4.408176422119141 Domain: biomedical Document type: Study Language: en Pressure-natriuresis is associated with significant increase in vasa recta blood flow 39 ) . It has been postulated that the mechanism, through which changes in medullary hemodynamics result in pressure-dependent increase in urinary sodium excretion, is due to transmission of pressure from the systemic circulation to the renal interstitium through the renal microcirculation 40 ) . In SHR, papillary blood flow in response to increased renal perfusion pressure is reduced, and pressure-natriuresis is blunted 41 ) . Hence, the attenuated response of renal medullary hemodynamics to change in renal perfusion pressure has been postulated to be causally linked to the abnormal pressure natriuresis in the SHR 41 , 42 ) . The restoration of the vasa recta hemodynamic response with L-arginine in SHR supports a role for NO in the regulation of medullary blood flow 17 ) . Ikenaga et al 43 ) reported that intravenous administration of L-arginine increase urinary excretion of NO metabolites in normotensive animals, and in SHR to a similiar degree, suggesting that, in the SHR, the ability to synthesize NO is preserved but the renal response to NO is impaired. Our study also demonstrated that the basal production of NO was higher in the kidney in SHR compared with that in WKY. bNOS proteins were expressed higher in the outer medulla and cortex, and iNOS proteins were higher in the inner medulla, outer medulla and cortex in SHR. These results suggest that impaired effect of NO or activation of other vasoconstricting factors may be responsible for the blunted pressure natriuresis in SHR rather than defective NO production. Section title: DISCUSSION Educational score: 3.9048702716827393 Domain: biomedical Document type: Study Language: en In conclusion, the NO generation may not be impaired, but rather increased in the kidney of SHR. It is likely that an altered expression of NOS isozymes plays a counter-regulatory role secondary to the hypertension in SHR. | Study | biomedical | en | 0.999997 |
10063311 | Section title: INTRODUCTION Educational score: 3.9577443599700928 Domain: biomedical Document type: Review Language: en Although the prevalence rate of pulmonary tuberculosis is decreasing due to the National Tuberculosis Control Programme, drug resistant pulmonary tuberculosis has been a longstanding public health problem in Korea. 1 ) The major concerns over drug resistance were a fear of spread of drug-resistant organisms and the ineffectiveness in chemotherapy of the patients infected with them. In general, the prevalence of drug resistance shows a close inverse relationship with the efficacy of antituberculosis treatment regimens, even though there were several reports that the patients with initial drug resistance responded fairly well with the conventional triple combined regimens and as good as sensitive cases with the intensive, short course regimens 2 , 3 ) . Also, multidrug resistant tuberculosis is a fatal disease because of the high mortality rate reported as 20% to 70%, depending on underlying diseases, especially AIDS, which is equivalent to the outcome for untreated tuberculosis 4 – 6 ) . Section title: INTRODUCTION Educational score: 3.1149566173553467 Domain: biomedical Document type: Study Language: en The nation-wide tuberculosis prevalence survey (NTPS) in Korea was performed at 5-year intervals since 1965, and the results show a decreasing tendency but still rather high in both prevalence rate and drug resistance 7 ) . From a practical point of view at university hospital level, the 3rd referral center, drug resistant pulmonary tuberculosis is somehow different from that of the nation-wide survey of tuberculosis based in various settings. So, we conducted this investigation, prospectively, to estimate resistance rate and to correlate the clinical characteristics of resistant tuberculosis with the patients of pulmonary tuberculosis who were referred to the university hospital. Section title: M. Tuberculosis isolates Educational score: 2.774794816970825 Domain: biomedical Document type: Study Language: en The study population consisted of 92 patients who were diagnosed as pulmonary tuberculosis by sputum culture with sensitivity test at Chungnam National University Hospital, from January, 1995 to June, 1996. Section title: Drug sensitivity tests Educational score: 4.065823078155518 Domain: biomedical Document type: Study Language: en When M. tuberculosis was identified by routine culture, multi-drug sensitivity test was done at the laboratory of the Reasearch Institute of Korean National Tuberculosis Association (KNTA). The procedure of drug sensitivity test was based on the absolute concentration method described by Canetti et al 8 , 9 ) , with a little modification of inoculum preparation and size 10 ) . The tests were done in the Lowenstein-Jensen Medium and the drugs were added before inspissation at the concentration shown on the table. We have defined multi-drug resistance (MDR) as resistance to both INH and RFP or more drugs. Section title: Severity and chest X-ray Educational score: 2.377250909805298 Domain: biomedical Document type: Study Language: en Severity was classified by NTA method 11 ) . We interpreted chest radiographs taken at the time of diagnosis. Presence of cavity was determined by only simple chest radiographs. Section title: Anti-tuberculosis chemotherapy Educational score: 2.7272789478302 Domain: biomedical Document type: Study Language: en We administered standard regimen (2HREZ+4HRE or 9HRE) to all patients with or without previous therapy. Follow-up chest X-ray and sputum smear were examined completely. We defined treatment failure as smear positive after 6 months or more therapy. Section title: Statistical analysis Educational score: 2.6046886444091797 Domain: biomedical Document type: Study Language: en Data are presented as number and percentage. Resistance ratio was compared using the student’s t-test or chi square test, when appropriate. A p value < 0.05 was considered significant. Section title: RESULTS Educational score: 3.886979818344116 Domain: biomedical Document type: Study Language: en We diagnosed 92 patients as active tuberculosis by either sputum AFB smear or culture. The patients characteristics are shown in Table 1 . Among 92 patients, 62 were men and 30 were women, with a mean age of 49 and 35 years, respectively. The 66 patients without previous anti-tuberculosis therapy were made up of 26 minimal, 25 moderately advanced and 15 far advanced disease patients. Of 26 patients with previous therapy, only 1 patient was minimal and the others were moderately advanced 19 ) or far advanced 6 ) . Cavitary lesion was found in 16 patients (24.2%) of the group without previous therapy, and in 16 patients (61.5%) of those with previous therapy. Section title: RESULTS Educational score: 4.074851989746094 Domain: biomedical Document type: Study Language: en Overall, 24 (26.0%) of the 92 patients had resistance to at least one drug. Among 66 patients without previous therapy, 11 (16.7%) patients had resistance to at least one drug, and among 26 patients with previous therapy, 13 (50.0%) patients had resistance. Rate of multi-drug resistance is 3.0% in patients without previous therapy, and significantly high (23.0%, p<0.05) in patients with previous therapy ( Table 2 ). Section title: RESULTS Educational score: 3.920809030532837 Domain: biomedical Document type: Study Language: en The rate of resistance to five first-line drugs are summarized in Table 3 . For all 92, resistance to INH was most common (19.5%) followed by SM (11.9%), RFP (9.7%), EMB (9.7%), PZA (5.4%). Generally, resistance of patients with previous therapy is higher than those of patients without therapy. Section title: RESULTS Educational score: 3.136476993560791 Domain: biomedical Document type: Study Language: en Resistance to 2 or more drugs are shown in Table 4 . One patient had resistance to all 5 first-line drugs. Patients with resistance to RFP (n=9) also have resistance to other first-line drugs (INH 8, other drug 1). Resistance to second-line drugs are summarized in Table 5 . Section title: RESULTS Educational score: 2.017920732498169 Domain: biomedical Document type: Study Language: en Resistance according to the radiologic severity are shown in Table 6 . The difference in resistance rate according to the severity was not significant Section title: RESULTS Educational score: 2.892279863357544 Domain: biomedical Document type: Study Language: en Resistance in patients with cavity is higher than in patients without cavity, in case of INH ( Table 7 ). Section title: RESULTS Educational score: 2.774915933609009 Domain: biomedical Document type: Study Language: en Outcome of chemotherapy was shown in Table 8 . More extended therapy is needed in patients with previous therapy and with presence of cavity. Treatment failure was found only in patients with previous therapy (11.1%). All patients associated with treatment failure have one or more drug resistance. However, treatment failure in patients with primary resistance was not found. Section title: DISCUSSION Educational score: 2.787142038345337 Domain: biomedical Document type: Other Language: en Drug resistant tuberculosis is one of the most important factors in treatment failure. Treatment of patients with tuberculosis resistant to RFP, INH and other medications is risky, and results in limited efficacy. In western countries, introduction of RFP in 1971 gave short-term treatment to anti-tuberculosis therapy. It is very hopeful but, with the advent of AIDS, not only increasing prevalence of tuberculosis but also emergence of the drug resistant tuberculosis became an important health care issue even in western countries 12 – 15 ) . Section title: DISCUSSION Educational score: 1.6643640995025635 Domain: biomedical Document type: Other Language: en The nationwide survey of tuberculosis in Korea showed a decreasing tendency of prevalence. But drug resistance tuberculosis is still high, and has been a major problem. Section title: DISCUSSION Educational score: 1.6636370420455933 Domain: biomedical Document type: Other Language: en Reports on drug resistance were so different because of difference in study area, in time of study and in study population etc. Section title: DISCUSSION Educational score: 3.3302619457244873 Domain: biomedical Document type: Study Language: en By NTPS in 1995, overall drug resistance was 9.9%, and resistance in patients without previous therapy was 5.8%. In patients with previous therapy, resistance was up to 25.0% 7 ) . Overall resistance rate in our study (26%) was higher than in NTPS. This difference may be due to difference in severity of patients. By NTPS, patients made up 40% of moderately advanced disease and 10% of far advanced disease, while patients of this study made up 48% of moderately advanced and 23% of far advanced disease. So, patients visited at our tertiary referral center were more likely to have severe disease. Section title: DISCUSSION Educational score: 3.9153926372528076 Domain: biomedical Document type: Study Language: en Among 66 patients without previous therapy, 11 (16.7%) patients had resistance to at least one drug and, among 26 patients with previous therapy, 13 (50.0%) patients had that. Rate of MDR is 3.0% in patients without previous therapy and 23.0% in patients with previous therapy. So, the prevalence of the single-drug and multi-drug resistance in patients with previous treatment is significantly higher than in patients without previous therapy. Section title: DISCUSSION Educational score: 2.595958709716797 Domain: biomedical Document type: Other Language: en For prevention of emerging resistance, maintaining good compliance of the patients and appropriate prescription are needed. If retreatment is required, a susceptibility test should be done. Futhermore, introduction of directly observed therapy may be helpful in lowering resistance rate 16 , 17 ) . Section title: DISCUSSION Educational score: 3.843656301498413 Domain: biomedical Document type: Study Language: en Resistance to INH is the most frequent. The reported prevalence of INH resistance was variable from nation to nation and time to time. Recently, M. Demissie reported it as 8.4% in Ethiopia 18 ) , and M.T. Mendoza found that 17% of Phlippine patients 19 ) had it. In our patients, an average of 19.5% had resistance to INH. Primary INH resistance was found to be 12.1% and highest, followed by streptomycin, ethambutol, pyrazinamide, rifampicin. Section title: DISCUSSION Educational score: 3.2257511615753174 Domain: biomedical Document type: Study Language: en Resistance to RFP is most harmful and closely related to treatment failure. Cauthen reported the rate of RFP resistance as 0.6% with previously untreated patients and 3.3% with previously treated patients in the US 20 ) . In our study, resistance to RFP is 3.0% in patients without previous therapy and 26.9% in patients with previous therapy. Moreover, all patients with RFP resistance also have MDR. So, RFP resistance was a more serious problem in Korea. Section title: DISCUSSION Educational score: 2.5331966876983643 Domain: biomedical Document type: Study Language: en Presence of cavity may be associated with a slow regression of the lesion and with more emerging of resistant strain 21 ) . In our study, cavity-positive patients had higher resistance to INH. More extended therapy is needed. Section title: DISCUSSION Educational score: 2.3439128398895264 Domain: biomedical Document type: Study Language: en Treatment of primary resistant patients or selection of the drug is not firmly established 17 ) . In our study, a small but significant percentage of the patients without previous therapy also had drug resistance, but no treatment failure is developed in the observed period. So, somehow, extended therapy is needed in some patients and regimen change is not needed in any patients. But the follow-up period is short and further long-term observation of patients with primary resistance will be performed. Section title: DISCUSSION Educational score: 3.6243410110473633 Domain: biomedical Document type: Study Language: en Sensitivity tests are strongly recommended in all culture-positive patients with previous therapy. But recommendation of a sensitivity test for patients without previous therapy is still debatable. Treatment failure may be seen in these patients 22 ) . A guide for optimal drug selection may be needed. For this reason, a susceptibility test is also needed in case of no history of previous therapy. In our study, however, treatment failure was not observed in patients with primary resistance, so a susceptibility test may not be required. Further large studies for recommendations of susceptibility tests of primary resistance are also needed. Section title: DISCUSSION Educational score: 3.2279441356658936 Domain: biomedical Document type: Other Language: en In conclusion, sensitivity tests are strongly recommended in all culture-positive patients in those with previous therapy but, in cases of primary resistant tuberculosis, sensitivity tests are not required and proper combination chemotherapy should be given under careful surveillance. | Review | biomedical | en | 0.999995 |
10063312 | Section title: INTRODUCTION Educational score: 4.235065460205078 Domain: biomedical Document type: Study Language: en Diabetic nephropathy is the leading cause of end-stage renal disease 1 ) . In addition, the development of diabetic nephropathy has a dramatic impact on the morbidity and mortality in diabetic patients 2 , 3 ) . The early stages of both clinical and experimental diabetes mellitus are characterized by abnormal glomerular hemodynamics, such as glomerular hyperperfusion and hypertension, which result in glomerular hyperfiltration 4 – 6 ) . This glomerular hyperfiltration has been proposed as one of the important factors in the pathogenesis of diabetic nephropathy 7 , 8 ) . Multiple agents or mediators for the hyperfiltration of early diabetes have been proposed 9 ) . Nitric oxide (NO) plays a critical role in the regulation of renal hemodynamics 10 , 11 ) , and is likely to be involved in the development of glomerular hyperfiltration in diabetes mellitus 12 , 13 ) . However, other studies 14 , 15 ) showed impaired nitric oxide release from diabetic kidneys and a role of nitric oxide in the pathogenesis of diabetic nephropathy has not been clear yet. The present study was aimed at investigating the role of nitric oxide in the pathogenesis of glomerular hyperfiltration in streptozotocin-induced diabetic rats. In the present study, we sought to investigate; (1) whether nitric oxide synthesis might be increased in the early stage of diabetic mellitus.; (2) nitric oxide might be one of the important mediators responsible for diabetic hyperfiltration.; (3) inhibition of excess nitric oxide synthesis by nitric oxide synthase (NOS) inhibitor might ameliorate or attenuate diabetic renal hyperfiltration. Section title: MATERIALS AND METHODS Educational score: 4.100257396697998 Domain: biomedical Document type: Study Language: en Male Sprague-Dawley rats, weighing 200 to 250 g, were used. Diabetes mellitus was induced by tail vein injection of streptozotocin (55 mg/kg body wt: Sigma Chemical Co., St. Louis, MO, USA). Body weight-matched control (normal) rats received a sham injection of the vehicle (0.9% NaCl). Blood glucose levels were measured with a blood glucometer (Lifescan Inc, Milpitas CA, USA) in the morning, 2 days and 28 days (during experiment) after streptozotocin injection, and the rats with blood glucose levels less than 300 mg/dL were excluded from the study. A group of diabetic rats was chronically treated daily in the afternoon with ultralente insulin (Iletin II, Eli Lilly, Indianapolis, IN, USA), with the dose (6–10 U) adjusted individually to normalize blood glucose concentration, 3 days after induction of diabetes. Another group of diabetic rats was treated with nitro-L-arginine methyl ester (L-NAME, Sigma), an inhibitor of NOS, dissolved in the drinking water at 10 mg/L, 3 days after streptozotocin injection. The animals were housed in individual cages, given free access to water and standard chow ad libitum. Section title: Study design Educational score: 4.08931303024292 Domain: biomedical Document type: Study Language: en As an index of synthesis of nitric oxide (NO), we measured plasma and urine concentrations of NO 2 − /NO 3 − , the stable metabolic products of NO, and protein expressions of 3 isoforms of nitric oxide synthases (NOS; bNOS, iNOS and ecNOS) in the kidney (Study I), and also performed renal hemodynamic studies (Study II). Section title: Studies of nitric oxide synthesis (Study I) Educational score: 4.038685321807861 Domain: biomedical Document type: Study Language: en To investigate whether nitric oxide synthesis alters in the early stage of diabetes mellitus, rats were divided into four groups and fed the same diet described previously. Group I (11 normal rats) and group 2 (14 diabetic rats) were given tap water. Group 3 (9 diabetic rats) was treated with insulin therapy, as described before. Group 4 (11 diabetic rats) was given tap water containing L-NAME. At day 28 following the streptozotocin injection, anesthesia was induced by i.p. injection of thiopental (50mg/kg body wt). Section title: Studies of nitric oxide synthesis (Study I) Educational score: 4.059998512268066 Domain: biomedical Document type: Study Language: en The urinary bladder was exposed through a small lower midline abdominal incision and the bladder was cannulated with PE 50 tubing for timed urine collection, and collected urine was stored at −20°C. After urine collection, the rats were sacrificed and trunk blood was collected and the obtained plasma was stored at −20°C. The kidneys were also taken, immediately frozen in liquid nitrogen and stored at −70°C until used for Western blot analysis. Section title: NO 2 − /NO 3 − assays Educational score: 4.0523681640625 Domain: biomedical Document type: Study Language: en The stable metabolic products of nitric oxide, NO 2 − /NO 3 − , were determined in deproteinated plasma and urine by the Griess reagent 16 ) . All samples were kept at −70°C until analysis. To a 500 μ L sample was added 100 μ L 35% sulfosalicylic acid, and this was reacted for 30 min at room temperature for deproteination. Section title: NO 2 − /NO 3 − assays Educational score: 4.127600193023682 Domain: biomedical Document type: Study Language: en For assay of nitrite and nitrate of samples, two hundreds μ L samples containing 100 μ L of 200 mM ammonium formate (including 100 mM HEPES, Sigma Chemical Co.) were reduced to nitrite at 37°C for one hour by adding 100 μ L nitrate reductase [ E coli , American Type Collection, Rolville, Md], followed by centrifugation to precipitate nonreacting E. coli for five minutes, and then the nitrite was quantified. Nitrite production was quantified colorimetrically after the Griess reagent 16 ) (0.1% naphphyl ethylene diamine dihydrochloride and 1% sulfanilamide in 5% concentrated phosphoric acid, Sigma Chemical Co.) in duplicate microtiter wells at room temperature. Chromophore absorbency at 540 nm was determined. Nitrite concentration was calculated using sodium nitrite (BDH Chemical Co.) as a standard. Section title: Western blot analysis Educational score: 4.076233386993408 Domain: biomedical Document type: Study Language: en The protein levels of 3 isoforms of NO synthases (bNOS, iNOS, and ecNOS) were determined by Western blot analysis in the kidneys of control and diabetic rats. The importance of post-transcription of ecNOS has been appreciated rather than transcriptional regulation 17 ) . Post-transcriptional regulation of iNOS also appears to be important 18 , 19 ) . Therefore, we used Western blot analysis to assess the changes in NOS expression. Section title: Western blot analysis Educational score: 4.105901718139648 Domain: biomedical Document type: Study Language: en Whole protein extracts were prepared and Western blotting was analyzed according to the method of Mattson and Higgins, with a slight modification 20 ) . Protein samples (100 μ g) were electrophoretically size-separated with a discontinuous system, consisting of a 6% polyacrylamide resolving gel and 5% polyacrylamide stacking gel, and transferred to a nitrocellulose membrane at 20 V and 100 mA overnight. The membranes were washed, blocked, incubated with a 1:2500 dilution of monoclonal mouse anti-NOS (Transduction Laboratories, Lexington, KY, USA), and then incubated with a horseradish peroxidase-labeled goat anti-mouse IgG . The bound secondary antibody was detected by enhanced chemiluminescence (Amersham, Buckinghamshire, UK). The protein levels of the three isoforms of NOS were determined by analyzing the signals of Western blot autoradiograms using the transmitter scanning videodensitometer. Section title: Renal hemodynamic studies (Study II) Educational score: 4.2020955085754395 Domain: biomedical Document type: Study Language: en We investigated the renal hemodynamic effects of acute and chronic inhibition of NO synthesis in streptozotocin-induced diabetic rats 28 days after streptozotocin (STZ) injection. We studied with 4 groups of experiment in 9 normal rats (group 1), 11 diabetic rats (group 2), 8 diabetic rats with insulin-treated (group 3) and 9 diabetic rats with chronic inhibition of NO synthesis with L-NAME (group 4). Anesthesia was induced by i.p. injection of sodium thiopental (50 mg/kg body wt). To determine the rate of urinary albumin excretion, the rats, treated as described above, were housed in individual metabolic cages at 27 days of diabetes. Urine was then collected during 24 h and albumin concentrations were measured by radioimmunoassay. The animals were prepared for renal hemodynamic studies at 28 days after streptozotocin injection. Animals were anesthetized with an intraperitoneal injection of sodium thiopental (50 mg/kg body wt). Polyethylene catheters (PE-50) filled with normal salines were inserted in the left jugular vein and the left carotid artery. After vessel cannulation, the left kidney was exposed through a small midline abdominal incision, extended laterally, and then a PE-50 polyethylene tubing was inserted into the left ureter. A continuous infusion of [ 3 H] inulin (4 μ Ci·h −1 ·kg body wt −1 ; Amersham, Bucks, U.K.) and 125 I-labelled hippurate (4 μ Ci·h −1 ·kg body wt −1 ; Amersham), diluted in normal saline, was instituted via a jugular vein catheter at a rate of 1 ml/h. After 120 min of equilibration, two 20-to-30 min control periods were performed. Urine was collected throughout the periods via a left ureter catheter connected to preweighed 5-ml plastic tubes, and the volume was measured gravimetrically. Blood samples (300 μ L) were taken from the arterial catheter at the midpoint of each period, and blood loss was replaced with an equivalent volume of normal saline. We also evaluated the effects of acute inhibition of nitric oxide synthesis by L-NAME on the renal hemodynamics in control and diabetic rats. Control (baseline) measurements were conducted as described previously. After completion of control periods, to determine to what extent diabetes related glomerular hyperfiltration is caused by enhanced NO synthesis, 9 control and 11 diabetic rats were infused with NO synthesis inhibitor, L-NAME (Sigma). If the elevated GFR in diabetic rats is caused by an increased NO synthesis, L-NAME should normalize or decrease the GFR between control and diabetic rats. Section title: Renal hemodynamic studies (Study II) Educational score: 4.19195556640625 Domain: biomedical Document type: Study Language: en After the collection of baseline clearances, L-NAME was infused at 50 μ g/kg body wt per min for 60 min, and three additional 20-min clearances were then collected. Blood samples (approximately 100 μ L) were collected after the first baseline clearance and at the end of the study (approximately 50 μ L). Glomerular filtration rate (GFR) of the left kidney was calculated from the expression GFR = U/P inulin × V where U/P inulin is the urine/plasma [ 3 H]-inulin concentration ratio, and V is the urine flow rate in ml/min. The concentrations of 125 I-hippurate were determined, as described below, and the extraction factor was calculated as 1 – (Cv/Ca), where Cv and Ca are the iodohippurate concentrations in renal vein and renal artery, respectively. Using this method, the extraction factor in the control rats was 0.86 ± 0.03 and 0.89 ± 0.05 in the diabetic rats (p>0.05). These extraction factors were subsequently used for calculations of renal plasma flow (RPF). Plasma and urine [ 3 H] inulin concentrations were determined by β -counting of 10 μ L aliquots for 5 min, and concentrations of 125 I-hippurate were determined in 75 μ L samples counted for 5 min by γ -counter . Beta activity of 125 I was subtracted from 3 H counts. RPF was determined as the quotient of hippurate clearance and the extraction factor. Filtration fraction (FF) was calculated as the quotient of GFR and RPF. Section title: Statistical Analysis Educational score: 2.7799956798553467 Domain: biomedical Document type: Study Language: en Data are expressed as mean ± SEM. The significance of the difference was analyzed by the Student’s t test of unpaired data. P values < 0.05 were considered significant. Section title: Effects of diabetes mellitus on nitric oxide synthesis (Study I) Educational score: 4.092414855957031 Domain: biomedical Document type: Study Language: en Body weight and blood glucose levels are shown in Table 1 . There was no significant difference in body weight between the groups of rats at the time of the study. However, diabetic rats exhibited severe hyperglycemia. The blood glucose levels in the diabetic rats with insulin treatment were significantly lower compared with those in diabetic rats without insulin treatment (p<0.001), but still higher compared with those in control rats (p<0.05). However, L-NAME did not affect the blood glucose levels in diabetic rats. Section title: Effects of diabetes mellitus on nitric oxide synthesis (Study I) Educational score: 4.126239776611328 Domain: biomedical Document type: Study Language: en Plasma NO 2 − /NO 3 − levels were significantly higher in streptozotocin-induced diabetic rats at day 28 than in control rats (p<0.05). However, plasma NO 2 − /NO 3 − levels in insulin-treated diabetic rats were not different from those in diabetes without insulin treatment. In addition, plasma NO 2 − /NO 3 − levels in diabetic rats with L-NAME treatment were not different from those in controls . The higher plasma levels of NO 2 − /NO 3 − and the higher GFR, filtered NO 2 − /NO 3 − , were twice more higher in the diabetic rats. Diabetic rats exhibited significantly elevated urinary NO 2 − /NO 3 − levels at 28 days of diabetes, when compared with control rats (p<0.001), and the total excretion of NO metabolites (NO 2 − /NO 3 − ) was approximately five-fold higher in diabetic rats than controls. Insulin and L-NAME therapy prevented the increment of the urinary levels of NO 2 − /NO 3 − in diabetic rats, respectively, as shown Fig. 2 . Section title: Effects of diabetes mellitus on nitric oxide synthesis (Study I) Educational score: 4.116782188415527 Domain: biomedical Document type: Study Language: en Three isoforms of NOS (bNOS, ecNOS, and iNOS) were determined in the renal cortex, and outer and inner medullas of control and diabetic rats by Western blot analysis. Anti-bNOS, anti-ecNOS and anti-iNOS anti-bodies recognized protein bands with molecular sizes of 155, 140 and 130 kDa, respectively. The three isoforms were all increased in the cortex, whereas they remained unaltered in the medulla at day 28 . Section title: Renal hemodynamic studies (Study II) Educational score: 4.133813858032227 Domain: biomedical Document type: Study Language: en Diabetic rats exhibited severe hyperglycemia (549±81 mg/dL ; control, 96±11 mg/dL; p<0.001). Insulin treatment significantly decreased the blood glucose levels, but did not normalize them to control levels (174±36 mg/dL; control, 96±11 mg/dL; p<0.05). L-NAME therapy did not affect the blood glucose levels (503±97 mg/dL; diabetic, control, 549±81 mg/dL; p>0.05). Section title: Renal hemodynamic studies (Study II) Educational score: 4.086897850036621 Domain: biomedical Document type: Study Language: en Table 2 shows the effects of diabetes mellitus, insulin-treated diabetes mellitus and chronic inhibition of NO synthesis by L-NAME in diabetes on the renal hemodynamics at 28 days of diabetes. GFR and RPF were significantly elevated in the diabetic rats compared with control rats (p<0.01, p<0.001, respectively). However, filtration fraction (FF) between the groups did not have statistically significant difference. There were no significant differences in renal hemodynamic parameters, such as GFR, RPF and FF, between the insulin-treated diabetic and normal rats. GFR and RPF in diabetic rats with L-NAME treatment were significantly lower than those in the diabetic rats were (p<0.05, respectively), but still higher than those in the normal rats (p<0.05, p<0.01, respectively). Section title: Renal hemodynamic studies (Study II) Educational score: 4.117720603942871 Domain: biomedical Document type: Study Language: en In the next series of experiments, we examined the effects of acute inhibition of nitric oxide synthesis by L-NAME administration at day 28 on the renal hemodynamics in rats of the control and diabetes mellitus groups ( Table 3 ). The mean arterial pressure rose significantly after acute L-NAME administration in both control and diabetic rats (p<0.05, respectively). The GFR remained stable after acute L-NAME treatment in normal rats. In contrast, the GFR of diabetic rats fell significantly after acute L-NAME infusion, compared with that of baseline (p<0.05), but was still higher, compared with that of control rats (p<0.05). Likewise, acute L-NAME treatment did not affect significantly the RPF in normal rats, but decreased the RPF in diabetic rats (p<0.05). However, the RPF in diabetic rats was still higher than that in normal rats (p<0.01). Section title: Renal hemodynamic studies (Study II) Educational score: 4.0952887535095215 Domain: biomedical Document type: Study Language: en Fig. 5 shows urinary albumin excretion rate for 24 h at day 28 of diabetes. Diabetic rats had increased urinary albumin excretion as compared with normal rats (p<0.01). Insulin treatment in diabetic rats effectively prevented the increased urinary albumin excretion. L-NAME treatment in diabetic rats significantly retarded the increased urinary albumin excretion at day 28 of diabetes (p<0.05), but could not return it to the levels of normal rats (p<0.05). Section title: DISCUSSION Educational score: 4.294288158416748 Domain: biomedical Document type: Study Language: en The present study shows that plasma and urinary excretion levels of NO 2 − /NO 3 − are significantly higher in diabetic rats of 28 days duration than in normal rats. Since NO 2 − /NO 3 − is the stable oxidation product of NO, which has a very short half-life, NO 2 − /NO 3 − can be taken as an index of NO production. Furthermore, measurement of urinary excretion of NO 2 − /NO 3 − provides a marker for assessment of the renal NO production 26 ) . It is reasonable to assume that the higher blood levels are the result of a generalized increase in synthesis of NO throughout the body. The blood samples for these measurements were collected from the trunk and, therefore, the central circulation, although regional differences in various organs may exist. Because of the higher blood levels and the higher GFR in the diabetic rats, the filtered load of NO 2 − /NO 3 − was approximately two-fold greater in the diabetic rats than in the normal rats and urinary excretion was about five-fold greater. The kidney is therefore a major organ for excretion of NO 2 − /NO 3 − in both normal and diabetic rats. These results agree with other studies 12 , 13 ) . Blood glucose levels in insulin-treated diabetic rats were significantly lower than those in diabetic rats without insulin therapy, but still higher than those in normal rats. Insulin therapy effectively prevented the increment of the blood and urinary excretion levels of NO 2 − /NO 3 − in diabetic rats, although blood glucose levels reduced markedly, but were still higher than normal levels. L-NAME, nonspecific inhibitor of nitric oxide synthase, also completely prevented the diabetes-related increases in blood and urinary NO 2 − /NO 3 − levels in diabetic rats. Section title: DISCUSSION Educational score: 4.2982096672058105 Domain: biomedical Document type: Study Language: en In the current study, NO synthesis was increased in diabetes, and insulin or L-NAME effectively prevented the enhanced NO synthesis in diabetes, respectively. These results are similar to the other studies 21 , 22 ) . In contrast, a number of studies 23 – 27 ) have shown that vascular relaxation in response to agents which release NO is impaired in diabetic animals and humans, and it has been postulated that diabetes interferes with synthesis or release of NO by endothelial cells. In addition, previous studies demonstrated an impairment of NO generation and/or stability in glomeruli from diabetic rats 14 ) , and attenuated glomerular cGMP production and renal vasodilation in diabetic rats 15 ) . The precise findings of altered NO synthesis or action in diabetes is uncertain and may differ depending on duration and severity of diabetes. Several lines of evidence suggest that NO generation and action may be increased early in diabetes 28 , 29 ) . Our data support this possibility. By contrast, stability and action of NO may progressively decline later in diabetes, due to increases in the production of reactive oxygen species, oxidized LDL and advanced glycosylation endproducts combined with impaired antioxidant defense systems 30 – 38 ) . Thus, both a reduction in NO action and an increase in toxic products of NO such as peroxynitrite may contribute to diabetic glomerular injury in later stage 39 ) . The mechanisms whereby diabetes mellitus may argument NO production are unclear. Excessive NO biosynthesis could result from either the increased availability of its precursor, L-arginine, or the enhanced activity of NOS. Diabetic rats usually intake much more food, which may contain L-arginine. NO has been identified as a mediator of protein-induced hyperfiltration 40 – 42 ) . We did not examine the L-arginine concentrations in plasma and tissues, and so can not exclude the possibility of the increased availability of NO precursor, L-arginine, due to increase in food intake or altered amino acids metabolism in diabetes. Section title: DISCUSSION Educational score: 4.177910327911377 Domain: biomedical Document type: Study Language: en Our studies showed increases in nitric oxide synthase by Western blot analysis. In these studies, the three isoforms of NOS (bNOS, iNOS, and ecNOS) proteins were all increased in the renal cortex, whereas they remained unaltered in the renal medulla at day 28 of diabetes. Whether NOS proteins expression is altered, and, furthermore, what kind of isoforms of NOS proteins in diabetic rats is altered, is not clear yet. No changes 43 ) or increases 44 , 45 ) in iNOS protein were detected in diabetes. Recent study 44 ) suggests that glycated albumin modulates endothelial cell iNOS activity by stimulating TNF- α synthesis. NO changes 46 ) or decreases 45 ) , or increases 12 ) in ecNOS activity or expression, were also reported in diabetes. Our data support that NO production is increased in early diabetes and increased NO production is due to increases in all isoforms of NOS. Section title: DISCUSSION Educational score: 4.181689262390137 Domain: biomedical Document type: Study Language: en In the present studies, GFR and RPF were significantly increased in diabetes compared with controls. However, filtration fraction between the groups did not have significant difference. Insulin treatment effectively prevented the glomerular hyperfiltration and hyperperfusion in early diabetes, and chronic inhibition of NO synthesis by L-NAME did not completely prevent the altered renal hemodynamics, including increases in GFR and RPF, but attenuated the diabetic hyperfiltration and hyperperfusion. The mediators of glomerular hyperperfusion and hyperfiltration in early diabetes have not been confirmed yet 9 ) , but mediators, such as growth hormone 47 ) and insulin-like growth factors 48 , 49 ) , glucagon 50 ) , atrial natriuretic peptide 51 , 52 ) , kinins 53 ) and glucose itself 54 ) , have been proposed. Nitric oxide is one of the important vasodilators in vasculature and, furthermore, in the kidney participates in several vital processes, including the regulating glomerular and medullar hemodynamics, the tubuloglomerular feedback response, renin release and the extracellular fluid volume 10 , 11 ) . Section title: DISCUSSION Educational score: 4.246634483337402 Domain: biomedical Document type: Study Language: en Recent studies 12 , 13 ) proposed that NO is a mediator of diabetic hyperfiltration and hyperperfusion. In our studies, the renal hemodynamic responses of diabetic rats to NO inhibitor are consistent with the hypothesis that excessive NO production participates in the pathogenesis of diabetic hyperfiltration and hyperperfusion. Acute administration of L-NAME partially reversed hyperfiltration and hyperperfusion in previously untreated diabetic rats, as opposed to unaltered GFR and RPF in controls. These enhanced responses support that excessive NO production may participate in the glomerular hyperfiltration and hyperperfusion in early diabetes. Data obtained in rats with chronic NO inhibition were consistent with those from acute experiments, since diabetic hyperfiltration and hyperperfusion were partially prevented by L-NAME treated, suggesting that NO participates in diabetic hyperfiltration and hyperperfusion. Chronic NO inhibition did not completely prevent glomerular hyperfiltration and hyperperfusion, rather attenuated them in diabetic rats. These results suggest that NO is one of the important mediators in altered hemodynamic changes in diabetes, and other mediators may participate in diabetic hyperfiltration and hyperperfusion. Our results are consistent with other studies 12 , 13 , 22 ) . Section title: DISCUSSION Educational score: 4.368906497955322 Domain: biomedical Document type: Study Language: en There is general agreement that the macula densa is the principal site of bNOS gene expression in the kidney 55 , 56 ) . While both tubuloglomerular feedback (TGF) and myogenic mechanisms contribute to autoregulation, the TGF mechanism is essential for the highly efficient autoregulation that is characteristic of the renal circulation 57 ) . Most if not all autoregulatory responsiveness is vested in preglomerular arterioles. NO exerts vasodilating influences on both afferent and efferent arterioles 10 , 11 , 58 , 59 ) , although the effects on the afferent arteriole appear to predominate 60 ) , and neuronal NO may have a unique role in counteracting TGF-mediated afferent arteriolar constriction 57 , 61 – 63 ) . Tilton et al 29 ) suggested that elevated glucose induces increases in intracellular levels of diacylglycerol, leading to activation of protein kinase C, which has been shown to activate NOS. Our studies demonstrated the increased expression of bNOS proteins, as well as ecNOS and iNOS proteins. Therefore, these results suggest that increased NO synthesis, due to increases in three isoforms of NOS, may participate in the pathogenesis of diabetic hyperfiltration and hyperperfusion. Lastly, our studies showed that chronic NO inhibition with L-NAME attenuated the urinary albumin excretion compared with diabetic rats, associated with attenuated hyperfiltration and hyperperfusion. Section title: DISCUSSION Educational score: 4.155712127685547 Domain: biomedical Document type: Study Language: en In summary, NO synthesis is increased due to enhanced NOS expression in diabetic rats and chronic NO blockade attenuated hyperfiltration and hyperperfusion in experiments of diabetes mellitus. In addition, diabetic rats exhibited enhanced renal hemodynamic responses to acute NO inhibition and excreted increased urinary NO 2 − /NO 3 − . These results suggest that excessive NO production may contribute to renal hyperfiltration and hyperperfusion in early diabetes. | Study | biomedical | en | 0.999994 |
10063313 | Section title: INTRODUCTION Educational score: 4.596819877624512 Domain: biomedical Document type: Study Language: en Cisplatin is one of the most active anticancer agents for the treatment of lung cancer and the cisplatin-based chemotherapeutic regimens have produced a statistically significant and clinically relevant improvement in survival. The long-term survival is directly linked to the degree of clinical response to chemotherapy 1 , 2 ) . However, more than one-third of patients do not achieve an appreciable clinical response to the cisplatin-based chemotherapy. Therefore, it is important to understand the molecular genetic features that can determine the response or resistance to cisplatin, which could permit the selection of the most suitable patients for the cisplatin-based chemotherapy and enhance the development of an innovative treatment for patients most likely to be refractory to cisplatin. The precise mechanisms responsible for cisplatin-mediated cytotoxicity are not fully understood, but evidence obtained in the last few years indicates that cisplatin inhibits DNA synthesis by double-strand breaks. It reacts readily with the N7 position of purines to form a variety of lethal platinum-DNA adducts, which trigger programmed cell death (apoptosis). Indeed, cells treated with cytotoxic levels of cisplatin display the biochemical and morphologic features of apoptosis 3 – 7 ) . Thus, the resistance to cisplatin can be caused by the loss of the regulation of apoptosis. Section title: INTRODUCTION Educational score: 4.169001579284668 Domain: biomedical Document type: Study Language: en p53 is involved in the activation of apoptosis induced by DNA-damage, such as cisplatin. As a transcriptional activator, p53 increases the transcription of a number of genes and the pattern of transcriptional regulation is critical in determining the cellular response to DNA damage. It is known to activate the transcription of death agonist, bax, but to repress the expression of death antagonist, bcl-2 8 – 14 ) . Given the roles of bcl-2, bax and p53 in cell death and survival, we examined the expression of these genes in small cell and non-small cell lung cancer cell lines to investigate the effect of their expression on the response to cisplatin in an attempt to understand the molecular events contributing to the development of the cisplatin-resistance in lung cancers. Section title: 1. Cell lines Educational score: 4.075738906860352 Domain: biomedical Document type: Study Language: en NCI H69 human small cell lung carcinoma, PC9 human lung adenocarcinoma, PC14 human lung adenocarcinoma and their in vitro selected cisplatin-resistant sublines, H69/CDDP, PC9/CDDP, PC14/CDDP, were kindly provided by Dr. N. Saijo (National Cancer Center Research Institute, Tokyo, Japan). All the cisplatin-resistant cells were cultured in cisplatin-free medium for at least 4 weeks before being used for the experiments. Cells were cultured in RPMI-1640 medium supplemented with 2 mM L-glutamine, 10% fetal calf serum, 10 μg/ml penicillin and 10 μg/ml streptomycin and grown in a humidified incubator with 5% CO 2 at 37°C. All media and chemical reagents were purchased from Gibco Co.(BRL, Paisley, UK). Section title: 2. MTT assay for cytotoxicity Educational score: 4.154129981994629 Domain: biomedical Document type: Study Language: en In vitro cisplatin-induced cytotoxicity was determined by the MTT dye reduction assay. Cells were plated out at a density of 3,000–4,000 cells per well into a 96-well microtiter plates and allowed to attach overnight. The next day, cells were treated with 0.1 to 1,000 μg/ml concentrations of cisplatin (Platosin®; Pharmachemie, Haarlem, Netherlands) dissolved in sterile water and incubated for 4 days. After this treatment, 20μl of MTT (3, (4, 4-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, final concentration of 5 mg/ml, Sigma, MO, USA) were added in each well and incubated at 37°C for 4 hr. After incubation, cells were centrifuged at 200 g for 5 min and the supernatant was aspirated. 200 μl of Dimethyl sulfoxide (DMSO) were added in each well to solubilize the formed formazan crystals. The absorbance was recorded at 540 nm on a Titertek Multiskan® MCC (EFLab, Finland). Wells containing only RPMI-FBS and MTT were used as control. The IC 50 (Inhibitory Concentration 50%) values were the drug concentrations inducing 50% reduction in the absorbance. Each experiment was performed using 6 replicate wells for each drug concentration and three independent experiments were carried out with consistent results. The relative resistance to cisplatin was obtained by comparing IC 50 values of cisplatin-resistant sublines with those of the parental cell lines. Section title: 3.A. Quantitation of cell viability by acridine orange/ethidium bromide uptake Educational score: 4.111479759216309 Domain: biomedical Document type: Study Language: en Approximately 5 × 10 5 cells were harvested after various times post-treatment with cisplatin (10 μg/ml) and washed with phosphate buffered saline (PBS). Cells were stained with acridine orange (100 μg/ml) and ethidium bromide (100 μg/ml) and viewed immediately under a fluorescence microscope. The staining clearly distinguished between viable cells and cells showing condensed chromatin staining characteristic of apoptosis. A minimum 200 total cells were counted and the percentage of apoptotic cells was calculated as follows: % apoptotic cell=(total number of apoptotic cells/total number of cells counted) × 100 Section title: 3.A. Quantitation of cell viability by acridine orange/ethidium bromide uptake Educational score: 2.119690179824829 Domain: biomedical Document type: Study Language: en Three independent experiments were performed with consistent results. Section title: 3.B. DNA isolation and gel electrophoresis Educational score: 4.183547496795654 Domain: biomedical Document type: Study Language: en For the electrophoretic characterization of DNA fragmentation, 3 × 10 6 cells were harvested after 12, 24, 48 and 72 hr post-treatment with 3.3, 10 and 100 μg/ml concentrations of cisplatin and washed twice with PBS, and the pellet in a 1.5 ml eppendorf tube was resuspended in 100 μl lysis buffer containing 10 mM EDTA (pH 8.0), 10 mM Tris-HCl (pH 7.4), 0.5% Triton-X 100 and lysed at 4°C for 10 min. The suspension was centrifuged at 25,000 g for 20 min and the resulting supernatant was harvested in a new eppendorf tube. Each sample was added with 2 μl RNase (20 μg/ml, Sigma) and incubated at 37°C for 1 hr. Each sample was added with 2 μl Proteinase K (20 μg/ml, Sigma) and incubated at 37°C for 1 hr. Then, 20 μl of 5M NaCl and 120 μl of isopropyl alcohol were added to each sample, which was stored at −20°C overnight. The next day, samples were centrifuged at 25,000 g, 4°C for 15 min. The pellets were dissolved with 20 μl TBE buffer containing 10 mM Tris-HCl (pH 7.4), 1 mM EDTA (pH 8) and electrophoresed through 2% agarose gel after adding 4 μl loading buffer (40 % sucrose, bromophenol blue 0.25%) at 100 volts for 90 min. 100bp DNA markers (Bio-Rad, CA, USA) were run in parallel. The gels were visualized by 1μg/ml ethidium bromide staining under ultraviolet light. Section title: 4. p53 Sequencing Educational score: 4.08074426651001 Domain: biomedical Document type: Study Language: en Sequencing of exon 5 to 8 of p53 was performed by polymerase chain reaction (PCR) amplification followed by direct automated sequencing of double-stranded DNA using biotinylated terminators. Section title: 4.A. PCR amplification Educational score: 4.078420162200928 Domain: biomedical Document type: Study Language: en PCR was performed using 200 ng of DNA, 1.5 mg MgCl 2 , 200 mM/liter of each dNTPs, 1 mM/liter of each primer, 0.4 ml of Taq polymerase enzyme and 5 ml of 10 X Taq buffer ((Promega, WI, USA) in a final volume of 50 ml. The cycling profile comprised 30 cycles of denaturation (95°C for 50 sec), annealing (58°C for 90 sec) and extension (72°C for 90 sec). Section title: 4.B. DNA nucleotide primers Educational score: 4.119824409484863 Domain: biomedical Document type: Study Language: en Exon 5 to 8 of p53 were amplified using the Human p53 Exons 5–8 Amplifier Panel™ (Clontech, CA, USA) in accordance with the manufacturers instruction. Their sequences were as follows: exon 5 sense strand, 5 CTC TTC CTG CAG TAC TCC CCT GC 3; exon 5 antisense strand, 5 GCC CCA GCT GCT CAC CAT CGC TA 3; exon 6 sense strand, 5 GAT TGC TCT TAG GTC TGG CCC CTG 3; exon 6 antisense strand, GGC CAC TGA CAA CCA CCC TTA ACC 3; exon 7 sense strand, 5 GTG TTG TCT CCT AGG TTG GCT CTG 3; exon 7 antisense strand, 5 CAA GTG GCT CCT GAC CTG GAG TC 3; exon 8 sense strand, 5 ACC TGA TTT CCT TAC TGC CTC TGG C 3; exon 8 antisense strand, 5 GTC CTG CTT GCT TAC CTC GCT TAG T 3. Section title: 4.C. Direct DNA sequencing of PCR products Educational score: 4.226238250732422 Domain: biomedical Document type: Study Language: en PCR products were primarily purified using 50 ml 7.5 M NH 4 AC and 300 ml 98% ethanol mix extraction and precipitation with 150 ml 70% ethanol. The same primers used for generating the PCR products were also used for the sequencing reactions. Other reagents were supplied in the GATC-BioCycle Sequencing kit (GATC GmbH, Konstanz, Germany). Both strands were sequenced for each exon. GATC-BioCycle sequencing protocol was followed for cycle sequencing of PCR products. 1 μl of the purified PCR product (approximately 40 ng), 5 pM/ml primer, 2μl reaction-buffer, 2μl dITP-Mix and 5 units of Thermo-Sequenase™ (Amersham, Bucks, UK) were well mixed with 20μl distilled water for extension. 5μl of extension-mix was transferred into a 0.5 ml PCR tube and 1μl ddGTP-Mix was added. G-A-T-C reaction tubes were placed on Thermo Cycler (ERICOM INC. CA, USA) and run the following program: denaturation for 3 min 95°C, 30 cycles, and final 4 min 60°C. 2 ml samples were loaded in a 7% denaturating polyacrylamide gel and the electrophoresis was performed on a GATC 1500 unit. Biotinylated terminator of 55 μl NBT (4-nitro blue tetrazolium chloride) and of 55 μl BCIP (X-phosphate/5-bromo-4-chloro-3-indolyl-phosphate; Boehringer Mannheim) in 20ml reaction buffer was used for visualization of DNA colorimetrically. All PCR products were sequenced in both directions. Section title: 5. Western blot analysis of Bcl-2, Bax and p53 Educational score: 4.2303009033203125 Domain: biomedical Document type: Study Language: en Cells were removed from tissue culture flasks using a cell-scraper and centrifuged at 300 X g for 5 min. The pellet was resuspended in ice-cold lysis buffer consisting of 20 mM Tris-HCl buffer (pH 7.4) containing 5 μg/ml aprotinin, 5 μg/ml leupeptin and 1 mM phenylmethylsulphonyl fluoride, and homogenized by the passage through a 26 gauge syringe needle. The suspension was centrifuged at 20,000 rpm for 15 min at 4°C and the supernatant was stored at −70°C until assay. Protein concentration was measured in each cell lysate by the BCA method (Sigma) and equal amounts of total protein were loaded for each blot. Cell lysates were mixed with equal volume of Tris-Glycine SDS-sample buffer and boiled for 2 min. 25 μl of each denatured sample was loaded and electrophoresed through 4–20% Tris-Gylcine gels in Tris-Glycine running buffer (all were Novex, San Diego, USA) for 2 hr at 100 volts. Electrophoresed gels were transferred to nitrocellulose membrane (Bio-Rad) in transfer buffer (12 mM Tris base 1.45 g, 96 mM glycine 7.2 g, methanol 200 ml, distilled water 1,000 ml) for 2.5 hr at 30 volts. Membranes were blocked in 5% skim milk solution overnight and probed with monoclonal mouse anti-human Bcl-2 Ab (1:500, 2 μg/ml of blocking solution, Santa Cruz, CA, USA), polyclonal rabbit anti-human Bax Ab (1:500, 2 μg/ml of blocking solution, Santa Cruz) or monoclonal mouse anti-human p53 Ab (1:500, 2 μg/ml of blocking solution, Zymed, CA, USA) for 90 min at 37°C. After washing with 0.05% Tween-20 three times, membranes were incubated for 45 min with peroxidase-conjugated goat IgG fraction to mouse or rabbit at 37°C. After several washings, they were added with 10 ml of tetramethyl-benzidine solution (Zymed) and shook for 10–30 min for visualization of immunoreactive materials. Immunobloting was repeated in all of the cell lines at least three times with consistent results. Section title: 6. Statistical analysis Educational score: 2.658747911453247 Domain: biomedical Document type: Study Language: en Differences between groups were tested for the significance using student’s two-tailed t-test (SAS Inst. INC, NC, USA), from which P values were calculated. Section title: 1. Cisplatin-induced cytotoxicity Educational score: 4.104742050170898 Domain: biomedical Document type: Study Language: en The cisplatin-induced cytotoxicity in lung cancer cell lines was evaluated by MTT colorimetric assay . The calculated IC 50 values and the relative resistance to cisplatin are presented in Table 1 . In this study, each paired in vitro cisplatin-resistant sublines had 3.1–4.7 fold more resistance to cisplatin than their parental cell lines had ( P <0.05). Among the parental cells, H69 was relatively resistant to cisplatin compared to other parental cell lines, in spite of its histologic type, small cell carcinoma (relative resistance 2.1–3.2, P <0.05). Section title: 2. Cell death induced by cisplatin is due to apoptosis Educational score: 4.0858154296875 Domain: biomedical Document type: Study Language: en In order to examine the nature of cell death induced by cisplatin, cells collected at various time points post-treatment with 10 μg/ml of cisplatin were processed for acridine orange and ethidium bromide staining for the detection of condensed or fragmented chromatin and internucleosomal DNA fragmentation, diagnostic of apoptotic cells. Since we could not observe the clear apoptotic features at the concentration of cisplatin near to IC 50 (3.3 μg/ml), we examined the cellular response to apoptosis at 10 μg/ml of cisplatin. Section title: 2. Cell death induced by cisplatin is due to apoptosis Educational score: 4.1696343421936035 Domain: biomedical Document type: Study Language: en Apoptotic patterns were observed in PC9 and PC14 cell lines as early as 12 hr after cisplatin exposure, and increased after 24 hr. In H69 cells, the accumulation of acridine orange positive cells in response to cisplatin was slower and lower than that of PC9 or PC14 cell lines. The percentage of apoptotic cells remained below 10% after 24 hr and more than 75% cells were viable even after 72 hr in PC9/CDDP, PC14/CDDP and H69/CDDP . Section title: 2. Cell death induced by cisplatin is due to apoptosis Educational score: 4.159857749938965 Domain: biomedical Document type: Study Language: en Examination of internucieosomal DNA fragmentation (DNA ladders) showed the similar pattern of response and DNA ladders were visualized in PC9 and PC14 cells after 24 hr post-treatment, whereas in H69 cells, it was visualized after 48 hr. In cisplatin-resistant sublines, DNA ladders were not visualized after 48 hr . However, at higher concentration of cisplatin (100 μg/ml), DNA ladders were observed after 48 hr in all of the cell lines . Section title: 3. Sequencing analysis of p53 gene Educational score: 4.080985069274902 Domain: biomedical Document type: Study Language: en There was no mutation of p53 in PC9, PC14 and their cisplatin-resistant sublines. The same point mutation was detected in H69 and H69/CDDP, localized in exon 5. As shown in Fig. 5 , the mutation in these cell lines was nucleotide substitution (transversion, GGA→GTA) at codon 171. Section title: 4. Relationship between bcl-2, bax and p53 protein expression Educational score: 4.219215393066406 Domain: biomedical Document type: Study Language: en Recent reports have suggested that p53 tumor suppressor gene product may not only regulate down bcl-2 but regulate up bax gene expression 8 – 14 ) . The endogenous levels of bcl-2, bax and p53 protein expression in lung cancer cells were assessed by Western blot analysis. In this study, the expression of p53 was reduced in H69 and H69/CDDP which had the mutation of p53 compared to other cell lines without p53 mutations. Bcl-2 protein had an inverse relationship with p53, which was detectable only in cells with very low expression of p53 protein such as H69 and H69/CDDP, but not detectable in cells with high expression of p53 protein such as PC9, PC9/CDDP, PC14 and PC14/CDDP. Bax protein was expressed in all cells with variable intensities and the relative amount of bax protein was high in cells with high p53 expression (PC9, PC9/CDDP, PC14, PC14/CDDP) comparing cells with very low p53 expression (H69, H69/CDDP). This indicates that the reduced expression of p53 protein contributes to up-regulation of bcl-2 as well as down-regulation of bax expression. Section title: 5. Regulation of cisplatin-resistance by the expression of p53, bcl-2 and bax Educational score: 4.117399215698242 Domain: biomedical Document type: Study Language: en We examined the relationship between bcl-2, bax and p53 expression and cisplatin-resistance. Among parental cell lines, H69 (small cell carcinoma) was more resistan to cisplatin in spite of its histologic subtype and lower apoptosis than other parental cell lines. These findings suggest that its intrinsic resistance to cisplatin and p53 mutation and up-regulation of bcl-2 may confer the intrinsic cisplatin-resistance in this cell line. Section title: 5. Regulation of cisplatin-resistance by the expression of p53, bcl-2 and bax Educational score: 4.146651744842529 Domain: biomedical Document type: Study Language: en In vitro selected cisplatin-resistant sublines had 3.1–4.7 fold resistance to cisplatin (P<0.05) and markedly reduced apoptosis compared to their parental cell lines. However, we observed no difference in levels of bcl-2, bax and p53 proteins between parental cell lines and their resistant sublines. This indicates that the acquired resistance made through cisplatin-exposure is related to the reduced susceptibility to apoptosis, but not to the alteration in the expression of p53, bax and bcl-2 proteins. Section title: DISCUSSION Educational score: 3.0331523418426514 Domain: biomedical Document type: Other Language: en Lung cancer is one of the most lethal cancers for both men and women in the world. The majority of patients present with advanced, inoperable diseases and overall cure rate for lung cancer approximates 13%. This low rate can be ascribed to the high propensity for metastasis and resistance to chemotherapy 5 , 7 ) . Section title: DISCUSSION Educational score: 4.0489935874938965 Domain: biomedical Document type: Study Language: en Cisplatin has a clinical activity against various solid tumors, especially lung cancers. The effectiveness of chemotherapy with cisplatin is restricted by the emergence of resistant cell populations, and defining of the molecular features that determine the resistance to cisplatin has an important clinical implication. The recent evidence suggests that the genetic regulation of apoptosis may affect the cellular response to DNA damages and, therefore, modulate the sensitivity of tumor cells to cisplatin 4 – 6 ) . Section title: DISCUSSION Educational score: 4.388956069946289 Domain: biomedical Document type: Study Language: en p53 tumor suppressor gene is most commonly mutated in human cancers. It is recognized as an important component of the pathway leading from DNA damage to apoptosis and several studies have suggested that p53 is involved in the activation of apoptosis induced by DNA-damaging anticancer agents 9 , 15 , 16 ) . Introduction of DNA strand breaks leads to a post-transcriptional increase in p53 protein levels 17 , 18 ) . However, whether p53 protein activity is altered or the levels are merely increased is not clear. The induced p53 protein is able to transcriptionally activate a number of different genes, including p21, gadd45 and mdm2 and the induction of p21 appears to be the major mechanism of G1 arrest 19 , 20 ) . However, in some cell types and some physiologic conditions, p53 induction can lead to apoptosis. Although the mechanism remains unclear, the upregulation of bax, death agonist, through transactivation by p53 could play an important role in apoptosis 10 – 13 , 21 , 22 ) . As the role in mediating DNA-damage induced cell cycle arrest was elucidated, it became clear that normal p53 function was also required for DNA-damage induced apoptosis 23 – 25 ) . In this study, we examined in vitro selected cisplatin-resistant sublines made by the continuous exposure of cisplatin. We expected the increasement in the level of p53 protein of these resistant sublines by cisplatin induced DNA-damage. However, we were not able to observe a significant difference in the level of p53 protein compared to parental cell lines. Mutation of p53 and a relatively reduced expression of p53 protein were observed in H69 and H69/CDDP, which showed markedly reduced apoptosis. This indicates that p53 mutation results in the functional loss and instabilization of p53 protein. However, other cisplatin-resistant cell lines, such as PC9/CDDP and PC14/CDDP, which had no mutation of p53 and a comparable level of p53 protein, showed markedly reduced apoptosis compared to their parental cell lines. Because the loss of p53 function can be resulted even with the wild type of p53, it is needed to evaluate the functional activity of p53 in these cell lines. Section title: DISCUSSION Educational score: 4.259176254272461 Domain: biomedical Document type: Study Language: en Although p53 is known to transactivate bax, it suppress bcl-2 expression and the magnitude of p53 suppression of bcl-2 expression may be tissue-specific 8 , 16 ) . The recent analysis on breast cancer shows that the reverse relationship between p53 and bcl-2 expression, and the overexpression of mutant p53, can induce down-regulation of bcl-2 both protein and mRNA level 8 ) . Immunohistochemical study on non-Hodgkin’s lymphoma shows the significant inverse relationship between p53 and bcl-2 protein expression 26 ) . Moreover, in vitro study of overexpression of bcl-2 in cancer cells delays accumulation of p53 and indirectly suppresses bax induction 16 , 27 , 28 ) . In this study, bcl-2 protein was only detectable in small cell lung cancer cells (H69 and H69/CDDP) which had p53 mutation and the decreased expression of p53, suggesting the reverse relationship between these proteins. Bax protein expression was also reduced in H69 and H69/CDDP, indicating indirectly suppression of induction by bcl-2. Section title: DISCUSSION Educational score: 4.525428771972656 Domain: biomedical Document type: Study Language: en Bcl-2 was originally identified at the chromosomal breakpoint of t(14;18)-bearing B-cell lymphomas. The bcl-2 gene encodes for the p26Bcl-2 protein, which suppresses apoptosis 4 , 27 – 34 ) . So, it is now known to belong to the growing family of apoptosis-regulatory gene products, which may either be death antagonist (Bcl-2, Bcl-X L , Bcl-w, Bfl-1, Brag-1, Mcl-1, and A1) or death agonists (Bax, Bak, Bcl-Xs, Bad, Bid, Bik and Hrk) 30 – 32 ) . Most of the proteins encoded by the bcl-2 gene family are predominantly localized in the outer mitochondrial membrane and they possess variable amounts of Bcl-2 homology (BH) regions, which determine their capacities to interact with each other or with other unrelated protein. Bax shows extensive amino acid homology with bcl-2 and forms homodimers and heterodimers with bcl-2 in vivo. When bcl-2 protein is expressed at high level in cells, it may form complexes with bax, preventing bax homodimerization and inhibiting cell death 29 – 32 ) . Thus, the ratio of death antagonist (bcl-2, bcl-X L , etc) to agonist (bax, bcl-Xs, bad, etc) is important in determining whether a cell will response to an apoptotic signal 29 – 32 , 14 , 36 , 37 ) . In this study, bcl-2 was expressed in H69 and H69/CDDP with p53 mutation and the level of bax protein was relatively decreased in these cell lines compared to other cell lines without p53 mutation. The delayed and reduced apoptosis by cisplatin in these cell lines may be attributed to bcl-2. Apoptosis in other cisplatin resistant cell lines was markedly reduced in spite of comparable expression of bax. However, the proapoptotic function of bax can be antagonized by other death antagonist, such as bcl-X L rather than bcl-2, which should be further evaluated. Section title: DISCUSSION Educational score: 4.147263050079346 Domain: biomedical Document type: Study Language: en Small cell lung cancer is well known to be more sensitive to chemotherapy than non-small cell lung cancer and the response approximates 90% with cisplatin-based chemotherapy, including 10–50% of complete response 1 , 2 ) . In this study, H69 is a small cell lung cancer cell. However, it is more resistant to cisplatin than other non-small cell lung cancer cells and it has a reduced and delayed response to apoptosis. Interestingly, p53 mutation and bcl-2 expression were observed in only small cell lung cancer cells (H69 & H69/CDDP). Since our experiment demonstrates that cancer cells with p53 mutation and bcl-2 expression are more resistant to cisplatin and they have a decreased susceptibility to apoptosis, cells naturally expressing bcl-2 and germline mutation of p53 may be intrinsically resistant to cisplatin. Section title: DISCUSSION Educational score: 4.224977493286133 Domain: biomedical Document type: Study Language: en In this study, we could not readily obtain apoptotic features at concentration close to IC 50 (3.3 μg/ml) in all of the examined cell lines. Although PC9 and PC14 cell lines had the wild type p53 and a comparable expression of p53 protein in this study, we could not obtain the clearly visualized DNA fragmentations, a morphologic feature of apoptosis at concentration close to IC 50 (3.3 μg/ml), but it was relevant at higher concentration of cisplatin (10 μg/ml). This indicates that the examined parental cells were not prone to apoptosis, even with a comparable induction of p53. With respect to the responsiveness of tumors to therapies, it is suggested to be derived from the tendency of the parental cells to undergo rapid apoptosis in response to DNA damage 38 ) . For example, the responsive tumor types, such as leukemia, lymphoma or germ cell tumors, are probably derived from cells that primarily use p53 induction as a signal to apoptosis, but the tumors derived from epithelial cells, such as lung carcinoma, do not rapidly signal to apoptosis in response to DNA damage, which use p53 for G1 arrest and which are relatively resistant to current treatments 31 , 41 , 42 ) . During G1 arrest, interfering with DNA-repair enzyme machinery or additional growth factors in environment may enhance the tolerance to DNA damage, provide a survival signal to cells and render the resistance to chemotherapy 3 , 7 , 39 , 40 ) . Section title: DISCUSSION Educational score: 4.116509437561035 Domain: biomedical Document type: Study Language: en In summary, we examined the reduced sensitivity to cisplatin treatment in the examined cell lines paralleled a resistance to apoptosis induction, and cancer cells with endogenous expression of bcl-2 and p53 mutation were more resistant to cisplatin, which might be useful as a factor in tailoring chemotherapy regimens. However, a larger prospective study is needed to confirm the findings of this report. We observed markedly reduced apoptosis in cisplatin-resistant cell lines, but could not find any differences in the expression of apoptosis-regulatory proteins, p53, bcl-2 and bax compared to parental cell lines. It is suggested that the evaluation of functional activity of p53, such as a signal to cell cycle arrest or to apoptosis, is needed to understand the reduced susceptibility to apoptosis in resistant cells. | Study | biomedical | en | 0.999998 |
10063314 | Section title: INTRODUCTION Educational score: 4.268034934997559 Domain: biomedical Document type: Study Language: en It is clear that in human cancer, including lung cancer, deregulation of cell cycle progression is a hallmark of neoplastic transformation and that genes involved in G1/S transition of the cell cycle are especially frequent targets for mutations. A cell-cycle related protein, the recently identified p16 INK4A gene product, plays an important role in the negative regulation of the kinase activity of cyclin dependent kinase(cdk) enzymes 1 ) . p16 INK4A gene is, thus, an important tumor suppressor gene and is also called MTS1(multiple tumor suppressive gene 1) 2 , 3 ) . In various malignancies, reports of alterations in the p16 INK4A gene are much more common than those involving any other oncogene or tumor-suppressive gene ever identified 2 , 4 , 5 ) It was particularly noticeable that there was no p16 expression in more than 70% of cell lines examined in the case of non-small cell lung cancer. It is speculated that p16 INK4A gene could exert a key role in the development of non-small cell lung cancer 6 ) . The purpose of this study was to investigate whether p16 INK4A gene is a suitable candidate for gene therapy in cases of non-small cell lung cancer. After the extraction of total RNA from normal fibroblast cell lines and subsequent reverse transcriptase and polymerase chain reaction, amplified p16 INK4A was subcloned into eukaryotic expression plasmid vector, pRC-CMV. Using lipofectin, the constructed pRC-CMV-p16 was transfected into NSCLC cell lines NCI-H441 and NCI-H157. After the extraction of proteins from cell lysates, the changes in G1 cell-cycle related proteins were investigated with Western blot analysis and immunoprecipitation and the tumor suppressive effect was observed by clonogenic assay. Section title: Cell lines Educational score: 4.031937599182129 Domain: biomedical Document type: Study Language: en Non-small cell lung cancer cell lines NCI-H441, NCI-H157 and NCI-H2009 were kindly donated by Frederic Kaye (National Cancer Institute, U.S.A.) and were grown in RPMI media supplemented with 10% fetal bovine serum and antibiotics. They were maintained at 37°C in a 5% CO 2 incubator and the media was changed twice a week. Section title: Construction of p16 expression plasmid Educational score: 4.112008094787598 Domain: biomedical Document type: Study Language: en p16 expression plasmid(pRC-CMV-p16) was kindly donated by Dr. Gregory Otterson (National Cancer Institute, U.S.A.). For the construction of p16 expression plasmid, RT-PCR was performed using mRNA from NIH3T3 fibroblast cell line (sense primer; 5′ AAG-CTT-ATG-GAG-CCT-TCG-GCT 3′, anti-sense primer; 5′ TTC-GAA-TCA-ATC-GGG-GAT-GTC 3′), and p16 cDNA was made. The lengh of p16 cDNA was 8 amino acids shorter than the natural full length. The PCR product was cloned into pRC-CMV plasmid, and the sequence was verified by sequencing. Using the cesium chloride method, an adequate quantity of pRC-CMV-p16 plasmid was obtained. Section title: Transfection Educational score: 4.043166160583496 Domain: biomedical Document type: Study Language: en Lung cancer cell lines, NCI-H441 and NCI-H157, were transfected via lipofectin, as described by the manufacturer (Life Technologies). Ten micrograms of pRC-CMV-p16 or control pRC-CMV plasmid were transfected into 100-mm-diameter subconfluent plates. Section title: Western blot analysis and Immunoprecipitation Educational score: 4.086613178253174 Domain: biomedical Document type: Study Language: en Cellular protein was extracted 60 hours after transfection by lysing cells with lysis buffer(50 mM Tris-HCl, pH 7.5/250 mM NaCl/5 mM EDTA/0.1% Nonidet P-40(NP-40)/50 mM NaF/1 mM phenylmethylsulfonyl fluoride(PMSF)) containing protease inhibitors(10 ug/ml leupeptin and 50 ug/ml aprotinin). The lysate was incubated for 20 min on ice and was centrifuged(14,000 X g for 20 min); the supernatant was frozen at −70°C until used. Section title: Western blot analysis and Immunoprecipitation Educational score: 4.047414302825928 Domain: biomedical Document type: Study Language: en For Western blot analysis, 200 ug of cellular proteins were electroblotted to nitrocellulose paper after separation on 7.5 – 15 % SDS-PAGE and then allowed to incubate overnight at 4°C with a 1:2000 dilution of primary antibody(anti-human RB, p16, cdk4 or cyclin D1) in PBS containing 5% powdered milk and 1% bovone serum albumin. Conjugation with secondary antibodies and visualization was performed using an ECL kit(Amersham) as recommended by the manufacturer. Section title: Western blot analysis and Immunoprecipitation Educational score: 4.041487216949463 Domain: biomedical Document type: Study Language: en For immunoprecipitation, 500 ug of cellular proteins were immunoprecipitated with 10 ug of anti-human CDK4 or anti-human cyclin D1 antibodies. The immunoprecipitated proteins, separated by protein A-sepharose beads, were immunoblotted with anti-human p16 or anti-human RB antibodies. Section title: Western blot analysis and Immunoprecipitation Educational score: 2.8341867923736572 Domain: biomedical Document type: Study Language: en Antibodies used for Western blot analysis and immunoprecipitation were mouse monoclonal anti-human RB, mouse monoclonal anti-human cyclin D1, rabbit polyclonal CDK4 and rabbit polyclonal anti-human p16(PharMingen, San Diego). Section title: Clonogenic assay Educational score: 3.8967549800872803 Domain: biomedical Document type: Study Language: en After transfection, transfected cells were selected using G418(150 ug/ml) containing media. When visible colonies were seen, the plates were fixed with methanol and stained with crystal violet, and the numbers of colonies were compared. Section title: Expression of p16 protein in transfected cells Educational score: 4.056980609893799 Domain: biomedical Document type: Study Language: en Transfection of pRC-CMV-p16 to p16(−) NCI-H441 cells resulted in p16 expression equal to that seen in non-transfected NCI-H2009, which expresses p16 normally. Control NCI-H441 cells did not express p16 protein, and this indicates that in non-small cell lung cancer cell lines, the pRC-CMV-p16 expression vector is very useful for transfection. Section title: p16:cdk4 complex formation of expressed p16 after transfection with pRC-CMV-p16 Educational score: 3.9587528705596924 Domain: biomedical Document type: Study Language: en p16 protein, expressed in NCI-H441 cells after transfection with pRC-CMV-p16, formed a complex with cdk4. p16 is known to inhibit cdk4 by forming a complex with it and inhibiting the phosphorylation of cdk4 substrate. The fact that p16 protein expressed after transfection formed this complex indicates that p16 functioned normally. Section title: Dephosphorylation of pRB protein after transfection with pRC-CMV-p16 Educational score: 4.067905426025391 Domain: biomedical Document type: Study Language: en To determine the effect of p16 protein expressed after transfection, changes in pRB protein in NCI-H441 cells were investigated. As expected, the amount of phosphorylated pRB protein was less than the amount of phosphorylated pRB protein in control cells . This means that p16 protein expressed after transfection of pRC-CMV-p16 inhibited the phosphorylation of pRB protein by cdk4. Section title: Changes in cyclin D1 and cyclin D1:cdk4 complex after transfection with pRC-CMV-p16 Educational score: 3.8219408988952637 Domain: biomedical Document type: Study Language: en Because p16 protein expressed after transfection inhibited the phosphorylation of pRB, we investigated changes in cyclin D1 and cyclin D1:cdk4 complex, known to be involved in the phosphorylation of pRB. No significant change was apparent in cyclin D1. However, the change in cyclin D1:cdk4 complex could not be determined because baseline cyclin D1:cdk4 complex band was too faint in our experiment. Section title: Change in the pRB:cyclin D1 complex after transfection with pRC-CMV-p16 Educational score: 3.638892889022827 Domain: biomedical Document type: Study Language: en In vivo , cyclin D1 is known to form a complex with pRB, thus affecting the phosphorylation of pRB. After transfection, no change in the pRB:cyclin D1 complex was noted , suggesting that p16 does not function through change in the pRB:cyclin D1 complex. Section title: Tumor suppressive effect of p16 Educational score: 4.076371192932129 Domain: biomedical Document type: Study Language: en The colony-forming ability of NCI-H441 and NCI-H157 cell lines after transfection with pRC-CMV-p16 was compared with that of control cell lines transfected with pRC-CMV. Significantly fewer colonies were observed in plates transfected with pRC-CMV-p16 than in those transfected with pRC-CMV, and this suggests that p16 may have a tumor suppressive effect on non-small cell lung cancer cell lines which do not express p16. Section title: DISCUSSION Educational score: 4.353045463562012 Domain: biomedical Document type: Study Language: en A variety of evidence suggests that p16 is a tumor suppressor gene. p16 INK4A is located in chromosome 9p21, where deletions have frequently been found in carcinomas of lung, kidney, ovary, pancreas, breast, bladder, head and neck and esophagus and in several other tumors, such as glioma, melanoma, leukemia and osteogenic sarcoma 2 , 3 , 7 – 9 ) . In addition, homozygous deletions or mutations of the p16 INK4A gene are also frequently observed in cell lines from these tumors 4 , 10 ) . Because deletion or mutation of p16 INK4A is significantly less frequent in primary cancer tissues than in cancer cell lines, it has been suspected, however, that p16 INK4A may not be a true tumor suppressor gene 5 , 11 ) . In cases of non-small cell lung cancer, about 70% of cell lines do not express p16 protein 6 ) , but mutation of the p16 INK4A gene is not frequent in primary tissues 12 ) . Section title: DISCUSSION Educational score: 4.327700138092041 Domain: biomedical Document type: Study Language: en To confirm the tumor suppressive effect and the mechanisms of p16 INK4A gene in non-small cell lung cancer, we transfected p16 INK4A gene into cell lines of this cancer via lipofectin. p16(−) NCI-H441 cell line transfected with pRC-CMV-p16 showed the formation of p16:cdk4 complex and decreased phosphorylated Rb protein, while the control cell line did not. These results are consistent with those of Sandig et al., who studied the expression of pRB in a hepatocellular carcinoma cell line 13 ) . pRB has been shown to play a protective role in cell apoptosis 14 ) . When its levels are sufficiently reduced, this role can no longer be maintained, and this may explain part of the tumor suppressor function of p16. Clonogenic assay demonstrated that the extent of colony formation was significantly less in p16(−) NCI-H441 and NCI-H157 cell lines transfected with pRC-CMV-p16 than in the control cell line. This study shows that the expression of p16 protein in gene-transfected NSCLC cell lines, in which p16 is absent, leads to p16:cdk4 complex formation and a subsequent decrease in phosphorylated pRb protein levels, and leads ultimately to tumor suppressive effects. Section title: DISCUSSION Educational score: 4.129489421844482 Domain: biomedical Document type: Study Language: en Using pRC/CMV plasmid vector, Okamoto et al. showed that when p16 INK4A gene transfer was involved, colony formation by esophageal cancer cell lines decreased 12 ) . Arap et al . demonstrated the growth suppressive effect of p16 INK4A gene in p16(−) glioma cell lines 15 ) , and this finding is in agreement with those of other reports. 16 , 17 ) Though this effect was not observed in glioma cell lines which express wild-type p16 protein 15 ) . It has been suggested that CDK4 or cyclinD may be amplified or overexpressed 18 , 19 ) , or a G1/S cell cycle pathway such as p53-p21 WAF1 may be inactivated in cases of glioma with wild-type p16 expression 15 ) . This suggests that, for the general use of gene therapy for the treatment of cancer, combination gene therapy involving p53 and p16, for example, may be necessary. Section title: DISCUSSION Educational score: 4.015342712402344 Domain: biomedical Document type: Study Language: en Tumor suppressor gene therapy requires efficient delivery of therapeutic genes and a high level of expression of the newly transduced gene in the tumor. The adenovirus is an efficient expression vector that can be produced in high titers 20 ) . Its expression time in the host cell is short, and it is thus suitable for use in gene replacement strategies involved in cancer treatment. For the clinical trial of p16 in cancer gene therapy, adenovirus vector is one of the suitable candidates. A few in vitro and animal studies on p16 in the adenovirus vector have been published recently 16 , 17 , 21 ) . Section title: DISCUSSION Educational score: 3.9120752811431885 Domain: biomedical Document type: Study Language: en It was difficult to obtain cell lines in which the expression of p16 protein after transfection of p16 INK4A gene in NSCLC cell lines was stable. Because p16-transfected cells which overexpress p16 do not usually survive, we could not obtain data relating to flow cytometric cell cycle analysis or quantitative cytotoxicity tests. This difficuty is a common problem in the study of tumor suppressor genes and, to overcome it, an inducible promotor is usually used in the cloning of expression vectors. Section title: DISCUSSION Educational score: 4.1055145263671875 Domain: biomedical Document type: Study Language: en In this study, we confirmed in vitro that after transfection of p16 INK4A gene to a cell line which has wild-type pRB but does not express p16, p16 formed a complex with cdk4, phosphorylation of pRB decreased, and there was decreased colony formation. This suggests that the p16 INK4A gene can be a candidate for gene therapy in cases of NSCLC in which p16 INK4A gene is inactivated. | Study | biomedical | en | 0.999996 |
10063315 | Section title: INTRODUCTION Educational score: 4.275685787200928 Domain: biomedical Document type: Study Language: en Rheumatoid arthritis is characterized by hyperplasia of synovial lining cells, excessive infiltration of mononuclear cells and extensive destruction of the articular cartilage 1 ) , and this abnormal proliferation of synovial cells is an important event in the pathophysiology of rheumatoid arthritis. Rheumatoid arthritis synovium shows aggressive invasiveness of rheumatoid pannus 2 ) , occurrence of newly formed blood vessels 3 ) and pleomorphic fibroblast-like cells with large nuclei and prominent nucleoli 4 ) . These features resemble those of preneoplastic conditions and suggest that dysregulation of the apoptosis may play a role in the pathogenesis of rheumatoid arthritis. Section title: INTRODUCTION Educational score: 4.226369857788086 Domain: biomedical Document type: Study Language: en The wild type of p53 gene is tumor suppressor oncogene that behaves as a negative growth regulator and regulator of cell survival, proliferation and apoptosis, but the mutant type of p53 gene behaves in an oncogenic maneuver 5 , 6 ) . Mutations of the p53 gene are the most frequently found of all the known tumor suppressor genes and oncogenes 7 ) , and such mutations prolong the half-life of the p53 protein that is normally short(<20 min) and permit its detection using immunohistochemistry 8 ) . Section title: INTRODUCTION Educational score: 4.006952285766602 Domain: biomedical Document type: Study Language: en Mutation of p53 may play a role in manifestation of rheumatoid arthritis synovium, but several studies on p53 expression in synovial tissues of rheumatoid arthritis showed conflicting and various results 9 – 13 ) . For that reason, we investigated the amount and pattern of p53 positive cells in rheumatoid arthritis synovium, in comparison with osteoarthritis synovium, by using immunohistochemistry with two other monoclonal antibodies for p53 to examine p53 expression in synovial tissues of rheumatoid arthritis. Section title: 1. Patients Educational score: 4.036752223968506 Domain: biomedical Document type: Study Language: en Synovial tissue specimens were obtained from nine patients with rheumatoid arthritis undergoing either synovectomy or joint replacement surgery. All patients included in this study satisfied the diagnostic criteria of the American College of Rheumatology 14 ) . They were receiving therapy with nonsteroidal anti-inflammatory drugs and four patients were treated with disease modifying anti-rheumatic drugs and four patients were receiving low dose prednisone. As a control, synovial tissue samples were also obtained from patients with osteoarthritis undergoing joint replacement surgery. The diagnosis of osteoarthritis was based on typical clinical and radiological features. All patients were receiving therapy with nonsteroidal anti-inflammatory drugs. Clinical characteristics of the patients were presented in Table 1 . Tissue samples were fixed in 10% formalin at room temperature, immediately after removal. Section title: 2. Immunohistochemistry Educational score: 1.4918804168701172 Domain: biomedical Document type: Other Language: en Commercial p53 monoclonal antibody(DO-1) was purchased from DACO(A/S, USA) and p53 monoclonal antibody(DO-7) was purchased from Santa-Cruz (Biotechnology, USA). They react with wild type and mutant type of p53 protein. We also purchased DACO LSAB plus kit, Peroxidase(DACO A/S, USA). Section title: 2. Immunohistochemistry Educational score: 4.142613887786865 Domain: biomedical Document type: Study Language: en Consecutive 5 μ m thick paraffin embedded sections were prepared, using standard methods. The avidin-biotin complex(ABC) immunoperoxidase staining were performed using the DO-1, DO-7 monoclonal antibodies for p53 and DACO LSAB plus kit according to the manufacturer’s protocol for paraffin sections. p53 positive reaction was recorded when nuclear or cytoplasmic staining was present. Intensity of staining in the synovial lining, inflammatory mononuclear cells, subsynovial fibroblast-like cells and vascular endothelial cells was judged on a 0 to 4+ scale as follows by the pathologist who was blinded to the clinical diagnosis and disease activity: 0, no staining; 1+, rare positive cells or trace staining; 2+, scattered clusters of positive cells; 3+, moderate staining in a specific region; 4+, extensive staining throughout a region. Section title: 3. Inflammation index Educational score: 4.0950398445129395 Domain: biomedical Document type: Study Language: en One section was stained with haematoxylin and eosin for histologic features of inflammation. Six variables were assessed, based on a protocol described previously 15 , 16 ) , with the modification that a grading system of 0–3 was used. Grading was based on polymorphonuclear cell infiltration, hyperemia, fibrin deposition, mononuclear cell infiltration, synovial proliferation and fibrosis. The assessment was made on the whole specimen, both at low power and high power by the pathologist who was blinded to the clinical data( Table 2 ). Section title: 4. Statistical analysis Educational score: 2.743990659713745 Domain: biomedical Document type: Study Language: en Data were expressed as mean ± SEM and statistical analysis was performed using Sperman rank order test. Differences were considered to be significant when p < 0.05. Section title: RESULTS Educational score: 4.091139316558838 Domain: biomedical Document type: Study Language: en In histologic appearance, synovial tissues, hyperemia, mononuclear cell infiltration, synovial proliferation and fibrosis were observed in the synovial tissues of rheumatoid arthritis more than in those of osteoarthritis, and polymorphonuclear cell infiltration and fibrin deposition were seen in the synovial tissues of osteoarthritis more than in those of rheumatoid arthritis, but there was no significant difference between the two groups(p > 0.05)( Table 3 ). Section title: RESULTS Educational score: 3.970435380935669 Domain: biomedical Document type: Study Language: en Immunohistochemical evaluation was made with p53 positivity defined as nuclear or cytoplasmic(with or without nuclear staining) patterns, as detected with the DO-1 and DO-7 monoclonal antibodies. In comparing tissue immunoreactivity by DO-1 and DO-7, all the samples showed similar results with both antibodies. Section title: RESULTS Educational score: 4.106542110443115 Domain: biomedical Document type: Study Language: en In the synovial tissues of patients with rheumatoid arthritis, p53 positive cells were detected as cytoplasmic pattern in 3 out of 9 samples(33%). One case showed trace staining and two cases showed scattered staining, and the positively stained cells were small and round with characteristics of lymphocytes. Synovial lining cells, subsynovial fibroblast-like cells and vascular endothelial cells were p53 negative. In order to compare p53 expression in the synovial tissues of osteoarthritis, we also investigated synovial samples obtained from patients with osteoarthritis. p53 positive cells were observed as cytoplasmic pattern in 2/5 OA samples(40%). They were inflammatory mononuclear cells that had characteristics of lymphocytes and showed trace staining. In the synovial samples of osteoarthritis, the distribution of p53 positive cells was comparable to that seen in the synovial tissues of rheumatoid arthritis. There were no striking differences in the amount and pattern of p53 positive cells between rheumatoid arthritis and osteoarthritis, and there was no demonstrable correlation between the two groups with respect to inflammation scores and expression of p53 protein in synovial tissues( Table 3 ). Section title: RESULTS Educational score: 3.950474500656128 Domain: biomedical Document type: Study Language: en These results indicate that p53 expression in RA synovial tissue is limited to inflammatory mononuclear cells, and the amount and pattern of p53 positive cells are not significantly different between the synovial tissues of rheumatoid arthritis and osteoarthritis. Section title: DISCUSSION Educational score: 4.040585994720459 Domain: biomedical Document type: Other Language: en The p53 gene acts as a molecular guardian for genomic integrity 5 ) . If DNA is damaged, p53 is induced, stabilized or activated and arrests the cell until the damage is repaired. If the damage cannot be repaired, p53 might initiate apoptosis. Wild-type p53 is thought to promote apoptosis, whilst mutant p53 has the inhibition of apoptosis 6 ) . Section title: DISCUSSION Educational score: 4.057706832885742 Domain: biomedical Document type: Study Language: en Mutant p53 protein, which takes on an abnormal conformation, has a half-life of several hours compared with 20 minutes for the wild-type p53 8 ) . It accumulates in cells and thus becomes immunologically detective. So, positive immunostainning is indicative of abnormalities of the p53 gene and its product. Although immunohistochemical detection of p53 can be influenced by variables, p53 immunohistochemistry is of value in detecting p53 mutation if appropriate care is taken 17 ) . In our study, we used the immunohistochemistry with two monoclonal antibodies for p53, and all the samples showed similar results with both anti-p53 antibodies, DO-1 and DO-7. Section title: DISCUSSION Educational score: 4.136318206787109 Domain: biomedical Document type: Study Language: en Rheumatoid arthritis is a chronic, inflammatory, autoimmune disease characterized by abnormal proliferation of synovial cells that is important in pathogenesis of rheumatoid arthritis 1 ) . The rheumatoid arthritis synovium resembles properties of malignant tumor cells 2 – 4 ) . However, the mechanism of abnormal proliferation of synovial cells is not clear. Most p53 mutations are not inherited and, instead, they arise from a coping error or an attack by a carcinogen. Thus, mutations in p53 may occur in rheumatoid arthritis synovium as a consequence of inflammation and may be associated with synovial proliferation. Section title: DISCUSSION Educational score: 4.234050750732422 Domain: biomedical Document type: Study Language: en To our knowledge, there were five studies on p53 expression in rheumatoid arthritis synovium, but their studies showed different and various results 9 – 13 ) . Firestein et al revealed that p53 protein was detected in both cytoplasmic and nuclei of lining cells, lesser amounts of immunoreactive p53 were detected in the sublining mononuclear cells and p53 expression in rheumatoid arthritis synovium was significantly more than that in osteoarthritis tissues. So, they suggest that somatic mutations in the p53 genes might occur as a consequence of pathogenesis of rheumatoid arthritis 9 , 10 ) . Gonagle et al revealed that p53 is not demonstrable in early RA but seen in three of thirteen established rheumatoid arthritis by immunohistochemistry, and they suggest that p53 expression in rheumatoid arthritis may be secondary to DNA damage as a consequence of chronic inflammation, like the results of studies by Firestein et al 11 ) . On the other hand, Sugiyama et al showed that p53 expression in rheumatoid arthritis synovium was seen only in the cytoplasm of the subsynovial cells. Synovial lining cells and infiltrated lymphocytes did not react with anti-p53 antibodies, and they suggest that p53 oncogene is probably not implicated in the apoptosis process of synovium of rheumatoid arthritis 12 ) . Moreover, Murata et al demonstrated that any mutations in p53 gene were not found from exon 4 through 10 in both CD14-positive synoviocytes and fibroblasts of rheumatoid arthritis and, on the contrary, in some patients(2/13) p53 mRNA expression was significantly decreased in CD14-positive cells. They suggest that loss of function of p53 due to decreased p53 gene expression may contribute to the overgrowth of the synovial tissues in rheumatoid arthritis 13 ) . Our study indicates that p53 expression was detected in 33% of the synovial tissues of rheumatoid arthritis and it was limited to cytoplasms of inflammatory mononuclear cells, and there was no significant difference in the amount and pattern of p53 positive cells between the synovial tissues of rheumatoid arthritis and osteoarthritis. The expression of p53 protein was also unrelated to the degree of histologic scores in both groups. We do not know the reason why several studies, including our study on p53 expression in RA synovium, showed conflicting results. Section title: DISCUSSION Educational score: 4.117792129516602 Domain: biomedical Document type: Study Language: en Evidence has been obtained of cytoplasmic p53 gene product localization, not necessarily ascribed to gene mutations 18 ) . p53 cytoplasmic localization may be due to binding to viral or cellular products, may be consequent to the ineffective nuclear translocalization and may depend on the conformational and phosphorylation status of the protein 19 ) . Therefore, in our study, there is the possibility that the expression of p53 cytoplasmic localization may be not due to mutations in RA and OA synovial tissues. The expression of other proto-oncogenes in rheumatoid arthritis synovial tissue has been documented in several studies, but it has remained unclear whether transforming oncogenes are also present 20 – 22 ) . Therefore, we also believe that the exact expression and role of p53 gene, as well as other proto-oncogenes, remain to be clearly established in the pathogenesis of rheumatoid arthritis. Section title: DISCUSSION Educational score: 2.6927974224090576 Domain: biomedical Document type: Other Language: en It has been reported that the presence of anti-p53 antibodies may have been associated with the accumulation of mutant p53 proteins in the tumor and they can be considered a marker for the presence of mutations in cancers 23 , 24 ) . It will be also necessary and interesting to examine anti-p53 antibodies in rheumatic diseases, and that will be also helpful in understanding the pathogenesis of rheumatic diseases. We are continuing the study on anti-p53 antibodies in rheumatic diseases. Section title: DISCUSSION Educational score: 4.093863487243652 Domain: biomedical Document type: Study Language: en In conclusion, our study showed that, in contrast to other studies, p53 expression was only restricted to inflammatory mononuclear cells, the amount and pattern of p53 positive cells are not significantly different between the synovial tissues of rheumatoid arthritis and osteoarthritis and that there was no correlation between inflammation scores and expression of p53 protein in synovial tissues of both groups. These findings suggest that altered p53 expression may not play a significant role in the manifestation of rheumatoid arthritis synovium. However, these data need to be strengthened by increasing the number of samples and molecular biology approaches, and the precise role of p53 remains the subject of ongoing study. | Study | biomedical | en | 0.999997 |
10063316 | Section title: INTRODUCTION Educational score: 3.457967519760132 Domain: biomedical Document type: Study Language: en Behcet disease is characterized by recurrent painful oral ulcer and genital ulcer, skin diseases and uveitis. It may involve cardiovascular, gastrointestinal and central nervous system, as well as joints. The etiology is unknown and its pathophysiology is incompletely understood. The diagnosis of Behcet disease primarily depends on the patient’s history and clinical findings. Arthritic manifestation is one of the minor manifestations and it is usually overlooked. The author analyzed 35 cases of Behcet disease patients, mainly for arthritic manifestations. Section title: 1. Patients Educational score: 3.909925699234009 Domain: biomedical Document type: Study Language: en Among the patients who visited the Rheumatology Division, Keimyung University Dongsan Medical Center, Taegu, Korea from March 1997 to February 1998, 52 patients were compatible for the diagnosis of Behcet disease according to the Shimizu criteria 1 ) . Forty seven patients with more than 3 months follow-up were enrolled in this study. Forty three patients were referred to the Rheumatology Division by primary care physicians, two from Department of Ophthalmology and two from General Surgery in Keimyung University Dongsan Medical Center. They had been diagnosed as having Behcet disease by careful history taking, medical record review and thorough physical examination. On initial visit, it usually took more than thirty minutes. All patients were followed-up closely and they visited the hospital every two weeks. A direct telephone number was given to the patients and, whenever they felt uncomfortable with ill-defined symptoms, they discussed with the examiner. The Shimizu criteria were retrospectively and strictly applied again, and five uncertain patients were excluded. Especially, regarding an oral ulcer, only the patients who complained of an evident, recurrent, painful one were included. “Possible” type of five Behcet cases were excluded and two patients, who carried another autoimmune diagnosis(SLE), were excluded, too. Thirty five patients were finally included in this study. Section title: 2. Clinical features Educational score: 3.829448699951172 Domain: biomedical Document type: Study Language: en The presence of various manifestations were evaluated and recorded as positive or negative. Recurrent aphthous ulceration in the mouth, various skin lesions(erythema nodosum-like eruptions, subcutaneous thrombophlebitis, hyper-irritability of the skin), eye lesions(recurrent hypopyon iritis or iridocyclitis, chorioretinitis) and genital ulcerations were evaluated. The presence of joint manifestations, gastrointestinal and vascular lesions, epididymitis and central nervous system involvements were evaluated too. Section title: 2. Clinical features Educational score: 1.6219538450241089 Domain: biomedical Document type: Other Language: en They were classified according to the subtypes. Section title: 3. Joint manifestations Educational score: 3.735628604888916 Domain: biomedical Document type: Study Language: en Arthropathy was defined as arthralgia with the presence of swelling and/or tenderness. Regarding the joint manifestations, the involved joints, tenderness, swelling and the pattern of the articular symptoms were evaluated. The pattern of involvement included the number of involved joints and the duration of arthropathy. Section title: 4. Laboratory and radiologic evaluation Educational score: 3.2708585262298584 Domain: biomedical Document type: Study Language: en Basic laboratory tests, including CBC, erythrocyte sedimentation rate (ESR, Westergren), C-reactive protein (CRP), complement, immunoglobulin, rheumatoid factor (RF) and antinuclear antibody (ANA), were done. HLA studies were done using Terasaki tray(One Lambda, Inc. U.S.A.). Synovial fluid analysis was tried whenever the patient had sufficient amount of swelling and agreed to aspirate. Section title: 4. Laboratory and radiologic evaluation Educational score: 1.7521134614944458 Domain: biomedical Document type: Other Language: en Simple radiologic studies of mainly involved joints were done. Section title: 5. Comparison with rheumatoid arthritis(RA) Educational score: 2.8041772842407227 Domain: biomedical Document type: Study Language: en The presence and the duration of morning stiffness, which is relieved by exercise, were also evaluated. The involved joints, tenderness, swelling and the number of involved joints were evaluated, as already mentioned above. RF and simple radiologic studies of mainly involved joints were evaluated again, as already mentioned above, for the purpose of comparing the data with those of rheumatoid arthritis. Section title: 5. Comparison with rheumatoid arthritis(RA) Educational score: 1.8145811557769775 Domain: biomedical Document type: Study Language: en The names of the initial diagnosis of the patients with joint manifestations were asked. Regarding oro-genital ulcers or skin lesions, the author asked whether they were asked of the presence of them by their primary care physicians. Section title: 1. Patient characteristics Educational score: 2.12443208694458 Domain: biomedical Document type: Study Language: en These are shown in Table 1 . Mean age was 37 and disease onset was 31. The range of duration between the first clinical manifestation and the diagnosis was from 1 to 25 years. Eighty percent of the patients were female. Section title: 2. Clinical features and interpretation according to the subtype Educational score: 4.052709102630615 Domain: biomedical Document type: Study Language: en All 35 patients had evident, recurrent, painful oral ulcers by the study definition. In 46%, they presented as multiple forms. Genital ulcers were found in 29%. Various skin lesions, including erythema nodosum-like eruption, subcutaneous thrombophlebitis and hyper-irritability of the skin were noted in 77%. Uveitis was evident in 9% of the patients. Joint manifestations appeared in 97%. Gastrointestinal ulcerations and vascular manifestations were found only in 6%, respectively. In vascular manifestations, one patient showed three vascular events (cerebral infarction and carotid, femoral aneurysms). Epididymitis and central nervous system involvement were not present. Section title: 2. Clinical features and interpretation according to the subtype Educational score: 1.8615422248840332 Domain: biomedical Document type: Study Language: en The complete type was found only in 3%. Most of them were incomplete and the suspected type. The possible type was 0%, since it was excluded originally. Section title: 2. Clinical features and interpretation according to the subtype Educational score: 1.7470120191574097 Domain: biomedical Document type: Study Language: en These are summarized as Table 2 . Section title: 3. Joint manifestations Educational score: 4.011696815490723 Domain: biomedical Document type: Study Language: en Joint manifestations appeared in 34 out of 35 patients(97%). Knee, proximal interphalangeal and metacarpophalangeal joints were the main sites. Other involved joints included wrist, elbow, shoulder, ankle, MTP and DIP. Tenderness was prominent in 91% and swelling was noted in 44%. In number of affected joints, monoarticular was 38%, oligoarticular 15% and polyarticular 47%. In most cases, the articular symptom was short-lasting. The duration of arthropathy was less than one week in 59%, one to four weeks in 18%, persisting more than 4 weeks in 18%. Section title: 3. Joint manifestations Educational score: 1.782099962234497 Domain: biomedical Document type: Study Language: en These are summarized as Table 3 . Section title: 4. Laboratory and radiologic studies Educational score: 3.321955680847168 Domain: biomedical Document type: Study Language: en Basic laboratory tests, including CBC, ESR, complement and immunoglobulin were nonspecific(data not shown) except CRP. CRP was positive in 12 patients and the mean concentration was 2.02 mg/dl (normal <0.32). It was positive in all complete type and in most of incomplete type. It was also positive in some of suspected type who had very active or advanced manifestations. These are summarized as Table 4 . Section title: 4. Laboratory and radiologic studies Educational score: 3.0381813049316406 Domain: biomedical Document type: Study Language: en RFs were found in 9% and they were all in low titer. ANA was not found at all. HLA B51 was positive in 46%(13/28). Synovial fluid analysis was available in 3 patients. All patients showed inflammatory synovial fluid with polymorphonuclear cells dominant. Section title: 4. Laboratory and radiologic studies Educational score: 3.4062232971191406 Domain: biomedical Document type: Study Language: en Radiologic studies of mainly involved joints were available in 85% (29/34) of patients. Among these, 90% were normal. Abnormal findings included minimal joint space narrowing and bony osteophytes. Section title: 5. Comparison with RA Educational score: 2.0399506092071533 Domain: biomedical Document type: Study Language: en Thirty five percent(12/34) of patients complained of morning stiffness. It lasted less than 15 minutes in all patients. Section title: 5. Comparison with RA Educational score: 2.0277931690216064 Domain: biomedical Document type: Study Language: en Polyarticular presentation was found in 47%. PIP, MCP, wrist, elbow, shoulder, ankle and knee were commonly involved. These were already shown in Table 3 . Section title: 5. Comparison with RA Educational score: 1.7291558980941772 Domain: biomedical Document type: Study Language: en Thirty one patients visited local medical clinics due to their joint problems. In Table 5 , initial diagnoses of the patients by their primary care physicians, later found as Behcet arthritis, were summarized. Section title: 5. Comparison with RA Educational score: 1.9419198036193848 Domain: biomedical Document type: Study Language: en Sixteen patients out of 31 with joint manifestations were carrying a diagnosis of rheumatoid arthritis. None of them were asked by their primary care physicians whether they had recurrent oro-genital ulcers or erythema nodosum-like skin lesions. No definite answer was obtained from their primary care physicians when the patients asked about the relation of skin lesion and the articular symptoms. Section title: DISCUSSION Educational score: 2.3002030849456787 Domain: biomedical Document type: Other Language: en Behcet disease is a systemic disease classified among vasculitis. The clinical picture is characterized by mucocutaneous and ophthalmologic manifestations. It is seen mostly in countries along the “Silk Road”, which include Turkey, Iran, Japan, China and, possibly, Korea. Section title: DISCUSSION Educational score: 4.0528717041015625 Domain: biomedical Document type: Study Language: en Behcet disease is diagnosed by a few set of diagnostic criteria, including the Shimizu diagnostic criteria 1 ) and the International Study Group criteria 2 ) . In this study, the Shimizu criteria were used since they were useful in dealing with minor manifestations, including arthritis. The Shimizu criteria are composed of major and minor criteria. Major criteria include recurrent aphthous ulceration in the mouth, skin lesions(erythema nodosum-like eruptions, subcutaneous thrombophlebitis, hyper-irritability of the skin), eye lesions(recurrent hypopyon iritis or iridocyclitis, chorioretinitis) and genital ulcerations. Minor criteria include arthritic symptoms and signs(arthralgia, swelling, redness), gastrointestinal lesions(appendicitis-like pains, melena, etc.), epididymitis, vascular lesions(occlusion of blood vessels, aneurysms), and central nervous system involvements(brain stem syndrome, meningo-encephalomyelitic syndrome, confusional type). It was proposed by them that if all four of the major symptoms appeared during the clinical course of the disease, the syndrome would be classified as “complete”. The “incomplete” type requires three majors or one major symptom and definite ocular involvement. The “suspected” type requires two major symptoms, and the “Possible” type requires one major symptom. Section title: DISCUSSION Educational score: 3.909177541732788 Domain: biomedical Document type: Review Language: en There are two comments that should be emphasized. First, in most cases with Behcet disease, various clinical manifestations occur in sequence 3 ) . Recurrent oral ulcer is the most common and the earliest symptom, and it is followed by genital and skin lesions. After the skin lesions, arthritis, neurological manifestations and ocular lesions occur which means, although ocular involvement tends to be the most serious problem, it usually does not occur very early in the course of the disease. In other words, although the appearance of uveitis makes the diagnosis of Behcet disease “complete”, it dose not necessarily mean that we should wait till the patient develops full systemic, lethal manifestations to confirm the diagnosis. Second, among the so-called minors, arthritic manifestation is known as the most common manifestation 4 – 7 ) . Section title: DISCUSSION Educational score: 4.03155517578125 Domain: biomedical Document type: Study Language: en The purpose of this study is to emphasize the clinical significance of Behcet disease, especially when the patients present with arthritic manifestations. In Korea, there were two reports focusing arthritic manifestations in Behcet disease among 150 papers for Behcet disease 8 , 9 ) . However, one 8 ) of them was done in Behcet Disease Specialty Clinic with already known Behcet disease patients, and the other 9 ) did not mention in detail how the patients presented to the doctors. The present study was not conducted at Behcet Disease Specialty Clinic, and the author did not intend to collect all Behcet patients from other departments. Most of them just came to the Rheumatology Division with arthralgia or arthritis, one among various early manifestations. That is why the arthropathy was the most common manifestation, excluding oral ulcer, since it was prerequisite to be enrolled in this study. Since most of them complained of arthritis, the earlier manifestation, the suspected type occupied most of this study group, instead of the clinically more advanced type, such as the incomplete type. The lesser presence of HLA B51 is also explainable by this reason. It was known that there was an association between the presence of HLA B5 and more severe types of Behcet disease 10 – 13 ) . Frequency of non-arthritic clinical features were similar to those of previous reports 4 – 7 ) , although the size of the present study group was too small to compare with. Section title: DISCUSSION Educational score: 4.040097236633301 Domain: biomedical Document type: Study Language: en Knee was the most commonly affected joint, as previously reported 8 , 9 , 14 ) . Sixteen patients with joint manifestations were carrying a diagnosis of RA when they were first enrolled in the present study. Most of them were notified by their primary care physicians as having seronegative RA. It is speculative that they are regarded as satisfying the classification criteria for RA by the articular involvement, morning stiffness 15 ) . It was likely that most doctors were aware of RA and they knew that rheumatoid factor might not be found in some RA patients. To be classified as RA, the joint swelling should be present and persistent, instead of simple tenderness or transient arthritic episode. Morning stiffness should be remarkable, instead of brief discomfort. By careful history taking and thorough physical examination, the author could find out quite easily that they had Behcet arthritis, after all possible rheumatic diseases had been excluded. With full and careful interpretation of all clinical manifestations, it may not be a problem for an experienced physician to differentiate RA with most of Behcet disease. However, for those who do not understand the heterogeneity of rheumatic diseases, Behcet disease may pose a diagnostic dilemma, especially when it presents very similar to RA with polyarticular involvement on PIP, MCP of hands and with equivocal morning stiffness. Section title: DISCUSSION Educational score: 4.103908061981201 Domain: biomedical Document type: Study Language: en Behcet disease was characterized by attacks and remissions 16 ) . It was known that the duration of attacks varied from a few days to a few weeks, and attacks were followed by remissions. Joint manifestations were known usually as non-erosive, even after extended periods of time. In the present study, the duration of each attack was less than 4 weeks in 76%, and this result was similar to the previous report 9 ) . Knee, PIP, MCP, wrist and shoulder were commonly involved, and the arthritic manifestation was present only for several days in more than half of the patients. In this context, it seemed feasible to say that arthritic manifestations in Behcet disease may present as palindromic rheumatism 17 – 19 ) . Palindromic rheumatism is characterized by recurrent acute episodes of arthritis. The joints involved in initial attacks in palindromic rheumatism are mostly knee, wrist, MCP, PIP and shoulder 18 ) . Each episode of palindromic rheumatism lasts, not infrequently, for a few weeks 19 ) , and there is no bone and cartilage destruction even after repeated episodes. Section title: DISCUSSION Educational score: 2.4519565105438232 Domain: biomedical Document type: Study Language: en CRP was positive in all complete type and most of incomplete type. It was also positive in some of suspected type who had very active or advanced manifestations, such as widespread erythema nodosum-like skin lesion, crippling arthritis, gastrointestinal ulceration or vascular aneurysm. It is likely that CRP may be positive in “active” Behcet diseases. Section title: DISCUSSION Educational score: 3.9160425662994385 Domain: biomedical Document type: Review Language: en In Korea, even after publication of about 150 papers mentioned above, it seems that Behcet disease is still regarded as a very rare disease to primary care physicians and patients, and especially, the arthritic manifestations in Behcet disease is usually overlooked. It is likely that many doctors do not completely understand the complex nature of Behcet disease, which includes the various clinical spectrum and clinical evolution that was mentioned above in detail. It is speculative that they usually understand Behcet disease as “strict” symptom triad which is composed of oral ulcer, genital ulcer and uveitis. Since they usually think that all manifestations should appear at the same time, at presentation to them, they will not diagnose a patient as Behcet disease if the patient has only one or two manifestations. Patients with oro-genital ulcers may not seek help since the clinical manifestation is sometimes tolerable or relieved by topical agents. Otherwise, they may feel uncomfortable in visiting a doctor’s office with genital problems which are not very urgent. Various skin lesions, including erythema nodosum-like eruptions, subcutaneous thrombophlebitis and hyper-irritability of the skin, manifest so diverse and are mostly subclinical. From these contexts, arthralgia or arthritis can be the first presenting problem in many Behcet disease patients. Section title: DISCUSSION Educational score: 3.377706289291382 Domain: biomedical Document type: Study Language: en The present study had a few limitations. The size of the sample was too small and the duration of follow-up was too short to discuss the entire clinical progression. Most patients were the incomplete or suspected type, instead of the complete type, since the author focused on early presenting manifestations and early detection. By the way, recently, there was a report focusing heel pain in Behcet disease patients which suggested that arthropathy in Behcet disease might be regarded as another entity of seronegative spondyloarthropathy 20 ) . In the present study, heel pain was present in one patient. The present study was focused only on articular manifestations and future study, including the enthesopathy, seems to be required. Section title: CONCLUSION Educational score: 2.057408332824707 Domain: biomedical Document type: Other Language: en In Behcet disease, various manifestations can be found. The arthritic manifestation seems quite common. It may present as seronegative rheumatoid arthritis. Otherwise, it may present as palindromic rheumatism. Much more attention to this disease and a nationwide survey in Korea seem to be required for better understanding, early detection and appropriate treatment. | Study | biomedical | en | 0.999995 |
10063317 | Section title: INTRODUCTION Educational score: 3.911609411239624 Domain: biomedical Document type: Clinical case Language: en Pylephlebitis is defined as septic thrombophlebitis of the portal vein or one of its tributaries, usually secondary to suppuration either in the region drained by the portal venous system or in structures contiguous to the portal vein (e.g., the common bile duct) 1 ) . Pylephlebitis, that previously well-characterized complication of appendicitis, is rare now, owing to core expeditious surgical management of intra-abdominal infections and the advent of antibiotic therapy 2 ) . Despite this rarity, mortality remains high and the number of case reports dealing with this entity seems to have increased over the past 15 years 3 ) . The current widespread use of abdominal ultrasonography and CT may, in part, explain the recent increase in reported cases. We report on a case of pylephlebitis and multiple liver abscesses secondary to appendicitis. Section title: CASE REPORT Educational score: 3.9368233680725098 Domain: clinical Document type: Clinical case Language: en The 37-year-old man had a 5-days duration of fever, chills, small amount of watery diarrhea and yellowish skin discoloration. His past medical and surgical history was unremarkable, but he recalled an episode of diffuse abdominal pain, nausea and vomiting more than 3 weeks earlier. He developed fever (38.8°C) without any focal symptoms. Bowel sounds were slightly increased and minimal right upper and lower quadrant tenderness was presented. Peripheral white blood cell count was 11,300/mm 3 (segment neutrophil: 66%, metamyelocyte: 20%). Hematocrit was 27.4% and platelet count was 76,000/mm 3 . Other values were SGOT 68 IU/L, SGPT 52 IU/L, gamma-GPT 68 IU/L, alkaline phosphatase 152 IU/L, total-bilirubin 16.79 mg/dL, direct—bilirubin 14.30 mg/dL, protein 5.54 g/dL and albumin 2.81 g/dL. Abdominal ultrasonography revealed a thrombus in the upper SMV, lower extrahepatic portal vein and dilated SMV. Hepatic vein and IVC were not remarkable. Doppler studies showed antegrade flow in the portal vein and space-occupying lesions in the liver. GB was collapsed with diffuse wall thickening. The pancreas and kidneys appeared normal. Bactroides fragilis was grown from blood culture. No micro-organism was cultured from stool and urine. Since intra-abdominal septic foci were suspected, abdominopelvic CT scan was taken. A contrast-enhanced CT scan confirmed the portal vein thrombosis and also demonstrated superior mesenteric vein thrombosis. Abnormal multiple focal densities located in the liver were multiple, small septic emboli. Dirty fat sign, with streaky infiltration and enlarged lymph nodes, were noted in the right lower abdomen, adjacent to the cecum. Normal appendix was not visible. An inflammatory condition around the cecum, such as chronic perforated appendicitis, was highly suggested. A small amount of abnormal fluid was seen around the liver, right paracolic gutter and in the pelvis. On the basis of the microbiological data, treatment was done with imipenem alone 0.5g/Iv q 6hours. On the 7th hospital day, the patient’s condition continued to improve and he was discharged 4 weeks later. Follow-up CT scan showed minor residual pericolonic inflammation, resolution of the multiple liver abscess and improvement of the portal vein thrombosis. After 14 days from treatment with imipenem, his bilirubin level was lowered to near-normal level (total-bilirubin 2.5 mg/dL, direct-bilirubin 1.3 mg/dL). He recovered. Further treatment was not needed. Section title: DISCUSSION Educational score: 2.6704626083374023 Domain: biomedical Document type: Other Language: en Pylephlebitis or Septic thrombophlebitis of the portal vein, a precursor of liver abscess, is an extremely rare and frequently fatal complication of diverticulitis. Section title: DISCUSSION Educational score: 4.328263282775879 Domain: biomedical Document type: Study Language: en In 1846, Waller documented autopsy findings in a patient with appendicitis, including multiple liver abscesses and pylephlebitis. He concluded that appendicitis was the direct cause of pylephlebitis and liver abscess. By the turn of the century, the incidence of pylephlebitis following appendicitis gradually diminished with the advent of antibiotics and early surgical treatment. Subsequently, acute colonic diverticulitis has become the leading cause of suppurative pylephlebitis 4 ) . The true incidence of pylephlebitis is difficult to estimate, however, since the diagnosis may be obscured by either the inciting focus of infection or the liver abscesses resulting from “portal pyemia”, such as acute diverticulitis, intestinal perforation, uterine infection, suppurative pancreatitis and pancreatic abscess formation, splenic abscess, perirectal abscess, hemorrhoid, epididymitis, omphalitis and inflammatory bowel disease 2 ) . In our case, appendicitis was the cause of pylephlebitis. Furthermore, the high morbidity and mortality of suppurative pylephlebitis and intrahepatic abscesses are, at least in part, attributable to the nonspecificity of the symptoms and signs and the limitations of available diagnostic modalities 5 ) . A clinician must be cognizant of both the nonspecificity of the clinical presentation and the limitations of the sensitivity of these diagnostic modalities. Nothing has superseded the necessity for a high index of clinical suspicion regarding these diagnoses. Diagnoses are based on clinical features, laboratory findings, blood culture and radiological examinations, or postmortem examination. Clinical feature includes fever, right upper quadrant pain, diarrhea, jaundice and hepatomegaly 2 ) . The clinical “pearl” that jaundice is rare in pylephlebitis, except in cases complicated by multiple liver abscesses, appears to be valid. Jaundice developed late (no earlier than 5 days after the onset of symptoms) since the relative absences of jaundice early in the illness helps distinguish pylephlebitis from ascending Escherichia coli , followed by Proteus mirabilis, Klesiella pneumoniae, Enterobacer species, Pseudomonas species and gram-positive cocci ( Staphylococcus aureus, Streptococcus species ). More recently, coloenteric anaerobic bacteria (Clostridia, Bacteroides, Fusobacterium and anaerobic streptococcus species) have been isolated with increasing frequency, as a consequence of both improved culture techniques and selective pressure by the prevalent use of antibiotics 5 ) . In our case, Bacteroides fragilis was grown from blood. Modern radiological imaging techniques provide supportive diagnostic evidence 7 ) . With regard to diagnostic imaging in pylephlebitis, the current series suggests that ultrasonography is a useful modality for demonstrating portal vein thrombosis. CT scanning also shows promise in this regard, and may be less operator-dependent than ultrasonography. In the setting of probable intra-abdominal infection, CT scanning may be the most reasonable initial choice for imaging, given its proven ability to detect not only thrombi but pericolonic abscesses as well 8 ) . Serial CT scans document both the outcome and temporal course of secondary hepatic infection. Early in the disease process, hepatic infection and inflammation are microscopic, and contrast CT may demonstrate patchy areas of attenuated hepatic parenchyma. Later in the clinical course, parenchymal hepatic infection may coalesce to form multiple microabscess, or a single macroabscess amenable to percutaneous CT-guided drainage 9 ) . Also in our case, inhomogenous patchy infiltration in the hepatic parenchyme was observed at the time of diagnosis, and resolution of these lesions were observed in follow-up film. Although modern imaging techniques have allowed the early diagnosis of pylephlebitis, the patient’s history and physical examination still provide clues to the diagnosis. On the basis of treatments, it appears that empirical antibiotic therapy for a patient with suspected pylephlebitis should include broad coverage for enteric facultative gram-negative bacilli and agents active against anaerobes, especially B. fragilis , and coverage for aerobic Streptococcus species. Specific coverage for enterococci does not appear to be a necessity. The ideal duration of antibiotic therapy for pylephlebitis is unclear 2 ) . Given the frequency of liver abscess as a complication of pylephlebitis, however, a minimum 4 weeks of therapy seems prudent since developing abscesses may not be visualized on CT scans. Patients with demonstrated macroscopic liver abscess complicating pylephlebitis should probably receive at least 6 weeks of antibiotic therapy, with or without drainage. In our case, imipenem (2.0g/d) monotherapy was performed for 2 weeks and resulted in complete resolution of hepatic lesions. No further treatment was performed. The role of anticoagulation in the treatment of pylephlebitis is controversial 10 ) . In prior writings, descriptions of its role have ranged from “unnecessary” to “probably useful” to “recommended”. All of these opinions appear to have been based on limited data and personal experience, since no formal study of anticoagulation in pylephlebitis has ever been done. Despite such evidence that heparin may not be critical for the survival of patients with pylephlebitis, the possibility exists that anticoagulation might benefit some patients by decreasing the chance of septic embolization to the liver from infected portal thrombi and pulmonary emboli 2 ) . Surgical intervention for pylephlebitis typically involves opening and drainage of the focus of infection, separation of large vessels of the portal system from the focus of infection (i.e., pericolonic or hepatic abscess) rather than surgery on the infected vessels themselves 11 ) . In our patient, a previously healthy patient presented with nonspecific symptoms and fever. Bacteroides fragilis was grown from blood, which clearly suggested an intra-abdominal origin. Appendicitis was the most likely cause of sepsis, confirmed by abdominal CT, with seeding to the portal vein and liver. Septic conditions were improved with early initiation of broad-spectrum antibiotics. No surgical intervention was performed, since the intra-abdominal phlegmonous process responded to antibiotic therapy. Section title: DISCUSSION Educational score: 3.375483751296997 Domain: biomedical Document type: Other Language: en Outcome depends on multiple factors, including age greater than 70 years 1 ) , multiple liver abscess 2 ) , combination of hepatic abscess and pylephlebitis 3 ) , concomitant intrahepatic and extrahepatic abscesses, usually subphrenic 4 ) , deterioration of liver function 5 ) due to intrahepatic infection, particularly jaundice, and hypoalbuminemia 1 ) . Section title: DISCUSSION Educational score: 3.2189857959747314 Domain: biomedical Document type: Other Language: en Although radiological imaging studies (e.g. contrast-enhanced abdominopelvic CT scan) facilitates diagnoses, early suspicion and prompt antibiotic therapy can lead to resolution of portal vein thrombosis and multiple liver abscesses thus resulting in recovery. Section title: DISCUSSION Educational score: 3.932603597640991 Domain: biomedical Document type: Other Language: en In conclusion, pylephlebitis is an uncommon complication of suppurative infections (most often diverticulitis) in the portal drainage area. Although rare, pylephlebitis is a treatable but often lethal complication of intra-abdominal sepsis, pylephlebitis should be suspected in patients with intra-abdominal sepsis with liver function abnormalities. A high index of suspicion is required for making an expedient diagnosis. The timely administration of broad-spectrum antibiotics appears to be the most critical component of therapy for pylephlebitis. | Clinical case | biomedical | en | 0.999997 |
10063318 | Section title: INTRODUCTION Educational score: 3.6276779174804688 Domain: biomedical Document type: Study Language: en Diabetic patients, who consume Vacor (N-3-pyridylmethyl-N’-p-nitrophenylurea, MW272), experience microangiopathy causing retinopathy and nephropathy 1 ) . The width of the capillary basement membrane in these patients thickens significantly compared to normal controls after 6 years of Vacor ingestion, on average. Section title: INTRODUCTION Educational score: 4.064386367797852 Domain: biomedical Document type: Study Language: en A number of animal models of diabetes have suggested that hyperglycemia can produce microangiopathic changes 2 ) . Rodents made hyperglycemic with streptozotocin (STZ) or alloxan developed glomerular changes that were similar to, but not strictly comparable to, those of humans 3 ) . Vacor-induced diabetic rats developed acute glomerulopathy within a few months and the glomerulus revealed prominent thickening of the basement membrane 4 ) . Section title: INTRODUCTION Educational score: 4.151544570922852 Domain: biomedical Document type: Study Language: en The glomerular filtration barrier consists of complex matrix constituents interposed between the glomerular endothelial and epithelial cells, which constitute the lining of the glomerular capillaries. They include collagen (mostly type IV), laminin, polyanionic proteoglycans (mostly heparan sulfate), fibronectin, entactin and several other glycoproteins 5 ) . These individual components of glomerular basement membrane (GBM) were immunohistochemically tagged using specific monoclonal antibodies and observed by light and electron microscopy 6 – 8 ) . Changes of four major components of GBM, (type IV collagen, laminin, fibronectin and proteoglycan), were studied in Vacor-induced diabetic rats in this study. Section title: MATERIALS AND METHODS Educational score: 3.8117034435272217 Domain: biomedical Document type: Study Language: en Adult male Wistar rats (n=42), weight 200–250g, were used for this study. A single dose of Vacor, 80 mg/kg of body weight, was administered to the experimental animals by an orogastric canule, while distilled water was given to controls. Blood glucose was measured by glucose oxidase before and after the treatment. Section title: MATERIALS AND METHODS Educational score: 4.108943462371826 Domain: biomedical Document type: Study Language: en The experimental animals were sacrificed 0.5, 1, 3, 7, 14, 28 and 56 days after the administration of Vacor. Animals were perfused through the jugular vein with 300 ml of physiologic saline followed by an equal amount of 10% buffered formalin. Both kidneys were taken, and then routine formalin-fixed, paraffin-embedded tissue blocks were made for the immunohistochemical studies of glomerular basement membrane (GBM) components. Tissue sections, 4 μm in thickness, were obtained, and stained with hematoxylin-eosin (H&E) and periodic acid Schiff (PAS) stain. Section title: MATERIALS AND METHODS Educational score: 4.212838172912598 Domain: biomedical Document type: Study Language: en For immunohistochemical studies of the GBM components, monoclonal antibodies for collagen type IV (Monosan, Netherlands), laminin (Monosan, Netherlands), fibronectin (Sigma Bioscience, U.S.A.) and proteogylcan chondroitin sulfate (CSPG, Biogenesis, U.K.) were used. Immunohistochemical studies were performed by using the peroxidase-antiperoxidase technique 9 ) . Paraffin sections were cut to 4μm in thickness. Deparaffined sections were soaked in absolute methanol containing 0.3% hydrogen peroxide for 30 minutes at room temperature in order to block endogenous peroxidase activity. After washing, the sections were incubated with 0.4% pepsin (Sigma) in 0.01 N HCI for 1 hour at 37°C, then treated with 0.05% protease type VII (Sigma) in phosphate-buffered saline (PBS), pH 7.2, for 15 minutes at 37°C. With these enzyme pretreatments, consistent visualization of collagen type IV in the formalin-fixed tissues was possible. Enzyme digestion was terminated with cold running tap water. After further washing in PBS, the sections were exposed to 1:20 diluted normal swine serum (DAKO-Immunoglobulins Ltd., Denmark) for 30 minutes at room temperature. Then sections were incubated with each primary antibody at a dilution of 1: 500 overnight at 4°C. After washing with PBS, treatment followed with anti-rabbit IgG swine serum (at a dilution of 1:20, DAKO), and PAP solution (at a dilution of 1:80, DAKO), for 30 minutes, respectively, at room temperature. Finally, the sections were soaked in 0.05 M Tris-HCI buffer, pH 7.6, containing 3,3′-diaminobenzidine hydrochloride (40 mg/100 ml) and hydrogen peroxide (0.015%) for 10 minutes, and counterstained with hematoxylin. Vascular walls and renal tubules in the specimens served as positive controls for the primary antibodies. As the negative control, the primary antibodies were replaced by nonimmune rabbit serum by the same procedure. Section title: Glomerular changes examined by H&E and PAS stains Educational score: 4.209031105041504 Domain: biomedical Document type: Study Language: en The glomeruli in control animals revealed patent capillary loops demarcated by a thin, delicate glomerular basement membrane. Mesangium was located among the capillary loops. The basement membrane and mesangium were positively stained by PAS stain. In the experimental groups, no histologic change was noted until 1 day after Vacor treatment. Mild mesangial widening was observed 3 days after treatment by PAS stain. Mild thickening of the GBM was evident at 7 days by H&E and PAS stains. Increased thickening of the GBM and mesangial widening were noted at 14 to 56 days by PAS and H&E stains . The width of the GBM had more than doubled at 28 and 56 days compared with normal controls. Focal nodular proliferation of both glomerular endothelial and mesangial cells in the glomerulus was observed . Section title: Changes of GBM components by immunohistochemical stains Educational score: 3.9349095821380615 Domain: biomedical Document type: Study Language: en The glomeruli in control animals show thin linear staining along the GBM after immunohistochemical staining for collagen type IV and laminin . Fibronectin and proteoglycan were also identified along the GBM, but they stained more faintly than collagen type IV and laminin. Section title: Changes of GBM components by immunohistochemical stains Educational score: 4.210982799530029 Domain: biomedical Document type: Study Language: en Immunohistochemical staining for collagen type IV was the most sensitive way to demonstrate GBM thickening, evident 3 days after a single, large dose (80 mg/kg) of Vacor treatment. The GBM was moderately thickened at 7 days, and was maximally thickened 28 to 56 days after the treatment by collagen type IV immunohistochemistry. The pattern was initially focal and global from the GBM peripheral loop, and became subsequently diffuse . Moderately thickened immunoreactivity with collagen type IV was noted after as many as 56 days. Laminin in the GBM also thickened after Vacor treatment, and behaved similarly to collagen type IV . Section title: Changes of GBM components by immunohistochemical stains Educational score: 4.086629867553711 Domain: biomedical Document type: Study Language: en Fibronectin in the GBM appeared faintly at 12 hours and 1 day after Vacor treatment. Mild, focal and segmental thickening of fibronectin was evident at 3 days. From 7 days after Vacor treatment, the fibronectin presence in the GBM began to resemble that of collagen type IV and laminin . Section title: Changes of GBM components by immunohistochemical stains Educational score: 4.005117416381836 Domain: biomedical Document type: Study Language: en Chondroitin sulfate proteoglycan (CSPG) in the GBM did not change so prominently as type IV collagen, laminin and fibronectin. Only a mild focal thickening of CSPG was noted along the GBM at 3 to 7 days after Vacor treatment. No identifiable changes of proteoglycan in the GBM were noted from 14 to 56 days after Vacor treatment . Section title: Changes of GBM components by immunohistochemical stains Educational score: 1.768243432044983 Domain: biomedical Document type: Study Language: en The results are summarized in Table 1 . Section title: DISCUSSION Educational score: 4.258063316345215 Domain: biomedical Document type: Study Language: en In humans, characteristic morphological changes of the glomeruli in diabetes mellitus include GBM thickening, diffuse glomerulosclerosis and nodular glomerulosclerosis 10 ) . Diffuse thickening of the GBM occurs in virtually all diabetics, irrespective of the presence of proteinuria, and constitutes a part of diabetic microangiopathy. GBM thickening can be detected only by electron microscopy. Careful morphometric studies demonstrate that this thickening begins as early as 2 years after the onset of type I diabetes and, by 5 years, develops to about a 30% increase 11 ) . Diffuse and nodular glomerulosclerosis are late events for diabetics, and become pronounced after 10 to 20 years of diabetes 10 ) . Section title: DISCUSSION Educational score: 4.158760070800781 Domain: biomedical Document type: Study Language: en In Vacor-treated rats, the glomeruli reveal prominent thickening of the GBM within a short period. The width of the GBM is more than twice that of normal controls, 28 and 56 days after Vacor administration. Increased expressions of collagen IV, laminin, fibronectin and neutral mucopolysaccharide engendered the thickening of the GBM in this study. Proteoglycan immunoreactivity was not significantly different between Vacor-treated and normal control groups. In a study of human diabetic diffuse glomeruloscleosis, the enlarged mesangial matrix also revealed increased staining reactions for collagen IV, V, laminin and fibronectin, whereas the staining pattern for heparan sulfate proteoglycan (HSPG) was markedly reduced 12 ) . These data suggest that Vacor-induced glomerulopathy in rats can provide a useful experimental model to study human diabetic glomerulopathy. Section title: DISCUSSION Educational score: 4.390855312347412 Domain: biomedical Document type: Study Language: en Expression of type IV collagen and its mRNA in glomeruli and the interstitium of diabetic rats increased at 3, 7 and 14 days after streptozotocin (STZ) administration 13 ) . The expression was associated with up-regulation of transforming growth factor (TGF)-β. Administration of TGF-β increased collagen synthesis in normal rat glomeruli in a dose-dependent manner up to 5 ng/ml 14 ) . These data indicate a regulatory role for TGF-β in renal glomerular collagen synthesis in normal and diabetic rats. Inhibition of TGF-β and type IV collagen expression by insulin treatment suggests that they may be useful structural markers for determining the efficacy of therapeutic intervention during early diabetic nephropathy 13 ) . Concomitant increased staining patterns for laminin and type IV collagen have been reported in human diabetic glomerular lesions 12 ) and experimental diabetic nephropathy 15 ) . Over-expression of TGF-β was also partly associated with the up-regulation of glomerular laminin gene expression in diabetic mice. The occurrence of collagen type III in late diffuse glomerulosclerosis could be interpreted as an irreversible change in glomerular structure 16 ) . Section title: DISCUSSION Educational score: 4.283076763153076 Domain: biomedical Document type: Study Language: en Fibronectin also increases in the GBM and mesangial matrix in human diabetic nephropathy 17 ) and STZ-induced diabetic rats 18 , 19 ) . Fibronectin is a multifunctional matrix protein important in wound healing, and is markedly increased in glomerular crescents 20 ) . There are two phases of fibronectin metabolism in anti-GBM models of rapidly progressive glomerulonephritis (RPGN) in the rabbit 20 ) . Phase I is associated with increased glomerular fibronectin content from plasma, and phase II with increased fibronectin mRNA in glomeruli at days 7 to 14. This finding suggests that fibronectin synthesis may take place in glomeruli and that it may also be a molecule regulating glomerular cell proliferation and fibrosis. Section title: DISCUSSION Educational score: 4.188495635986328 Domain: biomedical Document type: Study Language: en Two types of proteoglycan, HSPG and basement membrane-specific CSPG, have been studied in human diabetes 21 ) and diabetic rats 22 ) . The expression of HSPG decreases in the GBM 16 , 23 ) and other capillaries 24 ) in diabetes, while CSPG increases in STZ-diabetic rats 22 ) . A mildly increased expression of CSPG was noted only after 3 to 7 days of Vacor administration in this experiment. Other studies suggest that changes of CSPG displace the capillary endothelial cells from the GBM causing loss of fenestrae, while loss of the HSPG results in alteration of GBM charge-selectivity 23 ) . The abnormal levels of type IV collagen, laminin, fibronectin and proteoglycan may, in part, be the cause of diabetic nephropathy induced by Vacor administration. Section title: CONCLUSION Educational score: 4.051790237426758 Domain: biomedical Document type: Study Language: en To elucidate the biochemical component changes in the GBM in Vacor-induced diabetic glomerulopathy, immunohistochemical analyses of type IV collagen, laminin, fibronectin and CSPG were performed in adult male Wistar rats. Section title: CONCLUSION Educational score: 4.19473934173584 Domain: biomedical Document type: Study Language: en Mild thickening of the GBM was evident 7 days after Vacor administration, and the width of the GBM was more than twice that of normal controls at 28 and 56 days. Significantly increased expressions of type IV collagen, laminin, fibronectin and neutral polysaccharide in the thickened GBM was noted 14 to 56 days after the administration, and the mildly increased expression of CSPG was present after 3 to 7 days. These abnormal levels of GBM components might, in part, be the cause of diabetic nephropathy induced by Vacor administration. | Study | biomedical | en | 0.999997 |
10063319 | Section title: INTRODUCTION Educational score: 3.701921224594116 Domain: biomedical Document type: Review Language: en Low-dose Methotrexate (MTX) (5.0–20.0 mg/week), initially widely used to treat severe psoriasis and psoriatic arthritis, is one of the effective therapies for rheumatoid arthritis (RA) 1 ) . Its tolerability, prompt clinical response and relatively lack of serious side effects have all contributed to its widespread use in RA and other arthritides. Section title: INTRODUCTION Educational score: 3.950937032699585 Domain: biomedical Document type: Study Language: en Most reports on serious MTX toxicity have focused on hepatic abnormalities, while other effects, including hematologic reactions, have not been emphasized. The latter, however, are not an uncommon finding in RA patients receiving low-dose pulse MTX. The prevalence of hematologic toxicity, including leukopenia, thrombocytopenia, megaloblastic anemia and pancytopenia, is estimated to be 3% in MTX-treated RA patients 2 ) . There is an increasing number of reports of severe, and at times fatal, pancytopenia, reported to occur in these patients 3 – 7 ) . Section title: INTRODUCTION Educational score: 3.2952797412872314 Domain: biomedical Document type: Clinical case Language: en We experienced a case of pancytopenia secondary to MTX therapy in a patient with RA and renal insufficiency, and also reviewed the literature related to MTX-induced pancytopenia in RA patients, with clinical significance and associated risk factors, in this report. Section title: CASE HISTORY Educational score: 3.902958393096924 Domain: clinical Document type: Clinical case Language: en A 67-year-old woman with a 12-year history of active seropositive RA, that was a response to non-steroidal anti-inflammatory drugs(NSAIDs), hydroxychloroquinine and intra-articular steroid injections, had been followed up in our department since June 1993. Initial laboratory tests showed a hemoglobin level of 7.6 g/dl [mean corpscular volume(MCV) 77.3 fl mean corpscular hemoglobin(MCH) 24.2 pg mean corpscular hemoglobin concentration (MCHC) 31.3 g/dl], WBC count 6300/mm 3 and platelet count of 463000/mm 3 . The erythrocyte sedimentation (ESR) was 61 mm/hour. The serum creatinine level was 2.3 mEq/dl and the BUN level was 38 mEq/dl. Liver function profiles were normal. Recently, because of significant morning stiffness and polyarthralgia, the decision was made to institute MTX treatment. This was begun as a single oral dose of 5 mg/week in June 1997. After 2 doses, the patient was admitted to the hospital with general weakness. Physical examination revealed a mild-dehydrated and poor nourished woman. The blood pressure was 150/100mmHg and the temperature was 36.7°C. Laboratory tests showed a hemoglobin level of 7.9 g/dl (MCV 86.8 fl MCH 28.3 pg MCHC 32.5 g/dl), WBC count 1800/mm 3 and platelet count of 64000/mm 3 . The serum creatinine level was 6.1 mEq/dl and the BUN level was 82 mEq/dl. Liver function tests were normal, but the serum albumin level was 2.7 g/dl. Over 3 days, the patient developed fever. Laboratory data were as follows: hemoglobin 6.5 g/dl, WBC count 800/mm 3 (10% polymorphonuclear cells, 77% lymphocytes and 7% eosinophils), platelet count 90000/mm 3 . MTX and Sulindac were discontinued, and granulocyte colony stimulating factor and intravenous prophylactic antibiotic therapies were required. Because of weakness and easy fatiguability, she received 1 unit of packed red blood cells. On the twelfth hospital day, laboratory testing revealed the following values: hemoglobin 7.6 g/dl, WBC count 15400/mm 3 , platlet 126000/mm 3 . The BUN level was 84 mEq/dl, and the serum creatinine value was 6.6 mEq/dl. Her condition was improved. Section title: DISCUSSION Educational score: 2.146519422531128 Domain: biomedical Document type: Other Language: en Adverse drug reactions can lead to significnat morbidity and mortality. It is estimated that between 3% and 11% of hospital admissions can be attributed to drug side effects. Any drug can conceivably have a toxic or undesirable drug reaction which may be preventable 8 ) . Section title: DISCUSSION Educational score: 4.002843856811523 Domain: biomedical Document type: Study Language: en Since 1980, pancytopenia due to MTX therapy in RA has been reported in 70 patients. Sergio et al 9 ) reported that an estimate of the frequency was derived using data from long-term prospective studies, in which 7 of 511 patients(1.4%) exposed to MTX developed pancytopenia. In most cases, pancytopenia is transient and recovery occurs after discontinuation of MTX and treatment of the disorder. Seventeen percent of these patients, however, have died despite medical intervention. Section title: DISCUSSION Educational score: 4.028897762298584 Domain: biomedical Document type: Study Language: en Poor renal function 10 – 12 ) , or concomitant trimethoprim-sulfamethosazole(TMP/SMX) therapy 13 , 14 ) or a rising MCV 15 , 16 ) or increasing age 10 , 11 ) are all associated with an increased risk of pancytopenia. The excretion of MTX is primarily renal, through glomerular filtration and proximal tubular secretion, and it may be affected by changes in the glomerular filtration. Therefore, in the presence of renal impairment, precaution is needed in patients with RA receiving low-dose MTX therapy. As reported, several patients have developed severe hematologic reactions to small amounts of MTX (total cumulative dose as low as 10mg). This has led to the recommendation of a test dose of 5mg, which probably should be mandatory in patients with significant risk factors. Section title: DISCUSSION Educational score: 3.4783051013946533 Domain: biomedical Document type: Study Language: en Advanced age and perhaps NSAID therapy might be indirectly related to increased risk through their association with declining renal clearance. Even concurrent penicillin therapy may interfere with renal excretion of MTX, leading to severe pancytopenia 17 ) . Section title: DISCUSSION Educational score: 3.4989254474639893 Domain: biomedical Document type: Study Language: en The usefulness of the RBC MCV as a harbinger of pancytopenia has been suggested by Weinblatt and others 16 ) using smaller numbers of cases. An elevation of MCV coincided with the development of pancytopenia in most patients, but the exact mechanism for the development of pancytopenia remains unknown. In this case, the level of MCV was normal. Section title: DISCUSSION Educational score: 3.9841830730438232 Domain: biomedical Document type: Study Language: en As reported by others, the concurrent use of TMP/SMX appears to be an independent risk factor, presumably due to the antifolate effect of trimethoprim, although sulfamethosazole may be the offending component, as sulfamethosazole and other sulfa drugs are known to cause pancytopenia as single agents. Therefore, folinic acid therapy is the recommended therapy for MTX induced pancytopenia 18 ) . Section title: DISCUSSION Educational score: 2.8507628440856934 Domain: biomedical Document type: Other Language: en Other risk factors, such as infections, obesity, increased alcohol ingestion, diabetes mellitus, peptic ulcer disease and hypoalbuminemia may also contribute to the frequency and severity of bone marrow toxicity. Section title: DISCUSSION Educational score: 3.947694778442383 Domain: biomedical Document type: Other Language: en The most important point is to avoid the use of MTX therapy in a case where there are risk factors directly implicated in the development of pancytopenia, such as renal dysfunction, drug interaction and advanced age. In addition to the standard CBC with differential and platelet counts and liver function tests, monitoring of the renal function before and 2 weeks after initiation of MTX and monthly thereafter is strongly recommended. The role of serum levels of MTX in RA patients with or without renal insufficiency has not yet been well defined. Also, a recent report suggests granulocyte colony stimulating factor may be a useful adjunctive treatment for MTX induced pancytopenia. Section title: DISCUSSION Educational score: 3.837581157684326 Domain: biomedical Document type: Review Language: en In summary, MTX-related adverse hematologic side effects, including fatal pancytopenia, are relatively common, and a cause of increasing concern in patients with RA and renal insufficiency. Close monitoring of associated risk factors, particulary impaired renal function, should be mandatory for all patients who are receiving MTX therapy. | Review | biomedical | en | 0.999997 |
10063320 | Section title: INTRODUCTION Educational score: 3.4658641815185547 Domain: biomedical Document type: Other Language: en Leiomyosarcomas are uncommon malignant tumors that originate in smooth muscle, usually in the gastrointestinal tract, the retroperitoneum and the genitourinary tract 1 ) . Metastatic tumors in the omentum are not uncommon, but primary omental tumors are reported to be rare and usually malignant 2 , 3 ) . Section title: INTRODUCTION Educational score: 1.917237639427185 Domain: biomedical Document type: Other Language: en There were a few cases of primary omental leiomyosarcoma reported in the literature 4 ) . Mostly, these cases originated from the greater omentum. The case with leiomyosarcoma originated from the lesser omentum was extremely rare. Section title: INTRODUCTION Educational score: 3.4289228916168213 Domain: clinical Document type: Clinical case Language: en This report describes a rare case of primary leiomyosarcoma of the lesser omentum with a review of the literature. Section title: CASE REPORT Educational score: 2.343498468399048 Domain: clinical Document type: Clinical case Language: en A 72-year-old female was admitted to our hospital with palpable mass in the left upper quadrant of the abdomen and weight loss. The patient discovered the mass a month previously. She had no prior history of medical and surgical diseases. Her family was healthy with no genetic diseases. The patient denied any symptoms such as nausea, vomiting, diarrhea, constipation, melena, hematochezia, jaundice, hematemesis, flushing and palpitation. Section title: CASE REPORT Educational score: 2.577028512954712 Domain: clinical Document type: Clinical case Language: en On physical examination, blood pressure was 140/80 mmHg, pulse rate was 74/min, respiration rate was 20/min and the body temperature was 36°C. A hard, movable, man-fist-sized mass was palpated on the left upper quadrant of the abdomen. Section title: CASE REPORT Educational score: 3.4309709072113037 Domain: clinical Document type: Clinical case Language: en The hemoglobin was 10.1 g/dl and the hematocrit was 31%. The white cell count was 8,600/mm 3 with 60% polymorphonuclear cells. The platelet count was 220,000/mm 3 . The serum sodium was 141 mEq/L, potassium 4.3 mEq/L, and serum amylase was 121 IU/L. The protein was 6.7 g/dl, albumin 4.1 g/dl, cholesterol 189 mg/dl, total bilirubin 0.5 mg/dl, alkaline phosphatase 125 IU/L, AST 16 IU/L and ALT was 9 IU/L. Urinalysis did not reveal any evidence of abnormalities. Section title: CASE REPORT Educational score: 3.379751443862915 Domain: biomedical Document type: Clinical case Language: en X-ray of the chest and simple abdomen showed normal. Abdominal CT scan revealed a 12×8×8 cm-sized heterogeneous cystic mass in the left upper quadrant of the abdomen that had irregularly enhanced the solid portion and the large area of the central cystic portion. There was marked displacement of the stomach and omental vessels to the posterior and inferior side, due to compression by the mass . Section title: CASE REPORT Educational score: 2.5062766075134277 Domain: biomedical Document type: Clinical case Language: en Endoscopic ultrasonography showed a large cystic mass near the stomach and liver, which was 1.6 cm length in wall thickness. The wall echo of the stomach was normal . Section title: CASE REPORT Educational score: 3.321610689163208 Domain: clinical Document type: Clinical case Language: en Laparotomy and excision of the mass was performed. A pale gray-colored, lobulated 12×8×8 cm-sized mass, which originated from the lesser omentum, was discovered adjacent to the lesser curvature side of the high body of the stomach. The mass compressed the liver and displaced the stomach to the left lateral side. When the mass was bisected, it was mainly composed of cyst and hemorrhagic materials. But there was a small area of solid component, which was accompanied with focal hemorrhage and necrosis . Section title: CASE REPORT Educational score: 2.994086980819702 Domain: biomedical Document type: Clinical case Language: en Microscopic examination showed spindle-shaped cells with 7–8 mitoses per 10 high power field. She was diagnosed as rarely occurring leiomyosarcoma originating from the lesser omentum. Section title: DISCUSSION Educational score: 2.937551259994507 Domain: biomedical Document type: Other Language: en Even though the omentum is composed mostly of adipose tissue, tumors of smooth muscle origin are more common than those of adipose tissue origin. It is surmised that smooth muscle tumors arise from mesodermal elements present in the blood vessels, fibrous tissues and the nerves of the omentum 5 ) . We recently experienced this rarely occurring leiomyosarcoma originating from the lesser omentum in a 72-year-old woman. Section title: DISCUSSION Educational score: 3.688185930252075 Domain: biomedical Document type: Study Language: en In Dixon’s histologic diagnosis of omental tumors, nearly 33% were sarcomatous in nature, while only 18% were of adipose origin. They may affect any age group but occur most commonly in the middle-aged. The most common symptom of omental leiomyosarcoma is abdominal mass, distension or pain; ascites has also been reported 4 ) . Section title: DISCUSSION Educational score: 3.8781466484069824 Domain: biomedical Document type: Study Language: en CT scan is useful in diagnosing the tumor. In McLeod’s study of leiomyosarcomas, 6 ) 89 of 118 (75.4%) had abnormal findings on body CT scan, as most were large by the time of diagnosis. Barium enema may also be a useful diagnostic tool. A mass that displaces the stomach superiorly and posteriorly and the transverse colon inferiorly and anteriorly is pathognomonic 6 ) . Angiography may also be performed, although the hypervascularity of the tumor was the only identifying finding, as presented on Granmayeh’s series 7 ) . Section title: DISCUSSION Educational score: 3.8197052478790283 Domain: biomedical Document type: Study Language: en Leiomyosarcomas of the omentum were reported to be mitotically active, showing one to four mitoses per 10 high power field in one series and a more higher rate in the other series 8 ) . The present case showed active mitoses, accounting for 7–8 mitoses per 10 high power fields. Section title: DISCUSSION Educational score: 3.8967645168304443 Domain: biomedical Document type: Review Language: en Laparotomy is the procedure of choice for diagnosis, treatment and detection of possible metastases of the omental leiomyosarcoma. Especially, in the case with a primary omental tumor, a total omentectomy, including the mass, should be recommended because of the possible presence of microscopic metastases 9 ) . Before resection of the omental tumor, a careful search for any possible primary site in another organ is essential 10 ) . The long-term outcome of patients with omental leiomyosarcomas is unknown. Early reports, such as those by Sanes and Kenny 11 ) and Levy and Pund, 12 ) showed poor outcomes with a high post-operative death rate. No trials have been performed using chemotherapy or radiation therapy, but these do not seem to be efficacious based on other sarcomas’ durable response to these modalities 13 ) . | Other | biomedical | en | 0.999997 |
10063321 | Section title: INTRODUCTION Educational score: 4.07218599319458 Domain: biomedical Document type: Review Language: en Cytomegalovirus (CMV) disease is a major cause of morbidity and mortality in immunocompromised patients, particularly recipients of solid organ or bone marrow allograft, and patients with the acquired immune deficiency syndrome 1 ) . Although CMV pneumonia is well recognized as the commonest infectious cause of death in bone marrow allograft recipients, the morbidity associated with CMV gastroenteritis is sometimes overlooked. while chemoradiation treatment, graft-versus-host disease and various medications may lead to nausea and vomiting following bone marrow transplantation(BMT), persistent symptoms cannot be explained in many cases and some cases may represent unrecognized gastrointestinal(GI) infections 2 ) . Cytomegalovirus is the most prevalent infectious agent in the upper GI tract of symptomatic BMT recipients. Before the introduction of effective antiviral therapy, mortality of CMV infection was very high, but the use of ganciclovir has improved the prognosis. However, the appropriate method for treatment of CMV-associated enteritis is still unknown. Section title: INTRODUCTION Educational score: 3.555696964263916 Domain: clinical Document type: Clinical case Language: en We report here a case of CMV duodenitis which developed following allogenic BMT and was successfully treated with ganciclovir. Section title: CASE REPORT Educational score: 4.073122024536133 Domain: clinical Document type: Clinical case Language: en A 38-year-old male was diagnosed as acute lymphoblastic leukemia in December 1996. He received induction chemotherapy consisting of daunorubicin, vincristine, prednisolone and L-asparaginase. After two cycles of induction chemotherapy, complete remission was achieved and allogenic BMT was performed. Preparative chemotherapy for BMT consisted of busulfan (1mg/kg p.o every 6 hours for 4 days) and cytoxan (60mg/kg iv for 2 days). He received a bone marrow graft from a HLA matched sibling donor. Serologic tests for IgG CMV antibody were positive in both recipient and donor. For GVHD prophylaxis, cyclosporin and a short course of methotrexate were administrated. Granulocyte colony-stimulating factor (450 μ g/day) was given beginning on day 5. Enteral decontamination was done with ciprofloxacin and acyclovir was given for the prophylaxis of viral infection. CMV blood culture was performed weekly until 100 days post-BMT. The early post-BMT course was uneventful and the patient was considered to achieve engraftment by bone marrow aspiration and biopsy on post-transplant day 21. He had, however, prolonged nausea and vomiting for 5 weeks after BMT and mild fever and watery diarrhea was developed, all of which necessitated hyperalimentation persistently. Physical examination revealed no abnormalities except mild epigastric tenderness. Laboratory values included a WBC 4,200/mm 3 , hemoglobin 9.0 mg/dL, hematocrit 25.7%, platelet 34,000/mm 3 , bilirubin, liver transaminase, BUN and creatinine were normal and the stool was negative for C. difficile toxin . Weekly CMV blood cultures were all negative. For the evaluation of the prolonged nausea, vomiting and diarrhea, gastrofibroscopy and flexible sigmoidoscopy were performed on post-transplant day 39. By gastroscopic finding, the stomach showed multiple hyperemic patchy erosions mainly in the greater curvature of the body and, on the second portion of the duodenum, an octopus sucker-like elevated lesion with central hyperemic erosion and several hyperemic flat erosive lesions were noted. Sigmoidoscopic finding was normal. Gastric mucosal biopsy showed mild gastritis. Duodenal mucosal biopsy revealed cytomegalic cells which showed a large, densely staining nucleus and abundant cytoplasms with intracytoplasmic inclusion body. Immunochemical staining for CMV antigens using monoclonal antibodies confirmed the diagnosis of CMV duodenitis. The patient was treated with ganciclovir (5 mg/kg iv, twice a day for 3 weeks) for the CMV duodenitis. Nausea and vomiting were decreased and total parenteral nutrition could be stopped. At the end of 3 week’s treatment, follow-up gastrofibroscopy revealed healed erosions. Maintenance treatment with ganciclovir was not given. He was free of CMV infection until 288 days after allogenic BMT. Section title: DISCUSSION Educational score: 3.9436981678009033 Domain: biomedical Document type: Other Language: en The gastrointestinal disorders which are seen between day 0 and 20 post-transplant are generally related to toxicity of conditioning therapy, bacterial and fungal infection and side effects of drugs. Between day 20 to 30 post-transplant, the marrow graft becomes functional, but there is incomplete immunologic reconstitution. The abdominal symptoms at this time are commonly related to acute GVHD or viral infections, although venoocclusive disease may persist as well 3 ) . Thus, endoscopy with biopsy in symptomatic patients is an essential method for a definite diagnosis. Section title: DISCUSSION Educational score: 3.5248115062713623 Domain: biomedical Document type: Study Language: en Conditions that specifically predispose to CMV disease of the GI tract in BMT recipients have not been identified, but presumably are similar to those identified as predisposing to CMV infection of any type. The relationship between GI GVHD and GI CMV infection or disease is not well understood. Both of these conditions commonly coexist following BMT. Whether GVHD of the gut may predispose to secondary invasion with CMV is not proven 4 , 5 ) . In our patient, we could not find evidence of GVHD. Section title: DISCUSSION Educational score: 3.992995023727417 Domain: biomedical Document type: Study Language: en An indication of the incidence of GI CMV disease was provided in a study by Spencer et al 2 ) , in which they performed upper GI endoscopy to investigate unexplained nausea and vomiting in BMT recipients. Nineteen(38 %) of the 50 patients had CMV infections of the esophagus and/or small intestine. In another study, 3 (12.5 %) of 24 BMT recipients had GI involvement with CMV at autopsy. Section title: DISCUSSION Educational score: 4.229321479797363 Domain: biomedical Document type: Study Language: en All levels of the GI tract have been reported as the sites of CMV infection. BMT recipients have a much higher likelihood of having involvement of the upper GI tract (esophagus, stomach and duodenum) than the lower GI tract 6 ) . However studies that systematically examine the entire GI tract of these patient populations are not available. Einsele et al 7 ) reported that, because of the preferential involvement of the ascending colon and terminal ileum by CMV virus, a colonoscopy of the entire colon and terminal ileum, including biopsies from these sites, should be performed in all patients with severe diarrhea following allogenic BMT. Since endoscopic findings are variable, such as erythema, erosions, ulcerations, hemorrhage, nodules and polyps 8 ) , and radiological findings were nonspecific for CMV infection 9 ) , a biopsy was required for a definite diagnosis. The presence of cytomegalic cells on mucosal biopsy specimens stained with hematoxylin and eosin has been considered as the gold standard for establishing a diagnosis of CMV GI disease. To enhance the sensitivity and specificity of histopathologic analysis immunohistochemical staining methods for CMV antigens, using monoclonal antibodies, have been developed and utilized effectively. In our patient, we could be convinced of the diagnosis because the immunohistochemical staining confirmed the presence of CMV antigen in the biopsy specimen. Section title: DISCUSSION Educational score: 4.151167392730713 Domain: biomedical Document type: Clinical case Language: en Although ganciclovir, an antiviral agent with both in-vitro and in-vivo activity against CMV, has been used in treatment of CMV disease 10 ) , the appropriate method for treatment of CMV enteritis is currently unknown, and therapy with ganciclovir has been less successful in the treatment of GI CMV disease in the BMT population than in the solid organ transplantation population. A placebo-controlled trial by Reed et al 6 ) found no evidence of clinical or endoscopic improvement of GI CMV disease after 2 weeks of treatment with ganciclovir as compared with supportive care. However, on the basis of experiences in CMV pneumonitis, it is common practice to use ganciclovir. Immune globulin combined with ganciclovir may be helpful as in the CMV pneumonitis. The usual induction dose is 10 to 15 mg/kg per day administered in 2 to 3 divided doses daily for 3 weeks. Ganciclovir therapy for 3 weeks resulted in both a clinical and endoscopic remission of CMV duodenitis in our patient. It is not established whether maintenance therapy with ganciclovir should be given. In patients with severe and persistent immune deficiency, such as GVHD or AIDS, the relapse rate is high and some physicians advise maintenance therapy after induction therapy to prevent or delay relapse 11 ) . Our patient had no evidence of GVHD and his blood count recovered uneventfully. So, we decided not to give maintenance ganciclovir therapy and he was free of CMV infection post-transplant day 288. Section title: DISCUSSION Educational score: 3.687516450881958 Domain: biomedical Document type: Other Language: en In conclusion, CMV disease should be suspected in the BMT patients complaining of prolonged nausea and vomiting, and appropriate biopsies and cultures should be done to confirm the diagnosis. Prompt treatment with ganciclovir for the confirmed GI CMV disease may be helpful. | Review | biomedical | en | 0.999997 |
10063322 | Section title: INTRODUCTION Educational score: 4.268415927886963 Domain: biomedical Document type: Clinical case Language: en Juvenile dermatomyositis (DM) is a multisystem disease characterized by nonsuppurative inflammation of striated muscle, skin and the gastrointestinal tract, and also characterized early in its course by an immune complex vasculitis and, later, the development of calcinosis 1 ) . Although the etiology of juvenile DM remains unclear, it is suggested that IgE can be associated with autoimmune diseases, such as systemic lupus erythematosus (SLE) and juvenile DM, by mediating the release of chemical mediators and by faciliating the local deposition of immune complexes 2 ) . Intercurrent infections with elevated serum IgE level during the course of the disease give rise to problems in patients with juvenile DM. Atopic dermatitis, or development of calcinosis, may contribute to this problem 3 , 4 ) . HIE also has the characteristic findings of recurrent infections of the skin and sinopulmonary tract and high serum IgE level. Other findings of HIE include eosinophilia, presence of anti- S.aureus specific IgE, defect of neutrophil chemotaxis, poor delayed hypersensitivity responses and eczematoid dermatitis 5 ) . Herein, we describe a patient with juvenile DM complicating HIE. Section title: CASE REPORT Educational score: 4.076690196990967 Domain: clinical Document type: Clinical case Language: en A 13-year-old girl was admitted to the hospital because of multiple skin abscesses. Six years before admission, juvenile dermatomyositis was diagnosed at another hospital on the basis of symptoms of proximal muscle weakness, abnormal findings of muscle biopsy, elevated serum muscle enzyme and skin rash on her face. She had not been managed with regular follow-up. There was a history of recurrent skin infections, pneumonia and eczematous dermatitis over the whole body after the diagnosis of juvenile dermatomyositis. She denied any history of allergic diseases. There was no family history of specific diseases. The temperature was 38.0°C, the pulse was 116 and the respirations were 24. The blood pressure was 90/50 mmHg. On physical examination, the skin of her entire body showed multiple hyperpigmented lesions, lichenoid patches and eczemaoid confluent plaques. Oral thrush and subcutaneous cold abscesses of the left upper eye lid, back and right lower quadrant abdomen were found . No lymphadenopathy was observed. The lungs were clear. Both knee joints had flexion contractures with muscle atrophy. The following laboratory findings were recorded: hemoglobin 10.5gm/dl, white blood cell count 15,000/mm 3 (88,3% neutrophils, 6.6% lymphocytes, 3.5% monocytes, 0.2% eosinophils, 1.4% basophils), platelet count 288,000/mm 3 , erythrocyte sedimentaion rate 68mm/hour (Westergren method), The protein was 6.7g/dl (albumin 3.2g/dl; globulin 3.5g/dl). The values for glucose, urea nitrogen, creatinine, alanine aminotransferase, aspartate aminotransferase, creatine kinase, lactic dehydrogenase, aldolase, alkaline phosphatase, bilirubin, calcium, phosphorus, sodium, potasium, chloride and magnesium were normal. Urine analysis was normal except for proteinuria(+). Levels of C3, C4 and CH50 were normal. The tests for antinuclear antibody, rheumatoid factor, VDRL, hepatitis surface antigen, hepatitis C virus antibody and C-reactive protein were negative. Antibodies to Sm, RNP, Ro, La, Jo-1 and ds DNA were not detected. Serum immunoglobulin examination revealed normal IgG (1,970 mg/dl; normal 800–1,500 mg/dl), IgM (111 mg/dl; normal 45–150 mg/dl), elevated IgA (455 mg/dl; normal 90–325 mg/dl) and IgE (6,650 mg/dl; normal <200 mg/dl). Follow-up serum IgE levels decreased with the treatment of S. aureus infection, but still remained high (3,180 mg/dl, 3,120 mg/dl). Immunoelectrophoresis of serum revealed no evidence of paraproteinemia. Radiographs of the abdomen and both lower extremities showed multiple soft tissue calcifications. EMG and muscle biopsy findings were compatible with inflammatory myopathy . Antibodies to S. aureus of the IgE class were detected by direct ELISA method, as previously described, with some modifications 6 ) . The result of a delayed hypersensitivity skin test to purified protein derivative, tetanus, diphteria, streptococcus, candidia, trichophyton and proteus was negative. Radioallergosorbent tests to common allergens were negative except for Penicillium notatum. Bacteriologic cultures from skin abscesses were positive for S. aureus . Cultures from oral mucous lesion grew Candida albicans . The total numbers of T cells, T cell subpopulations (CD4+/CD8+) were within normal limits. The Nitroblue-Tetrazolium-test was normal. In vitro lymphocyte proliferation was normal exposure to nonspecific antigens (phytohemagglutinin, phorbol myristate acetate plus ionomycin). The polymorphonuclear leukocyte motility, assessed in a reversible Boyden chamber with fmet-leu-phe and patient’s serum as chemo-attractants, as prevoiusly described 5 ) , was impaired. She was successfully managed with surgical drainage and antibiotics. Section title: DISCUSSION Educational score: 4.376251220703125 Domain: biomedical Document type: Study Language: en Some diseases, including chronic granulomatous disease, Wiskott-Aldrich syndrome, icthyosis vulgaris, severe combined immunodeficiency and HIE, have similiar clinical findings of recurrent skin infections 7 ) . This patient was diagnosed as having HIE on the basis of recurrent infections of skin and pulmonary, extremely elevated levels of serum IgE and presence of anti- S.aureus specific IgE, chemotactic defect of neutrophile, eczematoid dermatitis and normal nitroblue tetrazolium test. The late onset of her recurrent infection and normal eosinophil count were atypical. But these have been observed in other reports 5 ) . S.aureus and C.ablicans were isolated from skin cold abscesses and oral cavity, respectively, in this case. These organisms are known to be most predominant pathogens in recurrent infections of HIE 5 ) . S. aureus infection with elevated serum IgE is rarely observed in juvenile DM, and this can be partially explained by the previous two reports. First, atopic dermatitis, which is frequently accompanied by S. aureus , has a higher tendency for developing juvenile DM 3 ) . Second, the development of calcinosis and granulocyte chemotactic defect in juvenile DM is associated with staphylococcal infections 4 ) . The causes of recurrent skin infections of S. aureus in this patient are presumed to have a somewhat different mechanism from those two. Hochreutener et al suggested that increased S. aureus specific IgE level, decreased S. aureus specific IgA level, diminished chemotaxis and soft tissue abscesses can be differential points between atopic dermatitis and HIE, but chemotactic defect and anti- S.aureus specific IgE were found to be not only HIE but also atopic dermatitis 8 , 9 ) . So, sometimes, it may be hard to differentiate HIE from atopic dermatitis in a patient with juvenile DM who has elevated IgE level and S.aureus infection. Differential diagnosis is important because treatment and prognosis is different. Atopic dermatitis does not have recurrent infections of the sinopulmonary tracts and peculiar cold abscesses and has a dermatitis that differs in character and distribution from lesions of HIE 5 ) . This patient denied any history of allergy, and RAST(radioallergosorbent test) to common allergens, except for Penicillium notatum, were negative. Moore et al emphasized the relationship between the development of calcinosis and recurrent staphylococcal infections with raised IgE in juvenile DM 4 ) . Although the level of serum IgE in Moore’s cases are elevated, most of them do not meet the definition of HIE (≥2,000 IU/ml). But this patient had extremely elevated IgE level (6,600 IU/ml) during infection and had constantly raised serum IgE levels(≥3,000 IU/ml) during follow-up. Anti- S.aureus specific IgE was detected only during S. aureus infection in this case. Calcinosis occurs commonly with scleroderma, as well as dermatomyositis, and rarely in association with SLE 10 ) . HIE has been rarely reported in patients with SLE 11 – 13 ) , but these cases had no association with calcinosis. Calcinosis develops in up to half the patients with juvenile dermatomyositis 1 ) . It must be clarified whether recurrent skin infection with elevated IgE occurs in most juvenile DM patients with calcinosis or not. Although the immunologic basis of elevated serum IgE in patients with HIE has not been defined, a deficiency of suppressor T cell to inhibit IgE production 5 ) , imbalances between IL-4 producing and IFN-γ producing helper T cells 14 ) and decreased metabolism of IgE 15 ) seem to be responsible for elevated levels of IgE. There were reports that CD8+ to CD4+ ratio was reduced in a patient with juvenile DM 16 ) . But this was not found in our case. The causes of susceptibility to infections have not been documented. Schopfer et al suggest that histamine is released on crosslinkng of mast cell-bound antistaphylococcal IgE by staphylococcal antigens and interferes with the activity of polymorphonuclear leukocytes, resulting in the failure of effective S. aureus clearing 17 ) . It is known that there is variability in both HIE and juvenile DM patient’s neutrophil and monocyte chemotaxis 5 ) . Whether the neutrophils were intrinsically abnormal or not was inconclusive in a patient with HIE, but chemotactic defect of neutrophil to fmet-leu-phe and patient’s serum as chemo-attractants was observed in this case. HIE has significantly lower proportions of circulating T cells that can produce IFN-γ and TNF-α, in comparison with normal controls, which also may contribute to recurrent infections 14 ) . In conclusion, similar findings, including elevated serum IgE, arti- S.aureus IgE and chemotactic defect of neutrophil, can be found in patients with HIE, juvenile DM and atopic dermatitis. HIE must be considered in the differential diagnosis of a patient with juvenile DM who has an elevated level of IgE and staphylococcus aureus skin infection. It will be interesting to define whether the underlying mechanism is the same or not in these diseases. | Clinical case | biomedical | en | 0.999996 |
10075972 | Section title: Mice. Educational score: 4.050711631774902 Domain: biomedical Document type: Study Language: en B6, transporter associated with antigen processing (TAP)1 −/− ( 29 ), β 2 m −/− ( 30 , 31 ), and TAP1/β 2 m −/− ( 32 ) mice of the H-2 b haplotype were bred at the Microbiology and Tumor Biology Center, Karolinska Institute. Animal care was in accordance with institutional guidelines. TAP1 −/− , β 2 m −/− , and TAP1/β 2 m −/− mice used were back-crossed to B6 background 6, 10, and 7 times, respectively. For thymectomy experiments, β 2 m −/− mice were 735 rad–irradiated and thymectomized or sham-thymectomized, and reconstituted with β 2 m −/− fetal liver hematopoietic cells. Section title: Cell Lines. Educational score: 4.21988582611084 Domain: biomedical Document type: Study Language: en RMA is a subline of the Rauscher virus–induced B6 lymphoma RBL-5, and RMA-S is a TAP2-deficient variant of RMA ( 33 ). T2 is a hybrid between the two human cell lines 0.174 and CEM, and has an antigen-processing deficiency due to a deletion on the MHC class II region including the TAP1 and TAP2 genes. T2D b is an H-2D b transfectant of T2. All cell lines were maintained at 37°C and 5% CO 2 in RPMI 1640 tissue culture medium supplemented with 5% FCS, 50 μg/ml streptomycin, 100 μg/ml penicillin, and 2 mM l -glutamine. Con A–activated blasts were generated by culturing erythrocyte-depleted splenocytes in 5 μg/ml of Con A for 2 d in tissue culture medium as described above with 10% FCS. Section title: Synthetic Peptides. Educational score: 4.065837860107422 Domain: biomedical Document type: Study Language: en The following synthetic H-2D b –presented peptides were synthesized using solid phase F-moc chemistry: LCMV GP33 KAVYNFATM ( 34 , 35 ); LCMV GP 33-4A (GP33-4A) KAVANFATM; LCMV GP 33-34A (GP33-34A) KAAANFATM; LCMV GP 33-348A (GP33-348A) KAAANFAAM; influenza PR8 NP 366-374 (NP366) ASNENMETM ( 36 ); and Yersinia enterocolitica Yop51 249-257 (Yop249) IQVGNTRTI ( 37 ). Section title: Generation of LCMV GP33-specific CTL Cultures and Clones. Educational score: 4.144627571105957 Domain: biomedical Document type: Study Language: en GP33-specific CD8 + CTLs were elicited in B6 and β 2 m −/− mice by peptide immunization ( 38 ). 100 μg peptide was dissolved in distilled water and mixed with IFA in a 1:1 ratio by sonication, then injected subcutaneously in the base of the tail. 12 d after immunization, 25 × 10 6 immune spleen cells were cocultured with 25 × 10 6 2,000 rad–irradiated B6 or β 2 m −/− splenocytes in the presence of 0.05 μM peptide in 12 ml complete medium at 37°C and 5% CO 2 . 6–7 d later, these cells were used as effector cells in a 51 Cr-release assay. CTL clones were generated by limiting dilution cloning. Long-term CTL clones and lines were maintained in complete medium (see above) based on MEM-α and further supplemented with Hepes buffer and 20 IU/ml IL-2. CTL clones and lines were restimulated in 12-d intervals with 2,000 rad–irradiated splenocytes in the presence of 0.05 μM peptide. Section title: mAbs and FACS ® Analysis. Educational score: 4.1237030029296875 Domain: biomedical Document type: Study Language: en B22-249.1 is a mouse mAb which binds to a conformation-dependent epitope on the α1 domain of the H-2D b molecule, and Y3 is a mAb which binds to conformed H-2K b molecules. For flow cytometry with these mAbs, cells were incubated with mAb for 30 min on ice, washed, and then stained with goat anti–mouse Oregon Green conjugate (Molecular Probes) on ice for 30 min. For measurement of CD8 and TCR expression, CTLs were stained for 30 min with the FITC-conjugated anti-CD8α mAb 53-6.7 ( PharMingen ) and the PE-conjugated anti–TCR-α/β mAb H57-597 ( PharMingen ), respectively. After washing, analysis was performed using a FACScan ® ( Becton Dickinson ) with CellQuest software. Section title: CTL Assay, Cold Target Competition, and CTL Blocking with mAb. Educational score: 4.189303398132324 Domain: biomedical Document type: Study Language: en CTL activity was measured in a standard 51 Cr-release assay. In brief, peptide-coated target cells were prepared by incubating cells with indicated concentrations of peptide for 1 h at 37°C. Coated cells were labeled with 10 μl 10 mCi/ml 51 Cr for 1 h at 37°C. Titrated amounts of effector cells were incubated with 3 × 10 3 51 Cr-labeled target cells for 4 h at 37°C, 5% CO 2 . After incubation, released radioactivity was measured and specific lysis was calculated according to the formula: % specific release = [(experimental release − spontaneous release)/(maximum release − spontaneous release)] × 100. Cold temperature–treated target cells were generated through culture of RMA-S at 26°C for 12 h ( 39 ). For cold target competition experiments, effector T cells and unlabeled (cold) competitor cells were mixed and preincubated at 37°C for 30 min before addition of labeled (hot) target cells. The assay was then run as a standard 51 Cr-release assay for 4 h. For blocking of CTL recognition with the B22-249.1 mAb, titrated amounts of mAb were included in the CTL assay medium. Section title: Expression of Properly Conformed H-2D b in β 2 m −/− Cells Is TAP Dependent. Educational score: 4.260021209716797 Domain: biomedical Document type: Study Language: en We first compared the levels of properly conformed H-2D b molecules on the cell surface of β 2 m −/− , TAP1/β 2 m −/− , and B6 (TAP1/β 2 m +/+ ) control cells stained with an mAb directed against H-2D b (B22-249.1) or H-2K b (Y3) conformation-sensitive epitopes. A substantial level of conformed cell surface H-2D b molecules was found on β 2 m −/− cells, whereas conformed H-2K b was detected at lower levels, in line with previously published results . This is compatible with H-2D b being more independent of β 2 m during folding and transport of MHC class I free heavy chains ( 27 , 28 ). On Con A blasts deficient for both β 2 m and TAP1, the staining with B22-249.1 was virtually at background levels, which implies that the pool of conformed H-2D b molecules on β 2 m −/− cells is dependent on a functional TAP complex. Section title: β 2 m −/− Mice Have Thymus-dependent CD8 + T Cells that Mount an H-2D b –restricted and Peptide-specific CTL Response against the LCMV GP33 Epitope. Educational score: 4.232985496520996 Domain: biomedical Document type: Study Language: en B6 and β 2 m −/− mice were immunized with antigenic peptides restricted to either H-2D b or H-2K b . All tested peptides primed responses in B6 mice (data not shown), whereas only one, the H-2D b – restricted LCMV GP33, primed a response in β 2 m −/− mice. This is in line with the low but significant levels of folded H-2D b molecules on the surface of β 2 m −/− cells . CTLs from both B6 and β 2 m −/− mice primed with the GP33 peptide killed RMA-S cells pulsed with the GP33 peptide used for priming but not a control influenza NP366 peptide (Table I ). CTL responses were mediated by CD8 + cells as determined by mAb- and complement-mediated depletion of effector populations in vitro (data not shown). FACS ® analysis of both polyclonal bulk populations and clones confirmed the TCR + CD8 + phenotype of the responding CTLs in β 2 m −/− as well as B6 mice . Cell surface expression levels of both TCR and CD8 were similar in CTLs from β 2 m −/− and B6 mice. However, a marginal increase in CD8 expression levels was detected in some β 2 m −/− CTLs compared with B6 CTLs. This increase may be due to the low levels of H-2D b expressed during selection and priming in the absence of β 2 m. However, lysis performed by β 2 m −/− CTLs did not show increased dependency on CD8 compared with B6 CTLs, as determined by mAb blocking of CD8 (data not shown). Section title: β 2 m −/− Mice Have Thymus-dependent CD8 + T Cells that Mount an H-2D b –restricted and Peptide-specific CTL Response against the LCMV GP33 Epitope. Educational score: 4.193158149719238 Domain: biomedical Document type: Study Language: en No priming of CTLs was observed in thymectomized β 2 m −/− mice that had been irradiated and reconstituted with fetal liver (Table I ). Control mice (receiving irradiation and fetal liver reconstitution without thymectomy) generated a normal antipeptide CTL response, showing that the responding CD8 + T cell population in β 2 m −/− mice was dependent on the thymus for development. In addition, a normal antipeptide response was also found in mice that had been thymectomized but not irradiated, showing that the thymus was not necessary during the priming of the response (Table I ). The latter control indicated that the generated CTLs were not positively selected in the thymus in response to the peptide injected for immunization. We conclude that β 2 m −/− mice have a peripheral pool of CD8 + T cells which are thymus dependent, H-2D b restricted, and peptide specific despite expressing very low levels of H-2D b molecules, corresponding to a few percent of those expressed by B6 mice. Section title: GP33-specific β 2 m −/− CTLs Have an Increased Avidity for H-2D b Molecules. Educational score: 4.246007919311523 Domain: biomedical Document type: Study Language: en Alloreactive CTLs from mice with low MHC class I expression have an increased avidity to self-MHC ( 23 ). By priming peptide-specific, self-MHC– restricted CTLs in β 2 m −/− mice it is possible to study the role of self-MHC avidity for T cell specificity in MHC- restricted T cells. To compare the avidity for self-MHC class I in B6 and β 2 m −/− CTLs specific for the GP33 peptide, these CTLs were tested for their ability to kill RMA target cells (MHC high ) in the absence of loaded peptides. B6 CTLs failed to kill RMA cells, whereas β 2 m −/− CTLs efficiently killed these target cells . Cold target competition experiments made with bulk CTLs showed that the β 2 m −/− CTL killing of labeled RMA cells was completely inhibited by RMA-S pulsed with GP33 but not by RMA-S without peptide . This showed that a majority of the clones in the β 2 m −/− CTL population were specific for both self-MHC expressed at high levels and the peptide antigen presented by MHC at low levels. Section title: GP33-specific β 2 m −/− CTLs Have an Increased Avidity for H-2D b Molecules. Educational score: 4.158220291137695 Domain: biomedical Document type: Study Language: en Three GP33-specific β 2 m −/− CTL clones were generated, all of which also killed RMA cells. However, the clone C10 was more efficient in killing RMA-S loaded with GP33 compared with RMA , whereas clone 27/30 killed RMA slightly better than RMA-S plus GP33 , and clone 3C5 killed both of these target cells to a similar extent . This pattern is compatible with a clonal variation in TCR avidity for peptide versus MHC within the β 2 m −/− CTL population. In line with the results obtained with bulk cultures, the GP33-specific B6 CTL clone 2C10 did not recognize RMA in the absence of GP33 peptide . Section title: GP33-specific β 2 m −/− CTLs Have an Increased Avidity for H-2D b Molecules. Educational score: 4.261838436126709 Domain: biomedical Document type: Study Language: en To test the reactivity of the CTLs against MHC class I in the absence of peptides, we used RMA-S cells incubated at cold temperature (26°C), which induce high levels of functionally “empty” MHC class I molecules ( 39 ), and T2D b cells expressing D b molecules mostly devoid of peptides. Clone 27/30 also killed RMA-S cells after cold temperature incubation . Furthermore, T2D b was recognized irrespective of loaded peptide, whereas T2 control targets were not killed . Thus, the β 2 m −/− CTL clone 27/30 displays an avidity for the restriction element of the GP33 peptide, even in the absence of specific peptide. It should be noted that these CTLs were primed to respond only against GP33, indicating that the ability to recognize self-MHC had been selected for during thymic development and priming. Although these results do not exclude that β 2 m −/− CTLs also have an increased avidity for self-peptides, we use the term “peptide independent” to describe the specificity for the restriction element displayed by the GP33-specific β 2 m −/− CTLs. Section title: Triggering of GP33-specific β 2 m −/− CTLs Uses a Higher Number of MHC Ligands to Compensate for Absence of Specific Peptide. Educational score: 4.270101070404053 Domain: biomedical Document type: Study Language: en We next compared the avidity of β 2 m −/− CTLs for self-MHC with and without added GP33, by blocking of the H-2D b ligands. We used an mAb specific for properly conformed H-2D b molecules (B22-249.1), the binding of which is not affected by GP33 (data not shown). RMA target cells were killed at similar levels regardless of the presence or absence of specific peptide . However, in the absence of GP33 peptide the CTL killing activity was efficiently blocked by 5.0 μg/ml of B22-249.1, whereas virtually no blocking was observed when the RMA target cells were prepulsed with the GP33 peptide at 37°C . FACS ® analysis of B22-249.1 binding to RMA cells did not allow accurate quantitation of the fraction of free H-2D b ligands at half-maximal lysis, since half-maximal blocking of CTL lysis was achieved at a concentration at which staining was close to maximal . However, these experiments clearly demonstrate that triggering of β 2 m −/− CTLs in the absence of GP33 peptide requires a considerably higher number of H-2D b ligands. Section title: Triggering of GP33-specific β 2 m −/− CTLs Uses a Higher Number of MHC Ligands to Compensate for Absence of Specific Peptide. Educational score: 4.321598052978516 Domain: biomedical Document type: Study Language: en We next made a similar series of experiments using T2D b cells as targets. These cells express the B22-249.1 epitope at levels <10% those of RMA (Table II ) but are still killed by the β 2 m −/− GP33-specific CTL clone 27/30 in the absence of specific peptide . Lysis of T2D b cells was blocked already at a 1.0 μg/ml concentration of B22-249.1 in the CTL assay , indicating that a large fraction of cell surface H-2D b molecules is required for triggering of this CTL clone. Pulsing of T2D b cells with GP33 peptide prohibited blocking at all mAb concentrations tested . This effect was peptide specific, since neither NP366 nor Yop249 had any effect on avidity as determined by blocking with mAb. By titration of B22-249.1 in FACS ® analysis of T2D b , the fraction of free H-2D b ligands on T2D b at half-maximal peptide-independent lysis was estimated at ∼80% . Thus, ∼80% of the H-2D b molecules on T2D b were necessary for peptide-independent recognition by the high-avidity CTL clone 27/30, which corresponds to about four times the level of folded H-2D b on β 2 m −/− cells (Table II ). Taken together, these data indicate that β 2 m −/− CTLs use peptide-independent recognition of a high number of H-2D b ligands to compensate for the absence of specific peptide. Section title: GP33-specific β 2 m −/− CTLs Are Less Dependent on the Exact GP33 Peptide Sequence. Educational score: 4.410985946655273 Domain: biomedical Document type: Study Language: en Finally, we analyzed the consequences of self-MHC avidity in GP33-specific B6 and β 2 m −/− CTLs in terms of peptide specificity, when MHC was expressed at lower levels. Neither B6 nor β 2 m −/− CTLs kill RMA-S (MHC low ) in the absence of specific peptide, and RMA-S cells require a pulse of ∼100 pM GP33 peptide to become sensitive targets to both B6 and β 2 m −/− CTLs . This shows that B6 and β 2 m −/− CTLs require roughly equal amounts of H-2D b /GP33 complexes to get a triggering signal. However, when using GP33 peptide variants alanine-substituted at one or several TCR contact residues (reference 40 , and data not shown), the β 2 m −/− CTLs recognized RMA-S cells pulsed with up to 1,000-fold less peptide than B6 CTLs . Substitution at one position (GP33-4A) already drastically reduced the efficiency of B6 CTLs against peptide-pulsed RMA-S targets, whereas β 2 m −/− CTLs could still recognize peptides with alanine substituted at two positions. Thus, although β 2 m −/− CTLs were still peptide specific, they were less dependent on the TCR contact residues in the peptide when the peptide was loaded on RMA-S target cells (which express about three times the levels of H-2D b found on β 2 m −/− cells [Table II ]). These data indicate that the avidities for peptide and MHC both contribute functionally to the triggering of CTLs, and that they can be considered separately. Further, increased avidity for the restriction element compensates for a reduced avidity for the peptide. Section title: Contribution of Peptide-independent Recognition of MHC to the Specificity of T Cells. Educational score: 4.383723258972168 Domain: biomedical Document type: Study Language: en In this paper we have investigated the influence of TCR interaction with self-MHC in recognition of MHC-bound peptides. To address this issue, we used MHC class I–restricted CD8 + T cells selected and primed in an environment with high (B6) or low (β 2 m −/− ) MHC expression ( 25 ). We find that self-MHC can deliver a triggering signal independently of specific peptide, provided there is an increase in self-MHC density. The low surface density of H-2 expressed on RMA-S cells was not sufficient to trigger either β 2 m −/− or B6 CTLs in the absence of the specific antigen, but increasing the ligand density by cold temperature incubation could sensitize RMA-S to the β 2 m −/− CTL clone 27/30. Furthermore, since T2D b cells were also killed irrespective of loaded peptide, recognition of syngeneic class I was most probably peptide independent. These results indicate that the interaction between TCR and self-MHC as observed in crystals not only provides the structural framework of specific T cell recognition, it also contributes a part of the avidity required for CTL triggering. Section title: Contribution of Peptide-independent Recognition of MHC to the Specificity of T Cells. Educational score: 4.344555377960205 Domain: biomedical Document type: Study Language: en Recognition of MHC high targets by β 2 m −/− CTLs was blocked by mAb against properly conformed D b molecules, whereas the presence of specific peptide prohibited blocking. These results indicated that the lack of GP33 was compensated by using an increased number of low-avidity interactions with self-MHC. The results further suggest that a minimum of four times the level of MHC class I expression present during selection and priming in vivo were necessary for activation by self-MHC to occur in vitro. RMA-S expresses only about three times the level of MHC found on β 2 m −/− cells (Table II ), which may explain why β 2 m −/− CTLs were peptide specific when tested against RMA-S targets. In the presence of specific GP33 peptide, no mAb-mediated blocking of MHC high target cell killing was observed. This supports the notion that MHC-restricted CTLs have a high avidity for the peptide antigen and a low avidity for self-MHC. Section title: Contribution of Peptide-independent Recognition of MHC to the Specificity of T Cells. Educational score: 4.2312517166137695 Domain: biomedical Document type: Study Language: en Interestingly, we found that T cells with an increased avidity for MHC class I molecules had a more relaxed peptide specificity, i.e., they were less dependent on the exact peptide sequence. While both B6 and β 2 m −/− GP33-specific CTLs had similar sensitivity for GP33, β 2 m −/− CTLs had superior sensitivity for peptides lacking one or several TCR contact residues of the GP33 peptide. Thus, increased recognition of the restriction element compensates for lack of TCR peptide affinity in recognition of low- affinity peptide ligands. Section title: Contribution of Peptide-independent Recognition of MHC to the Specificity of T Cells. Educational score: 4.124942779541016 Domain: biomedical Document type: Study Language: en Our data argue that recognition of the two entities of the MHC complex, peptide and MHC heavy chain, can be considered separately ( 41 ). Increased recognition of MHC could substitute for lack of peptide recognition. The indication that TCR avidity for its ligand can be subdivided in this way opens the possibility of extending the differential avidity model of T cell selection and recognition ( 42 ). Section title: Implications for the Avidity Threshold in T Cell Recognition. Educational score: 4.454220771789551 Domain: biomedical Document type: Study Language: en The present data can be interpreted within a model for T cell recognition based on the total avidity contributed by TCR affinity for MHC, affinity for peptide, and the number of MHC–peptide complexes. Increased avidity for MHC can compensate for lack of avidity for peptide, suggesting that these binding forces are functionally interchangeable and can be considered separately . This would suggest a variation in the avidities for MHC and presented antigens among mature CTLs . Interestingly, mice deficient in terminal deoxynucleotidyl transferase (TdT), which lack N-region additions in the TCR, display increased promiscuity in T cell recognition of peptide ligands ( 43 ). In this situation, MHC was suggested to provide compensatory avidity in recognition of peptide antigen by TdT −/− CTLs. Further, the data in this paper support an inverse correlation between the number of MHC–peptide ligands necessary for triggering and TCR avidity for the ligand . Lower avidity for an antigen would then be compensated by an increase in antigen density. This feature becomes most important for CTLs with a relatively high MHC avidity, since self-MHC can be regarded as a high-density and low-affinity ligand. Section title: Implications for the Avidity Threshold in T Cell Recognition. Educational score: 4.190797805786133 Domain: biomedical Document type: Study Language: en By combining Fig. 7 , A and B, a hypothetical three- dimensional diagram can be generated in which the surface depicts how the TCR affinities for peptide and MHC, and the number of ligands necessary for triggering, are interchangeable . The diagram proposes that when the TCR avidities for both peptide and MHC are low, the number of ligands necessary to achieve triggering will be high. We would like to suggest further that the same model would apply for thymic selection, where the threshold coordinates for positive selection would be considerably lower on all axes while coordinates for negative selection would be closer to the threshold for triggering of effector functions. Section title: Aspects of CD8 + T Cell Selection in the Absence of β 2 m. Educational score: 4.4828104972839355 Domain: biomedical Document type: Study Language: en Selection of H-2D b –restricted T cells specific for the GP33 epitope in β 2 m −/− mice is thymus dependent, and results in a peripheral repertoire biased towards recognition of the restriction element expressed with and without self-peptides. However, peptide-independent triggering occurs only when MHC is expressed at levels considerably higher than those encountered by the T cells in vivo. This indicates repertoire calibration during thymic selection to fit the self-MHC ligand density in the periphery. Our results fit very well with the notion that T cells being selected in the thymus view self-MHC as a low-affinity ligand. Self-ligands capable of triggering induce deletion, and in the periphery self-MHC never reaches the ligand density to trigger CTLs in the absence of triggering antigens. It should be noted that in addition to the increased recognition of MHC, the β 2 m −/− CTLs may also display an increased avidity for self-peptides. There may be a clonal variation in recognition of MHC and peptide within the β 2 m −/− CTL population, where the clone 27/30 represents the most peptide-independent phenotype. Section title: Aspects of CD8 + T Cell Selection in the Absence of β 2 m. Educational score: 4.206479072570801 Domain: biomedical Document type: Study Language: en Considering the low expression of MHC present during selection in the β 2 m −/− mice, it is possible that the CD8 + T cells use upregulation of (co)receptor expression to achieve positive selection. Indeed, we have observed a marginal increase in CD8 expression in some of the β 2 m −/− CTLs, which potentially could also contribute to the elevated recognition of self-MHC. However, in functional experiments based on recognition of GP33 and alanine-substituted variants, we have observed that β 2 m −/− CTLs are less susceptible to CD8 blocking with anti-CD8α mAbs compared with B6 CTLs. This result would suggest, rather, a decreased dependency on CD8 in triggering of β 2 m −/− CTLs (data not shown). Section title: Aspects of CD8 + T Cell Selection in the Absence of β 2 m. Educational score: 4.613008975982666 Domain: biomedical Document type: Study Language: en Accumulating evidence suggests that the CD8 + T cells present in the β 2 m −/− mice have retained important characteristics of the β 2 m +/+ wild-type concerning development and recognition of MHC. First, β 2 m −/− mice can generate in vivo CTL responses against MHC class I–restricted peptide (44; and this study) and viral ( 45 ) antigens, and reject skin grafts over a minor histocompatibility barrier ( 46 ). Also, β 2 m −/− CTLs can recognize peptide antigens on β 2 m +/+ targets (this study). Second, development of β 2 m −/− CD8 + T cells is dependent on the thymus (this study), and results in a CD8 + T cell repertoire biased towards recognition of syngeneic class I (23; and this study). Third, H-2D b heavy chains expressed in the absence of β 2 m are recognized by conformation-dependent mAbs (27, 28, 47; and this study), and expression is TAP dependent (32; and this study). Note also that all detectable β 2 m −/− CTL reactivity against self-MHC could be blocked by an mAb specific for properly conformed H-2D b molecules, which reduces the likelihood for a role of aberrantly conformed free D b heavy chains in this system. Fourth, wild-type CTLs can recognize endogenously processed antigens ( 48 ) and alloantigens ( 27 , 48 ) on β 2 m −/− cells. Taken together, these data suggest that T cell selection follows the normal rules in β 2 m −/− mice, although the selection window is dramatically shifted towards low ligand density. The confrontation of such T cells with cells expressing normal MHC levels allows detection of the T cell avidity for self-MHC. However, the functional avidity must be established already during selection and must exist during priming. Thus, we propose that T cells in normal mice have a similar avidity for self-MHC which would be detectable by exposing them to cells with supraoptimal MHC levels. Section title: Aspects of CD8 + T Cell Selection in the Absence of β 2 m. Educational score: 4.247137069702148 Domain: biomedical Document type: Study Language: en We have demonstrated that avidity for self-MHC can trigger MHC-restricted T cells independently of specific peptide if ligand density of self-MHC is sufficiently increased. This interaction compensates for lack of TCR affinity for peptide, and it depends on a TCR–MHC interaction of relatively low affinity that requires high numbers of MHC ligands. The data and the avidity threshold model discussed contribute to our understanding of T cell specificity in the periphery and during thymic selection. | Study | biomedical | en | 0.999997 |
10075973 | Section title: Cell Lines. Educational score: 4.287715911865234 Domain: biomedical Document type: Study Language: en The human melanoma cell lines NA8-MEL (provided by Dr. F. Jotereau, Institut National de la Santé et de la Recherche Médicale, Nantes, France) and SK23-MEL were cultured in RPMI medium ( GIBCO BRL ) supplemented with 10% FCS, antibiotics, and 20 mM NaHepes (pH 7.3). The CTL line LAU 198 NS (normal stimulation) specific for peptide MAGE-3 271–279 was established by stimulating CD8 + -enriched cells from a LAU 198 melanoma patient with peptide-pulsed, autologous PBL as described ( 18 ). The CTL line was restimulated weekly with irradiated (3,000 rads) autologous PBL. Before irradiation, PBL were incubated for 2 h at 37°C in serum-free medium (X-VIVO 10; BioWhittaker) in the presence of the synthetic peptide MAGE-3 271–279 (1 μg/ml) and 3 μg/ml human β 2 -microglobulin ( Sigma Chemical Co. ) and then washed extensively to remove unattached peptide. The CTL clone 3C5 used in Fig. 7 was isolated as described previously ( 19 ). In brief, PBL were stained with tetramers of HLA-A*0201–β 2 -microglobulin containing the peptide MAGE-3 271–279 . After cell sorting by FACS ® ( Becton Dickinson ), positive cells were cloned in the presence of irradiated PBL, 5 μg/ml PHA, and 50 U/ml IL-2 in human serum containing IMDM. The human lymphoblastoid cell lines T2 and LCL721.45 were used as targets in 51 Cr release assay. These cells were cultured in DMEM ( GIBCO BRL ) supplemented with 10% FCS, 0.55 mM arginine, 0.24 mM asparagine, and 1.5 mM glutamine. The TNF-α–sensitive cell line WEHI-164 clone 13 was maintained in RPMI medium ( GIBCO BRL ) supplemented with 10% FCS, 0.55 mM arginine, 0.24 mM asparagine, and 1.5 mM glutamine. Section title: Construction of Plasmids and Ubiquitin/Protein/Reference (UPR) Technique. Educational score: 4.205641746520996 Domain: biomedical Document type: Study Language: en All plasmid constructs described in this study were based on the plasmid pS65T-C1 ( Clontech ). This vector carries, under the control of the cytomegalovirus immediate early promoter, a mutated version of the green fluorescence protein (GFP), resulting in brighter fluorescence. The stop codon of the GFP moiety has been replaced by a multicloning site allowing COOH-terminal fusions to GFP. A fragment encoding yeast ubiquitin (Ub) carrying a lysine 48 to arginine mutation (to preclude that the COOH-terminal Ub moiety could serve as a ubiquitylation/degradation signal) and an influenza hemagglutinin epitope (ha) recognized by a monoclonal antibody was obtained by PCR, using the plasmid pRc/dUb–Met–βgal as template ( 20 ). This PCR fragment was digested with KpnI and SmaI and inserted in frame between the KpnI and SmaI sites present in the multicloning site of pS65T-C1, resulting in the vector pGFP AvaI /Ub expressing the fusion GFP–ha–Ub. The ha epitope is located between the GFP and Ub moieties. The plasmid pGFP AvaI /Ub was further digested with XhoI and EcoRI, blunted using Klenow PolI, and religated, thereby eliminating the AvaI site between GFP and Ub and rendering the site AvaI at the 3′ end of Ub unique. This latter construct, termed pGFP/Ub, served as vector for all constructs described below. Section title: Construction of Plasmids and Ubiquitin/Protein/Reference (UPR) Technique. Educational score: 4.248826503753662 Domain: biomedical Document type: Study Language: en Construct I was obtained by PCR amplification of a fragment encoding the tumor antigen MAGE-3 (a gift from T. Boon, Ludwig Institute for Cancer Research, Brussels, Belgium). This PCR fragment was digested with SacII and AvaI and inserted in frame between the SacII and AvaI sites of pGFP/Ub, resulting in the plasmid pGFP/Ub–MAGE-3 1–314 . The protein MAGE-3 includes the natural NH 2 -terminal methionine. Constructs II, III, and IV were obtained by annealing complementary synthetic oligonucleotides encoding the MAGE-3–derived peptide fragments MAGE-3 271–279 , MAGE-3 256–279 , and MAGE-3 271–285 , respectively. The oligonucleotides were designed so as to reconstitute the SacII site at the 5′ end and the AvaI site at the 3′ end of the fragment and included a stop codon immediately upstream of the AvaI site. Upon annealing, the fragments were inserted between the SacII–AvaI sites of pGFP/Ub, resulting in the plasmids pGFP/Ub–MAGE-3 271–279 , pGFP/Ub– MAGE-3 256–279 (construct III), and pGFP/Ub–MAGE-3 271–285 (construct IV). The sequences of the critical regions of all constructs were confirmed by DNA sequencing. Section title: Transfection and Metabolic Labeling. Educational score: 4.250083923339844 Domain: biomedical Document type: Study Language: en Cells were transiently transfected using the Lipofectamine reagent ( GIBCO BRL ) according to the manufacturer's protocol. For metabolic labeling, 3 × 10 6 cells were transfected with 4 μg plasmid DNA. After 8 h, an equal volume of DMEM containing 10% FCS was added to the cells. After a further 12-h incubation at 37°C, the culture medium was replaced with 1 ml DMEM lacking methionine and cysteine (ICN Biomedicals, Inc.), and the cells were incubated for 1 h at 37°C. Cells were labeled for 90 min at 37°C in the presence of 200 μCi of [ 35 S]Express ( New England Nuclear ). The cells were then lysed in 1 ml lysis buffer (1% Triton X-100, 0.15 M NaCl, 5 mM EDTA, 20 mM Tris–HCl [pH 7.3], and 0.2 mg/ml phenylmethylsulfonyl fluoride) and maintained on ice. The lysate was cleared by centrifugation at 12,000 g for 10 min. The volumes of supernatant were adjusted to contain equal amount of 10% TCA-insoluble, 35 S-labeled material and immunoprecipitated using a mixture of a saturating amount of a monoclonal antibody anti-ha epitope (Berkeley Antibody Co.) and a monoclonal antibody against MAGE-3 (21; a gift from G. Spagnoli, University of Basel, Basel, Switzerland). The samples were incubated with rotary shaking at 4°C for 30 min, followed by the addition of 20 μl protein G–Sepharose and another 30-min incubation at 4°C. The immunoprecipitate was washed three times in lysis buffer containing 0.1% SDS, resuspended in 20 μl SDS–sample buffer (100 mM Tris–HCl, pH 8.8; 1.2 M sucrose; 0.01% bromophenol blue; 2% SDS; and 90 mM dithiothreitol), and boiled at 100°C for 3 min. The samples were subjected to SDS–12% PAGE followed by autoradiography. Section title: TNF-α Release Assay. Educational score: 4.145618438720703 Domain: biomedical Document type: Study Language: en Cells were transfected using the same protocol as for the metabolic labeling with the following modifications: 10 4 cells were transfected in 96-well, round bottom microtiter plates with 200 ng plasmid DNA and 1 μl Lipofectamine in a final volume of 100 μl DMEM. After 6 h at 37°C, 100 μl DMEM containing 10% FCS was added to each well, and the cells were maintained at 37°C for another 14 h. At this point, the transfected cells were tested for their ability to stimulate the release of TNF-α by the MAGE-3 271–279 –specific CTL line. In brief, CTLs were added at the appropriate effector-to-target cell ratio (E/T) in 100 μl of IMDM supplemented with 10% human serum and 20 U/ml human rIL-2 (Glaxo Wellcome) provided by Dr. M. Nabholz (Swiss Institute for Experimental Cancer Research, Epalinges, Switzerland). After a 24-h incubation at 37°C, supernatants were collected and the TNF-α content was determined in a functional assay using WEHI-164 clone 13 cells ( 22 ) as described ( 23 ). Section title: Proteasome Purification. Educational score: 4.231812953948975 Domain: biomedical Document type: Study Language: en Proteasome was purified from human outdated blood by affinity chromatography using the monoclonal antibody MCP 21 (European Collection of Animal Cell Cultures) immobilized on CnBr-activated Sepharose (6 mg antibody/mg Sepharose; 24). ∼150 ml blood was washed five times with cold PBS, and the cells were pelleted at 2,000 g for 10 min. This pellet was resuspended in 1/5 volume of sterile H 2 O and mechanically disrupted by douncing in a Dounce homogenator. Immediately after this treatment, sucrose was added to the homogenate to a final concentration of 250 mM. Unsolubilized material was removed by centrifugation. Cleared lysate was incubated for 3 h at 4°C under rotary shaking with the immobilized mAb MCP 21 as a batch preparation. The material was then loaded onto a column and washed extensively with 20 mM Tris–HCl (pH 7.6), and 50 mM NaCl to remove unattached material. 20S proteasome was eluted with 20 mM Tris–HCl (pH 7.6) and 2 M NaCl. The eluate was dialysed for 24 h at 4°C against 20 mM Tris–HCl (pH 7.6) and concentrated to 4 mg/ml. The average yield was ∼1 mg purified proteasome/150 ml blood. Homogeneity of the eluted material was confirmed by analysis of an aliquot by SDS–12% PAGE and Coomassie blue staining of the gel. Section title: Enzymatic Assays. Educational score: 4.162396430969238 Domain: biomedical Document type: Study Language: en The activity of the proteasome was assayed using the following fluorogenic peptides: Z–Gly–Gly–Arg–βNA (Z–GGR–βNA; Z = benzyloxycarbonyl and βNA = β-naphtylamide) for tryptic-like activity, Z–Leu–Leu–Glu–βNA (Z–LLE– βNA) for PGPH activity, and Suc–Leu–Leu–Val–Tyr–AMC (Suc–LLVY–AMC; Suc = succinyl, and AMC = 7-amido-4-methylcoumarin) for chymotryptic-like activity. All peptides were purchased from Bachem. Each degradation assay contained 5 μg proteasome plus 100 μM peptides Z–GGR–βNA and Z–LLE– βNA or 10 μM Suc–LLVY–AMC in a final volume of 100 μl. The reaction was incubated at 25°C for 20 min, at which time the reaction was quenched with cold 100% ethanol. Fluorescence emission was measured using a Perkin Elmer fluorometer. Fluorescence excitation/emission wavelengths were 335:410 nm for βNA and 380:440 nm for AMC. Where indicated, the proteasome inhibitor lactacystin (Biomol) was incubated for 15 min with proteasome at 25°C before the addition of the peptide substrates. The final concentration of lactacystin was 50 μM. Section title: Peptide Synthesis. Educational score: 4.157487869262695 Domain: biomedical Document type: Study Language: en Peptides were synthesized using standard solid-phase F-moc chemistry on an Applied Biosystems synthesizer. After side chain deprotection and release from the resin, the peptides were purified by reverse-phase preparative HPLC using a Vydac C18 column. Fractions containing the expected product, as judged by mass spectrometry, were pooled and lyophilized. The purified material was then subjected to matrix assisted laser desorption ionization-time of flight (MALDI-TOF) and analytical HPLC analysis. All peptides were >95% pure as indicated by analytical HPLC. Section title: Peptide Digestion and Mass Spectrometry. Educational score: 4.165614128112793 Domain: biomedical Document type: Study Language: en For analysis by mass spectrometry, 4 nmol peptide was incubated for 40 min at 37°C with 16 μg purified proteasome in a final volume of 10 μl. Where indicated, lactacystin was added to a final concentration of 50 μM. The samples containing proteasome and lactacystin were incubated for 15 min at 37°C before the addition of peptide. The reaction was terminated by the addition of 5% TFA and immediate immersion in liquid N 2 . The samples were subsequently lyophilized. The lyophilized samples were dissolved in 20 μl 1% TFA/ H 2 O. An aliquot of this solution (1.5 μl) was mixed with 24 μl of matrix solution (5 mg/ml α-cyano-4-hydroxyl-cinnamic acid; Sigma Chemical Co. ) in 1% TFA/H 2 O:acetonitrile (1:1, vol/vol) and 1 μl of this mixture was deposited on a gold plated target and vacuum dried before transferring it into the source of the mass spectrometer. Section title: Peptide Digestion and Mass Spectrometry. Educational score: 4.189554214477539 Domain: biomedical Document type: Study Language: en MALDI mass spectra were obtained on a Perseptive Biosystems Voyager RP spectrometer using a 337-nm nitrogen laser, a 25-kV accelerating potential, and a delayed extraction of 150 ns. Each spectrum was the result of an accumulation of 128 single laser shots. External calibration of the MALDI spectra in the linear mode was carried out using a low molecular weight standard (Perseptive Biosystems). The mass determination was based on the mass-to-charge (m/z) ratio. The instrument error ranges between 0.01 and 0.1% of the molecular mass. Section title: Peptide Digestion and 51 Cr Release Assay. Educational score: 4.21911096572876 Domain: biomedical Document type: Study Language: en Samples used in subsequent CTL assay included 100 pmol peptide and 4 μg purified proteasome. Where indicated, trypsin (1 mg/ml) was added to the peptide and incubated for 20 min at 37°C in 10 mM Tris– HCl, pH 8.0. The reaction performed was identical to the reaction for the mass spectrometry analysis. Target cells were labeled with 51 Cr for 1 h at 37°C and washed twice. Labeled target cells (1,000 cells in 50 μl RPMI medium/5% FCS) were added to serial dilutions of the various peptide preparations (100 μl) in v-bottomed microwells for 15 min at room temperature. The CTL were then added at the appropriate E/T in 50 μl RPMI medium/5% FCS, and the release of 51 Cr was measured after incubation for 4 h at 37°C. Specific lysis was calculated as follows: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}\%\;specific\;lysis=[(Experimental\;release-spontaneous\;release)/\;(Total\;release-spontaneous\;release)]{\times}100.\end{equation*}\end{document} Section title: Infection with Recombinant Vaccinia Virus and Treatment of Cells with Proteasome Inhibitors. Educational score: 4.147646903991699 Domain: biomedical Document type: Study Language: en Approximately 10 6 LCL721.45 cells ( 25 ) were treated with 100 μM lactacystin for 1 h, washed, and incubated overnight in 1 μM lactacystin at 37°C. Cells were then infected with recombinant vaccinia virus (rec. v.v.) expressing the MAGE-3 1–314 protein at a multiplicity of infection of 10 and simultaneously labeled with 100 μCi of 51 Cr for 2 h. The cells were washed and incubated at 37°C for another 4 h before being exposed to the MAGE-3–specific CTL clone 3C5 at the E/T indicated in Fig. 7 . Rec. v.v. encoding the MAGE-3 protein was generated by recombination with a pSc11 derivative plasmid as described previously ( 26 ). The cDNA coding for MAGE-3 was digested with PstI and XbaI, blunted using Klenow PolI, and inserted into the SmaI site of the vector pSc11. Section title: Infection with Recombinant Vaccinia Virus and Treatment of Cells with Proteasome Inhibitors. Educational score: 4.129683494567871 Domain: biomedical Document type: Study Language: en The HLA-A*0201 + MAGE-3 + melanoma cell line SK23-MEL ( 18 ) was treated with 10 μM lactacystin or 100 μM LLnL for 17 h. Subsequently, 10 6 cells were labeled with 100 μCi of 51 Cr for 2 h, washed, and incubated with the MAGE-3–specific CTL clone 3C5 for 8 h at 37°C. Section title: Experimental Strategy. Educational score: 4.122111797332764 Domain: biomedical Document type: Study Language: en We have previously derived a MAGE-3 271–279 –specific CTL line from PBL of a melanoma patient that, although able to readily recognize HLA-A*0201 + cells exposed to exogenously added synthetic MAGE-3 271–279 peptide, was unable to lyse cells expressing the MAGE-3 protein ( 18 ). To address this discrepancy, we assessed in the present study the recognition of HLA-A*0201 + melanoma cells transfected with cDNA or cDNA fragments encoding the MAGE-3 protein. Expression of the various MAGE-3 gene products in transfected cells was determined by the UPR technique, originally developed to increase the accuracy of protein decay measurements ( 20 ). Section title: Experimental Strategy. Educational score: 4.320769786834717 Domain: biomedical Document type: Study Language: en Ub is a highly conserved protein that, in many instances, serves as a proteasomal degradation signal by forming a multiubiquitin chain on lysine residue(s) of a protein ( 27 ). Ub is naturally synthesized as a fused protein—either to itself or to other cellular proteins—and is rapidly cleaved by a member of the Ub-specific proteases. The UPR technique exploits this natural linear arrangement of the Ub gene and relies on a linear fusion with Ub located between a reference protein (here GFP) and a protein of interest . The rapid cleavage of the fusion protein by Ub-specific proteases after the last residue of Ub yields equimolar amounts of the protein of interest and the reference protein bearing a COOH-terminal Ub moiety. It is noteworthy that the use of Ub fusion also enabled us to bypass the need for a methionine residue at the NH 2 termini of the MAGE-3 peptide fragments. Finally, a sequence encoding a peptide tag derived from the influenza ha was inserted between the GFP and Ub moieties to permit the detection and quantitation of GFPha–Ub fusion. Section title: Experimental Strategy. Educational score: 4.160889625549316 Domain: biomedical Document type: Study Language: en NA8-MEL cells were transiently transfected with the plasmids pGFP/Ub–MAGE-3 1–314 , pGFP/Ub–MAGE-3 271–279 , pGFP/Ub–MAGE-3 256–279 , or pGFP/Ub–MAGE-3 271–285 coding for the reference GFPha–Ub and either MAGE-3 1–314 , MAGE-3 271–279 , MAGE-3 256–279 , or MAGE-3 271–285 , respectively . The transfected cells were labeled with [ 35 S]methionine/cysteine for 60 min, lysed, immunoprecipitated using saturating amounts of monoclonal anti-ha antibody and monoclonal anti–MAGE-3 antibody, and analyzed by SDS-PAGE and autoradiography. The radioactive band corresponding to the reference GFPha–Ub could be detected in all lanes where cells had been transfected with UPR-based plasmids but not in mock-transfected cells (lane e). The protein MAGE-3 was only detected in cells transfected with the full length MAGE-3 gene (lane a) and migrated as a doublet. The upper band of the doublet is the result of a posttranslational modification other than phosphorylation or N-glycosylation (data not shown). The presence of the reference protein in the other lanes confirmed the expression of the MAGE-3 protein fragments. Using the GFP-based UPR technique, we estimated that the transiently transfected constructs were expressed in ∼2% of the melanoma cells (data not shown). Section title: CTL Recognition of Cells Expressing MAGE-3 1–314 , MAGE-3 271–279 , and MAGE-3 271–279 Extended Precursors. Educational score: 4.324427127838135 Domain: biomedical Document type: Study Language: en NA8-MEL cells transfected with pGFP/Ub–MAGE-3 1–314 and pGFP/Ub–MAGE-3 271–279 (construct II) encoding the reference protein GFPha–Ub and either MAGE-3 1–314 or MAGE-3 271–279 , respectively, were incubated with cells from the CTL line specific for MAGE-3 271–279 at a lymphocyte-to-target cell ratio of 30:1. After 24 h of coculture, supernatants were assayed for the presence of TNF-α released by activated CTL. The amount of TNF-α produced by CTL incubated with cells expressing MAGE-3 1–314 was not significantly higher than that produced by CTL incubated with mock-transfected cells . In contrast, cells expressing MAGE-3 271–279 were efficiently recognized by CTL as indicated by the 50-fold increase in the amount of TNF-α produced. As expected, addition of a saturating amount of the corresponding MAGE-3 271–279 synthetic peptide to mock-transfected cells resulted in a strong production of TNF-α. Therefore, the impaired presentation of MAGE-3 271–279 by NA8-MEL cells transfected with MAGE-3 1–314 was not caused by an inefficient transport or loading of the peptide on HLA-A*0201 molecules but rather by an inaccurate processing of the protein MAGE-3. Section title: CTL Recognition of Cells Expressing MAGE-3 1–314 , MAGE-3 271–279 , and MAGE-3 271–279 Extended Precursors. Educational score: 4.221897602081299 Domain: biomedical Document type: Study Language: en To locate the region of the protein affecting the generation of the antigenic peptide, we extended the sequence of MAGE-3 271–279 at the NH 2 terminus by 15 amino acids and at the COOH terminus by 6 amino acids (construct IV). NA8-MEL cells were transfected with the plasmids coding for GFPha–Ub and either MAGE-3 256–279 or MAGE-3 271–285 , respectively. Cells expressing the fragment MAGE-3 256–279 were well recognized by CTL . On the contrary, cells transfected with the plasmid encoding MAGE-3 271–285 were not recognized by CTL. The length of the COOH-terminal extension did not influence the recognition of the transfected cells, as cells expressing MAGE-3 271–279 with a COOH-terminal extension of 15 amino acids did not lead to a recognition of the cells, either (data not shown). Therefore, the processing protease was unable to generate the MAGE-3 271–279 peptide from a COOH-terminally extended precursor but could generate it from a precursor containing a preprocessed COOH terminus as the result of genetic engineering. Section title: Analysis of Peptide Fragments Generated after Digestion with Purified Proteasome In Vitro. Educational score: 4.194080829620361 Domain: biomedical Document type: Study Language: en The human proteasome has been implicated in the generation of antigenic peptides presented by HLA class I molecules to CTL ( 11 – 13 ). We tested whether the peptide fragments obtained after exposure of NH 2 - and COOH-terminally extended MAGE-3 271–279 precursors to purified human proteasome correlated with the results obtained using transfected cells. Synthetic peptide MAGE-3 271–285 was incubated for 40 min at 37°C in the presence of proteasome purified from blood and was subsequently analyzed by mass spectrometry . A major peak corresponding to the original peptide MAGE-3 271–285 (theoretical mass 1,766 daltons) was detectable at time 0. Several additional peaks of lower mass appeared after incubation with the proteasome. These degradation products were unambiguously identified as the 11-mer MAGE-3 271–281 (1,288 daltons), the 10-mer MAGE-3 271–280 (1,187 daltons), the 8-mer MAGE-3 271–278 (959 daltons) and the 7-mer MAGE-3 272–278 (812 daltons). No peak corresponding to the 9-mer MAGE-3 271–279 (1,058 daltons) was detected. Section title: Analysis of Peptide Fragments Generated after Digestion with Purified Proteasome In Vitro. Educational score: 4.178970813751221 Domain: biomedical Document type: Study Language: en A similar analysis was performed using peptide MAGE-3 256–279 . The major peak detected at time 0 corresponded to the original peptide . After incubation with proteasome, the 9-mer corresponding to MAGE-3 271–279 (1,058 daltons) was clearly detected . Moreover, several other peaks were detected and identified as the 10-mer MAGE-3 256–265 (1,163 daltons), the 8-mer MAGE-3 272–279 (911 daltons), and the 7-mer MAGE-3 272–278 (812 daltons). The relative signal intensity of peptide MAGE-3 256–279 was reproducibly lower than that of peptide MAGE-3 271–285 and may reflect the chemical properties of this peptide. Thus, results obtained with in vitro digestion of synthetic MAGE-3 peptide extensions not only confirmed the results obtained with DNA- encoded peptides expressed intracellularly but also enabled precise identification of the degradation products. Section title: Selective Inhibition of Proteasomal Activity In Vitro Influences the Production of Peptide MAGE-3 271–279 . Educational score: 4.143495082855225 Domain: biomedical Document type: Study Language: en To ascertain the role of the different enzymatic activities of the proteasome in the generation of the proteolytic fragments in vitro, we performed experiments similar to the one described above in the presence of lactacystin, a specific inhibitor of the proteasome. Lactacystin has been shown to irreversibly block the trypsin-like and chymotrypsin-like activities, reversibly inhibit the PGPH activity, and moderately block the BrAAP activity of the proteasome ( 28 , 29 ). Section title: Selective Inhibition of Proteasomal Activity In Vitro Influences the Production of Peptide MAGE-3 271–279 . Educational score: 4.1904802322387695 Domain: biomedical Document type: Study Language: en Purified MAGE-3 256–279 and MAGE-3 271–285 peptides were incubated for 40 min in the presence of proteasome and 50 μM lactacystin and analyzed by mass spectrometry . Digestion of peptide MAGE-3 271–285 (C) resulted in the appearance of several peaks with masses similar to those obtained after the digestion without lactacystin (compare with B). Unexpectedly, the peptide species corresponding to the 9-mer MAGE-3 271–279 (1,058 daltons) was generated to a detectable level. The peak corresponding to the 7-mer MAGE-3 272–278 (812 daltons) was not detectable in the sample incubated with the proteasome inhibitor. Digestion of peptide MAGE-3 256–279 in the presence of lactacystin (F) yielded new peaks corresponding to the 12-mer MAGE-3 256–267 (1,331 daltons) and the 6-mer MAGE-3 256–261 (807 daltons), in addition to the 9-mer MAGE-3 271–279 (1,058 daltons) and the 8-mer MAGE-3 272–279 (912 daltons) already detected in digestion without lactacystin (E). It is interesting to note that the relative intensity of the peak corresponding to the antigenic 9-mer was reproducibly higher in digestions containing lactacystin than in those performed in the absence of lactacystin (compare E and F). Section title: Lactacystin Inhibits Two of the Three Tested Proteasomal Activities. Educational score: 4.159789085388184 Domain: biomedical Document type: Study Language: en To identify the lactacystin-mediated effect on the enzymatic activity of the proteasome, we performed a digestion of the fluorescent substrates Z–GGR–βNA (for trypsin-like activity), Z–LLE–βNA (for PGPH activity), and Suc–LLVY–AMC (for chymotrypsin-like activity) in the presence or absence of 50 μM lactacystin . Although all fluorescent substrates were cleaved by the proteasome in the absence of lactacystin as detected by increased fluorescence, two of the three activities of the proteasome could be efficiently blocked by the addition of lactacystin. The PGPH activity was only marginally affected by the presence of lactacystin. Further purification of the proteasome by size chromatography did not alter this effect, ruling out the contribution of other low molecular weight proteases present in the purified preparation (data not shown). Section title: Specific CTL Can Recognize Target Cells Pulsed after Digestion of Peptide Precursors. Educational score: 4.1254143714904785 Domain: biomedical Document type: Study Language: en Since the generation of the 9-mer MAGE-3 271–279 from peptides MAGE-3 271–285 and MAGE-3 256–279 was enhanced in the presence of lactacystin, we tested whether the peptide generated under this condition was able to sensitize target cells for lysis by MAGE-3 271–279 –specific CTL. Peptides MAGE-3 271–285 and MAGE-3 256–279 were either added directly to target cells or were first incubated with purified proteasome for 20 min at 37°C in the absence or presence of lactacystin. After 20 min, the reaction was stopped by the addition of 5% TFA and lyophilized. The lyophilized material was resuspended in medium and added in different dilutions to 51 Cr-labeled T2 cells. These HLA-A*0201 + cells are TAP deficient but can be efficiently sensitized with exogenous peptides for recognition by specific CTL. Section title: Specific CTL Can Recognize Target Cells Pulsed after Digestion of Peptide Precursors. Educational score: 4.374526023864746 Domain: biomedical Document type: Study Language: en Direct addition of the peptide MAGE-3 271–285 resulted in lysis of T2 cells only at very high peptide concentration . This activity was probably caused by the presence of a small amount of peptide MAGE-3 271–280 produced by the hydrolysis of peptide MAGE-3 271–285 . Indeed, the MAGE-3 271–279 –specific CTL used in the present study was able to recognize the synthetic decapeptide MAGE-3 271–280 (50% maximal lysis at 100 nM), although much less efficiently than the 9-mer MAGE-3 271–279 (50% maximal lysis at 0.1 nM). No additional activity was observed after digestion of peptide MAGE-3 271–285 with proteasome for 20 min . In contrast, incubation of peptide MAGE-3 271–285 with proteasome and lactacystin for 20 min resulted in the generation of an antigenic peptide recognized ∼20-fold more efficiently by the MAGE-3 271–279 – specific CTL (B). Based on the specificity of the CTL and on the degradation products detected by mass spectrometry, we concluded that the antigenic peptide MAGE-3 271–279 was indeed produced in the presence of lactacystin. Digestion of peptide MAGE-3 271–285 with trypsin completely abrogated recognition of the target cells by specific CTL. Section title: Specific CTL Can Recognize Target Cells Pulsed after Digestion of Peptide Precursors. Educational score: 4.201079368591309 Domain: biomedical Document type: Study Language: en A similar assay was performed using peptide MAGE-3 256–279 as substrate. Again, direct addition of the peptide precursor produced low but detectable lytic activity at high concentration. Digestion of the extended peptide for 20 min in the absence of lactacystin led to a 10-fold increase in lytic activity , an effect compatible with the detection of peptide MAGE-3 271–279 in the digestion products analyzed by mass spectrometry . This activity was completely abolished by the addition of trypsin. However, digestion of peptide MAGE-3 256–279 in the presence of 50 μM lactacystin resulted in 100-fold–increased recognition . This effect was most likely caused by the increased amount of MAGE-3 271–279 generated after proteasomal digestion in the presence of lactacystin as suggested by the increased relative intensity of the MAGE-3 271–279 peak detected by mass spectrometry . Section title: Treatment of Cells Expressing MAGE-3 1–314 with Proteasome Inhibitors Results in Efficient Presentation of MAGE-3 271–279 . Educational score: 4.303990364074707 Domain: biomedical Document type: Study Language: en To ascertain the relevance of the positive effect of lactacystin on the generation of peptide MAGE-3 271–279 by the proteasome, we infected HLA-A*0201 + lymphoblastoid cells with rec. v.v. coding for MAGE-3 1–314 . Infected cells expressing the full length protein MAGE-3 1–314 were not efficiently recognized by MAGE-3 271–279 –specific CTL. However, treatment of the same cells with lactacystin prior to and during the infection lead to the efficient lysis of the infected cells. To determine whether the lactacystin-mediated effect observed in lymphoblastoid cells upon infection and high level expression of MAGE-3 could also be observed in uninfected melanoma cells, lysis of HLA-A*0201 + MAGE-3 + melanoma cells was measured after treatment with lactacystin. As expected, lysis of untreated target cells was very low . In contrast, treatment with lactacystin led to efficient lysis of the melanoma cells. A similar effect was also observed after treatment of the target cells with the calpain (and proteasome) inhibitor I LLnL. Taken together, these results strongly support the findings obtained after in vitro digestion of peptide precursors by purified proteasome and lead us to conclude that the generation and presentation of peptide MAGE-3 271–279 detected after treatment with lactacystin is a direct consequence of the partial inhibition of the proteasome in cells. Section title: Discussion Educational score: 4.221011161804199 Domain: biomedical Document type: Study Language: en Two major findings are reported in this work: First, melanoma cells transfected with cDNA encoding either the preprocessed antigenic peptide MAGE-3 271–279 (FLWGPRALV) or the same peptide with an NH 2 -terminal extension were recognized by MAGE-3 271–279 –specific CTL. In contrast, melanoma cells expressing the full length protein MAGE-3 1–314 or peptide MAGE-3 271–279 with a COOH-terminal extension were not recognized. This discrepancy stems from the inappropriate proteasomal cleavage at the COOH terminus of the antigenic peptide. Second, treatment of MAGE-3 + melanoma cells with lactacystin resulted in the recognition of these normally unrecognized cells by MAGE-3 271–279 –specific CTL. This effect strongly correlates with the partial inhibition of proteasomal activity in vitro and the lactacystin-induced generation of peptide MAGE-3 271–279 from the COOH-terminally extended precursor. Section title: Discussion Educational score: 4.282085418701172 Domain: biomedical Document type: Study Language: en Expression of a minigene product in transfected cells has been difficult to assess by means other than functional assays. However, use of the UPR technique in this study not only allowed us to confirm the presence of the various minigene products via the detection of the reference protein GFP–Ub but also to quantitate the various MAGE-3 gene products, as both the reference protein GFP–Ub and the MAGE-3 protein, or protein fragments, were produced in equimolar amounts. Thus, the observed differences in CTL recognition of target cells transfected with the minigene encoding peptide MAGE-3 271–279 or the full length protein MAGE-3 1–314 could not be attributed to different expression levels of the two products but rather to the generation, or lack thereof, of the antigenic peptide. Cells expressing a short-lived variant of the MAGE-3 1–314 protein were not recognized by specific CTL, either (data not shown), eliminating the possibility that the lack of recognition was caused by a slow degradation of the MAGE-3 protein leading to a suboptimal concentration of the antigenic peptide. Finally, reciprocal transplantation of the MAGE-3 271–279 sequence into unrelated proteins indicated that the impaired generation of the antigenic peptide was not caused by residues flanking peptide MAGE-3 271–279 (data not shown). Altogether, these results suggested that the sequence of peptide MAGE-3 271–279 itself contributed to the improper processing of the MAGE-3 protein and led us to study the role of the proteasome in the deficient production of the antigenic peptide. Section title: Discussion Educational score: 4.477384090423584 Domain: biomedical Document type: Study Language: en The involvement of the proteasome in the processing of antigenic peptides has been inferred from two sets of data: First, treatment of target cells with specific proteasome inhibitors abolished CTL-mediated recognition of antigenic peptides derived from intracellular proteins without affecting recognition of preprocessed endogenous or exogenous antigenic peptides ( 13 ). Second, analysis of proteasome- mediated degradation of synthetic peptide substrates in vitro revealed that the structural features of the digested products were compatible with those of peptides naturally associated with MHC class I molecules ( 30 ). However, several reports have recently demonstrated that the presentation of certain antigenic peptides, derived mostly from protein fragments, were insensitive to the effects of proteasome inhibitors ( 28 , 31 , 32 ). Moreover, cell surface expression of assembled MHC class I proteins in cells lacking proteasomal function suggests that other proteases may be involved in the generation of antigenic peptides ( 33 ). Therefore, the participation of two complementary and possibly overlapping proteolytic systems in antigen processing could be envisaged and would be compatible with our results obtained after treatment of melanoma cells with lactacystin. To address this question in greater detail, we isolated human proteasome and performed in vitro digestion studies using synthetic peptides. After digestion, the samples were immediately analyzed by mass spectrometry so as to uncover all possible fragments generated by the proteasome. Using this approach, we obtained strong evidence in favor of the involvement of the proteasome in the final proteolytic step leading to the generation of the antigenic peptide MAGE-3 271–279 , notwithstanding the possibility that distinct cellular proteases may be involved at other stages in the processing of the MAGE-3 protein into precursor peptide fragments. Section title: Discussion Educational score: 4.1847004890441895 Domain: biomedical Document type: Study Language: en Five proteolytic activities of the proteasome have been described using short synthetic fluorogenic peptides: a tryptic-like activity, a chymotryptic-like activity, a PGPH activity, a BrAAP activity, and a SNAAP activity ( 3 ). Lactacystin has been shown to irreversibly block the trypsin-like and chymotrypsin-like activities and only partially block the PGPH and the BrAAP activities ( 28 , 29 , 34 ). No information is available on the inhibition of the SNAAP activity by lactacystin. In agreement with these results, we also found that the proteasome inhibitor lactacystin did not completely abrogate certain proteasomal activities but resulted in an altered degradation of synthetic peptides. Although previous results indicated that I LLnL blocks all five activities, recent reports have shown only selective inhibition of the chymotryptic-like activity ( 10 , 13 , 34 ). Section title: Discussion Educational score: 4.435063362121582 Domain: biomedical Document type: Study Language: en In the presence of lactacystin, the degradation of MAGE-3 peptide precursors by purified proteasome was only partially and selectively inhibited, even if the tryptic and chymotryptic activities on short fluorogenic peptides were completely abrogated . Indeed, we could still detect fragments generated by cleavage after leucine, valine, glutamic acid, alanine, and threonine. Therefore, we envisage two possibilities: First, the remaining PGPH activity not only cleaves at the COOH terminus of glutamic acid but may be more permissive and also cleave after nonacidic amino acid residues, or, second, the catalytic subunits responsible for the BrAAP and SNAAP activities of the proteasome, in addition to those responsible for the PGPH activity, are not affected by lactacystin. In light of recent results obtained after the analysis of peptide fragments generated by purified yeast proteasome ( 34 ), we favor the first hypothesis and suggest that the functionally defined PGPH activity is mediated by the same catalytic subunit as are the SNAAP and/or BrAAP activities. It is noteworthy that, in some cases, the PGPH activity of mammalian proteasome has been shown to result in cleavage after aromatic amino acids ( 35 ). Section title: Discussion Educational score: 4.440247058868408 Domain: biomedical Document type: Study Language: en Close comparison of the fragments detected by mass spectrometry in the absence or presence of lactacystin suggests that, in the case of peptide MAGE-3 271–285 , the antigenic fragment MAGE-3 271–279 is only detected in the absence of the fragment MAGE-3 272–278 . The production of the latter appears to be inhibited by lactacystin. Assuming the presence of two competing proteolytic activities of the proteasome, one dominant under normal conditions and producing peptide MAGE-3 272–278 and the other resulting in peptide MAGE-3 271–279 , it is possible that the inhibition of the dominant activity by lactacystin favors the lactacystin-insensitive activity, yielding only the antigenic peptide MAGE-3 271–279 . Alternatively, the positioning of the peptide in the inner cavity of the proteasome may favor the cleavage, generating peptide MAGE-3 272–278 under normal conditions. The covalent binding of lactacystin to the subunit responsible for the chymotryptic activity ( 29 ) may reorient the peptide substrate in the cavity and lead to the generation of peptide MAGE-3 271–279 . Section title: Discussion Educational score: 4.640718460083008 Domain: biomedical Document type: Study Language: en It has been reported that antigenic peptides from the same protein may be processed with different efficiencies ( 36 , 37 ). In this context, it is noteworthy that the protein MAGE-3 contains, in addition to the poorly presented peptide studied here, other antigenic peptides which are well presented by MAGE-3 + melanoma cells in association with HLA-A1 or HLA-B44 molecules ( 15 , 16 ). In a survey of antigenic peptides carrying the motif Ala–Leu–Val present at the COOH terminus of peptide MAGE-3 271–279 , we identified two such peptides. The first one, derived from the influenza nucleoprotein NP 147–155 (H-2 K d –restricted), has been shown to be inefficiently produced in H-2 K d –positive infected cells (∼30 peptides/cell) in comparison to another antigenic peptide NP 50–57 (1,800 peptides/cell; 36). Interestingly, recent results have indicated that the presentation of peptide NP 147–155 in infected cells was enhanced by the addition of lactacystin ( 38 ). Although it has been shown that amino acids at position 156 could positively influence the generation of the antigenic peptide NP 147–155 ( 39 ), we did not observe any effect of the amino acid at the COOH terminus of the cleavage site of peptide MAGE-3 271–279 . Indeed, transplantation of the MAGE-3 271–279 sequence into two unrelated proteins (carrying either the residue histidine or isoleucine immediately after the COOH-terminal valine) did not result in the presentation of peptide MAGE-3 271–279 (data not shown). The second peptide is derived from the melanosomal TRP-2 protein . Preliminary results suggest that this peptide is not presented by cells expressing TRP-2 but is efficiently presented when expressed as a minigene (Noppen, C., G. Spagnoli, and F. Lévy, unpublished results). All of these observations can be explained by the presence of unfavorable amino acids surrounding the cleavage site and acting as “negative processing signals.” Elucidation of the exact nature of the postulated negative processing signal and determination of other putative signals is underway and should further refine the current prediction algorithms used to select potential antigenic peptides. More importantly, understanding the proteolytic machinery controlling the generation and presentation of tumor-specific antigenic peptides in tumor cells should facilitate the development of efficient peptide-based vaccines aimed at inducing or enhancing the generation of tumor-reactive CTL in cancer patients. In parallel, demonstration that proteasome inhibitors like lactacystin or LLnL lead to the recognition of a peptide tumor antigen by specific CTL in melanoma cells opens the way to the development of specific proteasome inhibitors able to induce the presentation of antigens recognized by CTL and, hence, broaden the spectrum of CTL-defined tumor antigenic peptides to be considered for cancer vaccines. In this respect, the recent demonstration that the drug ritonavir, originally described as an inhibitor of HIV-1 protease and administered to HIV-1–infected patients, can act as proteasome inhibitor suggests that this drug may also favor the generation of new peptide tumor antigens efficiently recognized by specific CTL ( 40 ). | Study | biomedical | en | 0.999997 |
10075974 | Section title: Special Reagents. Educational score: 2.232832193374634 Domain: biomedical Document type: Study Language: en TAQ polymerase was from Boehringer-Mannheim , radiolabeled γ-[ 32 P]ATP was from New England Nuclear , PMA, dimethylacetamide, DMSO, and polymyxin B sulfate were from Sigma Chemical Co. , human recombinant TNF-α was provided by Genentech , and neutralizing polyclonal rabbit anti–human TNF-α was from Genzyme Corp. LPS from Escherichia coli 055:B5 was obtained from List Biological Labs., Inc., and LTA from S. aureus was obtained from Sigma Chemical Co. According to the suppliers, both the LPS and LTA were prepared by hot, aqueous phenol extraction using modifications of the methods of Westphal and Jann ( 6 ) and Fischer et al. ( 7 ), respectively. The LPS preparation was reported by the manufacturer to contain <1.7% protein. Section title: Cells. Educational score: 4.165619373321533 Domain: biomedical Document type: Study Language: en A stable transfectant of THP-1 cells (THP-1 LTR luc ) containing the luciferase reporter gene under the control of the HIV-1 LTR was prepared as previously described ( 3 ). IG5, a Jurkat T cell–derived cell line containing a stably integrated HIV-1 luciferase construct (Jurkat LTR luc ) ( 8 ) was obtained through the Research and Reference Reagent Program, Division of AIDS, NIAID, National Institutes of Health, Bethesda, MD. The HL-60 and U937 cell lines were obtained from the American Type Culture Collection. The cells were maintained in RPMI-1640 containing 10 mM Hepes buffer, 2 mM l -glutamine, 50 U/ml penicillin, 50 mg/ml streptomycin sulfate, and 10% heat-inactivated FBS (RF-10) (BioWhittaker). Human monocytes were isolated from peripheral blood as previously described ( 9 ). Cell viability was >98% as determined by trypan blue exclusion. Monocytes (5 × 10 5 ) were added to each well of 24-well plates and allowed to adhere for 2 h. Nonadherent cells were removed by washing with PBS and the adherent cells overlaid with RF-10 plus 10% heat-inactivated human AB serum (BioWhittaker). The monolayer was incubated overnight at 37°C in a CO 2 incubator and just before use was washed twice with RPMI without serum. Section title: Activation of the HIV-1 LTR. Educational score: 4.093814849853516 Domain: biomedical Document type: Study Language: en The components of the reaction mixture were incubated at 37°C in a CO 2 incubator (5% CO 2 /95% air) for 6 h, and the luciferase activity was determined as previously described ( 3 ) and designated as relative light units (RLU). For studies with large numbers of samples, for example column fractions with serial dilutions of each fraction, the reaction was conducted in 96-well filter plates (MultiScreen GV; Millipore ) with the stimulus added to 2.5 × 10 5 THP-1 LTR luc in a final volume of 250 μl RPMI. The cells were harvested, rinsed, and lysed in the wells before determination of luciferase activity as described above. Section title: Isolation of Crude Phenol-soluble Modulin. Educational score: 4.268198013305664 Domain: biomedical Document type: Study Language: en S . epidermidis UW-3 (University of Washington hospital strain 3; reference 3 ) was grown overnight at 37°C with shaking in 10 liters of IMDM with Hepes and l -glutamine (BioWhittaker) that had been brought to 1% glucose and filter-sterilized (0.22 μm Zap Cap; Schleicher and Schuell). All subsequent steps were conducted at 4°C except as noted. The bacteria were removed by centrifugation and filtration, and the supernatant was concentrated by tangential (Prep/Scale CDUF 02.5LB; Millipore ) and centrifugal (Centriprep 10; Amicon) ultrafiltration to ∼20 ml. The preparation was dialyzed extensively against 0.4 M NaCl in 25 kD mol wt cutoff (MWCO) tubing (Spectra/Por CE; Spectrum) and then against distilled water in 12 kD MWCO tubing ( Sigma Chemical Co. ). 50 ml of buffer-saturated phenol ( GIBCO BRL ) and enough 1 M sodium acetate (pH 4.7) was added to the retentate to bring the aqueous portion to 0.1 M. The mixture was maintained as a single phase by raising the temperature to 65°C and agitated for 1 h. After cooling and centrifugation for 15 min, the phenol layer was removed and the aqueous layer was extracted twice more with 25 ml of phenol. The pooled phenol layers were extensively dialyzed against distilled water at 4°C in 12 kD MWCO tubing. A fine precipitate formed which was vigorously mixed into the aqueous retentate. This material, designated phenol-soluble modulin (PSM), was lyophilized and stored at −20°C. Section title: Chromatography. Educational score: 4.108384609222412 Domain: biomedical Document type: Study Language: en Gel chromatography was performed to determine size using a fast protein liquid chromatography apparatus and an HR 10/30 Superose 12 column ( Pharmacia ). After equilibrating the column in PBS, 100 μl of concentrated bacterial supernatant or 100 μg of phenol-extracted material was injected and subsequently eluted with PBS at 0.4 ml/min. 2-ml fractions were collected and assayed. Molecular weight was estimated by comparing the elution time of standard proteins to the elution time of active fractions, as per manufacturer's instructions. Section title: Chromatography. Educational score: 4.142446517944336 Domain: biomedical Document type: Study Language: en HPLC was performed on an LKB (now Pharmacia ) 2150 instrument equipped with a Vydak 0.46 × 15 cm C4 column and a flow cell detector measuring absorbance at 214 nm. For the rapid (10 min) HPLC procedure, a 10 min gradient was run from 36 to 100% 1-propanol, and the aqueous portion was buffered with 2.5 mM ammonium acetate, pH 6.7. 100 μg of dialyzed, lyophilized PSM was injected in 20 μl 20% 1-propanol, and fractions were collected, lyophilized, and frozen. Slow (57 min) HPLC was done on pooled peak 1 material from several rapid HPLCs with a gradient from 28 to 48% 1-propanol, buffered as above. Slow (57 min) HPLC of crude material was done similarly except that the gradient was from 25 to 45% 1-propanol. For assay, the lyophilized material was dissolved in PBS with vigorous vortexing, and dilutions of these fractions were compared with a standard curve made from material that was similarly processed (in terms of lyophilization and solvent exposure) but not subjected to the HPLC separation. Section title: Chemical Analysis of PSMα, PSMβ, and PSMγ. Educational score: 4.266740322113037 Domain: biomedical Document type: Study Language: en Endotoxin was measured by a Limulus Amebocyte Lysate kit (BioWhittaker). Total phosphorous content after ashing of the specimen was determined by the ascorbic acid/ammonium molybdate method ( 10 ). Amino acid composition was done by AAA Laboratory on a Beckman 7300 Analyzer after 20 h of hydrolysis in 6 N HCl, 0.05% mercaptoethanol, and 0.02% phenol at 115°C. Mass spectrometry and NH 2 -terminal sequencing was performed by the University of Washington Biochemistry Core Facility. Mass spectral ionization was via MALDI-TOF (matrix-assisted laser desorption ionization–time of flight) on a Voyager Elite (PerSeptive Biosystems) instrument. Sequencing was done by Edman degradation on an Applied Biosystems 470A sequencer with on-line 120A phenol-thio-hydantoin analyzer. COOH-terminal sequencing was performed by partial digestion with carboxypeptidase Y and subsequent mass spectrometry, as previously described ( 11 ). Peak 1 from the rapid HPLC (see above) was rechromatographed using acetonitrile/0.05% TFA in a second, rapid HPLC, and the single peak was submitted to the Harvard Microchemistry Facility (Cambridge, MA) for tryptic digestion, separation of the fragments by HPLC, and sequencing of the fragments by Edman degradation. Fragment sequences were confirmed by mass spectrometry using a Finnigan TSQ Triple Quadrupole Mass Spectrometer. Sequence alignment was performed using the Basic Local Alignment Search Tool (BLAST). Section title: Chemical Analysis of PSMα, PSMβ, and PSMγ. Educational score: 4.057742118835449 Domain: biomedical Document type: Study Language: en SDS-PAGE was performed using a 12.5% gel and stained for protein with silver stain and Coomassie blue and for polysaccharide with Alcian blue–silver ( 12 ). High percentage tricine gels (16%) used the method of Schagger and von Jagow ( 13 ) and had a stacking gel of 12% and a resolving gel of 16%. Gels were fixed with 20% TCA. Section title: Chemical Analysis of PSMα, PSMβ, and PSMγ. Educational score: 4.117609024047852 Domain: biomedical Document type: Study Language: en Proteinase digestion of PSM was performed using proteinases immobilized on beaded agarose (proteinase K, Streptomyces griseus protease, trypsin [ Sigma Chemical Co. ]). Beads were washed three times with PBS and resuspended in 1 ml PBS containing 20 μg PSM. After incubation for 3 h at 37°C with agitation, the proteinases were removed by low speed (325 g ) centrifugation and the supernatant was tested for its ability to activate the HIV-1 LTR in THP-1 cells. Section title: Cytokine Production. Educational score: 4.0686445236206055 Domain: biomedical Document type: Study Language: en HL-60, U937, THP-1 (THP-1 LTR luc ), and human monocytes were incubated with LPS, LTA, and the PSM preparations, as described in the figure legends, for 6 h, and the supernatant fluid collected and frozen at −20°C. At intervals the samples were thawed and TNF-α, IL-1β, and IL-6 levels were determined using commercially available ELISA kits (TNF-α, Endogen ; IL-1β, Genzyme ; IL-6, Immunotech). Section title: Electrophoretic Mobility Shift Assay. Educational score: 4.153012752532959 Domain: biomedical Document type: Study Language: en The nuclear extraction protocol is essentially that of Dignam et al. ( 14 ) as modified by Brophy et al. ( 15 ). For the preparation of nuclear extracts, 10 7 THP-1 LTR luc were incubated with 100 ng/ml PSM, PSM peak 1, or PSM peak 2 in RPMI at a final volume of 2.0 ml for 1 h at 37°C in 5% CO 2 /95% air. The protein concentration of the extracts was determined using the Bradford assay (Bio-Rad). The extracts were stored in aliquots at −70°C until use. The double stranded κB consensus oligonucleotide (5′-AGT TGA GGG GAC TTT CCC AGG C-3′), obtained from Promega , was end-labeled with γ-[ 32 P]ATP and T 4 polynucleotide kinase using the Promega Gel Shift Assay kit. For supershifts, 1 μl of NF-κB p65 rabbit antiserum (provided by Dr. Karol Bomsztyk, University of Washington, Seattle, WA), 4 μl (4 μg) of NF-κB p50 rabbit polyclonal IgG antibody ( Santa Cruz Biotechnology ), or 2 μl (2 μg) of NF-κB c-Rel rabbit polyclonal IgG antibody ( Santa Cruz Biotechnology ) were preincubated with the nuclear extracts in binding buffer for 30 min before addition of the labeled probe. For studies with cold probe, 50× cold probe was added at the time of addition of the labeled probe. Electrophoresis of the DNA-protein complexes was performed with 6% nondenaturing polyacrylamide gels as described in the Promega Gel Shift Assay System protocol. Section title: Genetic Analysis. Educational score: 4.218781471252441 Domain: biomedical Document type: Study Language: en S . epidermidis UW-3 DNA was obtained by incubating the bacteria with 50 μg/ml lysostaphin ( Sigma Chemical Co. ) for 2 h at 37°C and then using the standard isolation procedure for Gram-negative bacteria ( 16 ). Southern blots of genomic DNA digested with an array of restriction endonucleases were probed with radiolabeled, degenerate oligonucleotides based on Edman degradation data ( 17 ). Plasmids were isolated using a Quiagen kit, and sequencing was done by fluorescent dye termination on an ABI Prism Model 377 at the University of Washington Biochemistry Facility using ABI ( Perkin-Elmer ) dRhodamine or BigDye kits. The genetic sequence downstream of each PSM gene was obtained by anchor PCR ( 18 , 19 ). PCR was performed using established procedures ( 20 ) with an Eppendorf MicroCycler. All PCR was done with a 94°C presoak for 4 min and a final extension step at 55°C for 9.5 min, and, except where noted, used 35 cycles of 15 s at 94°C, 15 s at 55°C, and 90 s at 72°C. A “doubled” PCR is one in which 1 μl from a completed PCR reaction is used as template in an identical PCR reaction. Section title: Genetic Analysis. Educational score: 4.07090950012207 Domain: biomedical Document type: Study Language: en The anchor PCR for PSMα incorporated a single-strand extension and Exonuclease III digestion as previously described ( 21 ) except that the final PCR was doubled. Sequence obtained in this manner generated the primers for direct PCR of PSMα (upstream: 5′-CGA ATA ATA CTA TTA ATA TAT TTT AAA TGA GCA AGA GTG TCA ATG G) (downstream: 5′-CAT GGT TGT AAA ACA TAT AAA AAT GCT AAT GAG TGT GAC AAT AAT TGA TGA TAA ACT GG). Section title: Genetic Analysis. Educational score: 4.117140769958496 Domain: biomedical Document type: Study Language: en Anchor PCR for PSMβ revealed that the gene occurred as two copies with ∼50 bases in between. A size-selected (5 kB) library of EcoRV cut genomic DNA in pBS was screened via colony hybridization to an oligomer specific for this PSMβ intergenic region (5′-TCT TAG TTT TTT AAA ATA TAA ATT TAA ATA ATT AAT TAG GGA GAG ATA) as well as two degenerate oligomers based on the amino acid sequence. Positive colonies were picked, grown out, and sequenced. Section title: Genetic Analysis. Educational score: 4.144908905029297 Domain: biomedical Document type: Study Language: en Anchor PCR for PSMγ yielded ∼500 bases of downstream sequence. From this sequence was generated reverse (5′-TGC TTC TCA CTT GCT TAG TTT ATA TTA GTA AAT TAT TAA GTT GGG ATG GCT CAA CAA CTC) and forward (5′-TTT GCT AGT AAC TGT AGT TTC CTT GGA CTC AGT GTT ACG TAT TAT TCT TAG CTA CCT TAA) primers for inverse PCR ( 22 ) on a Sau 3AI digested template. This inverse PCR provided sequence for the creation of upstream (5′-GAT ATT TTA CCA TAT TTA GTT TTA CAG TTG AGT ACT AAA TAT TGC TAT) and downstream (5′-CCA CAT CTT TAT AAA TAG CAT AGT TAA AGC CGT GAG C) primers for directly amplifying PSMγ. Section title: Genetic Analysis. Educational score: 3.9922380447387695 Domain: biomedical Document type: Study Language: en To compensate for errors introduced by TAQ, the sequences of several direct PCR products using primers for PSMα and for PSMγ were compared, and at least three identical assignments were found for each nucleotide in the sequence shown. The gene for PSMβ was isolated as two identical, 5-kB recombinant clones sequenced in their entirety. Section title: Statistical Analyses. Educational score: 2.834338665008545 Domain: biomedical Document type: Study Language: en The results are presented as the mean ± SEM. The Mann-Whitney U rank-sum test (unpaired, two-tailed) was used to analyze differences for significance unless otherwise indicated. P > 0.05 was considered not significant. Section title: S. epidermidis PSM. Educational score: 4.390402793884277 Domain: biomedical Document type: Study Language: en S . epidermidis strain UW-3 was grown overnight in glucose-enriched IMDM with Hepes and l -glutamine. This medium supported the rapid growth of the organism and, being a defined medium lacking large mol wt components (>3.5 kD), could be subsequently removed by dialysis. The culture medium, free of microorganisms, was concentrated ∼500-fold and dialyzed. About half the activity was lost if dialysis was carried out with 25 kD MWCO tubing against distilled water; these losses were prevented by dialysis against 0.4 M NaCl. A two-step dialysis program was therefore used, with 25 kD MWCO tubing in 0.4 M NaCl followed by 12 kD MWCO tubing in distilled water. Dialysis suggested a mol wt for the active material of >25,000 daltons and this was supported by gel chromatography using a Superose 12 column which indicated an effective mass for the concentrated and dialyzed material of 34,500 ± 5,900 daltons (mean ± SD, n = 3) Hot aqueous phenol extraction was performed as used in the purification of LPS ( 6 ) and LTA ( 7 ). In contrast to LPS and LTA, which partition into the aqueous layer, the S . epidermidis factor was found to be entirely in the phenol layer, thus the term phenol-soluble modulin (PSM). The mass of the active material, as measured by Superose 12 chromatography, remained ∼35 kD, allowing the phenol to be removed by dialysis. However, MALDI-TOF mass spectroscopy of the PSM did not reveal any component in the 35 kD region, but rather indicated the presence of four low mol wt components of 2,489, 2,648, 2,849, and 4,668 daltons (M + I) . The recovery of activity in two large scale purifications, beginning with 10 liters of culture medium and proceeding through concentration, dialysis, and three phenol extractions was 190% (wt 74.8 mg) for preparation A and 87% (wt 230 mg) for preparation B. In five smaller scale purifications using 500 ml of culture medium and a single phenol extraction, recovery was 71 ± 29% (mean ± SD). The total amount of material was determined by weight after dialysis and lyophilization, and the recovery was determined by comparing the activity of a weighed sample of the product to that of the starting material. Section title: S. epidermidis PSM. Educational score: 4.181286811828613 Domain: biomedical Document type: Study Language: en The S . epidermidis PSM had a small but significant activating effect on the HIV-1 LTR in THP-1 cells at 1 ng/ml with the activation increasing to a maximum at 30 ng/ml and remaining high as the PSM concentration was increased to 1 μg/ml . PSM was routinely used at 100 ng/ml. In contrast to PSM, LPS did not activate the HIV-1 LTR in THP-1 cells at comparable concentrations; however, a small but significant activation was observed at 10 and 100 μg/ml. LTA from S. aureus was ineffective over the entire concentration range used. In contrast to the LTR in THP cells, the HIV-1 LTR introduced into Jurkat T cells in association with the luciferase reporter gene was unaffected by PSM as well as by LPS and LTA under comparable conditions. The background luciferase activity (29,379 ± 6,538, n = 4) remained unchanged on the addition of 0.001–1 μg/ml PSM or 0.01–100 μg/ml LPS or LTA. Section title: S. epidermidis PSM. Educational score: 4.141233921051025 Domain: biomedical Document type: Study Language: en The activation of the HIV-1 LTR in THP-1 cells by the S. epidermidis PSM was not due to the release of TNF-α, since blocking antibody to TNF-α (diluted 1:2,000), although it inhibited activation via exogenously added TNF-α (100 U/ml), did not affect activation by PSM (100 ng/ml) (background, 1,189 ± 509 [RLU ± SEM]; PSM, 363,987 ± 95,583; PSM + anti–TNF-α antibody, 429,532 ± 105,532, NS versus PSM alone; TNF-α, 15,048 ± 5,585; TNF-α + anti–TNF-α antibody, 2,278 ± 332, P < 0.05 versus TNF-α alone; n = 4). The low activity of TNF-α as compared with PSM further supported the absence of an association. Section title: S. epidermidis PSM. Educational score: 4.1423139572143555 Domain: biomedical Document type: Study Language: en The S . epidermidis PSM was heat resistant, requiring 15 min at 100°C for a partial loss of activity and 60–120 min for complete loss of activity . Exposure to three different proteinases (proteinase K, Streptomyces griseus protease, trypsin) immobilized on agarose beads resulted in a loss of activity , suggesting that the active material is proteinaceous. Section title: S. epidermidis PSM Peak 1 and Peak 2. Educational score: 4.202032089233398 Domain: biomedical Document type: Study Language: en Separation of PSM by HPLC with a C4 column, a 36–100% 1-propanol gradient, an elution rate of 1 ml/min, and an elution time of 10 min yielded two peaks of activity in the THP-1 LTR luc assay, which were designated PSM peak 1 and peak 2 . Peak 1 had a lower absorbance at 214 nm but considerably more activity than peak 2. Approximately 25% of the activity applied to the column was recovered. Neither active peak contained phosphorous as assessed by a modified Fiske-Subborow assay. SDS-PAGE of each peak revealed a band that ran at the dye front even with high percentage tricine gels that could resolve down to 6 kD. These low mol wt bands stained positively for protein with Coomassie blue and silver stains. Silver staining was not intensified by Alcian blue pretreatment, suggesting the absence of polysaccharide. Section title: S. epidermidis PSM Peak 1 and Peak 2. Educational score: 4.117667198181152 Domain: biomedical Document type: Study Language: en The effect of PSM, PSM peak 1, and PSM peak 2 on THP-1 cells was not limited to the activation of the HIV-1 LTR. Fig. 5 demonstrates the stimulatory effect of 100 ng/ml PSM, PSM peak 1, and, to a lesser degree, PSM peak 2 on TNF-α and IL-1β production by THP-1 cells. Little IL-6 was produced by untreated THP-1 cells, with PSM producing a small but significant increase by paired analysis. As with activation of the HIV-1 LTR, LPS or LTA had no effect on the production of these cytokines by THP-1 cells. In contrast to its effect on THP-1 cells, PSM as well as LPS and LTA did not induce TNF-α production by HL-60 (cells alone, 32 ± 23; LPS, 17 ± 10; LTA, 17 ± 10; PSM, 15 ± 15 pg/ml; n = 4, NS) or U937 cells (cells alone, 14 ± 8; LPS, 11 ± 7; LTA, 11 ± 6; PSM, 9 ± 9 pg/ml; n = 4, NS). Section title: S. epidermidis PSM Peak 1 and Peak 2. Educational score: 4.15615701675415 Domain: biomedical Document type: Study Language: en TNF-α, IL-1β, and IL-6 production by human monocytes also was stimulated by PSM, PSM peak 1, and, to a lesser degree, PSM peak 2 . In contrast to THP-1 cells, LPS and LTA also induced cytokine production by monocytes, although LTA did not affect monocytic production of IL-1β. PSM was less effective than LPS, but more effective than LTA as a stimulant of TNF-α production by monocytes . A significant effect of LPS was observed at concentrations down to 0.1 ng/ml with the effect at 0.01 ng/ml just not significant by the Mann-Whitney U test ( P = 0.07), and just significant by the Student's t test ( P = 0.04). PSM was effective at concentrations down to 10 ng/ml, whereas LTA was stimulatory only at 100 ng/ml. Section title: S. epidermidis PSM Peak 1 and Peak 2. Educational score: 4.2671637535095215 Domain: biomedical Document type: Study Language: en The passage of PSM into the phenol layer upon hot phenol extraction and the low activity of LPS or LTA suggests that the activation of the HIV-LTR in THP-1 cells by PSM is not due to the presence of LPS or LTA. Further evidence against LPS or LTA involvement was the absence of inhibition by polymyxin B. Polymyxin B alone at 1 μg/ml had no effect on the LTR in THP-1 cells (background, 1,088 ± 154 [RLU ± SEM]; polymyxin B, 1,388 ± 320; n = 4, NS), nor did it decrease the activation by 100 ng/ml PSM (PSM, 243,012 ± 29,691; PSM + polymyxin B, 259,048 ± 27,766; n = 4, NS). Polymyxin B at 1 μg/ml also had no effect on TNF-α production by monocytes induced by 100 ng/ml PSM, whereas strongly inhibiting production by 100 ng/ml LPS or LTA (PSM without polymyxin B, 2,781 ± 997 pg/ml; with polymyxin B, 2,070 ± 779; n = 5-6, NS; LPS without polymyxin B, 3,160 ± 1,074; with polymyxin B, 358 ± 140; n = 5-6, P < 0.005; LTA without polymyxin B, 1,088 ± 421; with polymyxin B, 88 ± 71; n = 5-6, P < 0.05). The endotoxin level of a reaction mixture containing 100 ng/ml of PSM in RPMI was below the level of detection (<1 pg/ml) by the Limulus Amebocyte Lysate assay. Section title: S. epidermidis PSM Peak 1 and Peak 2. Educational score: 4.174727439880371 Domain: biomedical Document type: Study Language: en Both the HIV-1 LTR and the promoter region of the cytokine genes contain binding sites for the transcription factor NF-κB. The activation of NF-κB in THP-1 cells by S . epidermidis PSM, PSM peak 1, and PSM peak 2, as determined by the electrophoretic mobility shift assay, is shown in Fig. 7 . Nuclear extracts obtained from THP-1 cells pretreated with PSM, PSM peak 1, and PSM peak 2 produced a retardation in the migration of the 32 P-labeled kB consensus oligonucleotide, which was not seen when extracts from untreated THP-1 cells were used. Antibodies to the NF-κB components p65 and p50 caused a further retardation in migration (supershift) whereas antibody to c-Rel had no effect. The specificity of the mobility shift assay for NF-κB was further supported by competition studies in which unlabeled NF-κB oligonucleotide prevented the appearance of the retarded band. Section title: S. epidermidis PSMα, PSMβ, and PSMγ. Educational score: 4.367476463317871 Domain: biomedical Document type: Study Language: en MALDI-TOF mass spectroscopy of PSM peak 1 revealed two components, a minor component with a mass of 2,503.8 daltons (designated PSMα) and a major component of 4,683.8 daltons (designated PSMβ), whereas mass spectroscopy of PSM peak 2 indicated a single component with a mass of 2,864.8 daltons (designated PSMγ). The two components in PSM peak 1 could be separated by a second, slow HPLC procedure using the same column and solvents but a longer, shallower gradient . The composition of these peaks was verified by mass spectrometry. It was observed that the second slow HPLC of peak 1 resulted in a considerable loss of activity; <5% of the activity applied would typically be recovered. In every instance, PSMα was more active than PSMβ and in some instances PSMβ was inactive. A single HPLC separation of the S . epidermidis PSM using a slow HPLC yielded three major peaks of 214 nm-absorbing material . The first peak, PSMα by mass spectrometry, contained most of the activity. The two later peaks were not completely separated from each other. Mass spectroscopy indicated that the peak eluting at 29–31 min contained PSMβ as its major component and that the peak eluting at 31–33 min was largely PSMγ. The luciferase assay indicated a single peak of activity that overlapped both of these later peaks. Section title: Amino Acid Sequence Analyses. Educational score: 4.278027057647705 Domain: biomedical Document type: Study Language: en PSMα derived from the single, slow (57 min) HPLC separation using the 28–48% 1-propanol gradient was subjected to Edman degradation, which revealed the NH 2 -terminal 11 amino acids. Edman degradation of peak 1 provided the NH 2 -terminal 24 amino acids of PSMβ. PSMα was present in peak 1 in relatively small amounts that did not interfere with the sequencing of PSMβ. The complete sequence of PSMα and PSMβ was determined by NH 2 -terminal Edman degradation of tryptic digests. No fragments were found that did not correspond to PSMα or PSMβ. Verification of the COOH-terminal 8 amino acids of PSMβ was obtained by carboxypeptidase Y digestion and sequential mass spectroscopy. PSM peak 2 was used for the derivation of the NH 2 -terminal 16 amino acids of PSMγ. Section title: Genetic Characterization of PSMα, PSMβ, and PSMγ. Educational score: 4.329677581787109 Domain: biomedical Document type: Study Language: en The amino acid sequence enabled the creation of degenerate oligonucleotides and the isolation of the genes for the three PSMs. Anchor PCR allowed for the determination of sequence downstream from each gene. The isolation of the entire gene resulted from additional anchor PCR (for PSMα), colony hybridization (for PSMβ), and inverse PCR (for PSMγ). Although PSMβ could be readily cloned in E . coli , PSMα and PSMγ could not, so the gene sequence for these two was determined via several directly sequenced PCRs. All coding regions were preceded by a putative Shine-Delgarno sequence and followed by a stop codon. The deduced amino acid sequence was in agreement with that found by Edman degradation and was consistent with amino acid analysis. Section title: Genetic Characterization of PSMα, PSMβ, and PSMγ. Educational score: 4.350045204162598 Domain: biomedical Document type: Study Language: en PSMα is a 22-amino acid polypeptide without a high degree of homology to any known protein, although some similarity to delta toxin is evident . PSMβ is a 44-amino acid polypeptide whose gene was found to exist in two copies with an intervening 58 nucleotides. The deduced amino acid sequence for the two copies was identical, although slight codon variation was noted. Homology to S . hemolyticus antigonococcal polypeptides 1, 2, and 3, and S . lugdunensis SLUSH polypeptides A, B, and C is shown. PSMγ is a 25-amino acid polypeptide with the sequence MAADIISTIGDLVKWIIDTVNKFKK, which is identical to that of S . epidermidis delta toxin ( 23 ). The gene sequence of PSMγ with 100 flanking bases was identical to that reported by Otto et al. ( 24 ) except for two base differences in the flanking regions . Section title: Genetic Characterization of PSMα, PSMβ, and PSMγ. Educational score: 4.262019157409668 Domain: biomedical Document type: Study Language: en The mass of PSMα, PSMβ, and PSMγ calculated from the amino acid sequence was 2,460.0, 4,639.3, and 2,820.4, respectively. Mass spectrometry of PSM before HPLC indicated the presence of components of mass 2,487.9, 4,667.2, and 2,848.5, which exceeded the calculated mass of PSMα, PSMβ, and PSMγ by ∼28 daltons in each instance, a difference compatible with formylation of the polypeptides. After HPLC, the mass of each PSM was further increased by 16 daltons, consistent with the oxidation of methionine. A fourth component of 2648.0 daltons found at low levels in the postphenol extraction PSM was not detected on the subsequent HPLC separations. Section title: Genetic Characterization of PSMα, PSMβ, and PSMγ. Educational score: 2.3785433769226074 Domain: biomedical Document type: Other Language: en The gene sequence data, including 100 bases of flanking sequence, for PSMα, PSMβ, and PSMγ, are available from EMBL/GenBank/DDBJ under accession numbers AF068632 , AF068633 , and AF068634 , respectively. Section title: Discussion Educational score: 4.3161115646362305 Domain: biomedical Document type: Study Language: en We describe here modulins from S . epidermidis that partition into the phenol layer on hot aqueous phenol extraction. A number of lines of evidence indicate that the active components were neither LPS nor LTA. Both LPS and LTA partition into the aqueous layer on hot phenol extraction, whereas PSM partitioned into the phenol layer. LTA from S . aureus did not activate the HIV-1 LTR in THP-1 cells at concentrations up to 100 μg/ml and List E . coli 055:B5 LPS was only weakly active requiring concentrations of 10–100 μg/ml. In contrast, PSM was active at concentrations down to 1 ng/ml. Furthermore, polymyxin B, which binds to and inactivates LPS and LTA, had no effect on the activation of the HIV-1 LTR in THP-1 cells nor did it inhibit TNF-α production in monocytes induced by PSM. In contrast, polymyxin B strongly inhibited LPS- or LTA-induced production of TNF-α by monocytes. Active material also was devoid of sugars and phosphorous, and proteinase inactivation suggested that it was a protein or polypeptide. Finally, LPS could not be detected in the PSM preparation. Section title: Discussion Educational score: 4.726912975311279 Domain: biomedical Document type: Study Language: en The S . epidermidis PSM could be separated into three active polypeptides, designated PSMα, PSMβ, and PSMγ. PSMα is a 22-amino acid polypeptide whose amino acid sequence shows 40% identity to S . epidermidis delta toxin when a single residue gap is placed in each polypeptide . PSMβ is a 44-amino acid polypeptide with considerable structural homology to previously described gonococcal inhibitor 1, 2, and 3 from S . hemolyticus ( 25 ) and to a lesser degree to three structurally related peptides from S . lugdunensis with synergistic hemolytic activity (SLUSH A, B, and C) ( 26 ) . Closest homology was to S . hemolyticus gonococcal inhibitor 2 (73% identity). The polypeptides from the three staphylococcal strains had in common similar molecular weights ( S . epidermidis and S . hemolyticus , 44 amino acids; S . lugdunensis , 43 amino acids), strong hydrophobicity, and the absence of arginine, cysteine, histidine, proline, and tyrosine. It has been proposed on the basis of its primary structure that the S . hemolyticus antigonococcal proteins are signal sequences for secreted or membrane-associated proteins ( 25 ). Antibody to the S . hemolyticus polypeptides recognized a 51-kD protein in the cytoplasmic fraction of S . hemolyticus , and it was proposed that this protein may contain a signal peptide with homology to the S . hemolyticus antigonococcal proteins ( 27 ). As all three S . epidermidis PSMs have a stop codon immediately after their COOH terminus, such a role is unlikely for these molecules. Two copies of the gene for PSMβ were detected. PSMγ is identical to delta toxin of S . epidermidis ( 23 ). Delta toxin from S . epidermidis consists of 25 amino acids that share a high degree of homology with delta toxin from S . aureus . The latter consists of 26 amino acids with the substitution of glutamine for alanine at position 3 and the presence of threonine at position 24 which was absent in the S . epidermidis toxin ( 28 ). S . aureus delta toxin (delta hemolysin) is an amphipathic, helical peptide which can insert into hydrophobic membranes to form cation-selective ion channels with each channel consisting of six delta toxin alpha helices oriented with their hydrophilic face lining a central aqueous pore ( 29 – 33 ). Under some experimental conditions, lysis of the cell can occur. Helical wheel analysis of PSMα also suggests its orientation as an amphipathic alpha helix, with hydrophobic amino acids on one face and hydrophilic amino acids on the other. Section title: Discussion Educational score: 4.355654716491699 Domain: biomedical Document type: Study Language: en PSMα, PSMβ, and PSMγ are all strongly hydrophobic polypeptides that lack arginine, cysteine, histidine, proline, and tyrosine. Two (PSMα and PSMγ) have some structural homology to each other. Based upon their absorption at 214 nm and normalized for molecular weight, the three components occur in a molar ratio of ∼1:2:5 PSMα/ PSMβ/PSMγ. Their low molecular weight confirms the findings with SDS-PAGE and MALDI-TOF mass spectroscopy which indicated mol wt <5 kD. However, sephadex gel chromatography either of the crude extract or the S . epidermidis PSM suggested an active product with mol wt ∼35 kD, which is consistent with its retention during dialysis and ultrafiltration. Both S . aureus delta toxin ( 34 , 35 ) and S . hemolyticus gonococcal growth inhibitor ( 36 ) have been reported to form complexes or aggregates of various sizes and similar aggregation or complexing of S . epidermidis PSM appears to occur as well. It is not clear whether the polypeptides assemble into a highly ordered complex or whether they aggregate due to hydrophobic interactions with little or no order. We do not know the degree to which each polypeptide participates in the aggregate nor whether other components, proteinaceous or not, also are involved. Section title: Discussion Educational score: 4.220221519470215 Domain: biomedical Document type: Study Language: en Although our routine assay for the S . epidermidis PSMs is the activation of the HIV-1 LTR in THP-1 cells, their effects are not limited to this reaction or to this cell type. Cytokine production (TNF-α, IL-1β, IL-6) by either THP-1 cells or peripheral blood monocytes also is increased, as is the activation of NF-κB in THP-1 cells. Both the HIV-1 LTR and the cytokine genes contain NF-κB binding sites in their promoter region, suggesting that activation of NF-κB by the S . epidermidis PSMs may be central to the other effects observed. Cell-free supernatants of Gram-positive bacteria have been reported to stimulate TNF-α production from whole human blood and, of 63 strains tested, supernatants from coagulase-negative staphylococci were the most effective ( 37 ). The S . epidermidis PSMs did not activate the HIV-1 LTR in Jurkat T cells, indicating some selectivity for cells of macrophage lineage. Section title: Discussion Educational score: 4.328223705291748 Domain: biomedical Document type: Study Language: en We have concentrated here on the release from S . epidermidis of three small hydrophobic polypeptides which can induce gene activation through an effect on the transcription faction NF-κB. S . epidermidis can induce septic shock in humans ( 38 – 46 ) and a similar shock-like state in animals ( 47 ). Although most attention has focused on the role of lipoteichoic acid and peptidoglycan as the inducers of cytokine release and shock in this condition, evidence for the involvement of other factors has been presented. Thus, S . epidermidis has been reported to be a significant pathogen in neonatal necrotizing enterocolitis (NEC) ( 48 , 49 ) and to be a common pathogen in a milder form of enterocolitis in infants ( 50 ). In one study ( 48 ), >90% of the S . epidermidis isolates from the stools of patients with NEC produced a toxin resembling delta toxin, and delta toxin was detected by ELISA in the stools of 11 out of 35 patients with NEC and toxin-positive staphylococci. Delta toxin was shown to be enteropathic in infant rats, producing mucosal necrosis and hemorrhage ( 48 ). These findings suggest that coagulase-negative staphylococci may be enteropathic in humans through the formation of delta toxin. Delta toxin is produced by most clinical isolates of coagulase-negative staphylococci, including strains of S . epidermidis , S . saprophyticus , and S . hemolyticus ( 51 , 52 ), and a PSMβ-like molecule is produced by S . epidermidis , S . hemolyticus , and S . lugdunensis ( 25 , 26 ). Section title: Discussion Educational score: 4.245833396911621 Domain: biomedical Document type: Study Language: en Strains of S . aureus also release factors that activate the HIV-1 LTR in THP-1 cells ( 3 ) and induce the release of TNF-α and IL-1β by human monocytes ( 53 ). Essentially, all strains of S . aureus produce delta toxin; however, it is not yet known whether a PSMα- or PSMβ-equivalent molecule is produced by this organism. An S . aureus component that binds CD14 and induces IL-6 production by U373 cells and human PBMCs has been described ( 54 , 55 ). This factor differs from S . epidermidis PSM in that the active material distributes into the aqueous phase on hot phenol extraction. Delta toxin has been shown to induce the release of TNF-α from human monocytes ( 56 ). Section title: Discussion Educational score: 4.293715953826904 Domain: biomedical Document type: Study Language: en In summary, the pathogenesis of Gram-positive septic shock remains unclear. In 1996, Henderson et al. ( 2 ) described the issue as follows: “Gram-positive bacteria can produce a septic shock-like condition and as many people die each year from Gram-positive as from Gram-negative sepsis. LPS is obviously a very potent inducer of cytokine synthesis. Peptidoglycans and teichoic acids are believed to be the equivalent components, inducing cytokine-induced shock in patients with Gram-positive sepsis. However, given the relatively weak cytokine-inducing activity of peptidoglycan and teichoic acids in the studies described above, perhaps we have to look at other Gram-positive bacterial components as the inducers of the shock-like state.” The staphylococcal PSM is of interest in this regard in that it is more effective than lipoteichoic acid as an inducer of cytokine release by THP-1 cells or human monocytes. Further, it is normally shed or secreted by the bacteria as indicated by its release into the culture medium on overnight growth and into the extracellular fluid by vortexing of intact organisms. These properties make it a prime candidate for the initiation of a systemic effect contributing to septic shock in vivo. | Study | biomedical | en | 0.999997 |
10075975 | Section title: Mice. Educational score: 1.7254506349563599 Domain: biomedical Document type: Other Language: en Female C57BL/6 mice 6–8 wk of age were purchased from Harlan Sprague Dawley, Inc. Female C57BL/6 (nu/nu) mice were purchased from Taconic Farms, Inc. IL-4 and IL-10 gene–disrupted C57BL/6 mice were purchased from The Jackson Laboratory . The mice were maintained in accordance with the guidelines of the Committee on Animals of Harvard Medical School and the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council, as stated in the Department of Health and Human Services publication 85-23 . Section title: Cell Lines. Educational score: 4.159435272216797 Domain: biomedical Document type: Study Language: en The tumor cell lines used in this study were obtained from the American Type Culture Collection (ATCC). The carcinogen-induced lymphoma EL4 is of C57BL/6 (H-2 b ) origin. The EL4–B7-1 cells were provided by Dr. James Allison (University of California, Berkeley, CA). EL4–B7-2 cells were generated by introducing murine B7-2 cDNA into a pCDM8 vector by electroporation into EL4-wt cells (ATCC) as described ( 22 ). The transfectants were selected in the presence of 1 mg/ml G418 (Life Technologies). In another series, EL4–B7-1 cells (obtained from Dr. James Allison) were transfected with murine B7-2 cDNA to produce EL4–B7-1+B7-2 as described above. These transfectants were selected in 800 μg/ml hygromycin B ( Boehringer Mannheim ). The mock transfectant EL4 line (EL4-neo/ hygro) was derived similarly but transfected only with the genes for the selection antibiotics (G418 and hygromycin B). After 3–4 wk, cells growing in the presence of the drugs were sorted for B7-2– expressing cells with anti–mB7-2 mAb (GL1) using a Becton Dickinson FACS Vantage TM cell sorter. The B7-2–expressing cells were then subcloned and clones showing stable B7-2 expression were used in the experiments. The clones were screened for B7 expression once per week. All cell lines were maintained at 37°C in 10% CO 2 in DMEM (Life Technologies) containing 10% FCS (Fetalclone I; Hyclone), 1 mg/ml G418 (Life Technologies), and/or 800 μg/ml hygromycin B ( Boehringer Mannheim ). Section title: Antibodies. Educational score: 2.627446174621582 Domain: biomedical Document type: Other Language: en Anti–B7-1 antibody (1G10) was provided by Dr. Nasrin Nabavi (Hoffmann-LaRoche Research Center, Nutley, NJ; 28). Anti–B7-2 antibody (GL1) was obtained from the GL1 hybridoma line (ATCC). The antibodies were purified from ascitic fluid on protein G columns (LKB/ Pharmacia ). The anti–B7-1 and anti–B7-2 antibodies are both of rat IgG2a isotype. The anti-murine CD3 hybridoma line 145-2C11 was provided by Dr. Jeffrey Bluestone (Ben May Institute, University of Chicago, Chicago, IL). Human CTLA4–Ig was provided by Dr. Peter Linsley (Bristol Myers Squibb, Seattle, WA). Section title: Flow Cytometry. Educational score: 4.158348083496094 Domain: biomedical Document type: Study Language: en Spleen cells and tumor cells from mice or those growing in culture were harvested and washed three times with cold 1% BSA/PBS, pH 7.2, and then incubated with either supernatant from antibody-producing hybridomas or purified antibody (5 μg/ml) diluted in 1% BSA/PBS for 30 min at 4°C. The cells were then washed two to three times with the 1% BSA/PBS solution before incubating with FITC- or PE-conjugated goat anti–mouse Ig, goat anti–rat Ig, or goat anti–human Ig secondary antibodies ( Zymed) . The secondary antibodies were diluted 1/50 in 1% BSA/PBS and then incubated with the cells for 30 min at 4°C in the dark. After this incubation, the cells were washed three times with PBS and then fixed with an equal volume of 1% paraformaldehyde/PBS solution. Analysis was performed using a FACScan TM ( Becton Dickinson ). FITC- and PE-conjugated antibodies used for direct staining were obtained from PharMingen and included the following: anti–B7-1–FITC (clone 16.10A1), anti–B7-2–PE (clone GL1), and anti-CD95L–PE (clone KAY-10). For binding studies with hCTLA4–Ig, PE-conjugated goat F(ab′) 2 anti–human IgG (Southern Biotechnology, Inc.) was used for indirect staining. Section title: Costimulation Assay. Educational score: 4.152498722076416 Domain: biomedical Document type: Study Language: en T lymphocytes freshly isolated from C57BL/6 mouse spleen cells were positively selected using MicroBeads bound with anti-Thy1.2 (CD90) on an appropriate column (MACS; Miltenyi Biotec). This purification method yielded >95% enriched T cells. The positively selected T lymphocytes were used in costimulation assays as responder cells at a concentration of 10 5 cells/50 μl/well of 96-well tissue culture plates (Costar Corp.). Untransfected and transfected EL4 cells were treated overnight with 40 μg/ml mitomycin C ( Sigma Chemical Co. ) and then washed thoroughly and added as stimulator cells at 10 5 cells/25 μl/well. Supernatant from anti-CD3–secreting hybridoma 145-2C11 cultures was added at 1/100 final dilution/ 100 μl/well. In some experiments, anti–B7-1 antibody, anti–B7-2 antibody, or hCTLA4–Ig was added at a final concentration of 2 μg/ml per 100 μl/well for blocking in vitro costimulation. The plates were then incubated at 37°C in a humidified CO 2 incubator for 48 h. The plates were then pulsed with 1 μCi of 3 H-TdR per well for 16 h and harvested using a Tomec Mach II 96 cell harvester and counted on a 1205 Betaplate liquid scintillation counter (Wallac, Inc.). Section title: Generation of T Cell Lines. Educational score: 4.141589164733887 Domain: biomedical Document type: Study Language: en To generate long-term T cell lines specific for each of the EL4 tumor cells, spleen cells (0.5–1 × 10 6 /well) from mice implanted with each of the tumors were removed on days 10 to 12 and stimulated with the corresponding mitomycin C–fixed (40 μg/ml overnight) EL4 cells (EL4–B7-1, EL4–B7-2, or EL4–B7-1+B7-2 cells; 2 × 10 5 cells/well) together with gamma-irradiated (5,000 rads) syngeneic spleen cells. The T cells were restimulated every 12–14 d with the corresponding mitomycin C–treated tumor cells that had been used to stimulate them in vivo, resulting in the generation of long-term T cell lines. The T cell lines were grown in DMEM supplemented with sodium pyruvate, l -glutamine, penicillin, streptomycin, gentamycin sulfate, nonessential amino acids (0.1 mM), MEM vitamin mixture (1×; BioWhittaker), asparagine (0.1 mM), folic acid (0.1 mg/ml), 2-ME (5 × 10 −5 M; Sigma Chemical Co. ), 10% FBS (Hyclone), and 2% T cell growth factor (T-STIM; Collaborative Biomedical Products). The T cell lines thus generated were tested for cytotoxicity, cytokine production, and in vivo functional effects on tumor growth. Section title: 51 Cr Release Assay to Test Cytolytic Activity. Educational score: 4.120814323425293 Domain: biomedical Document type: Study Language: en Effector cells were harvested, washed, and adjusted to 10 6 cells/ml, and varying numbers of effector cells (Ficoll-purified if necessary) were added to 5 × 10 3 51 Cr-labeled target cells in 150 μl of culture medium in 96-well v-bottomed plates. After a 4-h incubation, 50 μl culture supernatant was collected and measured in a gamma counter. The mean percentage specific lysis of triplicate wells was calculated as follows: % specific lysis = ([cpm experimental release − cpm spontaneous release]/[cpm maximum release − cpm spontaneous release]) × 100. The spontaneous release of the 51 Cr-labeled target cells was <20% in all experiments. Section title: In Vitro Cytokine Assay. Educational score: 4.153462886810303 Domain: biomedical Document type: Study Language: en Supernatants were collected from T cells 40 h after activation in vitro with the corresponding EL4 cell line in the presence of syngenic spleen cells as APCs (see described generation of T cell lines). The concentrations of IL-2, IL-4, IL-10, IFN-γ, and TNF-α were measured by quantitative capture ELISA according to the guidelines of the manufacturers ( PharMingen ). In brief, purified rat mAb to mouse IL-2 (clone JES6-1A12), IL-4 (clone BVD4-1D11), IL-10 (clone JES5-2A5), IFN-γ (clone R4-6A2), and TNF-α (clone MP6-XT22) were obtained from PharMingen and used to coat ELISA plates (Immulon 4; Dynatech Laboratories, Inc.). Recombinant mouse cytokines (IL-2, IL-4, IL-10, IFN-γ, and TNF-α; PharMingen ) were used to construct standard curves, and biotinylated rat mAb to mouse IL-2 (clone JES6-5H4), IL-4 (clone BVD4-24G2), IL-10 (clone SXC-1), and IFN-γ (clone XMG1.2; all PharMingen ) were used as the second Ab. Detection of TNF-α was performed with biotinylated polyclonal rabbit IgG ( PharMingen ). Plates were developed with TMB microwell peroxidase substrate (Kirkegaard & Perry Laboratories, Inc.) and read after the addition of stop solution at 450 nm using a microplate reader . Section title: Animal Studies. Educational score: 4.168843746185303 Domain: biomedical Document type: Study Language: en C57BL/6 syngeneic mice were prepared for intradermal EL4 cell implantation by shaving their hind flank regions followed by depiliation with Nair (Carter Wallace, Inc.) 24 h before intradermal implantation of tumor cells. EL4-wt and transfected EL4 tumor cells were harvested in log phase growth from tissue culture flasks and washed four times with PBS (Bio-Whittaker) and resuspended at 4 × 10 7 cells/ml in PBS for implantation. Each intradermal injection consisted of 2 × 10 6 cells in 50 μl PBS and was performed using a 1-ml syringe fitted with a 27-gauge needle. 5–7 d after implantation, a tumor could be observed at the implantation site. The mice were scored for tumor growth three times per week and tumor size was documented by direct measurement in three perpendicular directions using a Max-Cal caliper (Cole Parmer Instrument Co.) and a plastic ruler. The experiments were terminated when the tumors reached 20–22 mm in diameter, if severe ulceration and bleeding had developed, or the mice had died. The measurements were recorded as tumor volumes (mm 3 ) from groups of five mice each. For blocking of the B7 pathway in vivo, mice were injected with 2 × 10 6 EL4–B7-1+B7-2 cells that had been premixed with 50 μg of murine anti–B7-1 or anti–B7-2 antibodies for 10 min at 4°C. Mice were treated with anti-B7 antibody every other day for 20 d by i.p. injection at 150 μg/mouse following tumor implantation. Tumor growth was measured every other day. Section title: Adoptive Transfer of Spleen Cells and T Cells. Educational score: 4.094708442687988 Domain: biomedical Document type: Study Language: en To test the biological effects of the T cells on tumor growth, a T cell line generated from EL4–B7-1+B7-2–bearing mice (5 × 10 6 /mouse) were intravenously injected into EL4–B7-1–bearing mice 24 h after tumor implantation. To test the direct effect of the unmanipulated spleen cells from mice (4 × 10 6 cells/mouse) that were bearing EL4–B7-2 tumors for 12 d, spleen cells from these mice were also intravenously injected into EL4–B7-1–bearing mice 24 h after tumor implantation. The control group received either PBS or 4 × 10 6 spleen cells from normal C57BL/6 mice intravenously. Section title: Expression of Murine B7 Molecules on EL4 Tumor Cell Lines. Educational score: 4.169638633728027 Domain: biomedical Document type: Study Language: en The wild-type tumor, the vector-only–transfected EL4, EL4–B7-1, and EL4–B7-2, and the double transfectant EL4–B7-1+B7-2 were screened for surface expression of B7-1, B7-2, and CD95L. B7-1+B7-2 double transfectants were generated by retransfection of EL4–B7-1 cells with B7-2 cDNA. Cell surface expression was determined by flow cytometry after indirect immunofluorescent staining using specific mAbs to B7-1 (16.10A1), B7-2 (GL1), or the soluble ligand hCTLA4–Ig, which binds to both B7-1 and B7-2. Only clones showing stable expression of B7 molecules were selected for further experimentation. The EL4-wt cells and the vector neo/hygro–transfected EL4 cells did not show any detectable expression of B7-1 or B7-2 . EL4–B7-1 cells showed a strong expression level for B7-1 but no detectable B7-2, as demonstrated by binding of anti–B7-1 and CTLA4–Ig but not anti–B7-2 . B7-2–transfected EL4 cells stained brightly with anti–B7-2 antibodies and CTLA4–Ig but not with anti– B7-1 antibody . The double-transfected EL4 cells showed a fairly equivalent expression for both B7-1 and B7-2 when compared to the expression levels found on the single transfectants . None of the cells expressed CD95L (data not shown). Section title: B7-1+B7-2 Double Transfectants Induce Tumors In Vivo. Educational score: 4.211811065673828 Domain: biomedical Document type: Study Language: en The tumor growth potential of EL4 cells was assessed by intradermal implantation of 2 × 10 6 cells into syngeneic C57BL/6 mice. EL4-wt cells showed aggressive tumor growth, producing visible tumors in the mice after only 5–9 d. No significant differences were observed between tumors produced from vector-only–transfected cells (EL4-neo/ hygro) and EL4-wt cells (data not shown), demonstrating that the transfection process and vector did not have any effect on the ability of EL4 cells to produce tumors in syngeneic mice (data not shown). In the same experiment, the tumorigenicity of transfected EL4–B7-1, EL4–B7-2, and double-transfected EL4–B7-1+B7-2 cells were tested by implanting 2 × 10 6 cells into syngeneic C57BL/6 mice. B7-1–transfected EL4 cells completely regressed by 13–16 d after implantation . In contrast, the EL4–B7-2 cells continued to grow at a rate similar to that of the EL4-wt cells. The most aggressive tumor growth was observed with the double-transfected EL4–B7-1+B7-2 cells, which reached experimental limits between days 13 and 17, 5–6 d earlier than the EL4-wt control group . Cumulative data from all the experiments is presented in Table I . The majority of EL4–B7-1 tumors were rejected, whereas the B7-2– transfected and B7–1+B7-2 double-transfected EL4 cells were not rejected by syngeneic mice. Furthermore, the difference between EL4–B7-1 and EL4–B7-2 or EL4–B7-1+ B7-2 in the incidence of tumor rejection and tumor volume was highly significant . In nude mice, all of the EL4 cell lines (wild type and transfected) showed similar tumor growth rates . Therefore, the regression of EL4–B7-1 tumors was not due to different rates of growth of the transfected tumor cells but required the presence of T cells for the regression to occur. Section title: Expression of B7-1 and B7-2 on EL4 Cells Is Stable In Vivo. Educational score: 4.155796051025391 Domain: biomedical Document type: Study Language: en One of our major concerns in these experiments was whether the expression level of B7 molecules on the surfaces of the transfected cells was maintained in vivo for the complete term of the experiment or whether the cells lost or lowered their B7 surface expression. If the B7 expression of the transfectant was to decrease, it could result in an increased growth rate of the tumors in vivo. Progressively growing tumors from the syngeneic C57BL/6 mice were removed (explanted) after 20 d, made into single-cell suspensions, and analyzed by flow cytometry to determine their surface expression levels of B7. Mice implanted with EL4–B7-1 cells did not have tumors available to explant due to complete tumor regression. The explanted tumor cells were then stained for B7 expression immediately after explantation and also after 4 d in tissue culture . The explanted EL4–B7-2 cells showed the same expression as before implantation, and expression was stable when the cells were maintained in cell culture . The explanted EL4–B7-1+B7-2 cells also showed stability for the expression of both B7 molecules on its surface . The B7-1 expression level of the double-transfected tumor cells was the same as before implantation. This clearly demonstrates that, although the EL4–B7-1+B7-2 cells still expressed B7-1 on their surfaces, B7-1 was no longer able to induce regression and tumor growth killed the mouse. Section title: B7-2 Molecules on the B7-1 and B7-2 Double Transfectants Are Functional and Costimulate T Cell Responses In Vitro. Educational score: 4.193900108337402 Domain: biomedical Document type: Study Language: en To determine the mechanisms for the different immune responses to EL4–B7-1 and B7-2 tumors, we tested the transfectants for their ability to costimulate T cell proliferation. We have previously reported that, although EL4–B7-1 costimulates T cell proliferation, EL4–B7-2 does not costimulate an anti-CD3–induced T cell response. In this series of experiments, single- and double-transfected EL4 cells were used as costimulators in a proliferation assay using submitogenic concentrations of soluble anti-CD3 antibody. The data presented in Fig. 4 confirmed that EL4–B7-1 cells costimulated significant amounts of T lymphocyte proliferation. This proliferation was almost completely blocked by the addition of anti–B7-1 antibodies or hCTLA4–Ig but was unaffected by the addition of anti–B7-2 antibodies . EL4-wt, vector-only–transfected EL4, and EL4–B7-2 cells were unable to costimulate T cell proliferation . The EL4–B7-1+B7-2 double transfectants were able to costimulate T cell proliferation as well as the EL4–B7-1 cells. This costimulation could be 60% blocked by the addition of anti–B7-1 antibodies but more than 90% blocked with anti–B7-2 antibody or CTLA4–Ig . EL4–B7-1+ B7-2 cells explanted after 20 d from tumors growing in mice also retained the same costimulatory activity as the in vitro-cultured EL4–B7-1+B7-2 cells. Section title: Blocking B7-2 Molecules In Vivo Results in Inhibition of Tumor Growth of the B7-1+B7-2 Double-transfected EL4 Tumors. Educational score: 4.266333103179932 Domain: biomedical Document type: Study Language: en To study whether the lack of antitumor immunity induced by the double transfectant was due to a dominant negative immunoregulation induced by the B7-2 molecule, we implanted syngeneic C57BL/6 mice with the double-transfected EL4–B7-1+B7-2 cells and tested the effect of blocking either B7-1 or B7-2 molecules with monoclonal antibodies in vivo. Groups of mice were intradermally implanted with EL4–B7-1+B7-2 cells that were premixed with excess (50 μg) anti–B7-1 or anti–B7-2 antibody to block the B7 molecules expressed on the cell surface. The mice were further treated with i.p. injections of 150 μg of the anti-B7 antibody every other day for 20 d after implantation of EL4 cells. Anti–B7-1 and anti–B7-2 antibodies are of the same isotype and thus control for each other. A control group was injected i.p. every other day with 200 μl PBS. In the anti–B7-1 antibody–treated group, the EL4–B7-1+B7-2 tumor grew as quickly as the PBS-treated control group , reaching experimental limits between days 15 and 22. In other experiments, treatment with anti–B7-1 antibody made the tumors grow more quickly than the PBS treatment (data not shown). In contrast, in the anti–B7-2 antibody–treated group, tumors grew slowly or began to regress by day 15, indicating that when the B7-2 molecule on the surface of the EL4 cell was blocked, the signal provided by the B7-1 molecule became dominant and induced regression. The experiment was discontinued at day 21, as the tumors in the PBS control and anti–B7-1 antibody groups had reached experimental limits. The in vivo effects of anti–B7-2 antibody in inducing tumor regression could be due to its modification of the immune response of the host and not just due to blocking of B7-2 molecules on the surfaces of tumor cells. To address this issue, we implanted EL4-wt cells into C57BL/6 host mice that were treated with anti–B7-1 antibodies, anti–B7-2 antibodies, or a control Ig to determine if the antibody treatments themselves were affecting the experimental results. During the 20-d course of the experiment, none of the antibodies had any significant effect on the growth of the EL4-wt tumors in vivo . Furthermore, in vitro treatment of EL4-wt, EL4–B7-1, EL4–B7-2, and EL4–B7-1+B7-2 cells with anti–B7-1 or anti–B7-2 antibodies did not inhibit or slow the in vitro growth rate of the cells (data not shown). Section title: Transfer of Immune Suppression by a T Cell Line and Spleen Cells from EL4-B7–bearing Mice. Educational score: 4.372016906738281 Domain: biomedical Document type: Study Language: en To examine the role of T cells in tumor rejection and immune suppression, T cell lines from EL4–B7-1, EL4–B7-2, and EL4–B7-1+B7-2 tumor–bearing mice were established. The T cell lines were derived from spleens of mice bearing tumors implanted 10–12 d earlier. Because of the lack of costimulatory activity of EL4-wt and EL4–B7-2 cells, we have not been successful in establishing long-term T cell lines against these tumors. However, we were successful in establishing long-term T cell lines against EL4–B7-1 and EL4–B7-1/2 double transfectant tumor cells. Three cell lines were established from three mice bearing the EL4–B7-1 tumor, and each of the three T cell lines were CD8 + (>95%). All of these T cells showed the same cytokine profile as demonstrated for the T cell line No. 4 , and all of them lysed EL4-wt and EL4–B7-1 tumor cells in a CTL assay. Five T cell lines were established from six mice bearing the EL4–B7-1+B7-2 tumor. Of these five lines following four in vitro restimulations, four T cell lines were >93% CD4 + T cells. The fifth cell line was 65% CD4 + and 35% CD8 + . This cell line was positive in a CTL assay for EL4–B7-1 and the double-transfected tumor cells, but over time it lost the CD8 + T cells and CTL activity. Detailed analysis of two prototypic T cell lines, No. 4 generated against the EL4– B7-1 and No. 1 generated against EL4–B7-1/2 double transfectants, is shown . The No. 4 T cell line, generated against EL4–B7-1, was CD8 + (>98%) and showed a very strong and specific CTL activity for the different EL4 cells, including the EL4-wt cells, but did not lyse a control tumor target R1.1–B7-1 . After specific activation of T cell No. 4 with EL4–B7-1, it produced high levels of IFN-γ, low levels of IL-10, and no IL-2, IL-4, or TNF-α . This is completely opposite to the response of T cell line No. 1, which was derived from a mouse bearing the B7-1/2 double-transfected tumor. This T cell No. 1 (>99% CD4 + ) showed no CTL activity at all and produced high amounts of IL-4 and IL-10 but no IL-2, IFN-γ, or TNF-α in response to EL4–B7-1+B7-2 . Transfer of the T cell line No. 1 i.v. into mice with a EL4–B7-1 tumor resulted in tumor growth in 9/16 animals . Furthermore, the intravenous transfer of spleen cells from mice with EL4–B7-2 tumors resulted in growth of EL4–B7-1 tumors . Intravenous injection of PBS or normal C57BL/6 spleen cells did not affect the tumor growth of EL4–B7-1 cells. In contrast to the EL4–B7-1–expressing tumor cells, which induce CTL responses, the data suggest that expression of B7-2 on the EL4 cells results in the induction of CD4 + T cells that predominantly produce Th2 cytokines. To further confirm that it is CD4 + cells that are responsible for suppression of anti-tumor immunity, we also tested the growth of EL4–B7-1+B7-2 in B6 mice depleted of CD4 + cells. Groups of C57BL/6 mice were either treated with control Ig or anti-CD4 mAb (GK1.5) and then transplanted with EL4–B7-1+B7-2 double transfectants. The results showed that in the control Ig-treated group, all mice developed tumors of large size; all mice had to be killed because of the large tumor size. In contrast, in the anti-CD4–treated group, average tumor size was much smaller. In the latter group, 75% of mice were still alive on day 24 and 25% of mice were actively rejecting tumors. These data directly demonstrate that CD4 + cells play an active role in suppressing antitumor immunity. Section title: EL4–B7-2 and B7-1+B7-2 Cells Are Rejected in IL4 −/− Mice. Educational score: 4.122743129730225 Domain: biomedical Document type: Study Language: en Previous studies have reported that B7-2 may preferentially induce Th2 responses ( 25 , 26 ). B7-2 expression on EL4 cells probably results in the induction of IL-4–producing Th2 or NK1.1 responses, which may in turn suppress antitumor responses. To further explore whether IL-4 and IL-10 were involved in the suppression of antitumor immunity by EL4–B7-2 and EL4–B7-1+B7-2 tumor cells, we assessed the tumor growth of EL4-wt and transfectants by intradermal implantation of 2 × 10 6 cells into syngeneic IL-4 −/− , IL-10 −/− , and normal C57BL/6 mice. Section title: EL4–B7-2 and B7-1+B7-2 Cells Are Rejected in IL4 −/− Mice. Educational score: 4.214939117431641 Domain: biomedical Document type: Study Language: en EL4-wt and EL4–B7-1 tumor cells showed the same tumor growth or tumor rejection in IL-4– or IL-10–deficient mice as in normal C57BL/6 mice . EL4-wt cells produced visible tumors in the mice after 5–9 d . B7-1–transfected EL4 cells were rejected in all three groups by 13–20 d after implantation . In contrast, EL4–B7-2–transfected tumor cells progressively grew in normal C57BL/6 and IL-10 −/− mice but were completely rejected in IL-4 −/− C57BL/6 mice . The B7-1+B7-2 double-transfected EL4 cells showed aggressive tumor growth in the C57BL/6 control group, whereas in the IL-4 −/− and IL-10 −/− mice, the EL4–B7-1+B7-2 tumor cells were rejected . These data demonstrate that host-derived IL-4 and/or IL-10 plays a critical role in abrogation of antitumor immunity induced by EL4–B7 transfectants. Section title: Discussion Educational score: 4.156064510345459 Domain: biomedical Document type: Study Language: en We have examined the costimulatory molecules B7-1 and B7-2 on EL4 thymoma cells for their ability to induce antitumor immune responses. Our studies using B7-2– transfected EL4 cells confirm the previously reported results that B7-1 and B7-2 expressed on EL4 tumor cells differ in the way they affect antitumor immunity and tumor growth in vivo ( 22 ). Transfecting B7-2 into EL4 cells does not induce antitumor immunity but results in more vigorous tumor growth. This is in sharp contrast to the complete regression observed with the B7-1–transfected EL4 cells. Interestingly, we observed in this study that expression of B7-2 in B7-1–transfected EL4 cells does not result in tumor regression but rather more aggressive growth of the tumor cells. This suggests that the presence of B7-2 on the cell surface was dominant, as it could suppress or negate the ability of B7-1 to induce antitumor immunity and elimination of the tumor. Section title: Discussion Educational score: 4.23038911819458 Domain: biomedical Document type: Study Language: en Although some investigators have reported that both B7-1 and B7-2 have equivalent costimulatory abilities ( 27 ), increasing evidence suggests that the functional outcome of B7-1– and B7-2–mediated signaling appears to have both distinct and overlapping functions (26, 28, and 29). We and others have recently demonstrated that expression of B7-1 on the surfaces of malignant tumor cells results in their rejection and can reduce tumor burden and eliminate established metastases ( 17 , 18 ). B7-2 has been transfected into a number of different types of tumor cells with conflicting results. Transfecting B7-2 into P815 mastocytoma cells ( 21 ) or malignant melanoma tumors ( 30 ) resulted in tumor regression. In contrast are the results obtained by Yang et al. for the fibrosarcoma MCA 102 ( 21 ) and Gajewski ( 24 ), who have reported that B7-1, but not B7-2, can efficiently costimulate CD8 + T lymphocytes. Leong et al. have found that B7-1, but not B7-2, can induce immunity to murine-malignant mesothelioma ( 31 ). Matulonis et al. have also reported that B7-2 is less potent than B7-1 in inducing antitumor immunity and tumor regression in myeloid cells ( 23 ). Our results are consistent with the Leong et al. and Matulonis et al. reports. Section title: Discussion Educational score: 4.301704406738281 Domain: biomedical Document type: Study Language: en Why would the two B7 molecules behave differently, even though they bind to the same receptors on the surfaces of T cells? There is some indirect evidence that suggests that B7-1 is quantitatively superior to B7-2 in providing costimulation. The differences in affinity/avidity and length of interaction (on/off rates) between B7 molecules and the CD28/CTLA4 receptors may affect the intracellular signaling events induced in T cells ( 29 ). Mechanisms which could explain the B7-2 effect on the immune system include the induction of anergy by preferentially engaging CTLA4, activation of the fas/fas ligand apoptotic pathway, or the recruitment of regulatory T cells producing inhibitory cytokines. The induction of anergy through the CTLA4 pathway does not appear to be an important mechanism of the observed B7-2 effect, as in vivo blocking of CTLA4 using a specific monoclonal antibody (whole or Fab) did not result in the rejection of the EL4–B7-2 tumors (data not shown). Section title: Discussion Educational score: 4.592573165893555 Domain: biomedical Document type: Study Language: en Th2 cells have been shown to be induced by B7-2 signaling, leading to the downregulation of Th1 cells and their associated cytokines. Kuchroo et al. ( 25 ) and Freeman et al. ( 26 ) initially suggested that B7-1 and B7-2 may have differential roles in T cell differentiation. Whereas B7-1 was suggested to induce Th1 differentiation, more compelling evidence has been reported for the role of B7-2 in inducing Th2 differentiation. Although they are controversial, a number of studies have now confirmed this initial observation (28, 29, and 33) and have also shown the importance of B7-2 for IL-10 production ( 29 ). It is not yet clear whether B7-2 expressed on the EL4 cells is inducing differentiation of naive T cells into a Th2/Tc2 pathway or whether it is expanding a memory Th2/Tc2 population. If B7-2 expression does not directly result in Th2 differentiation of the naive T cells, EL4–B7-2 cells may preferentially expand a preexisting memory Th2/Tc2 cell population that is cross-reactive with the EL4 cells. Although EL4 tumor cells are MHC class II − , they induce CD4 + T cells, probably by indirect presentation in that the tumor antigen of the EL4 cells is presented by host APCs. The rejection of EL4–B7-2 and EL4–B7-1+B7-2 tumor cells in IL-4–deficient mice demonstrates the important role of this Th2 cytokine in the regulation of an antitumor response in this system. Thus, if B7-2–expressing EL4 cells predominantly induced a Th2 response, these EL4-specific Th2 cells may inhibit antitumor immune responses. The importance of CD4 + and not CD8 + T cells for the immune-suppressive effect of the EL4–B7-1+B7-2 cells could be further demonstrated in mice depleted of CD4 + cells, as described in the Results section: mice depleted of CD4 cells could reject tumors or showed slower tumor growth when compared with control Ig-treated mice. This data, together with the results from the IL-4–deficient mice, which still have functional CD4 compartments, supports the idea that Th2 cytokines produced by CD4 cells induced by B7-2–bearing tumor cells are responsible for suppression of antitumor immunity in vivo. A few earlier studies have shown that preferential induction of Th2 responses may suppress specific antitumor immunity. Ghosh et al. have suggested that Th2 cells dominate in progressive tumor–bearing animals, reducing the number of Th1 cells and their associated cytokines and thus allowing the tumor to grow in the host ( 34 ). IL-10 has been implicated in reducing the ability of CD8 + T cells (CTLs) to eliminate tumors in vivo ( 35 , 36 ). The in vivo data for IL-4 −/− C57BL/6 mice further supports the hypothesis that B7-2 may be delivering a dominant costimulatory signal which induces IL-4, producing Th2-like responses. It would then follow that IL-4 and IL-10 would be produced by these cells, leading to inhibition of Th1 cells and CTL activity and favoring tumor growth. Our data further supports this hypothesis, in that transfer of T cells from B7-1+B7-2 tumor–bearing mice inhibited antitumor immunity in mice challenged with EL4–B7-1 cells. This may be part of the explanation for the B7-2 effect we have observed. Section title: Discussion Educational score: 4.4743733406066895 Domain: biomedical Document type: Study Language: en Functional differences in the effect of B7-2 expression on different cell types might be due to quantitative differences in surface expression, cell-specific posttranslational modifications in the B7-2 molecule, different isoforms of B7-2 used for transfection, or mutation in the cDNA. The same murine B7-2 cDNA preparation was transfected into cells of different tissue types such as CHO, 3T3, and BW1100 cells. We have found that only the EL4 cells expressing B7-2 failed to costimulate, eliminating the possibility that the cDNAs are different ( 22 ). Freshly isolated splenic T cells from a number of strains of mice were also examined and found to be unable to costimulate T cells in in vitro allo-MLRs, although they all expressed low levels of B7-2 as determined by flow cytometry. These two pieces of data argue against there being a mutation in the B7-2 cDNA used for transfection. Quantitative differences in B7-2 expression on the surfaces of the cells is also not a likely explanation for the observed inability of EL4–B7-2 and EL4–B7-1+B7-2 cells to induce antitumor immunity. We tested low and high B7-2–expressing EL4 cells, and all showed similar tumor growth in vivo (data not shown). Also, in the case of EL-4–B7-1+B7-2 double transfectants, there is high expression of both costimulatory molecules yet tumors grow progressively in the host. Quantitatively lower expression of B7 may not, therefore, be responsible for the observed effect. Furthermore, overexpression of B7-2 on EL-4 cells does not lead to induction of antitumor immunity, suggesting that B7-2 expressed on EL-4 or other T cells may be qualitatively different. In support of this hypothesis, Höllsberg et al. ( 37 ) have reported that human T cells express a hypoglycosylated form of B7-2 that exhibits a reduced ability to bind CD28 and does not costimulate T cell proliferation. This also indicates that B7-2 expressed on the surfaces of T cells may be qualitatively different. It is possible that B7-2 exists in different forms in various cell types and that posttranslational modifications can affect the binding and functions of the B7-2 molecule. Section title: Discussion Educational score: 4.2196574211120605 Domain: biomedical Document type: Study Language: en The conflicting reports concerning B7-2 effects on the antitumor response may be due in part to sequence differences in the B7-2 construct used for transfection. Comparison of the published sequences of B7-2 by Freeman et al. ( 12 ) and Azuma et al. ( 5 ) indicates the presence of six additional residues at the amino terminus of the B7-2 sequence isolated by Freeman et al. It is possible that these six additional amino acids, which are encoded by a separate exon ( 38 ), can affect the function of the B7-2 molecule or shunt it into different intracellular processing pathways within the cell. This could result in conformational changes in protein folding or posttranslational modifications (e.g., glycosylation), leading to alterations in the function of the B7-2 molecule in some cell types, such as tumors of T cell origin. Section title: Discussion Educational score: 4.2045416831970215 Domain: biomedical Document type: Study Language: en In summary, data presented here demonstrates that, whereas B7-1 and B7-2 enhance immune responses by providing a potent costimulatory signal to the T cell, B7-2 may serve to inhibit immune responses under some circumstances. The Th2 cytokine, IL-4 appears to play an important role in abrogating the antitumor response induced by the B7-2 molecule on EL4 cells. B7 molecules may thus have evolved with the capability to both enhance and regulate the immune response, depending on which receptor they engage (CD28 vs. CTLA4) and the cell type in which they are expressed (professional APC vs. T cell). | Other | biomedical | en | 0.999996 |
10075976 | Section title: Hemorrhagic Shock Protocol. Educational score: 0.9134503602981567 Domain: biomedical Document type: Other Language: en This study was performed in accordance with the National Institute of Health guidelines for the use of experimental animals, and all animal protocols have been approved by the Institutional Animal Care and Use Committee (IACUC) of Thomas Jefferson University. Section title: Hemorrhagic Shock Protocol. Educational score: 4.155817031860352 Domain: biomedical Document type: Study Language: en Wild-type (C57BL/6) mice and P-selectin–deficient (C57BL/ 6J-Sel) mice were obtained from The Jackson Laboratory . Male and female mice (8–14 wk old and 20–30 g body wt) were anesthetized with sodium pentobarbital (120 mg/kg) injected intraperitoneally. A tracheotomy was performed to maintain a patent airway throughout the experiment. The left carotid artery was cannulated for continuous blood pressure monitoring, and the right jugular vein was cannulated for blood withdrawal and fluid or antibody administration. Mice were subjected to hemorrhage by withdrawal of blood to allow mean arterial blood pressure (MABP) 1 to be maintained at 40 mmHg for 45 min. The mean bleedout volume was 0.72 ± 0.12 ml and 0.81 ± 0.09 ml for the wild-type and P-selectin–deficient mouse, respectively. Blood was collected in a heparinized (5 U) syringe and kept at 37°C until reinfusion. Mice were then resuscitated by infusion of the shed blood and intravenous injection of 0.5 ml 0.9% NaCl alone or with either an anti–P-selectin mAb or rs.PSGL.Ig. Mice were killed by exsanguination 45 min after resuscitation, after completion of intravital microscopy studies. Control wild-type mice underwent cannulation and anesthesia for the same period of time as hemorrhaged mice, but were not bled ( n = 6). Wild-type and P-selectin–deficient mice were randomly assigned to one of four experimental hemorrhage groups: (i) wild-type mice receiving saline ( n = 7); (ii) P-selectin–deficient mice receiving saline ( n = 6); (iii) wild-type mice receiving 1 mg/kg anti–P-selectin mAb (RB40.34; PharMingen ) ( n = 6); and (iv) wild-type mice receiving 1 mg/kg high affinity mutant rs.PSGL.Ig (rsPSGL.47mutFc; Genetics Institute, Inc.) ( n = 6). Section title: Hemorrhagic Shock Protocol. Educational score: 3.8257625102996826 Domain: biomedical Document type: Study Language: en The total number of circulating white blood cells in all experimental groups of mice was determined by hemocytometric count of smears of blood, which was obtained through the jugular vein cannula. Section title: Intravital Microscopy of Mouse Peri-intestinal Venules. Educational score: 4.206727027893066 Domain: biomedical Document type: Study Language: en All intravital microscopy experiments were conducted in anesthetized mice, which were surgically prepared as reported in the above hemorrhagic shock protocol section. Intravital microscopy was performed on mouse peri-intestinal venules, after exteriorization of a loop of ileal tissue via a midline laparotomy. The ileum was placed in a temperature-controlled fluid-filled plexiglas chamber and transilluminated for brightfield observation of the peri-intestinal microcirculation according to a previously described procedure ( 15 ). The ileum and mesentery were superfused throughout the experiment with a buffered Tyrode solution (pH 7.4, 37 ± 1°C). Preparations were allowed to stabilize for 15 min. Observations of rolling and adherent leukocytes were made with a Microphot microscope and a 40× salt water–immersion lens ( Nikon Corp. ). Images were projected by a high-resolution color video camera (DC-330; DAGE-MTI, Inc.) onto a color Sony high resolution video monitor (Multiscan 200-sf), and the image was recorded with a videocassette recorder. All images were then analyzed using computerized imaging software (Phase 3 Image System; Media Cybernetics) on a Pentium based IBM-compatible computer (Micron Millenia Mxe; Micron Electronics Inc.). Red blood cell velocity was determined on-line using an optical Doppler velocimeter ( 16 ) obtained from the Microcirculation Research Institute, College Station, TX. This method gives an average red blood cell velocity, which is digitally displayed on a meter, and allows for the calculation of shear rates ( 17 ). The number of rolling and adhered leukocytes was determined off-line by playback analysis of the videotape. Section title: Histologic Assessment of Neutrophil Infiltration and Immunohistochemistry of P-selectin. Educational score: 4.15583610534668 Domain: biomedical Document type: Study Language: en The number of neutrophils (PMNs) infiltrating into lung, liver, and intestine was determined in both wild-type and P-selectin–deficient mice. At the end of the 45 min reinfusion period, mice were killed and the pulmonary circulation was flushed by injecting PBS into the right ventricle. Lungs were injected with formalin through the trachea, then removed and placed into 4% formalin. Similarly, the splanchnic organs were perfused through the superior mesenteric artery and fixed in vivo by infusion of formalin ( 15 ). Lungs, liver, and intestine were then cut into blocks and dehydrated in graded acetone washes at 4°C. Tissue blocks were embedded in plastic (Immunobed; Polysciences, Inc.), and 4-μm-thick sections were cut and transferred to Vectabond-coated slides (Vector Laboratories, Inc.). The slides were soaked in ethanol for 10 min to remove the plastic embedding material and to allow staining of the tissue. After the 10-min ethanol wash, the tissue sections were stained with hematoxylin and eosin and examined blindly. The number of PMNs was counted and tallied. Five fields from each of two slides were counted from each organ, and three mice were studied per group. In addition, grading of the histopathologic changes in the lung (i.e., neutrophil infiltration, interstitial edema, and intraparenchymal hemorrhage) was performed on a 0 to 3 scale (with 0 being normal and 3 being the most severe abnormality). Section title: Histologic Assessment of Neutrophil Infiltration and Immunohistochemistry of P-selectin. Educational score: 4.096838474273682 Domain: biomedical Document type: Study Language: en Immunohistochemical localization of P-selectin was determined in ileal samples after intravital microscopy was completed, according to previously described methods ( 15 ). mAb PB1.3 only recognizes surface expressed P-selectin ( 18 ). Quantification of P-selectin was accomplished using the avidin-biotin immunoperoxidase technique (Vectastain ABC Reagent; Vector Labs.) as previously described by Weyrich et al. ( 18 ). 50 venules were analyzed per tissue section, 20 sections were examined per group, and the percentage of positive staining venules was tallied. Section title: Quantification of P-selectin and PSGL-1 mRNA Expression by Ribonuclease Protection Assays. Educational score: 4.077235221862793 Domain: biomedical Document type: Study Language: en Immediately after intravital microscopy was performed, mouse lungs, liver, and small intestine were removed for total RNA extraction. Total RNA was extracted from these tissues using the acid guanidium-phenol-chloroform extraction method described by Chomczynski and Sacchi ( 19 ). The concentration of RNA suspended in ribonuclease-free water was determined spectrophotometrically. Section title: Quantification of P-selectin and PSGL-1 mRNA Expression by Ribonuclease Protection Assays. Educational score: 4.120919704437256 Domain: biomedical Document type: Study Language: en The plasmid containing the full-length mouse P-selectin cDNA was provided by Professor Dietmar Vestweber (Institut fur Zellbiologie, ZMBE, Westfalische Wilhelms Universitat Munster, Munster, Germany). The plasmid for synthesis of mouse tissue P-selectin probe for ribonuclease protection assays was created by recloning of a PstI–AvrII fragment of mouse P-selectin into a PstI–SpeI site of pBluescript II SK + vector (Stratagene). This plasmid was digested with XhoI and used as a template for in vitro transcription of a 474-base radiolabeled antisense probe containing a 393-base protected fragment using T3 RNA-polymerase ( Boehringer Mannheim ) in the presence of [ 32 P]UTP ( Amersham Corp. ). Section title: Quantification of P-selectin and PSGL-1 mRNA Expression by Ribonuclease Protection Assays. Educational score: 4.194468975067139 Domain: biomedical Document type: Study Language: en Mouse PSGL-1 cDNA was synthesized by reverse transcriptase PCR using mouse lung total RNA and oligo(dT), and was amplified using forward primer (5′-CCTGGGAATTCACCTGCCCC-3′) and reverse primer (5′-GAGAGTGGAGCTAGCAAAGG-3′). These oligonucleotides correspond to amino acid sequences 267–283 and 394–388 of mouse PSGL-1, respectively . A 384-bp PCR fragment was cloned using PCR 2.1-TOPO Cloning Kit (a gift from Invitrogen Corp.). The Sst I–XbaI fragment of this plasmid was recloned in pTRIPLEscript vector (pTRIamp 18; Ambion). This plasmid was digested with XbaI to make, with T7 polymerase, a 570-base radiolabeled antisense probe that contained a 384-base protected fragment. All constructs used in this investigation were verified by sequencing the insert in the plasmid. These sequences were found to be 100% identical to the published sequences. The expression of PSGL-1 mRNA was analyzed by reverse transcriptase PCR analysis. Amplified PCR products were analyzed by electrophoresis on a 1.5% agarose gel. The intensity of each P-selectin and PSGL-1 mRNA band was normalized for GAPDH (data not shown). Section title: Quantification of P-selectin and PSGL-1 mRNA Expression by Ribonuclease Protection Assays. Educational score: 4.084502220153809 Domain: biomedical Document type: Study Language: en Mouse β-actin antisense RNA probes were used to evaluate total RNA, which in turn were used for P-selectin and PSGL-1 mRNA expression analysis. The mouse β-actin plasmid for the synthesis of the antisense RNA probe was received from Ambion. For the ribonuclease protection assay, 2 μg of total RNA for detection of β-actin mRNA, and 60 μg for detection of PSGL-1 mRNA in the lung, liver, and small intestine have been used accordingly to previously described procedures ( 20 ). The intensity of each P-selectin and PSGL-1 mRNA band was normalized for β-actin mRNA levels. Section title: Statistical Analyses. Educational score: 3.038367986679077 Domain: biomedical Document type: Study Language: en All values for data listed in the text and figures are presented as means ± SEM of n independent experiments. Data were compared by analysis of variance using post-hoc analysis with Fisher's correct t test. P ≤ 0.05 was considered significant in all cases. Section title: Hemodynamic Changes Induced by Hemorrhagic Shock. Educational score: 4.183335304260254 Domain: biomedical Document type: Study Language: en Fig. 1 illustrates the time course of systemic MABP in the five experimental groups of mice. All groups of mice exhibited initial MABP values in the range of 110–120 mmHg . In control wild-type mice, MABP did not significantly change over the entire 90-min observation period . In hemorrhaged mice, MABP was maintained at 40 mmHg for 45 min. After reinfusion of the shed blood to hemorrhaged mice, MABP increased to values not significantly different from control wild-type mice at that time . In the wild-type hemorrhaged group receiving only saline, MABP progressively decreased to 90 ± 5 mmHg at the end of the experiment. In contrast, hemorrhaged P-selectin–deficient mice as well as hemorrhaged wild-type mice receiving either the anti–P-selectin mAb or the rs.PSGL.Ig maintained a significantly higher MABP at the end of the 45-min observation period in the range of 115–125 mmHg . This higher MABP was not due to decreased bleedout volumes, since the volume of shed blood was not significantly different among all groups of mice. These final blood pressures were also not statistically different from the initial MABP in these same groups of mice. Thus, either gene deficiency or functional inactivation of P-selectin expressed on the vascular endothelium limits the systemic hemodynamic consequences of hemorrhagic shock. Section title: Hemodynamic Changes Induced by Hemorrhagic Shock. Educational score: 4.157888412475586 Domain: biomedical Document type: Study Language: en Venular shear rates for the five experimental groups of mice are reported in Table I . No significant differences were observed in initial shear rates among the five groups of mice. After hemorrhage, shear rates in peri-intestinal venules abruptly decreased to less than half of the observed initial control values. Therefore, the present hemorrhagic shock model is characterized by a marked hypoperfusion of the splanchnic microvasculature during the oligemic phase. However, upon reinfusion of shed blood, venular shear rates returned to normal values (Table I ). This strongly suggests that blood flow was reestablished to control levels during the postoligemic phase. Since shear rates were normal after reinfusion, the adhesive interactions observed between leukocytes and the microvascular endothelium during resuscitation from hemorrhage could not be attributed to alterations in physical hydrodynamic forces brought about by perturbations in local hemodynamics. Section title: P-selectin Is Required for the Upregulation of Leukocyte– Endothelium Interaction in Hemorrhagic Shock. Educational score: 4.429737567901611 Domain: biomedical Document type: Study Language: en A low baseline number of rolling and adherent leukocytes was observed in the mesenteric microvasculature for all experimental groups of wild-type mice. Furthermore, neither rolling nor adherence of leukocytes increased in peri-intestinal venules of P-selectin–deficient mice at any time. Baseline leukocyte rolling and adherence were not significantly changed during the first 45 min of the hemorrhage period in all wild-type and P-selectin–deficient mice. However, the number of rolling and adherent leukocytes in untreated hemorrhaged wild-type mice exhibited a threefold ( P < 0.01) increase after reinfusion compared with normal control values . In contrast, no significant increase in the number of rolling or adhered leukocytes was observed in the peri-intestinal venules of P-selectin–deficient mice . Similarly, intravenous infusion of either 1 mg/kg of an anti–P-selectin mAb or 1 mg/kg of rs.PSGL.Ig significantly attenuated both leukocyte rolling and leukocyte adherence induced by hemorrhage and reinfusion of shed blood. In addition, no significant change in the total number of circulating leukocytes was observed in the five experimental groups of mice, so that the changes in rolling and adherence could not be attributed to leukopenia. The average number of circulating leukocytes in wild-type mice and P-selectin–deficient mice was 5.9 ± 0.6 and 7.2 ± 0.4 10 3 cells/mm 3 (mean ± SEM), respectively. These values are not significantly different from each other, nor was leukopenia observed at the end of the experimental protocol or after systemic administration of either anti– P-selectin mAb or rs.PSGL.Ig. Therefore, functional expression of P-selectin protein on the mouse splanchnic microvascular endothelium exerts a crucial role in triggering inflammatory events after hemorrhage and fluid resuscitation. Section title: Determination of P-selectin Surface Expression In Situ by Immunohistochemical Localization. Educational score: 4.235069751739502 Domain: biomedical Document type: Study Language: en Immunolocalization of P-selectin was studied in the venular endothelium of the mouse ileum immediately after completion of intravital microscopic measurements. The percentage of venules staining positively for P-selectin in ileal sections from control wild-type mice was consistently low . Moreover, virtually no surface P-selectin expression was detected by immunohistochemistry at any time in P-selectin gene–deficient mice . However, hemorrhage plus reperfusion resulted in a significant increase in P-selectin expression in wild-type mice . Intravenous infusion of either anti–P-selectin mAb or rs.PSGL.Ig did not attenuate the number of venules staining positively for P-selectin after hemorrhage and reinfusion (74 ± 4% and 70 ± 6 positive venules respectively; NS versus hemorrhage wild-type mice). This clearly indicates that inhibition of leukocyte–endothelium interaction induced by anti–P-selectin mAb or rs.PSGL.Ig is due to functional neutralization of P-selectin on the endothelial cell surface rather than to significant attenuation of P-selectin expression on the microvascular endothelium. Section title: Inhibition of Neutrophil Infiltration into Lung, Liver, and Intestine Is Associated with Decreased Organ Injury in P-selectin– deficient Mice. Educational score: 4.174564361572266 Domain: biomedical Document type: Study Language: en As an additional verification of organ injury, we performed histological analysis of lung, liver, and intestine in control and hemorrhaged mice. After hemorrhage and reinfusion, P-selectin–deficient mice had fewer infiltrated neutrophils in lung, liver, and intestine than did wild-type mice. Moreover, P-selectin–deficient mice subjected to hemorrhage and reinfusion developed less interstitial lung edema and intra-alveolar hemorrhage compared with hemorrhaged wild-type mice, as assessed by histological analysis (histopathological score 0.45 ± 0.08 and 2.7 ± 0.15, respectively; P < 0.01). This decreased inflammation and injury in lungs and splanchnic organs of P-selectin–deficient mice strongly demonstrates the crucial role exerted by selectin-mediated leukocyte recruitment during the early pathophysiologic events of hemorrhagic shock. Section title: Hemorrhagic Shock Increases PSGL-1 mRNA Expression in Several Organs in the Wild-type Mouse. Educational score: 4.243844509124756 Domain: biomedical Document type: Study Language: en Levels of mRNA codifying for endogenous PSGL-1 were assessed in hemorrhaged wild-type mice, using a ribonuclease protection assay. As shown in Fig. 6 , the intensity of each PSGL-1 mRNA band was normalized to that of β-actin. After the 45-min resuscitation period, PSGL-1 transcripts were significantly increased in the lungs of mice subjected to hemorrhagic shock . Similar results were also observed in the liver and small intestine of hemorrhaged wild-type mice. After hemorrhage and reinfusion PSGL-1 transcripts in both liver and intestine increased 34 ± 8.4% and 32 ± 2%, respectively ( P < 0.001 versus control mouse tissue). In contrast, no significant changes were observed for the P-selectin mRNA transcript in wild-type mice after hemorrhage and reperfusion. This suggests that in the early phase of hemorrhagic shock, increased de novo synthesis of the counter-ligands for P-selectin in marginated blood cells entrapped in vital organs, more than de novo synthesis of P-selectin protein in the vascular endothelium, is responsible for further exacerbation of selectin-mediated leukocyte– endothelium interaction. Section title: Discussion Educational score: 4.246745586395264 Domain: biomedical Document type: Study Language: en This study was undertaken to determine the role of P-selectin in the early inflammatory response occurring after resuscitation from hemorrhagic shock. Using either mice genetically deficient in P-selectin protein or functionally blocking P-selectin in wild-type mice, by either an anti–P-selectin mAb or rs.PSGL.Ig, we first demonstrate that P-selectin plays an essential role in the pathological recruitment of leukocytes observed in hemorrhagic shock. We also found that upon resuscitation from hemorrhage, an increased expression of mRNA codifying for endogenous PSGL-1 occurs in the lung as well as in the liver and intestine of hemorrhaged wild-type mice. These data provide compelling evidence that P-selectin plays a key role in the activation of the inflammatory cascades occurring in hemorrhagic shock, thus designating P-selectin as a possible strategic target in the therapy of hemorrhagic shock. Our findings are also consistent with an earlier report that an mAb against P-selectin markedly reduced the volume of fluid necessary for resuscitation of hemorrhaged rabbits ( 21 ), although no analysis of leukocyte–endothelium interactions was attempted. Section title: Discussion Educational score: 4.5719099044799805 Domain: biomedical Document type: Study Language: en A multistep series of adhesive and signaling events regulates inflammatory responses to infection or injury ( 22 , 23 ). To initiate these responses, circulating leukocytes must first roll along the endothelium, and then adhere to the vascular wall under shear forces. Selectins mediate the first adhesive step, which is characterized by tethering and rolling of leukocytes on endothelial cells, platelets, or other leukocytes ( 24 ). In particular, P-selectin, expressed on activated platelets and endothelial cells, binds to ligands on most leukocytes ( 25 ). The regulated expression of the selectins and their high affinity ligand (i.e., PSGL-1), helps modulate the inflammatory response. However, inappropriate expression of these molecules contributes to leukocyte-mediated tissue damage in a variety of acute inflammatory disorders ( 26 , 27 ). In this regard, several investigators have demonstrated that inhibition of the rolling phase of leukocytes plays a key role in attenuating the acute inflammatory response ( 28 , 29 ). Consistent with such findings, we now demonstrate that soon after resuscitation from hemorrhage, leukocyte– endothelium interactions are significantly upregulated in the microcirculation, an event that is associated with increased expression of P-selectin on the microvascular endothelium. Moreover, as confirmed in P-selectin–deficient mice, in the absence of P-selectin protein virtually no leukocyte–endothelium interaction occurs after hemorrhage and reinfusion of shed blood. This finding agrees with previous observations showing severe attenuation of leukocyte rolling and extravasation in P-selectin–deficient mice ( 14 ). In addition, we observed de novo synthesis of PSGL-1 in the lungs and splanchnic organs of hemorrhaged wild-type mice, as confirmed by quantification of PSGL-1 mRNA. This increase in PSGL-1 mRNA levels may contribute to widespread increases in cell-to-cell interaction during acute inflammatory conditions such as hemorrhagic shock. Section title: Discussion Educational score: 4.3746795654296875 Domain: biomedical Document type: Study Language: en The inhibitory effect on leukocyte–endothelium interaction exerted by blockade of P-selectin may contribute to normalization of the pathophysiologic events in hemorrhagic shock. One possible explanation is that inhibition of the initial P-selectin–mediated tethering of leukocytes to endothelium may diminish the localized production of proinflammatory cytokines, which subsequently induce expression of endothelial cell adhesion molecules. In this regard, other investigators have demonstrated that after hemorrhagic shock increased infiltration of blood cells into vital organs increases intraparenchymal cytokine expression, starting 2 h after reinfusion and reaching a peak value after 3 d ( 30 , 31 ). Moreover, inhibition of leukocyte extravasation exerts a key role during inflammation because activated neutrophils, which have adhered to the endothelium, release cytotoxic mediators including proteases, eicosanoids, cytokines, and oxygen-derived free radicals ( 1 , 32 ), each of which can promote tissue injury and exacerbate endothelial dysfunction in hemorrhagic shock. Section title: Discussion Educational score: 4.510622024536133 Domain: biomedical Document type: Study Language: en One may speculate on the mechanism triggering the upregulation of P-selectin to the vascular endothelial cell surface during hemorrhagic shock. Hemorrhagic shock represents a severe form of whole body ischemia/reperfusion. Several investigators have demonstrated that a common pathophysiologic event occurring during ischemia/reperfusion is the early occurrence of acute endothelial dysfunction characterized by impaired endothelial release of nitric oxide ( 8 , 9 , 11 , 28 , 33 ). In this connection, acute endothelial dysfunction associated with severe organ injury has been reported in myocardial ischemia/reperfusion ( 11 ), splanchnic ischemia/reperfusion ( 9 ), traumatic shock ( 10 ), and hemorrhagic shock ( 33 ). Moreover, a functional relationship between loss of endothelium-derived nitric oxide and the upregulation of P-selectin on the venular endothelium has been reported previously ( 12 ). Therefore, hemorrhage-induced loss of endothelium-derived nitric oxide release is probably responsible for increased expression of cell adhesion molecules in the microvascular endothelium. This conclusion is supported by the observation that endothelial cell dysfunction occurs very early after hemorrhage and persists despite fluid resuscitation ( 33 ), and that an mAb against P-selectin attenuated fluid leakage in hemorrhagic shock ( 21 ). Section title: Discussion Educational score: 4.127196311950684 Domain: biomedical Document type: Study Language: en This study provides the first clear in vivo evidence of a purely P-selectin–dependent leukocyte–endothelium interaction in hemorrhagic shock. This work also suggests that neutralization of P-selectin in the early phase of hemorrhagic shock may limit infiltration of leukocytes into inflamed organs. | Other | biomedical | en | 0.999997 |
10075977 | Section title: Protease Inhibitor. Educational score: 3.8506786823272705 Domain: biomedical Document type: Study Language: en KD-IX-73-4 (HO-NH-CO-CH 2 -CH(CH 2 -CH(CH 3 ) 2 )-CO-Nal-Ala-NH 2 ) was a gift from Dr. T.K. Kishimoto ( Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT). 0.5 mg of the protease inhibitor KD-IX-73-4 was solubilized in 50 μl DMSO and diluted with saline to a final concentration of 1 mg/ml. 150 μg of KD-IX-73-4 was used in in vivo experiments for an estimated initial serum concentration of 50 μg/ml. Section title: Protease Inhibitor. Educational score: 3.9780561923980713 Domain: biomedical Document type: Study Language: en A related hydroxamic acid–based compound (slower isomer [SI]) HO-NH-CO-CH 2 -CH(CH 2 -CH(CH 3 ) 2 )-CO-Nal-Ala-NH-CH 2 -CH 2 -NH 2 was obtained from Peptides International. SI was solubilized, diluted, and aliquoted as described for KD-IX-73-4. SI was used in the experiments as a negative control. SI did not show any inhibitory effect on downregulation of L-selectin from neutrophils after activation with PMA (100 ng/ml; flow cytometry). Section title: mAbs. Educational score: 3.6902973651885986 Domain: biomedical Document type: Study Language: en mAb RB40.34 (rat IgG1, 30 μg/mouse) is a blocking mAb against murine P-selectin and was purified from hybridoma supernatant ( 25 ). mAb 9A9 (rat IgG1, 30 μg/mouse) is a blocking mAb against murine E-selectin ( 26 , 27 ). TNF-α (0.5 μg/ mouse) was obtained from Genzyme Corp. Section title: Animals. Educational score: 3.883082151412964 Domain: biomedical Document type: Study Language: en Experiments were performed on a total of 30 male mice 8–10 wk old and weighing 22–26 g. Mice included wild-type C57BL/6 (Hilltop), gene-targeted mice deficient in L-selectin ( 28 ), and gene-targeted mice deficient in E-selectin ( 29 ). Both L- and E-selectin knockout mice were backcrossed into a C57BL/6 background for at least seven generations. Section title: Intravital Microscopy. Educational score: 4.089658260345459 Domain: biomedical Document type: Study Language: en For intravital microscopy, mice were anesthetized with an intraperitoneal injection of ketamine hydrochloride (100 mg/kg, Ketalar; Parke-Davis) after pretreatment with xylazine (0.05 mg/kg i.p.) and atropine (0.1 mg/kg i.p.; Elkins-Sinn). Animals were kept at 37°C with a thermo-controlled heating pad. Some mice were pretreated 2 or 6 h before surgery with an intrascrotal injection of 0.5 μg murine TNF-α ( Genzyme Corp. ) in 0.3 ml isotonic saline as described ( 26 ). In the 6-h TNF-α model ( 30 ), the intrascrotally injected volume contained 30 U Heparin (Elkins-Sinn). Also, these mice were injected with 100 μg of mAb RB40.34 and 100 μg of mAb 9A9 intraperitoneally before the TNF-α treatment. Section title: Local Catheter. Educational score: 4.162257194519043 Domain: biomedical Document type: Study Language: en For local injection of the protease inhibitor KD-IX-73-4 and the negative control SI into the microcirculation of the cremaster muscle, a heparinized catheter was placed into the proximal part of the right femoral artery and advanced towards the branching section of the internal iliac artery from the common iliac artery, as shown in Fig. 1 . The solutions (KD-IX-73-4 and SI) were injected as a 0.1-ml bolus containing 50 μg peptide. Upon injection of the bolus into the vasculature of the cremaster muscle, a brief hemodilution of the blood flowing through the observed venule indicated passage of the injected fluid. Centerline velocity and hematocrit returned to normal shortly after the passage of the bolus through the venule. Section title: Cremaster. Educational score: 4.105022430419922 Domain: biomedical Document type: Study Language: en The cremaster muscle was prepared for intravital microscopy as described ( 31 ) and superfused with thermo-controlled 35°C bicarbonate-buffered saline saturated with 95% N 2 / 5% CO 2 ( 26 ). The exposed cremaster microcirculation remained well perfused. Time 0 was set at the beginning of the cremaster surgery, which took ∼15 min. In the case of TNF-α–pretreated animals, time 0 was set at the time of scrotal injection with 500 ng TNF-α in 0.3 ml saline. Section title: Cremaster. Educational score: 4.183194160461426 Domain: biomedical Document type: Study Language: en Microscopic observations were made on an Axioskope intravital microscope ( Carl Zeiss ) with a saline immersion objective (SW 40/0.75 numerical aperture). Each venule was observed for 90–120 s. Venules with diameters between 25 and 45 μm were observed, and video recordings were made through a CCD camera system on a Panasonic S-VHS recorder. Microvascular centerline red blood velocity was measured using a dual photodiode and a digital on-line cross-correlation program ( 32 ). Centerline velocities were converted to mean blood flow velocities by multiplying with an empirical factor of 0.625 ( 33 ). Shear rates were determined as γ w = 2.12 × 8 V b / d , where γ w is the wall shear rate, V b is the mean blood flow velocity, d is the in vivo diameter of the vessel, and 2.12 is a median empirical correction factor obtained from actual velocity profiles measured in microvessels in vivo ( 34 ). Section title: Cremaster. Educational score: 4.174230575561523 Domain: biomedical Document type: Study Language: en For continuous blood pressure monitoring and blood sampling, the carotid artery was cannulated with a heparinized PE-10 tubing. Blood samples of 10 μl volume each were taken before and 10 min after the systemic application of the protease inhibitor KD-IX-73-4 and SI to analyze systemic leukocyte concentrations (1:9 dilution with Kimura [11 ml of 5% (wt/wt) toluidine blue, 0.8 ml of 0.03% light green SF yellowish, 0.5 ml of saturated saponin, and 5 ml of 0.07 M phosphate buffer, pH 6.4; all reagents obtained from Sigma ]). Three different vessels in each cremaster preparation were selected for observations before and 1–15 min after the application of the protease inhibitor KD-IX-73-4 or the negative control SI. All vessels had calculated wall shear rates between 600 and 800 s −1 . Microvessel diameters and individual rolling leukocyte velocity were measured using a digital image processing system ( 32 ). Freeze-frame advancing was used to accurately monitor the movements of the individual rolling leukocytes. Each rolling leukocyte passing a line perpendicular to the vessel wall was followed for 0.5–1 s. Rolling velocities for individual leukocytes were calculated by dividing the traveled distance by the tracking time. Critical velocities were calculated for each vessel ( 35 ). The leukocyte rolling flux was determined from video recordings by counting all visible cells passing through a plane perpendicular to the vessel axis for 1 min before and 1 min after the systemic application of the protease inhibitor, starting 1 min after injection. Section title: Flow Cytometry. Educational score: 4.105034351348877 Domain: biomedical Document type: Study Language: en Mice were injected with KD-IX-73-4, or vehicle alone, and blood was obtained from the subclavian vein at 1 min after the injection. Mouse peripheral blood was stained with PE-conjugated anti–L-selectin mAb clone or PE-conjugated isotype control mAb 2 μl/ml at 4°C for 15 min. After lysis of the erythrocytes (lysing solution: 0.15 M NH 4 Cl, 0.01 M NaHCO 3 , and 0.001 M disodium EDTA), cells were washed once with BSA/PBS and fixed in 1% paraformaldehyde/PBS, and 0.5–1 × 10 4 cells/sample were analyzed by flow cytometry on a FACScan ® ( Becton Dickinson ). The results reported are for granulocytes, gated by their characteristic forward and side scatter. Section title: Statistical Analysis. Educational score: 2.2216429710388184 Domain: biomedical Document type: Study Language: en Statistical comparisons were carried out using Kolmogorov-Smirnov two-sample t test. NCSS statistical software ( http://www.icw.com/ncss ) version 6.0.11 was used for the statistical analysis. Statistical significance was set at P < 0.05 or P < 0.01. Section title: Inhibition of L-selectin Shedding Lowers Leukocyte Rolling Velocity under Physiologic Conditions In Vivo. Educational score: 4.125856399536133 Domain: biomedical Document type: Study Language: en Fig. 2 shows the results of inhibition of L-selectin shedding in wild-type mice. Velocities of 750 rolling leukocytes in 15 vessels of 5 wild-type mice were obtained before and after the application of KD-IX-73-4. Observations were made 1–8 min after the injection of the protease inhibitor and within the first 1 h after starting the cremaster preparation. The mean velocity of all observed cells before the inhibition of L-selectin shedding was 59 ± 5.6 μm/s and after the inhibition was 39 ± 3.6 μm/s, which represents a reduction of the mean velocity by 33 ± 2%. As an example, Fig. 3 shows a composite photomicrograph illustrating the change in rolling velocity in one representative vessel. Section title: Inhibition of L-selectin Shedding Lowers Leukocyte Rolling Velocity under Physiologic Conditions In Vivo. Educational score: 4.088221549987793 Domain: biomedical Document type: Study Language: en The related hydroxamic acid–based substance (SI) did not show any significant effect on the velocities of the rolling leukocytes. Interestingly, after the use of 150 μg SI (1 μg/ μl), the subsequent injection of 150 μg KD-IX-73-4 did not show any change in the velocity of rolling leukocytes, suggesting a possible competitive mechanism of interaction between the two hydroxamic acid–based substances and the L-selectin cleaving protease. Fig. 4 shows the results of the SI application followed by KD-IX-73-4 in wild-type mice. Section title: Inhibition of L-selectin Shedding Lowers Leukocyte Rolling Velocity under Physiologic Conditions In Vivo. Educational score: 4.114547252655029 Domain: biomedical Document type: Study Language: en To determine the duration of the effect of the protease inhibitor in vivo, single venule recordings were made in two wild-type animals for 15–20 min after the application of KD-IX-73-4. Rolling velocities of 10 cells were obtained in 1-min steps after the injection of 150 μg KD-IX-73-4. Within the first 1 min after the injection of 150 μg KD-IX-73-4, the reduction of rolling velocity could be observed and persisted for ∼9–10 min, after which the rolling velocity returned to control levels . Section title: Inhibition of L-selectin Shedding Lowers Leukocyte Rolling Velocity under Physiologic Conditions In Vivo. Educational score: 4.201093673706055 Domain: biomedical Document type: Study Language: en To assess whether the injected dose of KD-IX-73-4 resulted in complete inhibition of L-selectin shedding in vivo, we investigated the expression of L-selectin on neutrophils in peripheral blood by flow cytometry . All neutrophils in peripheral blood of untreated mice expressed L-selectin, which was almost entirely downregulated after PMA activation. In contrast, PMNs obtained from mice 1 min after KD-IX-73-4 injection did not shed L-selectin in response to PMA activation. The inhibitory effect of KD-IX-73-4 in vivo was no longer detectable at 15 min after the injection. SI, the peptide used as a control, did not show any inhibitory effect on L-selectin shedding after PMA activation of the cells. Taken together, these data show that KD-IX-73-4 transiently inhibits L-selectin shedding in vivo. The time course of this inhibitory effect parallels the time course of reduction of rolling velocity by KD-IX-73-4. Section title: Inhibition of L-selectin Shedding Lowers Leukocyte Rolling Velocity under Physiologic Conditions In Vivo. Educational score: 4.12744665145874 Domain: biomedical Document type: Study Language: en Systemic application of 150–300 μg KD-IX-73-4 did not cause an alteration of the systemic blood pressure, which remained between 80 and 95 mmHg throughout the experiments. Likewise, centerline velocities and diameters (and consequently the shear rates) of the observed venules were not affected by the systemic injections of KD-IX-73-4 or SI. Also, systemic white blood cell counts remained unchanged after systemic injection of KD-IX-73-4 (Table I ). Section title: Inhibition of L-selectin Shedding Lowers Leukocyte Rolling Velocity under Physiologic Conditions In Vivo. Educational score: 4.107469081878662 Domain: biomedical Document type: Study Language: en To determine whether the injected amount of KD-IX-73-4 (150 μg) in vivo was sufficient to cause maximal reduction of velocity, subsequent injections of KD-IX-73-4 (150–300 μg) were made in two wild-type animals to achieve an accumulation of the protease inhibitor in plasma. Such injections did not cause a further shift of velocities of the rolling cells compared with the cell rolling velocities after the first injection of 150 μg KD-IX-73-4 (data not shown). Therefore, an injection of 150 μg KD-IX-73-4 into a mouse produced complete inhibition of L-selectin shedding and maximal reduction of rolling velocity in vivo. Section title: Inhibition of L-selectin Shedding Lowers Leukocyte Rolling Velocity under Physiologic Conditions In Vivo. Educational score: 4.298944473266602 Domain: biomedical Document type: Study Language: en As another parameter relevant for leukocyte trafficking, the flux of rolling leukocytes (number of rolling cells passing per unit of time) was measured in eight vessels of wild-type mice. Inhibition of L-selectin shedding caused a significant 22% increase of rolling flux compared with the mean of the rolling flux of the same vessels before the KD-IX-73-4 treatment (118 cells/min). This finding suggests that inhibition of L-selectin shedding not only reduces the velocity of rolling leukocytes, but also aids in promoting leukocyte capture from the free stream and/or stabilizes their rolling along the venular tree. Furthermore, the number of firmly adherent leukocytes was significantly increased after inhibition of L-selectin shedding in mice treated with TNF-α for 6 h (data not shown). In this model, rolling is primarily L-selectin dependent ( 30 ). Concomitant with increased adhesion, inhibition of L-selectin shedding also increased the number of transmigrating cells, apparent by the characteristic change of shape during their transendothelial passage (data not shown). This suggests that, beyond its prominent and immediate effect on the velocity of rolling leukocytes, L-selectin shedding significantly impacts leukocyte recruitment. Section title: Inhibition of L-selectin Shedding Lowers Leukocyte Rolling Velocity under Physiologic Conditions In Vivo. Educational score: 4.142614841461182 Domain: biomedical Document type: Study Language: en To investigate whether the effect of KD-IX-73-4 on the leukocyte velocities in vivo is specific to the inhibition of L-selectin shedding or whether other variables contribute to the shift of velocities, experiments with L-selectin–deficient mice were made. Fig. 7 shows leukocyte rolling velocities obtained in L-selectin–deficient mice before and after systemic application of KD-IX-73-4. There was no significant change of the mean rolling velocities measured after application of KD-IX-73-4 (37 ± 1.4 μm/s before and 38.2 ± 1.5 μm/s after the inhibition; 100 cells from 2 animals). The absence of the reduction of velocity in L-selectin–deficient mice shows that inhibition of L-selectin shedding with KD-IX-73-4 does not alter the rolling velocity of leukocytes unless L-selectin is expressed. Flux also remained unchanged after injection of KD-IX-73-4 in L-selectin–deficient mice (data not shown). Section title: Inhibition of L-selectin Shedding Lowers Leukocyte Rolling Velocity under Physiologic Conditions In Vivo. Educational score: 4.200647354125977 Domain: biomedical Document type: Study Language: en The systemic application of the protease inhibitor into the jugular vein is likely to produce a homogenous serum concentration of the inhibitor throughout the vascular system. However, in order to be able to make a statement about the onset of the effect of KD-IX-73-4 on rolling velocity, it was necessary to observe rolling leukocytes from the moment of their first contact with the inhibitor. For this purpose, the protease inhibitor was injected as a small bolus (50 μg/0.1 ml) through a local catheter into the microcirculation of cremaster muscle. With the passage of the solution through the cremaster vasculature, the inhibitor washed over rolling leukocytes in the cremaster venules and might inhibit L-selectin shedding in these cells within the field of view. A brief hemodilution effect caused by injection of the bolus furnished evidence for the passage of the solution through the observed venules. Within the first 5 s after the passage of the bolus, velocities of 25 rolling leukocytes/venule were obtained. The mean velocity of 50 rolling leukocytes from 2 animals before contact with the protease inhibitor was 40.2 ± 3.1 μm/s, which was lowered to 29.9 ± 2.7 μm/s immediately after the injection of KD-IX-73-4. This equals a 26% reduction of leukocyte rolling velocity. Section title: Inhibition of L-selectin Shedding Lowers Leukocyte Rolling Velocity under Physiologic Conditions In Vivo. Educational score: 4.156106948852539 Domain: biomedical Document type: Study Language: en To explore the role of L-selectin shedding during cytokine-induced inflammation, we used intrascrotal injection of TNF-α 3 h before the observation period, as described previously ( 6 ). Treatment of wild-type mice with intrascrotal injections of 0.5 μg TNF-α 2 h before the experiments causes the expression of E-selectin and increased expression of P-selectin on the endothelial cells ( 36 ). Presence of E-selectin on the endothelium of the cremaster venules lowers the mean leukocyte rolling velocity by almost one order of magnitude to under 5 μm/s ( 37 ). Fig. 8 A shows results of inhibition of L-selectin shedding with KD-IX-73-4 in TNF-α–treated wild-type mice. The mean velocity of 300 rolling leukocytes (4.5 μm/s) before application of the protease inhibitor did not change significantly compared with the mean of 300 rolling leukocytes (4.1 μm/s) obtained between 1 and 6 min after the application of 150 μg KD-IX-73-4. Section title: Inhibition of L-selectin Shedding Lowers Leukocyte Rolling Velocity under Physiologic Conditions In Vivo. Educational score: 4.166074752807617 Domain: biomedical Document type: Study Language: en We hypothesized that E-selectin–mediated leukocyte rolling proceeded at such low velocities that inhibition of L-selectin shedding did not influence the actual velocity because the low off-rate of E-selectin ( 38 ) would determine and dominate the rolling velocity, no matter whether L-selectin was being shed or not. Previous studies had shown that rolling velocities were significantly higher in E-selectin– deficient mice treated with TNF-α than in wild-type mice ( 37 ). To explore the reason for the apparent absence of an effect of inhibiting L-selectin shedding in TNF-α–treated mice, we used TNF-α–treated E-selectin–deficient mice. In these mice, mean rolling velocity was 24 μm/s and was reduced to 17 μm/s after treatment with KD-IX-73-4 . Section title: Discussion Educational score: 4.284364700317383 Domain: biomedical Document type: Study Language: en Although proteolytic cleavage of L-selectin from the surface of leukocytes was described as early as 1989 ( 11 , 12 ), the physiologic relevance of L-selectin shedding in vivo has remained unclear and the subject of controversial speculations. The results of this study establish L-selectin shedding as a novel parameter, in addition to adhesion molecule expression and hemodynamic forces, relevant for regulation of leukocytes rolling. Continuous L-selectin shedding appears necessary for physiologic leukocyte rolling at typical velocities. Though Walcheck et al. have noted that inhibition of L-selectin shedding lowers the rolling velocity of isolated neutrophils on immobilized MECA-79 antigen ( 23 ), the results of other in vitro experiments using cultured endothelial cells seemed to contradict their conclusion ( 24 ). In experiments with TNF-α–treated HUVECs, a role for L-selectin shedding in neutrophil attachment, rolling, or transmigration could not be shown. Our results in untreated and TNF-α–treated wild-type mice can explain this discrepancy and establish the role of L-selectin shedding for leukocyte trafficking. Section title: Discussion Educational score: 4.22991418838501 Domain: biomedical Document type: Study Language: en When E-selectin is expressed on endothelial cells, inhibition of L-selectin shedding has no effect on leukocyte rolling velocity. However, when E-selectin is not expressed, either in wild-type mice not treated with an inflammatory cytokine, or in E-selectin–deficient mice, the modulatory role of L-selectin shedding is evident. In TNF-α–treated cultured HUVECs ( 24 ), E-selectin is likely to be expressed. This would explain why Allport et al. ( 24 ) did not find a velocity difference between human neutrophils with or without incubation with a zinc-dependent metalloproteinase inhibitor . In the experiment of Walcheck et al. ( 23 ), E-selectin is not present, because a purified L-selectin ligand (MECA-79) is used as the sole adhesive substrate. We investigated the effect of inhibiting L-selectin on rolling in vivo both in the presence and absence of E-selectin. In the absence of E-selectin, inhibition of L-selectin shedding causes a significant decrease of rolling velocity (as in reference 23 ), which is not detectable when E-selectin is involved in mediating leukocyte rolling (as in reference 24 ). Section title: Discussion Educational score: 4.210015296936035 Domain: biomedical Document type: Study Language: en As presented in Fig. 2 , a shift towards lower leukocyte rolling velocities can be seen with the inhibition of L-selectin shedding in wild-type mice without intentional induction of inflammation. The amount of protease inhibitor used in these animals provided a maximum inhibition of L-selectin shedding, since further injections of KD-IX-73-4 did not lower the rolling velocity of the cells further. The shift of velocity in these animals shows that normal leukocyte rolling requires continuous shedding of L-selectin. Consistent with this, Palecanda et al. detected soluble L-selectin in the plasma of healthy adults, whose peripheral blood leukocytes did not demonstrate any obvious signs of activation ( 39 ), and Schleiffenbaum et al. found high physiologic levels of soluble L-selectin in the serum of healthy volunteers ( 16 ). Since no soluble or secreted splice variants of L-selectin are known, we hypothesize that continuous L-selectin shedding during physiologic leukocyte rolling is a likely source for soluble L-selectin in the plasma of healthy individuals. Section title: Discussion Educational score: 4.183773994445801 Domain: biomedical Document type: Study Language: en The fact that no change in the rolling velocity can be measured with the protease inhibitor KD-IX-73-4 in L-selectin– deficient mice shows that the observed shift of velocities in wild-type mice was specifically and exclusively caused by the inhibition of L-selectin shedding. Other molecules such as P- or E-selectin, which also mediate leukocyte rolling, do not contribute to the shift of velocity caused by the application of KD-IX-73-4. Section title: Discussion Educational score: 4.338551044464111 Domain: biomedical Document type: Study Language: en As a negative control, we used the hydroxamic acid– based substance (SI), which is structurally closely related to the protease inhibitor KD-IX-73-4 but does not inhibit L-selectin shedding as shown by flow cytometry. Interestingly, after the use of SI, subsequent injections of KD-IX-73-4 did not show a shift of velocity. Therefore, SI may interact with the protease responsible for L-selectin shedding at the same binding site as KD-IX-73-4, thus competing with KD-IX-73-4 binding. PMNs from KD-IX-73-4– treated wild-type mice do not shed L-selectin in response to PMA activation. However, the inhibitory effect of KD-IX-73-4 in vivo is not permanent, as PMNs obtained 15 min after KD-IX-73-4 treatment responded to PMA activation by shedding L-selectin. This parallels our observations from the functional assay that the shift of velocity lasts for ∼10 min after one injection. The inhibition of L-selectin shedding in vivo and the velocity shift in the functional assay can both be observed after the injection of the protease inhibitor KD-IX-73-4. These data are consistent with a causal relationship between the injection of KD-IX-73-4, the inhibition of the L-selectin shedding, and the shift of rolling velocity. Section title: Discussion Educational score: 4.319692611694336 Domain: biomedical Document type: Study Language: en The current findings show that proteolytic cleavage of L-selectin shedding is relevant for physiologic leukocyte rolling. Since enzymatic processes often require metabolic energy, it may be worthwhile exploring whether L-selectin–mediated leukocyte rolling is modulated by cell metabolism or whether it is as independent of cell metabolism as formerly believed ( 40 , 41 ). Furthermore, in light of the present study, a revision of the calculated dissociation rate of L-selectin appears necessary, which was made with the assumption that during stable rolling the average number of bonds that form and break will be equal ( 9 ). This assumption may prove erroneous, since inhibition of L-selectin shedding decreases rolling velocity. Therefore, we believe that the 7.5–11.5-fold faster L-selectin–mediated rolling does not necessarily require a 7–10-fold more rapid bond dissociation, as proposed by Alon et al. ( 9 ). Rather, continuous cleavage of L-selectin and breakage of bonds are likely to occur at the same time and thus cause short duration of L-selectin–mediated tethering events. Section title: Discussion Educational score: 4.167973041534424 Domain: biomedical Document type: Study Language: en In conclusion, inhibition of L-selectin shedding decreases the leukocyte rolling velocity and increases the leukocyte rolling flux in vivo. A decreased rolling velocity increases the transit time of rolling leukocytes. Leukocyte transit times have recently been shown to be an important determinant of leukocyte recruitment in vivo ( 42 ). Modulation of L-selectin shedding is a novel and influential parameter in the leukocyte trafficking by virtue of its regulatory function on rolling velocity, rolling flux, and transit time of leukocytes. | Study | biomedical | en | 0.999996 |
10075978 | Section title: Mice. Educational score: 1.81117844581604 Domain: biomedical Document type: Other Language: en Male Balb/cByJ mice at 8–14 wk of age were obtained from The Jackson Laboratory . Mice were housed at least 1 wk before experimentation. Mice were cared for and handled at all times in accordance with the National Institutes of Health and our institutional guidelines. Section title: B Cell Purification. Educational score: 4.123534202575684 Domain: biomedical Document type: Study Language: en Splenic B cells from 8–12-wk-old naive Balb/cByJ mice were purified and depleted of T cells and macrophages as previously described ( 22 ). RBCs and nonviable cells were removed by sedimentation over Lympholyte M (Cedarlane Labs., Canada). The resulting B cells were cultured at 37°C with 5% CO 2 in RPMI 1640 medium (BioWhittaker) supplemented with 5% heat-inactivated fetal bovine serum ( Sigma Chemical Co. ), 10 mM Hepes (pH 7.2), 50 μM 2-ME, 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. Section title: Differential Display. Educational score: 4.234033107757568 Domain: biomedical Document type: Study Language: en Total RNA was prepared from primary murine splenic B cells stimulated with CD40L/CD8α fusion protein cross-linked with anti-CD8 antibody (CD40L) for 48 h, in the absence or presence of F(ab′) 2 fragments of polyclonal goat anti–mouse IgM (anti-Ig) added for the final 6 h of the culture period, using Phenol/GITC ( Sigma Chemical Co. ; 27). Reverse transcription and differential display were performed as previously described ( 28 ) using the RNAImage Kit (GenHunter). Putatively differentially expressed cDNA fragments were excised from dried sequencing gels, eluted in dH 2 O, and reamplified using the same primer pair originally used in differential display. These fragments were tested for differential expression by Northern blot analysis; PCR products confirmed by this assay were subcloned into a TA cloning vector (Invitrogen). Plasmid DNA from individual clones was radiolabeled and used to probe additional Northern blots in order to identify the insert responsible for differential expression. Subcloned Northern blot–positive cDNAs were subject to automated fluorescent DNA sequencing (Applied Biosystems) and analyzed by comparison to standard sequencing databases in the public domain (BLAST; National Center for Biotechnology Information). Section title: Northern Blotting. Educational score: 4.140289306640625 Domain: biomedical Document type: Study Language: en Total RNA was prepared from primary murine splenic B cells using UltraSpec RNA isolation reagent (Biotecx Laboratories). Purified RNA was electrophoresed on a 1% agarose/formaldehyde gel, transferred to GeneScreen Plus (NEN) in 10× SSC and hybridized to a 234-bp radiolabeled fragment of faim generated by PCR, using the primers CTGGATGGCGAGGACCTGAG (5′) and GGTGTCACTGAGTGAGCTCTG (3′). Initial Northern probing to confirm differential expression was performed as above except that differential display primers were used and the annealing step of PCR was performed for 2 min at 40°C. Autoradiography was performed using intensifying screens at −80°C for 1–3 d. A multiple tissue Northern blot was obtained from OriGene Technologies. Section title: cDNA Library Screening. Educational score: 4.1474103927612305 Domain: biomedical Document type: Study Language: en A radiolabeled probe generated as described above was used to screen a directional murine thymic cDNA library that was constructed in pBKCMV (Stratagene) and was provided by Dr. Adam Lerner (Boston University Medical Center, Boston, MA). Plaque lifts were performed using Protran membranes (Schleicher and Schuell). A number of individual clones from among 10 6 plaques were sequenced, leading to the isolation of a full-length clone as determined by an in-frame stop codon upstream of the start methionine. This clone encodes a novel 179–amino acid protein as discussed below. Section title: Transfection. Educational score: 4.134829998016357 Domain: biomedical Document type: Study Language: en Mid-log phase BAL-17 B lymphoma cells in suspension were transfected with 20 μg faim -containing plasmid or pBKCMV empty vector (plus 500 μg carrier salmon sperm DNA) by electroporation at 276 V and 550 μF using a Gene Pulser apparatus (Bio-Rad). Transfected cells were immediately plated to prewarmed medium and cultured at 37°C with 5% CO 2 as above ( 29 ). After 2 d, transfected cells were split 1:15 into fresh medium containing 2 mg/ml G418 ( Sigma Chemical Co. ) to obtain pools of transfectants. Separately, individual stably transfected clones were isolated by limiting dilution in medium containing G418. Section title: Fas-mediated Apoptosis. Educational score: 4.115907669067383 Domain: biomedical Document type: Study Language: en BAL-17 transfectants stimulated for 24 h with CD40L were tested as targets in standard 4-h, lectin-dependent 51 Cr-release assays with AE7 CD4 + Th1 effector cells at effector/target cell ratios of 0.3:1 to 9:1, as previously described ( 22 , 25 ), or with Jo-2 anti-Fas antibody ( PharMingen ) at 50, 5, or 0.5 ng/ml. Alternatively, nuclei obtained from CD40L-stimulated BAL-17 transfectants were stained with propidium iodide and the level of subdiploid DNA was determined by flow cytometry, as described ( 30 ). Section title: PARP Cleavage. Educational score: 4.091708183288574 Domain: biomedical Document type: Study Language: en B cell protein lysates were resolved by 15% SDS-PAGE, transferred to Hybond nitrocellulose membranes ( Amersham ), and blocked with 5% nonfat dry milk in TBS-Tween 20 (TBS-T) for 1 h at room temperature. Membranes were probed with anti-PARP antibody 2-C-10 ( Calbiochem ) at 1:500 in TBS-T. After washing, blots were incubated with horseradish peroxidase–conjugated goat anti–mouse IgG and developed by enhanced chemiluminescence (NEN), as previously described ( 31 ). Section title: Fas Expression. Educational score: 4.040744781494141 Domain: biomedical Document type: Study Language: en B cells were stained with PE-conjugated Jo-2 Fas-specific antibody or anti-TNP isotype control antibody ( PharMingen ) in the presence of 2% normal rabbit serum and 2.4G2 (anti-FcR) antibody, as previously described ( 32 ). Relative fluorescence intensity was detected by flow cytometry with a FACScan ® instrument ( Becton Dickinson ). Section title: FAIM Expression. Educational score: 4.140105247497559 Domain: biomedical Document type: Study Language: en Two peptides (amino acids 57–68 and 125–138) corresponding to predicted hydrophilic regions of the FAIM open reading frame ( 33 ) were synthesized by Research Genetics Corp. These peptides contain an NH 2 -terminal cysteine followed by an amino-caproic acid. Each peptide (2 mg) was separately coupled to KLH ( Pierce Chemical Co. ); the coupled peptides were combined and used to generate anti-FAIM peptide antibodies in chickens (Aves Labs.). B cell protein lysates were resolved by 15% SDS-PAGE, transferred to Hybond nitrocellulose membranes ( Amersham ), and blocked with 10% Blok-Hen blocking reagent (Aves Labs.) for 1 h at room temperature. The nitrocellulose filters were then probed with FAIM-specific antibodies diluted 1:1,000 in TBS-T containing 10% Blok-Hen for 1.5 h at room temperature. After washing, blots were incubated with horseradish peroxidase–conjugated goat anti–chicken IgY (Aves Labs.) for 1 h and developed by enhanced chemiluminescence (NEN) as described ( 31 ). Section title: Reagents. Educational score: 4.090341567993164 Domain: biomedical Document type: Study Language: en Affinity purified F(ab′) 2 fragments of polyclonal goat anti–mouse IgM were obtained from Jackson ImmunoResearch Labs. and used at 10 μg/ml. Soluble rCD40L was obtained from transfected J558L cells that secrete a chimeric CD40L/ CD8α fusion protein ( 34 ), which was collected and dialyzed against 25,000 mol wt cut-off dialysis tubing, as previously described ( 35 ). A similarly dialyzed supernatant containing anti-CD8 antibody from the 53-6-72 hybridoma was used to cross-link the fusion protein. CD40L and anti-CD8 containing supernatants were used at final dilutions of 1:10 and 1:80, respectively. G418 was obtained from GIBCO BRL . An expressed sequence tag (EST) encompassing putative human FAIM was obtained from the I.M.A.G.E. consortium ( 36 ). 51 Cr was obtained from NEN. Section title: Results Educational score: 4.195572853088379 Domain: biomedical Document type: Study Language: en In previous work we showed that B cell treatment with anti-Ig for only the final 1–12 h of a 48-h culture with CD40L produced a time-dependent increase in Fas resistance that was abrogated by cycloheximide ( 25 ). Additional experiments demonstrated that the induction of Fas resistance in CD40L-stimulated B cells by anti-Ig treatment for 6 h was completely blocked by the addition of actinomycin D (data not shown). These results strongly suggest that transcriptional activation and gene expression are required for the receptor-specific induction of the Fas-resistant state. For this reason, genes that oppose Fas-mediated apoptosis might be captured by identifying transcripts expressed uniquely in Fas-resistant B cells. Section title: Differential Display Detects Transcripts Specific for B Cells Rendered Fas Resistant. Educational score: 4.21323299407959 Domain: biomedical Document type: Study Language: en To identify genes expressed coordinately with the induction of Fas resistance, we used a differential display strategy ( 28 ). RNA was extracted from B cells stimulated with CD40L alone for 48 h (Fas-sensitive) and from B cells stimulated with CD40L (for 48 h) plus anti-IgM for the final 6 h of culture (Fas-resistant), and was reverse transcribed. Application of arbitrary decameric primer pairs to these cDNA populations permitted reproducible amplification of a number of transcripts present in Fas-resistant B cells but absent in their Fas-sensitive counterparts. These amplified gene fragments were excised and used as probes in Northern blots of RNA obtained from Fas-sensitive and Fas-resistant primary B cells to confirm differential expression . Of 40 such fragments, 8 failed to reamplify. Of the remaining 32, 8 displayed differential expression by Northern blot analysis. One of these recognized an ∼1.2-kb transcript on Northern blot that was widely expressed in multiple tissues, with the highest levels present in murine brain, thymus, kidney, and spleen . This transcript was chosen for further analysis. Section title: A Differentially Expressed Gene Encodes a Novel Protein. Educational score: 4.264622211456299 Domain: biomedical Document type: Study Language: en Using a radiolabeled probe generated by PCR, a murine thymic cDNA library was screened and the DNA from positive plaques was sequenced. A number of overlapping clones were identified whose consensus sequence was ∼1.2 kb, consistent with the expression data described above. Subsequently a full-length clone was identified that contained an in-frame STOP codon upstream of the START methionine, and possessed, in the 3′ UTR, an RNA instability motif, polyA + consensus motifs and a polyA + tail ( 37 ). This cDNA appeared to encode a novel 179–amino acid open reading frame . Structural analysis predicted a β-strand–rich, stable, soluble protein with a slightly acidic pI (pH 5.4). No substantial regions of homology with any other sequence are present. Section title: A Full Length Clone Functions as a Fas Apoptosis Inhibitory Molecule (FAIM). Educational score: 4.449055194854736 Domain: biomedical Document type: Study Language: en To determine the capacity of the isolated cDNA clone to produce resistance to Fas-mediated apoptosis, BAL-17 murine B lymphoma cells were transfected with the pBKCMV expression vector. BAL-17 cells were chosen because their activation responses mimic primary B cells in a variety of ways and they are readily transfectable ( 38 – 40 ). Like primary B cells, unstimulated BAL-17 B cells express little Fas, but treatment with CD40L induces Fas expression and sensitivity to Fas-mediated apoptosis (data not shown). After electroporation, pools of BAL-17 B cells stably transfected with either full-length cDNA or empty vector were selected in G418 for 2 wk. These two populations differed in their susceptibility to Fas killing induced by FasL-bearing Th1 effector cells: at each effector/target cell ratio tested, specific lysis of cDNA-transfected BAL-17 B cells, detected by Cr-release assay, was reduced by half or more in comparison to cells transfected with vector alone , despite equivalent levels of surface Fas expression (data not shown). These results suggest a level of protection of ninefold or more, in terms of the effector/target ratio required to produce equivalent levels of apoptosis in faim - and vector-transfected BAL-17 B cells. The reduction in Fas killing was also apparent when cytotoxicity was induced by lytic Jo-2 anti-Fas antibody (data not shown). In addition, stably transfected clones were isolated by limiting dilution and tested for susceptibility to Fas killing. The results obtained with individual clones completely mimicked those obtained with G418-resistant pools in that Fas-mediated apoptosis (produced by Jo-2) was reduced by one-half to two-thirds in stably transfected cDNA-expressing BAL-17 B cells in comparison to BAL-17 cells transfected with empty vector, as detected by propidium iodide staining for subdiploid DNA . These data indicate that the novel cDNA transcript initially identified in inducibly Fas-resistant B cells codes for a Fas apoptosis inhibitory molecule (FAIM) that counteracts Fas signaling for cell death when overexpressed. Section title: FAIM Expression Blocks PARP Cleavage. Educational score: 4.2110819816589355 Domain: biomedical Document type: Study Language: en To characterize the nature of the FAIM-induced block in Fas signaling for cell death, the fate of PARP, a terminal caspase cleavage product, was examined by Western blot of size-separated whole cell extracts ( 41 ). Proteolytic fragments of PARP were readily detected when vector-transfected BAL-17 B cells were treated for 40 min with Jo-2 anti-Fas antibody. In contrast, there was little or no PARP cleavage in faim -transfected BAL-17 B cells up to 60 min after anti-Fas treatment . Thus, FAIM blocks Fas apoptosis at a step proximal to the cleavage of the caspase substrate, PARP. Section title: FAIM Expression Blocks PARP Cleavage. Educational score: 4.137596607208252 Domain: biomedical Document type: Study Language: en As a control for these experiments, FAIM expression in faim - and vector-transfected BAL-17 B cells was determined by Western blotting with polyclonal anti-FAIM antibody. Antibody was prepared by immunizing chickens with two relatively hydrophilic peptides derived from the FAIM sequence (amino acids 57–68, DGKEEIRREWMF; and 125–138, RLDGEDLRVVLEKD) coupled to KLH; the resultant antibody (purified from the IgY fraction of egg yolks) specifically recognized a protein of the expected size, ∼20 kD, on Western blot, whereas pre-immune IgY did not. Using this antibody, the expression of FAIM protein was found to be much increased in faim -transfected, as opposed to vector-transfected, BAL-17 B cells . Section title: faim Expression Correlates Well with Inducible Fas Resistance. Educational score: 4.162311553955078 Domain: biomedical Document type: Study Language: en To further evaluate the association between FAIM expression and inducible Fas resistance, primary B cells were studied by Northern and Western blotting after stimulation with CD40L and anti-Ig. CD40L stimulation alone, which induces Fas expression and Fas sensitivity, elicited little or no increase in faim expression over the low basal level present in unstimulated B cells. However, addition of anti-IgM, which induces Fas resistance, to CD40L-stimulated B cells produced a marked, time-dependent increase in faim mRNA, beginning at 1 h and reaching a maximum after 6 h of anti-Ig treatment . Similar results were obtained when the expression of FAIM protein was monitored. FAIM was absent in unstimulated B cells and B cells stimulated for 48 h with CD40L alone; however, addition of anti-IgM to CD40L-stimulated B cells produced a marked increase in FAIM protein, first seen after 18 h of anti-Ig treatment . In some experiments FAIM protein expression was detected after 6 h of anti-Ig treatment (data not shown). Section title: faim Expression Correlates Well with Inducible Fas Resistance. Educational score: 4.195446014404297 Domain: biomedical Document type: Study Language: en The correlation between sIg-induced FAIM expression and Fas resistance was tested further by examining tolerant, autoreactive B cells obtained from double transgenic, anti– hen egg lysozyme (HEL)/HEL mice. In these B cells, in vitro studies showed that specific antigen (soluble [s]HEL) is an insufficient stimulus to produce Fas resistance, whereas Fas resistance is induced by more extensive sIg cross-linking with anti-Ig ( 42 ). In keeping with this, sHEL failed to induce upregulation of FAIM protein expression in B cells drawn from double transgenic anti-HEL/HEL mice, whereas FAIM protein was induced by anti-Ig in these B cells . In this situation, as with anti-Ig–treated B cells from normal mice, above, induction of FAIM expression correlates with production of Fas resistance. Section title: faim Is Evolutionarily Conserved. Educational score: 4.159538269042969 Domain: biomedical Document type: Study Language: en To evaluate the possibility that faim is phylogenetically conserved, public databases were searched for evidence of similar genes in other species. Human faim was obtained by identifying a consensus sequence from overlapping human ESTs with homology to mouse faim , followed by sequencing of a single EST clone that completely spanned putative human faim ( 36 ). The consensus/EST sequence was used to predict an amino acid sequence, which showed human FAIM to be 90% identical to the predicted amino acid sequence of mouse FAIM . These results are complemented by Southern blot analysis of genomic DNA showing hybridization by a mouse faim probe to all mammalian species tested (human, monkey, rat, mouse, dog, cow, and rabbit; data not shown). Section title: faim Is Evolutionarily Conserved. Educational score: 4.276463985443115 Domain: biomedical Document type: Study Language: en C . elegans FAIM was obtained by amplifying cDNA with primers based on the predicted exon structure of a random genomic sequence of unknown function, and then sequencing the resultant DNA. The predicted amino acid sequence of this C . elegans FAIM is 50% identical to the predicted amino acid sequence of mouse FAIM . The extensive evolutionary conservation manifest in the sequences of human, mouse, and C . elegans faim strongly suggests that the faim gene product is a key apoptotic regulatory molecule that has been retained with minimal change throughout phylogeny. Section title: Discussion Educational score: 4.542444229125977 Domain: biomedical Document type: Study Language: en Fas engagement contributes to cell fate determination in a variety of cell types, and plays an important role in modulating immune responses and regulating autoreactive B lymphocytes ( 23 , 42 ). The results reported here identify a novel gene, isolated by differential display ( 28 ) and termed faim , that opposes Fas-mediated apoptosis when overexpressed in a model B cell line, and whose expression in primary B cells is coordinately regulated with sIg signals that block Fas killing. Thus, FAIM is an inducible effector molecule that mediates Fas resistance produced in B cells by sIg engagement, and does so by blocking a step in the Fas signaling pathway before the activation of caspase 3, an effector caspase responsible for PARP cleavage ( 41 ). The influence of FAIM appears to be specific to Fas-induced cell death, because BAL-17 B cells stably transfected with faim are not more resistant to apoptosis produced by staurosporine or ultraviolet radiation than vector-transfected controls (data not shown). As FAIM bears no regions of homology with other gene products that modulate Fas killing, it appears to represent a distinct new class of antiapoptotic protein. Section title: Discussion Educational score: 4.650987148284912 Domain: biomedical Document type: Study Language: en Overexpression of FAIM in stably transfected BAL-17 B cells produced substantial, but less than complete, resistance against Fas-mediated apoptosis, and was less effective in blocking Fas killing than optimal sIg signaling in primary B cells. This suggests that receptor-mediated Fas resistance in primary B cells cannot be explained on the basis of FAIM induction alone. Recently we reported that Bcl-x L is induced in primary B cells after sIg engagement that produces Fas resistance, and that the Fas sensitivity of CD40L-stimulated B cells from Bcl-x L overexpressing transgenic mice was substantially reduced compared with littermate control B cells ( 26 ). These results infer a key role for Bcl-x L in sIg-mediated Fas resistance. However, further inhibition of susceptibility to Fas-mediated apoptosis in Bcl-x L transgenic B cells was produced by anti-Ig treatment, implicating additional antiapoptotic factors ( 26 ). Thus, sIg-triggered Fas resistance may result, at least in part, from the combined effects of inducible FAIM and Bcl-x L expression. In support of this, Fas-resistant faim transfectants express no more Bcl-x L than do vector-transfected controls (data not shown), so there is no direct linkage between FAIM and Bcl-x L , the only other antiapoptotic gene presently implicated in B cell Fas resistance. Thus, inducible resistance against Fas is multigenic in nature. Although FAIM and Bcl-x L may inhibit separate and distinct intracellular pathways leading from Fas to terminal apoptotic events ( 43 ), the possibility that these antiapoptotic molecules interfere with the same death mediators has not been ruled out. Notably, the multigenicity of inducible protection against Fas killing suggests the intricacy of regulatory controls asserted over apoptotic death by environmental cues. Section title: Discussion Educational score: 4.501778602600098 Domain: biomedical Document type: Study Language: en FAIM shares no regions of homology with known antiapoptotic proteins and contains no known protein–protein interaction motifs, implying that an as yet unidentified structure is responsible for its antiapoptotic function. Preliminary results suggest that the activity of this structure is broad and crosses species boundaries, in that human HeLa cells transiently transfected with mouse faim display Fas resistance in comparison to vector-transfected controls (data not shown). Moreover, FAIM-like genes are expressed in other species, including C . elegans . In fact, the degree of amino acid identity between C . elegans and mouse FAIM (50%) is much greater than that between C . elegans ced-3 and mouse ICE, and between C . elegans ced-9 and mouse bcl-2 (both <25%), two pairs of genes with accepted similar functions related to apoptosis ( 44 , 45 ). This remarkably high degree of evolutionary conservation across a broad phylogenetic chasm suggests that FAIM plays a key role in cellular physiology. Furthermore, three specific regions of FAIM, encompassing 21, 17, and 17 amino acids, respectively, exceed 75% identity between mouse, human, and C . elegans FAIM; one or more of these regions may well be indispensable to the antiapoptotic function of FAIM. | Other | biomedical | en | 0.999997 |
10075979 | Section title: Mice. Educational score: 3.179295063018799 Domain: biomedical Document type: Study Language: en p53 gene–deficient, Bcl-2 transgenic (Tg), DO.11.10 OVA TCR-α/β Tg, and P14 TCR-α/β Tg mice have been described previously ( 30 – 33 ). BALB/c mice were purchased from Taconic Farms. All mice were kept at the Animal Facility of the Ontario Cancer Institute in accordance with institutional guidelines. Section title: Apoptosis Induction and Inhibitors. Educational score: 4.154112815856934 Domain: biomedical Document type: Study Language: en Freshly isolated thymocytes from BALB/c mice were cultured in RPMI 1640 medium (10% FCS, 10 −5 M β-mercaptoethanol) in the absence or presence of dexamethasone ( Sigma ), heat shock, γ-irradiation, anti-CD95 Ab (clone Jo91; PharMingen ), anti-CD3ε (clone 145-2C11; PharMingen ), PMA (12.5 ng/ml), or etoposide (2.5 μg/ml) for different time periods and at different concentrations as indicated in the figure legends ( 34 ). Optimal concentrations and activation regimes for the induction of apoptosis were determined in pilot studies. The specific Cdk2 blockers olomoucine ( Calbiochem ) and roscovitine (gift of Dr. Meijer, CNRS, Roscoff, France) were dissolved in DMSO ( Sigma ). Titration experiments determined that 100 μM olomoucine and 50 μM roscovitine were the most effective concentrations for inhibiting Cdk2 activity and apoptosis in thymocyte cultures. DMSO had no effect on Cdk2 activity or the induction or prevention of apoptosis in response to all stimuli tested. The cell cycle blockers TGF-β (1 nM; R&D Systems) and rapamycin (2 ng/ml; Calbiochem ) were used at concentrations that optimally blocked cell cycle progression in T cell lymphoma cells. Section title: Detection of Apoptosis. Educational score: 4.1781086921691895 Domain: biomedical Document type: Study Language: en Total cell numbers of viable and apoptotic cells were determined by trypan blue exclusion. Relative percentages of viable and apoptotic CD4 + CD8 + thymocytes were determined by triple staining with anti-CD4–PE, anti-CD8–FITC, and the vital chromogenic dye, 7AAD ( 34 ). The results were expressed as the percentage of viable thymocytes remaining after 22 h, calculated as follows: (number of viable CD4 + CD8 + thymocytes after stimulation)/(number of viable CD4 + CD8 + thymocytes cultured under the same conditions in the absence of stimulation) × 100. For the detection of cycling and apoptotic cells, thymocytes were stained with propidium iodide (PI). After different periods of stimulation, thymocytes were harvested, washed once in PBS (0.5% glucose), and fixed in cold 70% ethanol overnight. Fixed cells were pelleted to remove ethanol and stained with PI (final concentration 50 μmol/ml) for 30 min at room temperature. Apoptosis-mediated membrane changes were determined via staining with Annexin V (R&D Systems). PI and Annexin V staining of thymocytes was determined by cytofluorometry using a FACSCalibur™ ( Becton Dickinson ). Section title: Kinase Assays. Educational score: 4.141665935516357 Domain: biomedical Document type: Study Language: en After different periods of stimulation, thymocytes were harvested and lysed, and proteins were immunoprecipitated using Abs against Cdk2 (amino acids [aa] 283–298), Cdk4 (aa 282–303), and Cdc2 (aa 278–297) (all from Santa Cruz Biotechnology ). Cdk2 and Cdc2 kinase activities in immunoprecipitates were assayed using [γ- 32 P]ATP (3,000 cpm/pmol) and histone H1 (2 μg/ml; Boehringer Mannheim ) or p53 (2 μg/ml; PharMingen ) as substrates. Cdk4 activity was determined using glutathione S -transferase (GST)-Rb as a substrate (2 μg/ml; PharMingen ). H1, p53, or GST-Rb phosphorylation was assayed by autoradiography after SDS-PAGE separation. The levels of immunoprecipitated Cdk2, Cdc2, and Cdk4 were determined by Coomassie blue staining and Western blotting. Section title: Immunoprecipitations and Western Blotting. Educational score: 4.119800567626953 Domain: biomedical Document type: Study Language: en Thymocytes were lysed in 1% NP-40 lysis buffer. Proteins were separated by SDS-PAGE, transferred onto nitrocellulose membranes, and incubated with Abs reactive to Cdc2, Cdk2, Cdk4, Cdk7, Pctaire-2, Cdc25A, cyclins A, E, D1, D2, B, and D3, E2F-1, p27 Kip1 , caspase 2, and p53 (clone 240) (all from Santa Cruz Biotechnology ), p21 ( Calbiochem ), Bcl-XL (Transduction Laboratories), Bcl-2 ( PharMingen ), caspase 3 (gift of Dr. R. Sekaly, McGill University, Montreal, Quebec, Canada), and Rb (clone G3-245 reactive to an aa 300–380 epitope of Rb, PharMingen ; and clone C-15 reactive against aa 914–928, Santa Cruz Biotechnology ). The anti-caspase 8–specific Abs were developed in our Institute and were a kind gift of Dr. R. Hakem (Amgen Institute). Immunoprecipitations were performed using protein A–Sepharose. Optimal Ab concentrations and conditions for immunoprecipitations were determined in pilot studies. Section title: ΔΨ m Disruption. Educational score: 4.173295021057129 Domain: biomedical Document type: Study Language: en The mitochondrial transmembrane potential (ΔΨ m ) results from the asymmetric distribution of protons across the inner mitochondrial membrane, giving rise to a chemical (pH) and electric gradient ( 35 , 36 ). The inner side of the inner mitochondrial membrane is negatively charged. As a consequence, the cationic lipophilic fluorochrome 3,3′-dihexyloxacarbocyanine iodide (DiOC 6 (3)) is distributed on the mitochondrial matrix as a function of the Nernst equation, correlating with ΔΨ m . DiOC 6 (3) can be used to measure variations in the ΔΨ m on a per-cell basis. Cells induced to undergo apoptosis manifest an early reduction in the incorporation of ΔΨ m -sensitive dyes, indicating a disruption of ΔΨ m . For DiOC 6 (3) staining, 10 6 thymocytes were incubated with DiOC 6 (3) (final concentration 20 nM in PBS) for 20 min at 37°C. DiOC 6 (3) staining was analyzed immediately using a FACSCalibur™. Section title: In Vitro Negative Thymocyte Selection. Educational score: 4.17108678817749 Domain: biomedical Document type: Study Language: en Thymocytes were purified from P14 Tg mice, which express an α/β TCR (TCR Vα2Vβ8) specific for a peptide (p33) of the lympholytic choriomeningitis virus (LCMV). P14 Tg thymocytes (10 6 /well) were cultured on a monolayer of confluent and adherent MC57/L fibroblasts (H-2 b/b ) in RPMI medium (5% FCS, 10 −5 M β-mercaptoethanol). MC57/L APCs were pulsed with different concentrations of the deleting LCMV-p33 peptide for 2 h before coculture with thymocytes. Thymocytes were harvested after 22 h incubation and stained with anti-CD4–PE, anti-CD8–FITC, and the dye, 7AAD. Percent survival was calculated as follows: (total number of viable CD4 + CD8 + thymocytes cultured at different LCMV-peptide concentrations)/(total number of viable CD4 + CD8 + thymocytes cultured with MC57/L cells at 37°C in the absence of peptide) × 100. Total numbers of viable and dead cells were determined by trypan blue exclusion. For detection of Cdk2 activity in peptide-activated thymocytes, P14 Tg thymocytes (10 7 /well) were cultured on a monolayer of confluent (noncycling) and adherent MC57/L fibroblasts pulsed with 10 −5 M of the deleting p33 peptide ( 37 , 38 ). After 5 h incubation, thymocytes were separated from adherent fibroblasts and subjected to Cdk2 kinase assays as above. Section title: Fetal Thymic Organ Culture. Educational score: 4.118200302124023 Domain: biomedical Document type: Study Language: en DO.11.10 males were mated with estrous BALB/c females. On day 16 of gestation, pregnant females were killed and embryonic thymi were harvested and placed in culture. Thymi were microdissected and placed on the surface of 0.8-mm filters (Nucleopore) resting on Gelfoam gelatin sponges (Upjohn) in RPMI 1640 medium supplemented with 10% FCS. Each sponge was placed into a 3.5-cm plastic dish in 2 ml of medium. Cultures were incubated at 37°C. Chicken (c)OVA protein was added at 1 mg/ml on day 1 of culture, and thymi were analyzed 20 and 40 h later. Cell numbers were determined by counting in the presence of trypan blue. Section title: Cdk2 Kinase Activity in Thymocyte Apoptosis. Educational score: 4.525474548339844 Domain: biomedical Document type: Study Language: en To determine whether the cell cycle machinery has a role in apoptosis of noncycling G1 CD4 + CD8 + thymocytes, we analyzed the activity of several Cdks in these cells. Freshly isolated thymocytes were treated with different death stimuli such as dexamethasone, heat shock, γ-irradiation, and CD95 for different time points, and Cdk activities of stimulated as well as control thymocytes were assessed in in vitro kinase assays. Surprisingly, increased activity of the cyclin-dependent kinase Cdk2 was detected within 30 min of dexamethasone activation and peaked at 5 h . Cdk2 activity was rapidly increased in response to all apoptotic stimuli tested, including dexamethasone, anti-Fas (CD95) cross-linking, heat shock, or γ-irradiation . No changes in the kinase activities of Cdk4 or Cdc2 were observed after induction of apoptosis . Cdc2 activation was also not observed using an anti-Cdc2 phosphorylation epitope-specific Ab indicative of Cdc2 activation. Cdc2 and Cdk4 activities were readily detectable in cycling T lymphoma cells (not shown). The expression levels of molecules involved in the cell cycle, such as Cdk2, Cdk4, Cdc2, Cdk7, Pctaire-2, Cdc25A, cyclins A, B, D1, D2, D3, and E, p21, p27 Kip1 , and E2F-1, did not change 5 h after stimuli (not shown). Cdk2 was found to bind to cyclin A and E thymocytes after treatment with dexamethasone and γ-irradiation. Immunoprecipitations of both cyclin A and cyclin E showed that after γ-irradiation or dexamethasone, histone H1 was phosphorylated, suggesting that both cyclin A and cyclin E have a role in Cdk2 activation . These results show that the induction of thymocyte apoptosis by dexamethasone, γ-irradiation, heat shock, or CD95 cross-linking leads to the activation of the cell cycle regulator Cdk2. Section title: Inhibition of Cdk2 Blocks Thymocyte Apoptosis. Educational score: 4.316617488861084 Domain: biomedical Document type: Study Language: en To test whether Cdk2 activity was required for the induction of thymocyte apoptosis, the effects of two specific inhibitors of Cdk2, olomoucine and roscovitine ( 39 , 40 ), were examined. These inhibitors are purine analogues that selectively inhibit the activity of Cdk2 and Cdc2 by specific binding to the ATP-binding pocket. Both molecules completely inhibited dexamethasone-induced Cdk2 activation in thymocytes , and blocked thymocyte apoptosis after stimulation with dexamethasone, heat shock, γ-irradiation, PMA, or the DNA damaging agent, etoposide . PI staining confirmed that thymocytes were in the G1 phase of the cell cycle and that induction of apoptosis did not correlate with cell cycle progression . A “point of no return” was reached between 15 and 30 min after treatment with the apoptotic stimulus such that a Cdk2 blocker added after this time was unable to prevent apoptosis . Addition of other cell cycle blockers such as TGF-β1 or rapamycin, used at the optimal concentrations, had no effect on the kinetics or extent of thymocyte death . Interestingly, although CD95 cross-linking led to strong Cdk2 activation , Cdk2 blockers did not inhibit CD95-mediated thymocyte death . In fact, CD95-mediated apoptosis was consistently enhanced in the presence of the Cdk2 blockers. These results imply that CD95 uses a pathway other than the one used by the other inducers, i.e., a receptor/ caspase 8 pathway instead of a nucleus/mitochondrial/ caspase 9 pathway ( 41 ). It should be noted that in our screen, CD95 activation is the only thymocyte death stimulus so far that cannot be blocked by Cdk2 inhibition. Section title: Cdk2 Kinase Activity Is Required for Peptide-specific Thymocyte Apoptosis. Educational score: 4.229155540466309 Domain: biomedical Document type: Study Language: en The process of clonal deletion and selection-triggered thymocyte death is a fundamental mechanism required for the maintenance of lymphocyte homeostasis and immunotolerance. CD4 + CD8 + thymocytes expressing TCRs which recognize self-antigens with high affinity/ avidity are clonally deleted via apoptosis, leading to the removal of T cells that express TCRs with potentially harmful self-reactivity (thymic tolerance ). To test whether Cdk2 is a physiological regulator of thymocyte apoptosis, we induced apoptosis of CD4 + CD8 + immature thymocytes by anti-CD3 cross-linking ( 42 ). Fig. 2 A shows that Cdk2 inhibitors were able to block anti-CD3–mediated apoptosis of immature thymocytes. Section title: Cdk2 Kinase Activity Is Required for Peptide-specific Thymocyte Apoptosis. Educational score: 4.326890468597412 Domain: biomedical Document type: Study Language: en To further investigate the role of Cdk2 in clonal deletion, we used an in vitro negative selection system using thymocytes from P14 Tg mice. P14 Tg mice express a rearranged TCR α/β chain reactive to the p33 peptide of LCMV. P14 Tg CD4 + CD8 + thymocytes undergo apoptosis after culture with APCs pulsed with different concentrations of the deleting p33 peptide. Thymocytes from P14 Tg mice underwent apoptosis in a p33 peptide dose- dependent fashion which was inhibited by the addition of Cdk2 blockers . Importantly, induction of peptide-specific apoptosis of P14 Tg thymocytes triggered Cdk2 kinase activity . Inhibition of Cdk2 did not interfere with TCR-mediated proximal signaling events or with TCR internalization, which is a functional measure of antigen receptor–mediated activation (not shown). Moreover, inhibition of Cdk2 by olomoucine blocked OVA-mediated negative selection of CD4 + CD8 + OVA-specific TCR Tg thymocytes in fetal thymic organ cultures . Cdk2 blockers did not interfere with positive thymocyte selection in reaggregation culture assays (not shown), indicating that Cdk2 has a specific role in peptide-specific thymocyte apoptosis. Section title: Cdk2 Acts Upstream of Mitochondrial Permeability Transition, Bcl-2, and Caspases. Educational score: 4.362143516540527 Domain: biomedical Document type: Study Language: en Where does Cdk2 function in the hierarchy of apoptosis? Disruption of ΔΨ m due to the opening of mitochondrial pores has been invariably associated with apoptosis and is an early common denominator of cell death ( 43 ). Alterations in mitochondria lead to the release through the outer mitochondrial membrane of molecules such as cytochrome c and the apoptosis-inducing factor (AIF), and the activation of the caspase cascade ( 7 , 35 , 36 , 44 – 48 ). Section title: Cdk2 Acts Upstream of Mitochondrial Permeability Transition, Bcl-2, and Caspases. Educational score: 4.205999374389648 Domain: biomedical Document type: Study Language: en To assess whether Cdk2 acts upstream or downstream of mitochondrial events, we examined changes in ΔΨ m using cytometry and the fluorochromic dye, DiOC 6 (3). Thymocytes were stimulated either with dexamethasone or anti-CD95, and the mitochondria changes of ΔΨ m were assessed at different time points. The first changes in thymocyte ΔΨ m were observed 2 h after dexamethasone treatment, and ΔΨ m was significantly disrupted after 5 h . Addition of Cdk2 inhibitors blocked dexamethasone-induced losses of ΔΨ m . CD95-mediated ΔΨ m disruption and apoptosis still occurred in the presence of Cdk2 inhibitors , implying that Cdk2 inhibition per se does not interfere with opening of mitochondrial pores. Since ΔΨ m is regulated by Bcl-2 family members ( 43 , 49 ), we also tested Cdk2 activation in Bcl-2 Tg thymocytes ( 31 , 50 ). Although overexpression of Bcl-2 protected thymocytes from dexamethasone- and irradiation-induced cell death and disruption of ΔΨ m ( 31 , 50 ), Cdk2 was still activated in Bcl-2 Tg thymocytes in response to these apoptotic stimuli (not shown). Section title: Cdk2 Acts Upstream of Mitochondrial Permeability Transition, Bcl-2, and Caspases. Educational score: 4.215342998504639 Domain: biomedical Document type: Study Language: en Caspase activation is a crucial event in apoptosis, and caspases can function upstream or downstream of mitochondrial ΔΨ m disruption ( 8 , 9 ). To determine where Cdk2 acts during apoptosis with regard to the caspase activation cascade, processing of different caspases was assessed in thymocytes after treatment with different apoptotic stimuli in the presence or absence of Cdk2 inhibitors. Within 2 h after dexamethasone and γ-irradiation, caspase 3 (Cpp32) and caspase 8 activation was observed in thymocytes whereas caspase 2 (nedd2) processing was first observed 3 h after death induction. Caspase activation peaked at 5 h after induction of cell death . However, activation of caspase 3 , caspase 2 , or caspase 8 (not shown) did not occur after blocking of Cdk2 kinase activity. These results show that Cdk2 acts upstream of Bcl-2, ΔΨ m , and caspases. Section title: Proteolytic Processing of the Retinoblastoma Protein in Thymocyte Apoptosis. Educational score: 4.448150157928467 Domain: biomedical Document type: Study Language: en During apoptosis, various cell cycle regulatory molecules such as p21 and Rb are proteolytically cleaved by caspases. In particular, proteolytic processing of the G1 to S cell cycle gatekeeper Rb (ΔRb) has been previously reported in TNF- and CD95-treated tumor cell lines ( 51 , 52 ). Rb and Cdk2 were found to coimmunoprecipitate in developing thymocytes (not shown). Induction of thymocyte apoptosis in response to dexamethasone, irradiation, heat shock, or anti-CD95 correlated with the appearance of a second smaller Rb protein . Although it has been shown that Rb is cleaved by caspase 3 ( 53 ) and in thymocytes ΔRb was found to be a proteolytic cleavage product of Rb mediated by caspases, ΔRb was still observed in caspase 3 gene–deficient mice (not shown). The earliest detectable Rb cleavage (ΔRb) occurred 5 h after dexamethasone treatment . Cdk2 inhibitors or transgenic overexpression of Bcl-2 in thymocytes prevented cleavage of Rb in response to dexamethasone . These results demonstrate that Cdk2 acts upstream of mitochondrial pore opening, Bcl-2, caspase activation, and proteolytic cleavage of the cell cycle regulator Rb. The functional consequences of Rb cleavage are not known. Since ΔRb can only be observed downstream of the caspase effector phase and still binds to Cdk2 (not shown), the generation of ΔRb might function as a regulatory feedback loop that could influence Cdk2 and/or E2F-1 activity. Section title: Cdk2 Is Upstream of p53 and Bax Expression in Irradiated Thymocytes. Educational score: 4.345082759857178 Domain: biomedical Document type: Study Language: en How is Cdk2 activity mechanistically linked to apoptotic mitochondrial events? Various members of the Bcl-2 family of mitochondrial gatekeepers are phosphorylated on serine/threonine residues ( 9 , 49 ). Although Bcl-2 and Bcl-XL contain consensus sites for Cdk2 activity, we could not detect Cdk2-mediated phosphorylation of either Bcl-2 or Bcl-XL in in vitro kinase assays (not shown). The tumor suppressor p53 is a substrate for Cdk2 in the DNA repair response ( 54 ), and thymocytes mutated in p53 are resistant to γ-irradiation–induced apoptosis but still susceptible to dexamethasone and antigen receptor–mediated cell death ( 22 , 23 ). The effect of the p53 mutation has been mapped upstream of apoptotic mitochondrial events ( 55 ). Section title: Cdk2 Is Upstream of p53 and Bax Expression in Irradiated Thymocytes. Educational score: 4.132319450378418 Domain: biomedical Document type: Study Language: en Therefore, we tested whether p53 is a target for Cdk2 activity during thymocyte apoptosis after γ-irradiation. In vitro kinase assays using immunoprecipitated Cdk2 from γ-irradiated and dexamethasone-treated thymocytes showed that Cdk2 can phosphorylate p53 . Moreover, p53 was found to associate with Cdk2 in thymocytes . To test the effect of Cdk2 activity on p53 expression, we analyzed the levels of p53 protein in γ-irradiated thymocytes in the presence or absence of Cdk2 inhibitors. Although p53 protein accumulated to significant levels after treatment of cells with γ-irradiation alone, little p53 accumulation was observed when cells were treated with γ-irradiation in the presence of Cdk2 blockers . Irradiation-induced p53 protein accumulation was caused by enhanced p53 protein stability but not by p53 gene transactivation (not shown). Section title: Cdk2 Is Upstream of p53 and Bax Expression in Irradiated Thymocytes. Educational score: 4.227167129516602 Domain: biomedical Document type: Study Language: en To further corroborate the regulation of p53 by Cdk2, we examined the expression of the p53-inducible death promoter Bax ( 56 – 58 ) by Northern blotting. Induction of thymocyte apoptosis by γ-irradiation led to an increase in Bax transcripts, and Bax transactivation was found to depend on Cdk2 activity . In dying thymocytes we found only induction of the p53-regulated death promoter Bax but not transactivation of the p53-regulated gene p21 (not shown). Cdk2 kinase activity was normally induced in γ-irradiated p53 −/− thymocytes (not shown), indicating that p53 is downstream of Cdk2 in the thymocyte death signaling cascade. Since thymocytes from p53 −/− mice are not resistant to dexamethasone or antigen receptor–mediated apoptosis, other molecules must exist that link Cdk2 activation to cell death. Section title: Discussion Educational score: 4.789665222167969 Domain: biomedical Document type: Study Language: en The identification of Cdk2 as a master regulator of cell death provides the first evidence for a shared signaling pathway that integrates multiple death signaling pathways into a common death effector cascade in developing thymocytes. The hierarchy of Cdk2 action suggests that Cdk2 is the earliest known common signaling element required for thymocyte apoptosis in response to environmental and developmental cues such as negative selection. This hypothesis is based on the following findings: (a) all nonspecific (γ-irradiation, heat shock, dexamethasone) and specific (peptide-mediated thymocyte cell death) apoptotic stimuli tested induce rapid activity of the cyclin-dependent kinase Cdk2 in noncycling thymocytes; (b) Cdk2 acts upstream of the opening of mitochondrial pores, Bcl-2 family proteins, caspase activation, p53, and proteolytic processing of Rb; (c) inhibition of Cdk2 completely protects thymocytes from γ-irradiation, heat shock, dexamethasone, PMA, anti-CD3, and peptide-mediated cell death; (d) Cdk2 and the tumor suppressor, p53, constitutively associate in thymocytes and activated Cdk2 isolated from apoptotic thymocytes can phosphorylate p53; (e) Cdk2 regulates p53 protein accumulation and transactivation of the p53-inducible death promoter, Bax, after γ-irradiation. These data provide the first link between the cell cycle machinery and apoptosis in normal development and differentiation and indicate that Cdk2 is a crucial kinase that mediates cell death in thymocyte maturation and thymocyte selection. Section title: Discussion Educational score: 4.495491981506348 Domain: biomedical Document type: Study Language: en Our results indicate that after γ-irradiation, Cdk2 phosphorylates and stabilizes p53, which then transactivates the death promoter Bax. Interestingly, in dying thymocytes we found only induction of the p53-regulated death promoter Bax but not transactivation of the p53-regulated gene p21 ( 59 ), suggesting that only certain gene loci are accessible for p53 transactivation or that other cofactors act in concert with p53 to modulate gene expression in a tissue- and lineage-specific manner. Although p53 protein levels were increased in thymocytes after dexamethasone and γ-irradiation, it has been shown in p53 gene–deficient mice that p53 protects thymocytes only from DNA damage, and not from dexamethasone or antigen receptor–mediated cell death ( 22 , 23 ), implying that other downstream molecules exist that link Cdk2 to apoptosis. Preliminary evidence from our laboratory implies that the glucocorticoid receptor which is required for dexamethasone-mediated cell death can be phosphorylated by activated Cdk2 and coimmunoprecipitates with Cdk2 in dying thymocytes. Besides association with the ligand, phosphorylation of the glucocorticoid receptor is required for its translocation from the cytoplasm into the nucleus ( 60 – 62 ). In addition to the glucocorticoid receptor, other orphan steroid receptors such as Nur77 might be molecular targets for Cdk2 kinase activity. Section title: Discussion Educational score: 4.578582763671875 Domain: biomedical Document type: Study Language: en It has been shown that mitochondria are early checkpoints that integrate multiple death signaling pathways into a common Ced4/caspase-regulated effector mechanism. Opening of mitochondrial pores, mitochondrial swelling, disruption of ΔΨ m , and release of proapoptotic molecules, including cytochrome c and apoptosis-inducing factor (AIF), from the mitochondrial intermembrane spaces into the cytoplasm have all been implicated as fundamental mechanisms that initiate and propel the effector phase of apoptosis. Posttranslational modification and the balance between death suppressors, such as Bcl-2 and Bcl-XL, and death promoters, including Bax and Bad, are crucial mechanisms of mitochondrial integrity and the apoptotic effector phase ( 9 , 49 ). Our results show that Cdk2 acts upstream of ΔΨ m disruption, Bax and Bcl-2, and caspase activation in developing thymocytes. Moreover, whereas loss of the mitochondrial transmembrane potential can only be observed 2 h after addition of death stimuli, Cdk2 kinase activity is induced very rapidly and a “point of no return” was reached between 15 and 30 min after treatment with the apoptotic stimulus, such that a Cdk2 blocker added after this time was unable to prevent apoptosis. Thus, our results indicate that Cdk2 is the earliest known common denominator that can integrate many independent apoptotic signals into one common effector pathway. Section title: Discussion Educational score: 4.3084611892700195 Domain: biomedical Document type: Study Language: en Although CD95 (Fas) stimulation induced Cdk2 activity in thymocytes, inhibition of Cdk2 did not block CD95-mediated apoptosis. In fact, Cdk2 inhibition enhanced the susceptibility to CD95 killing. So far, CD95-mediated apoptosis is the only death signal in thymocytes that does not rely on Cdk2 activation. Although apoptosis in response to γ-irradiation, heat shock, dexamethasone, or peptide-specific negative thymocyte selection requires active transcription of death genes, apoptosis after CD95 killing can occur in the presence of RNA or protein synthesis inhibitors ( 9 ). Moreover, enucleated cells can undergo apoptosis after CD95 activation, suggesting that all components necessary for CD95-mediated apoptosis are present in cells and that CD95 activation can directly trigger the apoptotic machinery. It should be noted that both Cdk2 inhibitors roscovitine and olomoucine do not prevent transcription or translation in noncycling neurons ( 63 ) and that inhibition of Cdk2 did not regulate transactivation of the FasL in thymocytes (not shown). Section title: Discussion Educational score: 4.431578636169434 Domain: biomedical Document type: Study Language: en The specific Cdk2 blockers olomoucine and roscovitine are purine analogues that inhibit Cdk2 kinase activity by specifically binding to the Cdk2 ATP-binding site ( 39 , 40 ). At concentrations at which olomoucine and roscovitine blocked apoptosis in in vivo thymocyte cultures, these inhibitors had no effects on in vitro kinase activity of PKCα-ζ, Cdk4, Cdk6, Abl, cAMP- or GMP-dependent protein kinases (PKA, PKG), mitogen-activated protein kinase (MAPK; extracellular signal regulatory kinase [ERK]1, ERK2), Src family kinases, glycogen synthase kinase 3 (GSK3), casein kinase, receptor tyrosine kinases, myosin light chain kinase, p38/HOG, or stress-activated protein kinase (SAPK)/c-Jun NH 2 -terminal kinases (JNKs) (39; and data not shown). Through their unique selectivity for Cdk2, roscovitine and olomoucine provide a unique opportunity to study the role of Cdk2 in thymocyte apoptosis. However, we cannot exclude that roscovitine and olomoucine inhibit a yet unidentified kinase, and our results need to be confirmed using genetic model systems. Thus, genetic systems for inducible and thymocyte-specific inactivation/activation of Cdk2 need to be developed in the future. However, our results showing that all death stimuli lead to Cdk2 kinase activity in thymocytes and that two different Cdk2 kinase inhibitors, but not other inhibitors that block G1 to S progression, inhibit thymocyte apoptosis strongly suggest that Cdk2 is a key kinase involved in thymocyte apoptosis. Section title: Discussion Educational score: 4.438792705535889 Domain: biomedical Document type: Study Language: en Cdk2 is crucial for the progression from the G1 to the S phase of the cell cycle. Inhibition of Cdk2 activity in vitro has been shown to protect cultured sympathetic neurons and heart muscle cells from apoptosis ( 26 , 27 ). Our results in noncycling CD4 + CD8 + thymocytes provide the first evidence that Cdk2 has a crucial role in the induction of cell death during normal development. However, it has been shown that Cdk2 inhibition can also lead to cell death in tumor cell lines, and Cdks are frequently deregulated in tumors ( 28 ). Similarly, we found that in contrast to developing, noncycling thymocytes, inhibition of Cdk2 in four different thymic lymphoma cell lines led to rapid apoptosis and sensitized T cell tumors to anti-CD3, dexamethasone, or γ-irradiation–mediated cell death (not shown). These results suggest that Cdk2 has functions in the apoptotic processes that regulate normal development which are distinct from those in tumorigenesis and transformation. Thus, specific inhibition of Cdk2 could be exploited to sensitize tumor cells to apoptosis by anticancer drugs, whereas molecular inhibition of Cdk2 might protect normal, noncycling cells from the adverse effects of the same drugs. | Study | biomedical | en | 0.999998 |
10075980 | Section title: Mice. Educational score: 2.7509965896606445 Domain: biomedical Document type: Study Language: en SJL/J and C57BL/6 mice were obtained from the National Cancer Institute. Breeding pairs of C57BL/6 IL-12 −/− (N6) and C57BL/6 IFN-γ −/− were originally provided by J. Magram (Hoffman-La Roche, Nutley, NJ) and D. Dalton and T. Stewart (Genetech Inc., South San Francisco, CA), respectively. B10.S mice were obtained from both McLaughlin Research Institute and Taconic Farms. All mice were housed under specific pathogen-free conditions. They were exclusively female and between 2 and 4 mo of age when experiments were started. Section title: Peptides. Educational score: 4.034692764282227 Domain: biomedical Document type: Study Language: en Peptides corresponding to residues 260–283 of Influenza A nucleoprotein (NP 260–283 , ARSALILRGSVAHKSCLPACVYGP), residues 87–106 of myelin basic protein (MBP 87–106 , VVHFFKNIVTPRTPPPSQGK), and residues 139–151 of proteolipid protein (PLP 139–151 , HSLGKWLGHPDKF) were synthesized and purified by HPLC by the Laboratory of Molecular Structure, Peptide Synthesis Laboratory (NIAID, National Institutes of Health [NIH]). Chicken OVA was purchased from Sigma Chemical Co. Section title: Immunization. Educational score: 3.967397928237915 Domain: biomedical Document type: Study Language: en Mice were immunized subcutaneously at four sites over the flanks with an emulsion consisting of equal volumes of CFA (DIFCO Labs.) and antigen dissolved in PBS. Doses of antigen used were as follows: MBP 87–106 , 100 μg; NP 260–283 , 5 μg; OVA, 100 μg; PLP 139–151 , 100 μg. Section title: Disease Induction. Educational score: 4.101441383361816 Domain: biomedical Document type: Study Language: en For disease induction by adoptive transfer, donor mice were immunized with 100 μg of MBP or PLP in CFA (1:1); 10–14 d later, draining LN cells were cultured for 96 h with MBP or PLP. Recovered cells (6 × 10 7 ) were injected intraperitoneally into naive syngeneic recipients that were examined daily for signs of EAE and rated on a five-point scale as previously described ( 20 ). Section title: Cell Cultures. Educational score: 4.242497444152832 Domain: biomedical Document type: Study Language: en 10–14 d after immunization, draining LN cells (axillary and inguinal) were removed and processed as previously described ( 20 ). In brief, single cell suspensions of spleen or LN tissue were prepared by passage through wire mesh and red blood cells lysed with ACK buffer (NIH Media Unit). Cells (4 × 10 6 /ml) were cultured in RPMI 1640 containing 10% FCS and standard supplements ( 25 ) for 24–72 h in the presence of MBP 87–106 (50 μg/ml), NP 260–283 (5 μg/ml), PLP (50 μg/ml), or OVA (100 μg/ml). To generate short-term lines, draining LN cells were cultured in MBP or NP for 4 d, washed extensively, and rested for 7 d in complete media. For maintenance of lines, T cells (10 6 /ml) were restimulated with antigen in the presence of irradiated, syngeneic splenocytes (4 × 10 6 /ml) every 10 d. For IL-12 assays, T cells (10 6 /ml) were restimulated with or without antigen in the presence of syngeneic peritoneal exudate macrophages (5 × 10 5 /ml). To generate peritoneal exudate macrophages, B10.S or SJL mice were injected with 3 ml fluid thioglycollate media (NIH Media Unit). 3 d later, macrophages were removed from the peritoneum of treated mice, washed, and used in experiments. In some experiments, cells were purified using T cell enrichment columns (R&D Systems). T and/or NK cell depletion was performed by treating cells with anti-Thy1.2 (clone HO-13.4) culture supernatants or anti-NK1.1 antibody (clone PK136) followed by treatment for 45 min at 37°C with rabbit complement (Cedarlane Labs.). Section title: Proliferation Assays. Educational score: 4.050719261169434 Domain: biomedical Document type: Study Language: en LN cells (5 × 10 5 /0.2 ml) were cultured with various concentrations of antigen or with media alone for 4 d in 96-well round-bottomed plates (Costar Corp.). Wells were pulsed for the final 16 h of culture with 1 μCi of [ 3 H]TdR ( Amersham ) and counted as previously described ( 20 ). Section title: Antibodies and Cytokines. Educational score: 4.024035453796387 Domain: biomedical Document type: Study Language: en Where specified, cytokines or neutralizing antibodies were added to the primary cultures as follows: IL-12, 20 ng/ml (gift of S. Wolf, Genetics Institute, Cambridge, MA); recombinant murine IFN-γ, 30 ng/ml ( PharMingen ); recombinant murine IL-10, 10 ng/ml ( PharMingen ); anti–mouse IFN-γ, 10 μg/ml (clone XMG 1.2); rat anti–mouse IL-12, 10 μg/ml (clone C17.8); rat anti–mouse IL-10, 10 μg/ml (clones SXC-1 and SXC-2); rat IgG, 10 μg/ml ( Sigma Chemical Co. ); and anti-CD3 (2C11), 1.0 μg/ml. In certain studies, mice were injected intraperitoneally on days 0, 3, and 6 with 1 mg of either control rat IgG or rat anti–mouse IL-10 (clones SXC-1 and SXC-2). Section title: Northern Blot Analysis. Educational score: 4.2034220695495605 Domain: biomedical Document type: Study Language: en Total RNA was isolated from LN cell cultures using RNAzol RNA isolation solvent (Tel-Test). Samples (10 μg of total RNA per lane) were run on a 1.2% agarose gel containing MOPS buffer and formaldehyde, and blotted onto a Hybond-N nylon membrane ( Amersham ). Membranes were baked for 2 h at 80°C, then probed for murine IL-12Rβ2 subunit, murine IL-12Rβ1 subunit, or β-actin. The following primer sets (Bio-Synthesis) were used to generate oligonucleotide cDNA probes: IL-12Rβ2 forward CTG CAC CCA CTC ACA TTA AC; IL-12Rβ2 reverse CAG TTG GCT TTG CCC TGT GG; IL-12Rβ1 forward GAG GAG GCG GCT CTC CTC AG; IL-12Rβ1 reverse ACA TTC CTC CTG CTC CAG GG. The β-actin primer set was purchased from Clontech . PCR was performed for 40 cycles with the following parameters: 94°C, 30 s; 58°C, 30 s; 72°C, 1 min. cDNA probes were run on a 1.2% agarose gel, and purified using the Wizard PCR DNA purification system ( Promega ). PCR fragments (50 ng) were labeled with [ 32 P]dCTP using an oligolabeling kit ( Pharmacia ). Blots were prehybridized for 1 h at 42°C, followed by overnight hybridization with labeled probe at 42°C. Blots were then washed for 30 min in 2× SSC, 0.1% SDS buffer (room temperature) followed by 30 min in 0.1× SSC, 0.1% SDS buffer (55°C for IL-12Rβ2 and IL-12Rβ1; 65°C for β-actin). Section title: PCR. Educational score: 4.149748802185059 Domain: biomedical Document type: Study Language: en cDNA was reverse transcribed from 5 μg of RNA using Superscript II ( GIBCO BRL ). The following CD40L primers have been previously described: forward CCC TTA AGC TTG CAT GAT AGA AAC ATA corresponding to residues 10–26; and reverse TAG AGC TCG AGG TTC AGA GTT TGA GTA AGC C corresponding to residues 778–786 ( 29 ). A competitive PCR MIMIC was constructed from a kit according to the manufacturer's instructions ( Clontech ). The pMus-3 plasmid (gift of Dr. Nancy Noben-Trauth, NIH) was used in conjunction with the following primers to quantitate β2 microglobulin: forward TGA CCG GCT TGT ATG CTA TC; reverse CAG TGT GAG CCA GGA TAT AG. PCR reactions were carried out using Ready To Go PCR beads ( Pharmacia ) with 1 μl of MIMIC and 2 μl of cDNA reaction. PCR was performed for 40 cycles with the following parameters: 94°C, 30 s; 58°C, 30 s; 72°C, 1 min. PCR products were separated on a 1.2% agarose gel, and densitometry performed on an Eagle Eye Gel Reader (Stratagene). A relative ratio of CD40L/β2 microglobulin was calculated, and data were expressed as the percentage change of stimulated levels of CD40L/β2 microglobulin compared with background levels. Section title: Cytokine ELISA. Educational score: 4.0343475341796875 Domain: biomedical Document type: Study Language: en IFN-γ was quantified using a sandwich ELISA technique based on noncompeting pairs of antibodies as previously described ( 28 ). IL-12p40 was quantified using the mAbs produced by clones C17.5 and C15.6 (gifts of G. Trinchieri, Wistar Institute, Philadelphia, PA) for capture and detection, respectively. Section title: Independent Effects of IL-12 and IFN-γ on the Expression of the IL-12R. Educational score: 4.410446643829346 Domain: biomedical Document type: Study Language: en Previous studies have focused either on the role of IFN-γ in the upregulation or the role of IL-4 in the downregulation of IL-12Rβ2 expression ( 25 , 30 ). However, as IL-12 is a potent inducer of IFN-γ production by T and NK cells, it was impossible to determine from these experiments whether IL-12 directly, or indirectly via IFN-γ, induced the expression of IL-12R. To distinguish between these possibilities, we immunized C57BL/6 wild-type, IL-12 −/− , and IFN-γ −/− mice with OVA in CFA. 10 d later, draining LN cells were cultured in vitro for 3 d. Total RNA was extracted and used to perform Northern blot analysis. LN cells from C57BL/6 wild-type mice upregulated IL-12Rβ2 subunit mRNA strongly in response to stimulation with OVA in vitro ; the level of IL-12Rβ2 mRNA was diminished by the addition of anti–IL-12 or anti–IFN-γ to the cultures. OVA-reactive LN cells from IFN-γ −/− mice also upregulated IL-12Rβ2 subunit mRNA to a level comparable with that induced on wild-type LN cells . The addition of anti-IL-12 to cultures of OVA-primed LN cells from IFN-γ −/− mice markedly reduced their expression of IL-12Rβ2; conversely, the addition of IL-12 to these cultures markedly enhanced the level of expression of IL-12Rβ2 subunit above that seen when the cells were stimulated with OVA alone. In contrast to the results with cells from IFN-γ −/− mice, only weak upregulation of IL-12Rβ2 subunit expression in response to stimulation with OVA was seen with LN cells from IL-12 −/− mice; IL-12Rβ2 expression was moderately enhanced by the addition of exogenous IFN-γ to the cultures . Taken together, these studies demonstrate that a low level of IL-12Rβ2 expression is induced by TCR signaling alone and that IL-12 and IFN-γ have independent as well as complementary effects in promoting/maintaining the expression of the IL-12Rβ2 subunit. Section title: B10.S MBP-reactive T Cells Fail to Induce IL-12p40 Production by Peritoneal Exudate Macrophages and Manifest a Deficiency in CD40L Expression. Educational score: 4.385500907897949 Domain: biomedical Document type: Study Language: en We have previously shown that B10.S, but not SJL mice, are resistant to the induction of EAE and that the major difference between these strains was that T cells from B10.S, but not SJL mice, demonstrated an antigen-specific defect in IFN-γ production upon activation in vitro ( 28 ). Since our data clearly demonstrated that endogenous IL-12 is required for optimal IL-12Rβ2 subunit expression, we questioned if the defect in the ability of B10.S MBP-reactive T cells to produce IFN-γ was secondary to a failure of APCs to produce IL-12. It is unlikely that an intrinsic defect in IL-12 production is present in B10.S APCs, as it would be manifested as impaired IFN-γ production in response to multiple antigens, whereas our results indicate marked antigen-specificity. Furthermore, purified dendritic cells and splenocytes from B10.S and SJL mice produced comparable amounts of IL-12p70 heterodimer when stimulated with either anti-CD40 or LPS/IFN-γ (data not shown). Therefore, it seemed more likely that any deficiency in IL-12 production must result from a defect of the T cell to prime the APCs to produce IL-12. We generated B10.S MBP- and NP-reactive T cell lines and stimulated them with antigen in the presence of syngeneic peritoneal exudate macrophages. After 48 h of stimulation, B10.S NP-reactive T cell lines induced high levels of IL-12p40 production by macrophages, whereas B10.S MBP-reactive T cell lines failed to induce any detectable IL-12p40 production . In contrast, SJL MBP- and NP-reactive T cell lines induced comparable amounts of IL-12p40 production from macrophages . Section title: B10.S MBP-reactive T Cells Fail to Induce IL-12p40 Production by Peritoneal Exudate Macrophages and Manifest a Deficiency in CD40L Expression. Educational score: 4.208415985107422 Domain: biomedical Document type: Study Language: en Because the interaction of CD40L on T cells and CD40 on APCs is a major stimulus for IL-12 production, these results raised the possibility that the failure of activated B10.S MBP-reactive T cells to express sufficient levels of CD40L upon activation is responsible for their failure to prime APCs to produce IL-12. We used a competitive PCR mimic to quantitate the relative levels of CD40L mRNA expressed upon antigenic activation by B10.S MBP- and NP-reactive LN cells. MBP-reactive LN cells failed to upregulate CD40L mRNA, whereas NP-reactive LN cells upregulated CD40L mRNA eightfold over unstimulated background levels . Thus, the relative deficiency in CD40L expression by B10.S MBP-reactive LN cells may account for their failure to induce IL-12 production by APCs. Section title: MBP-specific T Cells from EAE-resistant B10.S Mice Fail to Upregulate IL-12Rβ2 Subunit mRNA. Educational score: 4.219776153564453 Domain: biomedical Document type: Study Language: en To test directly whether the failure of the B10.S MBP-specific T cells to induce IL-12 leads to a deficiency in the expression of adequate levels of IL-12Rβ2 subunit, draining LN cells from SJL and B10.S mice that had been primed 12 d previously with a combination of MBP and NP in CFA were cultured in vitro for 72 h with either MBP or NP and evaluated for IL-12R expression. LN cells from SJL mice upregulated IL-12Rβ2 subunit mRNA strongly in response to stimulation with either MBP or NP . In contrast, LN cells from B10.S mice upregulated IL-12Rβ2 subunit mRNA only in response to stimulation with NP, but not MBP . Since NK cells and dendritic cells have been reported to express IL-12R ( 26 , 31 ), T cells were purified from LN cells from immunized B10.S mice and depleted of NK cells. These T cells were combined with T cell–depleted, NK cell–depleted splenocytes from naive B10.S mice and stimulated for 72 h, then evaluated for IL-12Rβ2 subunit expression. Results similar to those shown in Fig. 4 A were obtained, thus excluding dendritic cells or NK cells as the source of IL-12R observed . In contrast to the IL-12Rβ2 subunit, which was induced only upon antigen stimulation, IL-12Rβ1 subunit was constitutively expressed with no consistent patterns of antigen-induced upregulation , in agreement with previously published reports ( 25 , 26 ). Section title: IL-12Rβ2 Subunit mRNA Expression Correlates with Susceptibility to EAE. Educational score: 4.334231853485107 Domain: biomedical Document type: Study Language: en We have previously demonstrated that the addition of pharmacological concentrations of IL-12 to cultures of B10.S MBP-reactive LN cells restored their ability to produce IFN-γ and converted them into encephalitogenic effectors ( 28 ). Indeed, the addition of exogenous IL-12 restored the ability of the B10.S MBP-reactive LN cells to upregulate the expression of IL-12Rβ2 subunit mRNA in an antigen-specific, dose-dependent manner . Although the addition of IFN-γ failed to restore the ability of these cells to produce IFN-γ upon secondary stimulation ( 28 ), modest induction of IL-12Rβ2 subunit expression was seen (data not shown). The addition of neutralizing antibodies to IL–12 or IFN-γ to cultures of NP-reactive B10.S LN cells or MBP-reactive SJL LN cells inhibited the ability of these cells to upregulate IL-12Rβ2 subunit mRNA in response to antigenic stimulation. Thus, the complementary roles of endogenous IL-12 and IFN-γ for optimal expression of IL-12Rβ2 subunit seen in the response of C57BL/6 mice to OVA can be extended to a second foreign antigen (NP) and an autoantigen (MBP). Section title: Role of IL-10 in the Expression of IL-12Rβ2 Subunit mRNA. Educational score: 4.307933807373047 Domain: biomedical Document type: Study Language: en One possible explanation for the failure of B10.S MBP-specific T cells to induce IL-12 production and to upregulate IL-12Rβ2 expression is that a major component of the response to MBP in this strain is mediated by Th2 cells. However, we have previously failed to demonstrate production of IL-4 or IL-10 by B10.S MBP-specific T cells and treatment of B10.S mice with anti–IL-4 in vivo or anti–IL-10 in vitro did not result in the induction of IFN-γ production ( 28 ). Furthermore, addition of anti–IL-4 in vitro failed to restore the ability of these cells to express IL-12Rβ2 subunit (data not shown). However, our recent demonstration that IL-10 production by antigen nonspecific CD4 + T cells regulates the proinflammatory effects of IL-12 in vivo prompted us to more carefully examine the role of IL-10 in the priming of B10.S mice to MBP in vivo ( 20 ). B10.S mice were immunized with MBP/NP and simultaneously treated with neutralizing anti–IL-10 mAbs. Neutralization of IL-10 in vivo and in vitro did not restore the ability of MBP-reactive LN cells to express IL-12Rβ2 subunit or to produce IFN-γ (data not shown). However, it should be emphasized that IL-10 had a potent downregulatory effect on the expression the IL-12Rβ2 in vitro as the addition of exogenous IL-10 to cultures of either B10.S NP-reactive or SJL MBP–primed LN cells strongly inhibited upregulation of the expression of the IL-12Rβ2 subunit . Section title: The Failure of B10.S MBP-specific T Cells to Fully Differentiate into Th1 Effectors Does Not Appear to Be Secondary to a Decreased Functional Avidity of the TCR. Educational score: 4.262834548950195 Domain: biomedical Document type: Study Language: en One of the problems with the comparison of immune responses between B10.S and SJL mice at the population level even with a well-defined peptide antigen is that these two strains differ by many background genes including the TCR gene complex ( 32 ). Thus, as a result of distinct positive/negative selection events in the thymus, the B10.S MBP-specific T cell repertoire may be quantitatively smaller or exhibit an overall lower affinity for the MBP 87–106 peptide I-A s complex compared with similarly restricted SJL T cells. The average lower affinity of the B10.S MBP-specific TCRs might result in impaired induction of CD40L and the cascade of events leading to defective upregulation of the IL-12Rβ2 subunit. Targoni and Lehmann have shown that immunization with low doses of peptide would generate recall responses only from T cells with a high affinity TCR for their MHC–peptide ligand ( 33 ). We immunized B10.S and SJL mice with different amounts (50, 100, or 400 μg) of MBP 87–106 in CFA and measured T cell proliferation in response to a broad range of peptide concentrations in vitro . In several experiments of this type, no consistent differences between SJL and B10.S mice were seen in the magnitude of the proliferative responses or the shape of the dose–response curves. Thus, it appears that the expansion of MBP-reactive cells is similar in the two strains and that no obvious difference exists in their functional avidity. Section title: The Defect in IL-12Rβ2 Subunit Expression by B10.S MBP-specific T Cells Does Not Extend to Other Myelin Autoantigens. Educational score: 4.2678422927856445 Domain: biomedical Document type: Study Language: en Several studies have suggested that B10.S mice have a global defect in their responses to myelin autoantigens as the incidence and/or severity of EAE in B10.S mice immunized with PLP 139–151 or with whole spinal cord homogenate was significantly less than that seen in similarly immunized SJL mice. To examine whether a failure of IL-12Rβ2 subunit expression was also seen in response to PLP, we immunized B10.S mice with a combination of MBP and PLP in CFA. PLP- but not MBP-reactive LN cells upregulated IL-12Rβ2 subunit strongly in response to antigenic stimulation . Furthermore, PLP- but not MBP-reactive LN cells produced large quantities of IFN-γ in response to antigenic stimulation in secondary cultures . To further address the question of whether the encephalitogenicity of primed LN cells correlated with their ability to express the IL-12Rβ2 subunit, we immunized B10.S mice with PLP 139–151 or MBP 87–106 in CFA. 10 d later, we stimulated draining LN cells in vitro with PLP or MBP for 4 d, then transferred the cells into naive B10.S recipients. Transfer of PLP-reactive LN cells resulted in a 30% incidence of EAE, whereas transfer of MBP-reactive LN cells failed to induce EAE in any mice . Thus, the capacity of LN cells to transfer EAE was found to correlate with their ability to express IL-12Rβ2 subunit upon activation. Section title: Discussion Educational score: 4.429028511047363 Domain: biomedical Document type: Study Language: en Although CD4 + Th1 lymphocytes have been implicated as the effector cells in both animal models and human organ-specific autoimmune diseases, the failure to identify a single effector cytokine that is responsible for pathogenicity has suggested that cytokine-targeted therapeutic approaches to autoimmune disease should be directed against the primary cytokine responsible for the differentiation of Th1 cells, IL-12. Because the IL-12Rβ2 subunit has been identified as the critical molecule involved in maintaining IL-12 responsiveness and in controlling Th1 lineage commitment, we have focused our studies on critically examining the requirements for induction of the IL-12Rβ2 subunit on CD4 + T cells responding to autoantigens as well as conventional foreign antigens. We have used cytokine-deficient mice to establish a critical role for IL-12 itself, independent of IFN-γ, in the induction of IL-12Rβ2 expression. Although IFN-γ was capable of upregulating IL-12Rβ2 on T cells derived from IL-12 −/− mice, the level of expression was always less than that seen on T cells from wild-type and IFN-γ −/− mice. As IL-12 −/− mice have been found to have markedly deficient Th1 responses and to be resistant to Th1-mediated autoimmune diseases ( 34 ), the significance of this IL-12–independent, IFN-γ–dependent pathway of IL-12Rβ2 upregulation remains to be determined. More importantly, our studies with neutralizing antibodies in vitro have clearly shown that both IL-12 and IFN-γ play critical roles in the upregulation of IL-12Rβ2 on restimulation of antigen-primed T cells in vitro from wild-type mice. Section title: Discussion Educational score: 4.678592205047607 Domain: biomedical Document type: Study Language: en We have previously defined a critical role for IL-12 in the cytokine cascade needed for activation of encephalitogenic MBP-specific T cells from EAE-resistant B10.S mice ( 28 ). Although we could induce Th1 differentiation with pharmacological concentrations of IL-12, we did not determine whether the failure to normally generate MBP-specific Th1 cells in this strain involved deficient IL-12 production or deficient IL-12Rβ2 subunit expression. We have now shown that the primary defect in the immune response of the B10.S mouse to MBP is defective expression of CD40L on the MBP-specific T cell population with subsequent failure to generate IL-12 production from APCs. This in turn leads to defective expression of sufficient levels of IL-12Rβ2 needed for Th1 differentiation. The importance of CD40/CD40L interactions in the differentiation of encephalitogenic effectors is supported by the suppression of EAE induction by the administration of blocking CD40L mAbs ( 35 , 36 ). Our previous studies have ruled out excessive production of IL-4 as the mechanism responsible for defective MBP-specific Th1 differentiation in the B10.S mouse ( 28 ). Neutralization of IL-10 in vivo and in vitro also did not restore IL-12Rβ2 expression by the MBP-specific T cells. However, addition of exogenous IL-10 to the in vitro culture exerted a powerful downregulatory influence on expression of IL-12Rβ2 subunit by antigen-specific T cells primed in vivo under Th1 conditions. Presumably, IL-10 acted directly on the APCs in the cultures to inhibit IL-12 production and thereby mimicked the effects of the addition of anti–IL-12 ( 37 ). However, a direct effect of IL-10 on the responding T cells remains a possibility. In any case, it appears that once IL-12Rβ2 subunit expression is induced by IL-12 in vivo, IL-10 can act to limit the level of functional IL-12Rβ2 ultimately expressed. Indeed, this step may be the most important therapeutic effect of IL-10 in the treatment of autoimmune disease. Section title: Discussion Educational score: 4.628605365753174 Domain: biomedical Document type: Study Language: en The antigen-specific defect we have observed in the CD40L/IL-12/IL-12Rβ2 subunit pathway in MBP-specific B10.S T cells should be contrasted with the more global defects of the B10.S strain in the response to myelin- derived autoantigens reported by others ( 38 , 39 ). It is likely that this antigen-specific defect is superimposed over and above a more global defect. First, although the addition of exogenous IL-12 to cultures of B10.S MBP-reactive T cells restored their capacity to transfer EAE, the resultant disease was monophasic and less severe than the relapsing-remitting course manifested by SJL recipients of syngeneic MBP-primed T cells ( 28 ). Second, although we have demonstrated that B10.S PLP-reactive T cells can induce disease upon adoptive transfer, it should be noted that disease incidence and severity were much less than those observed in SJL mice. Furthermore, production of IFN-γ was only observed during secondary but not primary stimulation in vitro. The differences between our results and those of Encinas et al., who reported complete resistance of B10.S mice to PLP-induced EAE, may be secondary to the different disease induction protocols used (active induction versus passive transfer) or differences in the mouse colonies consequent to genetic drift ( 38 ). Thus, B10.S mice appear to have inherited a set of traits that confer a certain degree of protection against autoimmune phenomena in general, distinct from the antigen-specific defect in the IL-12 pathway. This set of traits is presumably mediated by the products of non–H-2 background genes and may also be responsible for the monophasic (as opposed to relapsing- remitting) course of EAE exhibited by B10.PL mice as well as for the resistance of C57BL/6 mice to autoimmune orchitis. In this context, it is interesting to note that one of the genetic loci found to be important in susceptibility to EAE (eae7) colocalizes to the same region of chromosome 11 as Orch3, a susceptibility locus in autoimmune orchitis ( 39 ). Hence, in the case of the B10.S response to MBP, we believe that absolute resistance arises as a result of the antigen-specific defect superimposed on a more global pattern of resistance to the manifestations of autoimmune disease. Section title: Discussion Educational score: 4.380139350891113 Domain: biomedical Document type: Study Language: en What then is the mechanism responsible for the generation of this antigen-specific defect in the capacity of a population of autoreactive T cells to differentiate into pathogenic Th1 effector cells? The simplest explanation is that it is secondary to the size of the T cell repertoire specific for MBP. We believe that this is unlikely as the magnitude of the proliferative responses and of IL-2/IL-3 production by B10.S mice to MBP does not differ significantly from that of SJL mice ( 28 ). However, this question will only be able to be addressed quantitatively when specific peptide-MHC binding T cells are measured in this model. It is also possible that the affinity of the interaction of the anti-MBP TCRs from B10.S mice with the MBP peptide–I-A s complex is too low to generate a sufficient signal to trigger high enough levels of CD40L expression to trigger the IL-12 production cascade. However, we detected no differences between the proliferative capacities of B10.S and SJL MBP 87–106 reactive populations after immunization with limited doses of peptide, suggesting that there are no major differences in TCR avidity. It is also unlikely that the failure of the B10.S T cells to differentiate is secondary to the delivery of a suboptimal costimulatory signal which potentially could influence CD40L expression, as the addition of anti-CD28 to B10.S cells in vitro failed to generate antigen- specific IFN-γ production (our unpublished observations). Section title: Discussion Educational score: 4.67777156829834 Domain: biomedical Document type: Study Language: en We propose that the defect in the B10.S MBP-specific population is generated during the process of negative selection during thymic differentiation. Two recent studies in shiverer mice, which have a mutation in the MBP gene, have suggested that those autoantigen-specific T cells that normally escape negative selection in the thymus to enter the peripheral T cell pool do so because of their low affinity for the autoantigen ( 33 , 40 ). Yet, MBP 87–106 –specific T cells from B10.S mice appear to have emerged from the negative selection process with an additional defect—a blunted capacity to become pathogenic effectors even when primed under conditions that readily generate such effectors in susceptible strains of mice such as the SJL or B10.PL. We have previously postulated that one mechanism by which autoreactive T cells may escape deletion in the thymus is secondary to complete downregulation of their TCR signaling properties with a major defect in their capacity to produce IL-2 ( 41 ). Such cells, which are characterized by expression of the CD25 antigen, appear to function as suppressor cells that prevent organ-specific autoimmunity after thymectomy early in life. The B10.S MBP–specific cells appear to be the products of a different fail-safe mechanism imposed on the autoreactive T cell repertoire during negative selection—the inability to upregulate the CD40L antigen and the subsequent IL-12 cascade. Thus, negative selection of autoreactive TCRs may be a more complex process than simple killing of high affinity cells and allowing low affinity cells to pass through. Intrathymically, some autoreactive T cells may recognize their target autoantigen in a qualitatively different way similar to the manner in which mature T cells recognize altered peptide ligands. However, at this stage of their development, this altered recognition of self may result in a variety of permanent defects in the TCR signal transduction cascade ranging from anergy to the partial defect we have described in the MBP-reactive population of the B10.S mouse. This model does not exclude the possibility that the expression of autoantigens, such as MBP, in the periphery may also be required for the maintenance of the nonpathogenic state of the autoreactive T cells. We suspect that other interesting phenotypes involving the inability of autoreactive cells to fully differentiate will be uncovered in the future. Section title: Discussion Educational score: 4.552633285522461 Domain: biomedical Document type: Study Language: en What are the implications of these results for the pathogenesis of organ-specific autoimmune disease in experimental animals and in man? B10.S MBP-reactive T cells defy simple categorization into the Th1/Th2 paradigm. They differ from Th1 cells by failing to upregulate IL-12Rβ2 to an appreciable degree upon activation. However, they also differ from Th2 cells by retaining the capacity to express IL-12Rβ2 when triggered under conducive environmental conditions. B10.S MBP-reactive cells fall within a distinct subset of Th cells, which we refer to as T pre-A cells, signifying a latent ability to develop into autoimmune effectors. We have found that IL-12–inducing microbial components or high doses of recombinant IL-12 can serve as triggers for the conversion of T pre-A cells ( 27 , 28 ). This may explain, in part, why infectious diseases are so effective in precipitating the onset and exacerbation of autoimmune diseases, including multiple sclerosis (MS). T pre-A cells may comprise a significant number of autoreactive T cells in the periphery of healthy individuals. Hence, defining those conditions that favor as well as suppress their conversion into mature autoimmune effectors could have important clinical implications regarding the prevention of autoimmune disease and its recurrences. Similar numbers of myelin protein–reactive T cells have been detected in the peripheral blood of MS patients and healthy HLA-matched controls ( 42 – 46 ). The antigen-specific inducibility of the IL-12Rβ2 subunit and/or CD40L may potentially serve as more reliable distinguishing characteristics of pathogenic myelin protein–reactive T cells from MS patients. If a recurrent pattern is established, IL-12Rβ2 subunit or CD40L expression may eventually serve as a marker to detect subclinical disease and measure active disease in MS. Furthermore, the development of immunotherapies that target subsets of T cells expressing high levels of the IL-12Rβ2 subunit may be warranted. | Study | biomedical | en | 0.999996 |
10075981 | Section title: Reagents. Educational score: 1.545371651649475 Domain: biomedical Document type: Other Language: en Type I collagen solution extracted from porcine skin (Cellmatrix I-A) was purchased from Nitta Gelatin Co. EC growth supplement (ECGS) and porcine heparin were purchased from Collaborative Research and Nakarai Chemical Co., respectively. Recombinant human IFN-γ was provided by Shionogi Pharmaceutical Co. Monocyte chemoattractant protein-1 (MCP-1) was provided by T. Kasahara (Kyoritsu College of Pharmacy, Tokyo, Japan; 19). Rhodamine-conjugated anti-CD26 mAb and phycoerythrin-Cy5–conjugated anti-CD3 mAb were obtained from Coulter Corporation. Anti-CD11a mAb and purified mouse IgG3 were obtained from Becton Dickinson and Zymed Laboratories, Inc. , respectively. FCS was purchased from Cell Culture Laboratories. BSA, Hepes buffer, gelatin, diisopropyl fluorophosphate (DFP), papain, L-cysteine, and collagenase (type 1-A) were obtained from Sigma Chemical Co. Pertussis toxin (PT) and M199 were obtained from Seikagaku Corporation and GIBCO BRL , respectively. Section title: Preparation of Cells. Educational score: 4.144032001495361 Domain: biomedical Document type: Study Language: en PBMCs were prepared from heparinized healthy human venous blood by Ficoll-Conray density gradient centrifugation as described previously ( 16 ). The T cell–enriched fraction was obtained by passing the mononuclear cells through a nylon wool column. CD3 + cells were negatively selected by inclusion of the fraction with magnetic anti-CD16 mAb (Advanced Magnetics, Inc.) The selected cells contained >96% CD3 + cells, as determined by flow cytometry. Neutrophils were prepared by dextran sedimentation, centrifugation with Ficoll-Conray, and hypotonic lysis of contaminating erythrocytes ( 20 ). Neutrophil fractions contained >95% neutrophils. Endothelial cells were obtained from human umbilical cord veins treated with 0.1% collagenase as described previously ( 16 ). Cells were grown on gelatin-precoated dishes in M199 containing 20% heat-inactivated FCS, 60 μg/ml ECGS, 100 μg/ml heparin, 1% penicillin and streptomycin solution, and 15 mM Hepes buffer. Culture medium was changed every 3 d. These experiments used cells in passages 2 and 3 only. Section title: Production of Anti-4C8 mAb. Educational score: 4.207555294036865 Domain: biomedical Document type: Study Language: en The anti-4C8 mAb was produced by standard techniques after immunization of BALB/c mice with PBMCs cocultured with HUVEC monolayers. In brief, after removal of nonadherent PBMCs from the cocultures, the cocultured adherent cells containing at least 10 7 lymphocytes were intraperitoneally injected five times at 2–3 wk intervals. The final immunization was performed by intravenous injection of 7 × 10 6 transmigrated T cells isolated by using an in vitro vessel model as previously described ( 16 ). 3 d later, the spleen was removed and cells were fused with NS-1 cell line. Hybridoma cultures producing antibodies that inhibited T cell migration across but not adhesion to HUVEC monolayers were selected, cloned, and recloned by limiting dilution methods in the presence of IL-6. Malignant ascites were then developed and further purified by an IgG purification kit ( Pierce Chemical Co. ). The anti-4C8 mAb was shown to be of the IgG3 subclass by an ELISA method for determining subclasses of mouse IgG. Fab fragments were produced by incubating purified IgG with 10 μg/ml papain, 5 mM L-cysteine, and 2 mM EDTA and then purified by passing over DEAE-cellulose. SDS-PAGE performed under nonreducing conditions proved that the fragments were properly cut and that no extraneous bands were present on Coomassie blue stain. Fluorescein-conjugated anti-4C8 mAb was prepared by using a fluorescein labeling kit ( Sigma Chemical Co. ). Section title: Adhesion and Transmigration Assays. Educational score: 4.1350603103637695 Domain: biomedical Document type: Study Language: en For the adhesion and transmigration assays, we modified the original system that was described elsewhere ( 16 ). HUVEC monolayers were grown to confluence on collagen gels (50 μl/well) in 96-well flat bottom plates ( Becton Dickinson ), followed by treatment for 48 h with or without IFN-γ (500 U/ml) before assay. Freshly isolated CD3 + T cells suspended in M199–0.1% BSA were or were not pretreated with mAbs for 20 min on ice. The cells were added to the wells without washing (3 × 10 5 cells/100 μl/well). The plate was centrifuged for 1 min at 50 g and incubated for 3–4 h at 37°C in a 5% CO 2 -humidified incubator. In the adhesion assay, unbound T cells were gently washed out, and then adherent cells were immediately fixed with 1% paraformaldehyde in PBS. The transmigration assay was performed simultaneously with the adhesion assay. To count migrated cells, adherent T cells and HUVECs were removed from the surface of collagen gels by 0.4% EDTA treatment. In some experiments, mAbs were added after unbound cells had been washed from the cultures. Adherent cells on the apical surfaces of HUVECs or cells that had transmigrated into the collagen gels were counted by phase–contrast microscopy in a blinded manner. The cells in a field of 0.25 mm 2 were counted at a magnification of 100. Adhesion or migration index (%) was calculated as follows: the number of cells with antibody/the number of cells without antibody × 100. All experiments were performed in triplicate. Section title: Release of Adherent and Transmigrated T Cells. Educational score: 4.1277570724487305 Domain: biomedical Document type: Study Language: en After T cells (2 × 10 7 cells) were cultured for 5 h with a confluent HUVEC monolayer on 2 ml of collagen gels (60 mm dish), adherent and transmigrated cells were collected as described ( 16 ). In brief, after unbound T cells were removed, T cells bound to HUVECs were incubated for 20 min with 0.4% EDTA. Almost all adherent T cells could be obtained by this treatment. The HUVEC monolayer was then removed from the surface of collagen gels by the EDTA treatment for another 30 min. The collagen gels containing transmigrated T cells were incubated with 0.05% collagenase in PBS for 3 min to release the cells. This collagenase treatment was repeated twice. No changes in the expression of surface proteins of T cells were found following the treatment. Section title: Chemotaxis and Checkerboard Assays Using Collagen Gels. Educational score: 4.099116325378418 Domain: biomedical Document type: Study Language: en We modified a chemotaxis assay using collagen gels as described by others ( 21 ). Resting and activated T cells were prepared by culturing freshly isolated T cells for 2 d in RPMI 1640 containing 10% FCS and for 6 d on anti-CD3–coated dishes (0.4 μg/ml) in medium with 100 U/ml of IL-2, respectively. The cells were washed, resuspended in M199 plus 0.1% BSA, and added directly onto collagen gels (50 μl/well), with or without impregnated MCP-1 (100 ng/ml), in 96-multiwell plates (1–4 × 10 5 cells/ well). After 1.5–2 h, unbound cells and cells attached on the surfaces of the gels were washed out with 0.4% EDTA in PBS. Cells that migrated into the gels were counted under a phase–contrast microscope at 200× as described above. In the checkerboard assay, mAbs were impregnated into collagen gels and/or added directly to freshly isolated T cells (4 × 10 5 cells/well) above the gels at varying concentrations in 96-multiwell plates. T cells were incubated for 4 h under these conditions. In these experiments, to reduce spontaneous migration of T cells, the collagen gels were prepared with collagen solution at a concentration of 3 mg/ ml, which is three times higher than in the transmigration assays. Migrated cells in the gels were carefully counted and the migration index was calculated as described above. All experiments were performed in triplicate. Section title: Flow Cytometric Analysis. Educational score: 4.049853324890137 Domain: biomedical Document type: Study Language: en Cells were treated for 20 min with saturating amounts of fluorescein-conjugated mAb and washed three times with PBS containing 0.1% BSA and 0.01% sodium azide. The stained cells (10,000 cells) were analyzed on a FACScan ® flow cytometer ( Becton Dickinson ) with gating on the lymphocyte, monocyte, or neutrophil population. All staining procedures were performed at 4°C. Lysis II software ( Becton Dickinson ) was used to analyze the data obtained. Section title: Western Blotting. Educational score: 4.148982524871826 Domain: biomedical Document type: Study Language: en PBMCs or neutrophils (10 7 cells) were incubated for 30 min with intermittent agitation with or without DFP (1 mM ) on ice. The pellets of the cells were resuspended for 60 min in 100 μl of ice cold extraction buffer containing 50 mM Hepes (pH 7.4), 2 mM sodium orthovanadate, 100 mM sodium fluoride, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 mM PMSF, 100 μg/ml aprotinin, and 10 μg/ml leupeptin. Cells were treated for 30 min with 1 mM DFP on ice before cell lysis. After centrifugation, the supernatant was mixed 1:1 with 2× sample buffer (4% SDS, 20% glycerol, 10% mercaptoethanol, and a trace amount of bromophenol blue dye in 125 mM Tris-HCl, pH 6.8), heated at 100°C for 5 min, and loaded onto an 8% SDS–polyacrylamide gel. After electrophoresis, proteins were transferred onto a nitrocellulose membrane ( Pierce Chemical Co. ). Residual binding sites on the membrane were blocked by incubating the membrane in Tris-buffered saline (pH 7.6) containing 0.1% Tween-20 and 5% nonfat dry milk for 2 h at room temperature. The membranes were incubated with anti-4C8 mAb and then with biotin-conjugated anti–mouse IgG antibody. After incubation, enzymatic development was performed by using peroxidase-conjugated streptoavidin ( GIBCO BRL ) and the ECL system ( Amersham ). Section title: Cell Morphology Assay and F-actin Staining. Educational score: 4.119372367858887 Domain: biomedical Document type: Study Language: en The anti-4C8– induced changes in cell shape and F-actin formation were visualized by staining with TRITC-labeled phalloidin ( Sigma Chemical Co. ) as described previously by others ( 22 ). Glass slides (uncoated eight-well CultureSlide; Becton Dickinson ) were coated with anti-4C8 mAb (10 μg/ml, 250 μl/well) or control IgG3 overnight. T cells (5 × 10 5 cells/well in M199 with 0.1% BSA) were added to the slides and incubated for 2 h at 37°C. After incubation, attached cells were fixed with 3.7% paraformaldehyde, permeabilized with 0.1% Triton X-100/PBS, and stained for F-actin with TRITC-labeled phalloidin. Microscopic analysis was performed using an Olympus microscope equipped with fluorescence accessories and photographed with an ×100 oil immersion objective. In some experiments, T cells (5 × 10 5 cells/500 μl of 0.1% BSA–M199/Eppendorf tube) were incubated for 3 h with anti-4C8 mAb or control IgG3 at 37°C. After fixation and permeabilization, the cells were double-stained with FITC-conjugated phalloidin and rhodamine-conjugated anti-CD26 mAb. The stained cells were then analyzed by a flow cytometer. Section title: Scanning Electron Microscopy. Educational score: 4.138179779052734 Domain: biomedical Document type: Study Language: en The transmigration assay was performed in the presence of anti-4C8 (1 μg/ml) in 24-multiwell plastic plates (Falcon Labware). After a 5-h incubation, cultures were fixed overnight in 2% electron microscopy–grade glutaraldehyde and 5% sucrose in 0.1 M sodium cacodylate buffer (pH 7.4), followed by postfixation with 1% OsO 4 . The fixed cells and collagen gels were removed from the plate and processed for scanning electron microscopy by critical-point drying and gold coating. Section title: Statistical Analysis. Educational score: 2.7672269344329834 Domain: biomedical Document type: Study Language: en All values are represented as means ± SD. When comparing two groups, P values were calculated by Student's t test. P values <0.05 were considered to indicate a significant difference. Section title: Expression of the 4C8 Antigen on Human PBLs and HUVECs and Its Structure. Educational score: 4.164090156555176 Domain: biomedical Document type: Study Language: en We first examined immunofluorescence profiles of 4C8 expression on PBLs and HUVECs by a flow cytometer. As shown in Fig. 1 , the 4C8 antigen was expressed intensely on CD3 + T cells and to a lesser extent on CD3 − cells (largely CD16 + cells). Staining of the cells gated on the monocyte population was positive and two peaks were seen. In contrast, anti-4C8 did not react with neutrophils or unstimulated HUVECs. The negative expression of neutrophils was unaffected by stimulation with LPS or TNF-α (data not shown). Immunofluorescence microscopy also showed no significant staining of confluent monolayers of HUVECs (not shown). To address the molecular weight of the 4C8 antigen, Western blotting analysis was performed using lysates from PBMCs and neutrophils . Anti-4C8 reacted with a single band of 80 kD in lysates from PBMCs but not neutrophils. It is possible that the 4C8 antigen was cleaved by numerous proteolytic enzymes released from neutrophils during the procedure of cell lysis. However, anti-4C8 did not react with the lysates in the presence of DFP, a serine protease inhibitor. There were no differences between blots from gels electrophoresed under reducing and nonreducing conditions (data not shown). In addition, immunoprecipitation failed to detect the 4C8 antigen by a standard method (not shown). This finding is consistent with fluorescence profiles of the 4C8 antigen on these cells and further suggests that the antigen is not present in the intracellular contents of neutrophils. Section title: Anti-4C8 mAb Inhibits T Cell Transmigration Subsequent to LFA-1–mediated Adhesion to a HUVEC Monolayer. Educational score: 4.389166831970215 Domain: biomedical Document type: Study Language: en In our system, 20–30% of total added CD3 + T cells adhered to unstimulated HUVEC monolayers after 3–5 h of incubation, and 10–20% of the adherent cells transmigrated during this period ( 16 , 17 ). IFN-γ augments the expression of intercellular adhesion molecule-1 (ICAM-1), the ligand for CD11a/CD18 (LFA-1 α/β), on HUVEC. Stimulation of HUVEC with IFN-γ increased T cell adhesion and transmigration to 1.5- and 3-fold the base line values, respectively . We then assessed the changes in adhesion and transmigration in the presence of antibodies. Anti-CD11a mAb (10 μg/ml) inhibited T cell adhesion to and transmigration through unstimulated and IFN-γ–stimulated HUVEC monolayers by 50–60 and 80%, respectively. This indicates that T cell transmigration is largely dependent upon LFA-1–mediated adhesion to HUVECs. On the other hand, anti-4C8 mAb (1 μg/ml) did not inhibit T cell adhesion but rather induced a small increase in adhesion to IFN-γ–stimulated HUVEC monolayers . The small increase was in accord with the number of T cells that were retained on HUVECs by the transmigration-blocking effect of anti-4C8. However, transmigration was strikingly inhibited: 79 and 87% with unstimulated and IFN-γ–stimulated HUVEC monolayers, respectively . Control IgG3 (the same isotype as anti-4C8) showed neither inhibition of T cell adhesion nor transmigration. To determine even more directly whether anti-4C8 mAb acts subsequently to LFA-1–mediated adhesion, the antibody blocking study was performed following removal of unbound T cells 1 h after coculturing T cells and IFN-γ–stimulated HUVEC monolayers. As shown in Fig. 4 , both anti-4C8 IgG (1 μg/ml) and Fab fragments (10 μg/ml), but not anti-CD11a or control IgG3, completely blocked subsequent migration of adherent T cells during a 5-h incubation. The blocking effect was not due to detachment of the adherent cells from the apical surfaces of HUVECs (data not shown). However, it is possible that the blockage is caused by a direct suppressive effect of anti-4C8 on cell motility. To examine this, we next performed chemotaxis assays using a three-dimensional collagen matrix (collagen gels), with or without impregnated MCP-1. Resting and anti-CD3–activated T cells were added to the gels and incubated for 1.5–2 h in the presence or absence of anti-4C8 at 1 μg/ml. Although spontaneous migration of both resting and activated T cells into the gels was enhanced two to three times by MCP-1, anti-4C8 had no effect on spontaneous or MCP-1–induced migration . Taken together, the data suggest that anti-4C8 mAb inhibits postadhesive transmigration of T cells without affecting adhesion or suppressing cell motility. Section title: Anti-4C8 mAb Inhibits T Cell Transmigration at the Intercellular Junctions of HUVECs. Educational score: 4.166765213012695 Domain: biomedical Document type: Study Language: en It has been reported that monocytes treated with anti-CD31 remained bound to the apical surface of HUVEC monolayers at the intercellular junctions ( 10 ). We therefore examined where the blockage of T cell transmigration by anti-4C8 occurs on the apical surface of IFN-γ–treated HUVEC monolayers. Although numerous T cells migrated across the monolayer into collagen gels below in the presence of control IgG3, the migration was strongly inhibited in the presence of anti-4C8 at 1 μg/ml . Compared to the controls, T cells that remained on the apical surface of the monolayer increased in number, and most of them appeared to be arrested at the intercellular junctions of HUVECs. Scanning electron microscopy revealed that these T cells were firmly attached and flattened on the EC surface, with pseudopods extending into the junction . Section title: Anti-4C8 mAb Impregnated into Collagen Gels Predominantly Stimulates Chemokinetic Migration of CD26 hi T Cells. Educational score: 4.301871299743652 Domain: biomedical Document type: Study Language: en Activation of cell motility is a critical event in the transmigration process of T cells through the EC junctions. If the 4C8 antigen plays an essential role in the process, stimulation of T cells via the antigen should promote cell motility and migration. To examine this hypothesis, we used collagen gels in which anti-4C8 had been impregnated to substitute for the 4C8 ligand. The impregnated anti-4C8 IgG increased T cell migration in a dose-dependent manner, whereas anti-4C8 Fab fragments, as well as control IgG3 and anti-CD11a, had no such effect (Table I ). In the checkerboard analysis, when soluble anti-4C8 was directly added to T cells above plain gels, migration was significantly enhanced only at the high dose of 10 μg/ml but not at doses ≤1 μg/ml. When the dose of the antibody was the same above and in the gels, the degree of migration was almost equivalent to that induced by anti-4C8 impregnated in the gels, irrespective of the presence of a gradient. Thus, stimulation via the 4C8 antigen appeared to induce migration with increased random motility resembling chemokinesis but not chemotaxis. In our transmigration assay system using HUVEC monolayers, most T cells that do not adhere to HUVECs express low CD26 (CD26 lo ), whereas adherent (but not migrating) cells are predominantly CD26 − and migrating cells are CD26 hi . If the 4C8 antigen is involved in the transmigration, its chemokinetic action should result in migration of CD26 hi cells. We therefore examined whether anti-4C8 stimulation actually causes selective migration of CD26 hi cells. After T cells were incubated for 5 h on collagen gels, with or without impregnated anti-4C8 (10 μg/ml), cells that migrated were isolated and analyzed for CD26 expression. The CD26 profiles of migrated T cells with or without anti-4C8 were different from profiles of the initial T cells . The proportion of CD26 − cells increased among cells that had spontaneously migrated without anti-4C8, whereas the proportion of CD26 hi cells substantially increased among cells that migrated after stimulation with anti-4C8. The CD26 profile of cells that migrated in response to anti-4C8 stimulation was similar to the profile of cells that migrated through HUVEC monolayers. Section title: Anti-4C8 Stimulation Induces Formation of Pseudopods Rich in F-actin and Increases F-actin Content in CD26 hi T Cells. Educational score: 4.449963092803955 Domain: biomedical Document type: Study Language: en Polarization of F-actin content and changes in cell shape are essential events in cell movement ( 23 – 25 ). Therefore, these changes should occur selectively in CD26 hi cells with cell movement induced by anti-4C8. When T cells were incubated for 1 h on the substrate with immobilized anti-4C8, the majority of T cells attached by the mAb displayed cell flattening and irregularities in cell shape. With fluorescence microscopy using TRITC-conjugated phalloidin staining, ∼15% of the cells revealed extreme cell polarization with lamellipodia or filopodia rich in F-actin, whereas with immobilized control IgG3, almost all T cells maintained spherical morphologies . We determined whether the anti-4C8–induced increase in F-actin content is characteristic of CD26 hi T cells. After stimulation for 3 h with antibodies in solution, T cells were fixed and double-stained with FITC-conjugated phalloidin and rhodamine-conjugated anti-CD26 mAb. Flow cytometry revealed that a definite increase in F-actin content was induced in CD26 hi cells, and to a lesser extent in CD26 lo cells, by 10 μg/ml of anti-4C8 but not by control IgG3 at the same dose . The small increase of F-actin in CD26 lo cells was consistent with the observation that anti-4C8 stimulation also induced minimal migration of CD26 lo cells as described above. However, 1 μg/ml of anti-4C8, which strongly blocked transmigration, had no such effect on CD26 hi cells. These data suggest that signaling via the 4C8 antigen induces an increase in F-actin content and morphologic polarization most strongly in CD26 hi T cells, resulting in activated motility and migration of the subset. Section title: G proteins Are Involved in Postadhesive T Cell Transmigration and Migration Induced by Anti-4C8 Impregnated in Collagen Gels. Educational score: 4.222480773925781 Domain: biomedical Document type: Study Language: en In general, chemoattractant receptor signaling is G protein linked and can be inhibited by PT ( 26 , 27 ). G proteins may mediate the signal via the 4C8 antigen, as anti-4C8 stimulation induced chemokinetic migration into collagen gels. Finally, we determined that PT-sensitive G proteins are involved in T cell migration, as well as adhesion and transmigration with IFN-γ–stimulated HUVEC monolayers. The anti-4C8–induced migration and the transendothelial migration were strongly inhibited by PT pretreatment of T cells . In contrast, spontaneous T cell migration and adhesion to the monolayers were insensitive to PT. The inhibitory effect of PT suggests that both anti-4C8–induced chemokinetic migration and transendothelial migration are mediated by signaling pathways that include G proteins. Section title: Discussion Educational score: 4.388772010803223 Domain: biomedical Document type: Study Language: en In our culture system, a restricted subset, ∼6%, of the added T cells migrate through resting HUVEC monolayers. However, the migrating subset mainly consists of CD26 hi cells, despite adhesion of both CD26 − and CD26 hi cells to the HUVECs . This strongly suggests that there are molecules (distinct from those mediating adhesion) that selectively stimulate the motility of CD26 hi cells, causing them to move toward the subendothelial space. The present data indicate that the antigen recognized by anti-4C8 mAb is one of the molecules, due to the following lines of evidence: first, anti-4C8 blocked postadhesive transmigration of T cells without interfering with adhesion and cell motility; second, anti-4C8 impregnated in collagen gels induced chemokinetic migration of T cells, predominantly CD26 hi cells; and third, anti-4C8 stimulation induced formation of large pseudopods rich in F-actin and increased F-actin content selectively in CD26 hi cells. These results suggest that the cellular interaction via the 4C8 antigen between T cells and HUVECs predominantly stimulates cell motility and transmigration of CD26 hi cells. Section title: Discussion Educational score: 4.5284881591796875 Domain: biomedical Document type: Study Language: en Previous studies by others have suggested the possibility that adhesion molecules, such as integrins or ICAMs, participate in transendothelial migration of adherent lymphocytes ( 28 – 31 ). For example, ICAM-2 peptide or cross-linking of LFA-1 (CD11a/CD18) receptors induces migration of NK cells and actin polymerization ( 28 ). In the present study, we have shown that anti-CD11a mAb blocked T cell transmigration. However, this blockage is due to the inhibitory effect of anti-CD11a on T cell adhesion, because the mAb did not inhibit subsequent transmigration of T cells after tight adhesion to HUVEC monolayers . Based on this finding, although stimulation of LFA-1 may be capable of triggering cell locomotion, the molecule seems unlikely to directly mediate T cell transmigration in our system. This conclusion is in line with a recent report showing that activated T cells can not migrate through ICAM-1–transfected monolayers in the absence of a chemotactic gradient ( 8 ). The authors speculated that the diffuse distribution of ICAM-1 on the apical cell surface may not support directed locomotion of T cells across the monolayers. In this regard, the concentrated distribution of CD31 at the EC junctions may be an appropriate stimulus for transmigration of leukocytes, as it has been reported that pretreatment of the EC junctions with anti-CD31 blocks transmigration of monocytes ( 10 ). A hypothesis is now proposed that homophilic interactions of leukocyte and EC CD31 mediate transmigration of adherent leukocytes, including T cells, monocytes, neutrophils, and NK cells ( 3 , 8 – 13 ). However, it should be noted that CD31 + T cells display a naive phenotype characterized by CD45RA + expression, whereas a majority of T cells that migrate through HUVEC monolayers express a memory type phenotype characterized by CD45RO + expression ( 15 – 18 ). Indeed, CD31 − CD45RO + cells have been shown to be the predominant transmigrated cells (15, 18, and our unpublished data). Thus, CD31 appears not to be required for transmigration of memory T cells across HUVEC monolayers. Section title: Discussion Educational score: 4.3052568435668945 Domain: biomedical Document type: Study Language: en Soluble anti-4C8 inhibited T cell transmigration up to 90% at a dose as low as 1 μg/ml. In contrast, it also promoted cell migration into collagen gels when impregnated in the gels at doses from 1 to 10 μg/ml or when used in solution at 10 μg/ml (Table I ). Since Fab fragments of anti-4C8 did not stimulate migration into collagen gels, it seems likely that anti-4C8, at the high dose of 10 μg/ml, cross-links the 4C8 antigen existing on T cells at high density and generates a signal to increase F-actin content, consequently activating cell movement. Thus, the blockage of transmigration might be due to random movement stimulated by anti-4C8, which is sufficient to overcome the force to transmigrate generated on HUVEC monolayers. However, soluble anti-4C8 at 1 μg/ml affected neither F-actin content in T cells nor the spontaneous and chemotactic motility of resting and activated T cells. Rather, these findings suggest that anti-4C8 at the low dose blocked the cellular interaction through the 4C8 antigen (which mediates T cell transmigration) between T cells and HUVEC monolayers without cross-linking of the antigen. Complete inhibition of postadhesive transmigration by Fab fragments strongly supports this notion. Microscopic observations imply that the interaction occurs at the EC junction and that the 4C8 ligand might be concentrated in the junctions like endothelial CD31. More studies are needed to elucidate the existence and the nature of the ligand. Section title: Discussion Educational score: 4.611201286315918 Domain: biomedical Document type: Study Language: en CD26 hi T cells actively transmigrate, but CD26 − T cells remain adherent to HUVEC. This finding strongly suggests that additional cytoskeletal changes should occur to support active cell movement of adherent CD26 hi cells, but not CD26 − cells, directed toward the subendothelial space. To migrate, cells must acquire the redistribution of actin-based cytoskeleton from symmetry around the cell rim to concentration in a particular region, followed by morphological polarization characterized by extension of lamellipodia and filopodia (23, 24, and 32). Interestingly, anti-4C8 caused these changes in resting T cells under culture conditions in which the mAb can stimulate T cells. Solid-phase– immobilized anti-4C8 induced changes in cell shape, including extreme morphological polarization and formation of lamellipodia or filopodia rich in F-actin . When impregnated in collagen gels, anti-4C8 promotes migration of T cells into the gels. The checkerboard analysis suggests that although a gradient is established with the mAb, the migration appears to depend upon the concentration of the antibody itself but not the gradient. Moreover, these effects were observed preferentially in CD26 hi cells . The 4C8 antigen thus appears to transduce a signal preferentially in CD26 hi cells for cell polarization, with protrusion of the membrane that is tightly coupled to polymerization of F-actin. A similar cellular polarization induced by mAbs other than anti-4C8 has been reported with use of T lymphoblasts ( 33 – 36 ). Those reports showed that, similar to chemokines, engagement of ICAM-3 or CD43 with specific mAbs activates the integrin-mediated adhesion and causes the development of a cytoplasmic projection at the tailing edge, termed the uropod, and also induces the redistribution of ICAM-1, -3, CD43, and CD44 to the uropod. These morphological and functional changes may contribute to lymphocyte locomotion and recruitment. However, the 4C8 antigen is definitely distinct from ICAM-3 and CD43 in structural characteristics and tissue distribution. Whether the cellular events caused by anti-4C8 are similar to those caused by anti-ICAM-3 and anti-CD43 mAbs remains to be studied. Section title: Discussion Educational score: 4.398916244506836 Domain: biomedical Document type: Study Language: en It is unclear why CD26 hi T cells are preferentially stimulated by anti-4C8, despite the intense expression of the 4C8 antigen on all CD3 + T cells. CD26 is a widely distributed cell surface glycoprotein of 110 kD with multiple functions ( 37 , 38 ). On human T cells, its expression is preferentially restricted to the CD4 + memory subset and strongly upregulated following cell activation. Recent studies indicate that CD26 has dipeptidyl–peptidase IV activity, is not only a functional receptor for collagen but also a receptor for adenosine deaminase, and acts as a costimulatory molecule for T cell activation. More recently, the chemokine RANTES (regulated on activation, normal T cell expressed and secreted) has been shown to be a natural substrate for CD26 ( 39 , 40 ). These findings suggest that CD26 plays an important role in immune system events such as T cell activation and migration. However, two different anti-CD26 mAbs failed to inhibit T cell transmigration (our unpublished data), suggesting that CD26 is not directly involved in the transmigration process. The avidity of LFA-1 for ICAM-1 is enhanced by a conformational change in the integrin upon activation ( 41 ). Similarly, the activation state of T cells as related to CD26 expression might regulate the affinity of the 4C8 antigen. Section title: Discussion Educational score: 4.574088096618652 Domain: biomedical Document type: Study Language: en We have shown that PT pretreatment of T cells inhibited both anti-4C8–induced chemokinetic migration and postadhesive transmigration. Thus, both of these types of cell motility are mediated via PT-sensitive, G protein–coupled pathways. This is consistent with the idea that the 4C8 antigen is involved in the process of transmigration after adhesion of T cells. The result also raises the question of whether the 4C8 antigen belongs to the chemokine receptor family, because chemoattractant signaling is generally mediated by G proteins and inhibited by PT ( 26 , 27 ). CC chemokines have been shown to attract a subset of CD26 hi CD45RO + T cells ( 42 – 44 ). More recently, it has been reported that transendothelial migration can be triggered by an agonistic mAb against the CC chemokine receptor 2 (CCR2) via a PT-sensitive signaling pathway ( 45 ). Therefore, the 4C8 antigen might be a receptor for these chemokines. However, the 4C8 appears to be different from chemokine receptors identified to date in the following ways: the molecular mass of the 4C8 antigen is 80 kD, whereas the molecular mass of a chemokine receptor was estimated to be ∼35 kD ( 45 , 46 ); in contrast to the 4C8 antigen, chemokine receptor levels on T cells are generally much lower than those on neutrophils and eosinophils ( 43 , 47 ); and the CCR2 expression is low on the CD26 hi T cells and undetectable on other T cells. Moreover, a novel membrane-bound chemokine with a CX 3 C motif that was recently identified has potent adhesive and chemoattractant activity for unstimulated lymphocytes, particularly CD16 + NK cells ( 48 , 49 ). The chemokine is induced on activated endothelial cells and thus presumed to regulate leukocyte trafficking. However, its receptor is expressed on only a small population (up to 14%) of CD3 + T cells ( 49 ). Thus, although chemokine receptors and the 4C8 antigen show functional similarities, they appear to be different molecules based on the differences described above. To our knowledge, the 4C8 antigen is a previously unknown molecule. Gene cloning and identification will define the structural nature of this molecule in the near future. Section title: Discussion Educational score: 4.1891770362854 Domain: biomedical Document type: Study Language: en Finally, we propose herein a hypothesis that the interaction via the 4C8 antigen between a T cell and a HUVEC transduces a signal to stimulate crawling locomotion of CD26 hi cells with profound changes in cell morphology and cytoskeletal assembly, resulting in preferential migration of this T cell subset. | Other | biomedical | en | 0.999997 |
10075982 | Section title: Animals Used in the Study. Educational score: 3.874772548675537 Domain: biomedical Document type: Study Language: en Seven SIV-infected and one uninfected rhesus macaque ( Macaca mulatta ) were used (see Table I ). Six infected and one uninfected animal were given OKT8F (gift from R. Knowles, R.W. Johnson Pharmaceutical Research Institute, Raritan, NJ). One other infected macaque received an isotype-matched (IgG2a) control antibody, P1.17 (TIB-10; American Type Culture Collection). All animal protocols were approved by the Institutional Animal Care and Use Committee at the Tulane Regional Primate Research Center. Section title: Depletion of CD8 + T Cells In Vivo. Educational score: 3.8716464042663574 Domain: biomedical Document type: Study Language: en Macaques were first anesthetized with 10 mg/kg ketamine-HCl, then given bolus injections of OKT8F or P1.17 at 2 mg/kg daily for three consecutive days. 5–6 ml of peripheral blood was obtained frequently before and at various time points after antibody injection from each monkey. Section title: Phenotypic Analysis of the T Cell Subsets. Educational score: 4.1158857345581055 Domain: biomedical Document type: Study Language: en Four-color flow cytometric analyses of whole blood samples were performed to monitor changes in T cell populations. In brief, 100 μl aliquots of whole blood were incubated with respective antibodies for 30 min at 4°C, then washed twice with PBS containing 2% FCS. The red blood cells were first lysed with FACS Lysing Solution ® ( Becton Dickinson ), and washed away. Ki67-FITC (Immunotech) or an FITC-conjugated control antibody was then added to respective tubes for intracellular staining for 30 min at 4°C, followed by further washes and resuspension in 2% formaldehyde (Tousimis Research Corp.) before analysis on a FACSCalibur™ flow cytometer ( Becton Dickinson ). The antibodies used in the study were RhCD3-FITC (Immunotech), CD3-PE ( PharMingen ), CD4-allophycocyanin (APC; Exlpha), CD8–peridinine chlorophyll protein (PerCP; Becton Dickinson ), CD8β-PE (Immunotech), CD45RA-FITC ( Becton Dickinson ), CD69-PE ( Becton Dickinson ), and control antibodies IgG1-FITC, IgG1-PE, IgG1-PerCP, and IgG1-APC (Caltag Laboratories). Section title: Quantifying Plasma Load of SIV by Real-time PCR. Educational score: 4.152405738830566 Domain: biomedical Document type: Study Language: en To measure the viral load of infected monkeys, plasma was separated from whole blood collected in EDTA-containing tubes. Plasma samples were initially spun at 5,000 rpm for 10 min to remove any cells. About 600 μl to 1 ml of plasma was ultracentrifuged at 17,000 rpm for 60 min at 4°C. Supernatant was removed leaving ∼140 μl of plasma and a viral pellet. Viral RNA was purified using the QIAamp viral RNA kit (QIAGEN Inc.). Reverse transcription of viral RNA was then performed in 96-well plates. Each 30-μl reaction contained 10 μl of viral RNA, 1× Taqman Buffer A ( Perkin-Elmer ), 5 mM MgCl 2 , 2.5 μM random hexamers ( Perkin-Elmer ), 0.5 mM each of dATP, dCTP, dGTP, and dTTP, 20 U RNAsin ( Promega ), and 20 U Moloney murine leukemia virus reverse transcriptase (Superscript; GIBCO BRL ). One round of reverse transcription (25°C for 15 min, 42°C for 40 min, 75°C for 5 min) was performed. Section title: Quantifying Plasma Load of SIV by Real-time PCR. Educational score: 4.1719160079956055 Domain: biomedical Document type: Study Language: en To quantify the copy number of viral RNA, a molecular beacon was used in combination with real-time PCR. This method of detection using molecular beacons ( 31 , 32 ) and a fluorescence detector system ( 33 ) has been described previously. Each 50-μl reaction contained 30 μl of cDNA from the reverse transcription reaction, and the final concentration of each component was as follows: 0.4× PCR buffer II, 0.6× Taqman buffer A, 3.5 mM MgCl 2 , 0.4 pmol/μl of molecular beacon, 0.4 pmol/μl of each primer, 1.25 U of AmpliTaq Gold DNA polymerase ( Perkin-Elmer ). The primers used were SL03 (5′-AGGGAAGAAAGCAGATGAATTA-3′) and SL04 (5′-GTTTCACTTTCTCTTCTGCGT-3′). The molecular beacon was designed to recognize a region within SIV gag (5′-FAM-CGCTGGAGAACAAAGAAGGATGTCACAGCG-DABCYL-3′) where FAM (6-carboxyfluorescein) serves as the reporter fluorochrome and DABCYL (4′ dimethylaminophenylazo benzoic acid) the quencher. One cycle of denaturation (95°C for 10 min) was performed, followed by 45 cycles of amplification (95°C for 15 s, 55°C for 30 s, 72°C for 30 s). The PCR reaction was carried out in an ABI 7700 PRISM spectrofluorometric thermal cycler (Applied Biosystems, Inc.) that monitors changes in the fluorescence spectrum of each reaction tube, while simultaneously carrying out programmed temperature cycles. Section title: Quantifying Plasma Load of SIV by Real-time PCR. Educational score: 4.107295989990234 Domain: biomedical Document type: Study Language: en The method of quantifying viral load was adapted from a previous report ( 33 ) with some modifications. For each run, a standard curve was generated from duplicate samples of purified RNA transcripts ranging from 10 6 copies to 50 copy equivalents per reaction (using serial log dilutions in RNase-free water). Each test specimen was reverse transcribed and amplified in duplicate. A third reaction was processed and amplified without the addition of reverse transcriptase to control for potential DNA contamination. Copy numbers were calculated by interpolation of the experimentally determined threshold cycle as described previously ( 33 ). The lower limit of detection was 50 copy equivalents per reaction with a linear dynamic range of >5 logs. When adjusted for the various dilution effects, this results in a sensitivity of ∼400 RNA copies per ml of plasma. The mean of replicate samples was reported. Section title: Results Educational score: 4.072421550750732 Domain: biomedical Document type: Study Language: en Eight macaques were included in this study. Seven were SIV-infected, and one was uninfected. The macaques had been inoculated with either SIVmac251 or 239 between 1 and 4 yr before these experiments (Table I ). The effects of administration of either a murine anti-CD8 mAb, OKT8F, or an isotype-matched control antibody, P1.17, on lymphocyte subsets and plasma viremia were examined. Section title: Inverse Association between CD8 + T Cell Count and Plasma Viremia after OKT8F Injection. Educational score: 4.159216403961182 Domain: biomedical Document type: Study Language: en Six SIV-infected macaques were given OKT8F (2 mg/kg) intravenously as a bolus injection daily for three consecutive days. No febrile episodes or adverse events were observed after each antibody administration. Blood was taken before and at various time points after OKT8F injection. After the third antibody injection, there was an average of 99.9% reduction of CD8 + T cells in peripheral blood (from a mean of 1,139 to 1/μl). In five of six macaques, the depletion was apparent as early as 1 h after the first dose of OKT8F. The number of CD8 + T cells stayed low for 8–10 d, then began to recover in all six animals . To ensure that the disappearance of CD8 + T cells from the blood was genuine, the difference between the percentage of CD3 + T cells and that of CD4 + T cells was determined as a measurement of the percentage of CD8 + T cells. Results showed that this difference was also substantially decreased in all animals (from a mean of 43 to 13%) after the administration of OKT8F. Section title: Inverse Association between CD8 + T Cell Count and Plasma Viremia after OKT8F Injection. Educational score: 4.244620323181152 Domain: biomedical Document type: Study Language: en The effect of CD8 + T cell depletion on plasma viremia was dramatic. An immediate increase in viral load in five of six macaques (AT-02, AT-22, AR-68, AR-71, and AR-93) was noted. The magnitude of the peak viral load increase varied from 20- to 2,500-fold . One macaque, AR-90, had a viral load below the level of detection initially (<400 SIV RNA copies/ml) and remained so during the course of the experiment . The rise and fall of viremia were temporally associated with the drop and then rebound of CD8 + T cell counts during the first 2 wk. After CD8 + T cell counts recovered to pretreatment levels, the viral loads also returned to near baseline levels in four of five macaques that had detectable viremia . The second rise in plasma viremia on day 36 in AR-68 is unexplained. In contrast, the injection of a control antibody, P1.17, to SIV-infected macaque AT-48 caused only a minor perturbation of CD8 + T cells and a relatively modest elevation of plasma viremia transiently . Section title: The Impact of OKT8F on Other Lymphocyte Subsets. Educational score: 4.128993988037109 Domain: biomedical Document type: Study Language: en Consistent with a depletion of CD8 + T cells, the total number of CD3 + T lymphocytes was also reduced in all macaques receiving OKT8F . The CD3 counts stayed lower than the baseline levels for ∼8–10 d, then began to recover in a majority of the animals (AT-22, AT-03, AT-02, AR-68, and AR-93), following kinetics very similar to those of CD8 + T cells. Macaques AR-71 and AR-90 had a slower rate of recovery. Section title: The Impact of OKT8F on Other Lymphocyte Subsets. Educational score: 4.193187236785889 Domain: biomedical Document type: Study Language: en The injection of OKT8F also had effects on CD4 + T cells in the six SIV-infected macaques. A 51% reduction of the absolute number of CD4 + T cells occurred, most noticeably on day 1 (from a mean of 505 to 250/μl). This was followed by a period of rapid fluctuation in cell counts over the next few days before the CD4 + T cell numbers recovered to pretreatment levels by day 15 in most macaques . Again, AR-71 and AR-90 had a slower rate of recovery. The reduction in the number of CD4 + T cells could be accounted for by either an increased destruction of CD4 + T cells as a consequence of elevated viral replication, or OKT8F-mediated cellular activation followed by apoptosis. To distinguish between these two possibilities, OKT8F was given to an uninfected macaque, AT-03. As expected, dramatic CD8 depletion (100%) was observed in the blood of this animal . But the injection of this antibody also led to a 50% reduction of CD4 + T cells, similar to that observed in infected macaques. Therefore, the partial CD4 depletion is unlikely to be caused by SIV replication. Section title: The Impact of OKT8F on Other Lymphocyte Subsets. Educational score: 4.1850104331970215 Domain: biomedical Document type: Study Language: en If antibody injection can lead to nonspecific cellular activation, then the observed viral load increase may have resulted from elevated viral production due to an expansion of activated CD4 target cells, rather than a simple loss of viral suppression by CD8 + T cells. To examine this possibility, an isotype-matched control antibody, P1.17, was given to an SIV-infected macaque, AT-48. P1.17 injection did not deplete CD8 + T cells, but fluctuations in CD4 and CD8 cells were observed . On the other hand, the viral load fluctuated, with a modest peak increase of only threefold, which then decreased to levels below the baseline value . Thus, the stimulation caused by large doses of a murine mAb is not sufficient to explain the dramatic increases in plasma viremia observed after OKT8F injection. Section title: The Impact of OKT8F on Other Lymphocyte Subsets. Educational score: 4.382029056549072 Domain: biomedical Document type: Study Language: en The observed increase in viremia after CD8 + T cell depletion could either be a reflection of diminishing viral control by CD8 + T cells, or an augmentation of viral production due to increased availability of CD4 + target cells, or both. Because activated or proliferating CD4 + T cells are better target cells for SIV infections ( 34 ), CD4 + T cells were examined by flow cytometry for the expression of a nuclear antigen, Ki67, that is indicative of cells in the process of cycling (not in G 0 ). Results from these experiments showed that there was an initial reduction in the absolute number of Ki67 + CD4 + cells after the first dose of OKT8F, and a later recovery during the period of CD4 cell rebound . On average, there was only a threefold increase (from 46 to 150/μl) in the number of CD4 + T cells expressing Ki67 in SIV-infected animals and less than a threefold increase in the SIV-negative monkey, AT-03, a level insufficient to account for the increase in plasma viremia. The first peak of Ki67 + CD4 + T cells appeared around day 3 in six of seven macaques receiving OKT8F (AT-02, AT-22, AT-68, AR-71, AR-90, and AR-93). The same was true for the macaque (AT-48) that received the isotype-matched control antibody . In several macaques (AT-02, AT-03, AT-22, and AR-68), there was a pronounced second peak of Ki67 + CD4 + T cells on days 12–24, coincident with the timing of CD8 + T cell regeneration. The precise mechanism responsible for this second peak is unclear. However, since it appeared much later than the peak of plasma viremia, this second wave of Ki67 + CD4 + cell rise is unlikely to have contributed to the one- to three-log increases in plasma viremia observed earlier. Section title: Theoretical Predictions on the Possible Mechanisms Responsible for the Observed Increase in Plasma Viremia after CD8 + T Cell Depletion in Infected Macaques. Educational score: 4.283979415893555 Domain: biomedical Document type: Study Language: en Several mechanisms could be responsible for the observed increase in viral load. The removal of CD8 + CTLs could increase the life span of productively infected cells, leading to greater virion production. Alternatively, the depletion of CD8 + T cells may reduce the production of β-chemokines, which block the entry of certain virions into target cells ( 26 ), or of inhibitory factors that suppress viral transcription, thereby increasing virion output from productively infected cells ( 36 ). To assess these possibilities, we used a standard model of HIV-1 dynamics ( 37 , 38 ) adjusted to account for the eclipse phase of the viral life cycle (39, and our unpublished observations). Specifically, we used a model involving target cells, T , productively infected cells, T *, virus, V , and τ which defines the number of days required for a generation of HIV in vivo. In addition, the following parameters are introduced: (a) the death rate of infected cells, δ ; (b) the viral production rate, p ; and (c) the infection rate constant, k . Based on these parameters, the model could be described by the following equations: ( 1 ) dT/dt = λ – d T T – kVT , ( 2 ) dT*/dt = ∫ k(t − τ)V(t − τ)T(t − τ)dτ – δT* , and ( 3 ) dV/dt = pT* − cV . Section title: Theoretical Predictions on the Possible Mechanisms Responsible for the Observed Increase in Plasma Viremia after CD8 + T Cell Depletion in Infected Macaques. Educational score: 4.11960506439209 Domain: biomedical Document type: Study Language: en The delay τ is given by a gamma distribution with a peak at 1.5 d and an SD of 0.31 d ( 39 ). Parameters initially chosen were δ = 0.5 d −1 , c = 30 d −1 (reference 39 , and our unpublished observations), p = 100 d −1 , and d T = 0.01 d −1 . The parameters λ and k were chosen to yield a steady state consistent with the measured baseline viral load and CD4 + T cell count. Giving anti-CD8 antibody was assumed to perturb the steady state of the system such that the parameter being examined changed immediately. Theoretical curves were then generated using nonlinear least-squares regression to find the new value of δ , k , or p that gave the best fit to the data. Section title: Theoretical Predictions on the Possible Mechanisms Responsible for the Observed Increase in Plasma Viremia after CD8 + T Cell Depletion in Infected Macaques. Educational score: 4.210901737213135 Domain: biomedical Document type: Study Language: en A representative analysis of the experimental data on two macaques is shown in Fig. 3 , where for AT-22, λ = 21.1 d −1 and k = 1.33 × 10 −3 μl −1 d −1 , whereas for AR-93, λ = 10.4 d −1 and k = 9.0 × 10 −4 μl −1 d −1 . To mimic the loss of CTL killing, the death rate of productively infected T cells, δ , was decreased. However, setting δ = 0 as the most extreme case did not cause the viral load to increase as rapidly as observed experimentally. Similarly, increasing the infection rate constant, k , to mimic the loss of β-chemokines from CD8 + T cells, could not explain the rapid increase in viral load seen during the first 24 h, since removing a block on infection has no effect on plasma viral load until after the eclipse phase of the viral life cycle ( 37 ). Finally, removing inhibitory factors on viral transcription is equivalent to increasing p , the rate of virion production by productively infected cells. As shown in the two examples in Fig. 3 , this gives a rapid increase in plasma virus, followed by a plateau period during which newly produced virions go on to infect cells and traverse the eclipse phase. It is evident from these preliminary theoretical analyses that neither changes in p or k fit the observed data particularly well, but an increase in p provides a better fit than an increase in k . Section title: Discussion Educational score: 4.175228595733643 Domain: biomedical Document type: Study Language: en The role of CD8 + T cells in the control of SIV replication in vivo awaits experimental confirmation. Here, we examined their importance by depleting these cells with an anti-CD8 mAb, OKT8F, and by following the subsequent virologic and immunologic changes. Although the extent of CD8 + T cell depletion was not examined in lymphoid tissues in our study, we found that depleting such T cells in peripheral blood led to a peak viral load increase of one to three orders of magnitude in five of six macaques studied. These results demonstrate that CD8 + T cells, at a minimum, must play a major role in the control of SIV replication during chronic infection. If OKT8F administration did not result in complete CD8 + T cell depletion in lymphoid tissues in our experiment, then the role of these cells in inhibiting viral replication in vivo would be even more significant than what was observed here. Thus, we concur with the conclusion drawn by Matano et al. ( 29 ), who studied the effect of CD8 depletion in primary SHIV infection. Section title: Discussion Educational score: 4.107237339019775 Domain: biomedical Document type: Study Language: en The depletion of CD8 + T cells by OKT8F was not unexpected; however, the speed at which CD8 + T cells disappeared from blood was surprising. Nevertheless, the kinetics of CD8 depletion observed in our experiments is consistent with previous depletion experiments done by others ( 28 , 29 ). Likewise, the OKT4 antibody cleared CD4 + T cells from peripheral blood within 10 min of injection in rhesus monkeys ( 40 ). The OKT3 antibody also depleted most of the peripheral T lymphocytes within 1 h after administration to humans ( 41 ). Section title: Discussion Educational score: 4.454436302185059 Domain: biomedical Document type: Study Language: en Several mechanisms may be responsible for the observed depletion of CD8 + T cells from the blood. First, the binding of OKT8F to CD8 molecules on cells may have led to complement-mediated cytolysis. Second, OKT8F may have only masked the epitope recognized by the staining anti-CD8 antibody, resulting in an artificially low number of CD8 + T cells. Third, there may have been an increased CD8 endocytosis after binding by OKT8F, thereby lowering surface CD8 expression without cellular depletion. Finally, there may have been a preferential trapping of antibody-bound CD8 + T cells in lymph tissues, leading to an apparent depletion of such cells in the circulation. To address the second and third possibilities above, we examined the difference in the number of CD3 + and CD4 + T cells. This difference declined in parallel with the number of CD8 + T cells measured directly, showing that there is a genuine depletion of CD8 + T cells in blood. Further experiments are necessary to examine the possibility of trapping of CD8 cells in lymphoid tissues after OKT8F injection. However, if the CD8 + T cells were trapped in the lymph nodes, where most of the viral replication takes place, they were probably nonfunctional given the substantial rise in plasma viremia. Irrespective of the underlying mechanism, the injection of OKT8F appears to have severely impacted the CD8 + T cells as to cause a dramatic increase in virus replication. However, it is still formally possible that the loss of control of viral replication is due partly to the elimination of NK cells that express CD8 on their surface. Section title: Discussion Educational score: 4.376595497131348 Domain: biomedical Document type: Study Language: en Several different mechanisms could potentially be responsible for the increase in viral load. The removal of CD8 + CTLs could increase the life span of productively infected cells, leading to greater virion production. Alternatively, the depletion of CD8 + T cells may reduce the production of factors, such as β-chemokines, that block the entry of certain virions into target cells ( 26 ), or of inhibitory factors that suppress viral transcription, thereby reducing virion output from productively infected cells ( 36 ). To quantitatively evaluate these hypotheses, we used a standard model of HIV-1 dynamics ( 37 , 38 ) adjusted to account for the eclipse phase of the viral life cycle (39, and our unpublished observations). For each monkey, a set of parameters was determined that corresponded to the animal being in a steady state with the measured baseline viral load and CD4 + T cell count. To mimic the loss of CTL killing, the death rate of productively infected T cells, δ , was decreased. Even lowering δ to 0 as the most extreme case did not cause the viral load to increase as rapidly as observed. Similarly, increasing the infection rate constant, k , to mimic the loss of β-chemokines from CD8 + T cells, could not explain the rapid increase in viral load seen during the first 24 h . Finally, removing inhibitory factors on viral transcription (increasing p ) gives a better fit to the data. Thus, the rapid rise in plasma viremia over the first 2–3 d is more consistent with a higher virus production rate than a higher infection rate. Moreover, even a maximal decrease in δ fails to give a good fit to the data, suggesting that decreased cytotoxic killing is not the primary mechanism responsible for the increased viremia. Overall, these conclusions on the mechanism of virus inhibition by CD8 + T cells should be viewed as preliminary and must be addressed in greater detail in future experiments. Section title: Discussion Educational score: 4.225239276885986 Domain: biomedical Document type: Study Language: en The depletion of CD8 + T cells was accompanied by reduction in the CD4 + T cells in all animals receiving OKT8F. Several possible explanations might be considered. There may have been increased destruction of CD4 + T cells due to rapid rise in SIV replication after OKT8F infection. However, when the same antibody was given to an uninfected macaque (AT-03), a similar magnitude of depletion of CD4 + T cells was also observed. Therefore, SIV is unlikely to have contributed significantly to this phenomenon. In agreement with our findings, it was observed in a previous study that treatment with anti-CD8 antibody also resulted in lowering of B cells and CD4 + T cells ( 42 ). It is worth noting that the injection of OKT3 also led to a depletion of non-T cells, such as B cells and NK cells, in humans ( 41 ). Thus, another explanation is that OKT8F injection may be equivalent to a large dose of foreign antigen, activating numerous cell types, including CD4 + T cells that later undergo activation-induced apoptosis. Finally, we have noted that there are greater numbers of lymphocytes (up to 14%) that coexpress CD4 and CD8 in SIV-infected macaques (our unpublished observations). The elimination of such cells by OKT8F may contribute to the lowering of CD4 + T cell counts, although they are numerically insufficient to be the sole mechanism. Further studies are necessary to understand the indirect impact on the CD4 + T cell population. Section title: Discussion Educational score: 3.869443655014038 Domain: biomedical Document type: Study Language: en In summary, we have demonstrated that CD8 + T cells do play a major role in controlling SIV in vivo. Therefore, we suggest that in the development of SIV or HIV vaccines, a stronger emphasis be placed on inducing specific CD8 + T cell responses. | Study | biomedical | en | 0.999999 |
10075983 | Section title: Cell Culture and Treatments. Educational score: 4.078702449798584 Domain: biomedical Document type: Study Language: en Mouse embryonic fibroblasts (MEFs) derived from RelA-deficient mice ( 9 ) were cultivated in DMEM supplemented with 10% calf serum and antibiotics. MEFs were stimulated in the presence of 10 ng/ml of TNF-α ( Genzyme ), 10 ng/ml of IFN-γ ( Genzyme ), or 10 μg/ml of LPS ( Sigma ) for the time periods indicated in the figure legends. Section title: Antibodies. Educational score: 2.9129037857055664 Domain: biomedical Document type: Study Language: en mAb against Fas receptor (Jo2) and PE-conjugated hamster Jo2 were purchased from PharMingen . Jo2 was used at 1 μg/ml for Fas-induced cell killing. Section title: Northern Blot Analysis. Educational score: 4.154746055603027 Domain: biomedical Document type: Study Language: en RNA was isolated from cells grown under normal conditions (untreated cells) or stimulated for 6 h with LPS and/or cytokines. Total RNA was extracted using the TRIzol reagent (Molecular Research Center) as recommended by the manufacturer. 10 μg of total RNA was size fractionated on denaturing formaldehyde gels for 4–5 h and transferred overnight to a nylon membrane. Different mRNAs were detected by hybridization to specific 32 P probes (reverse transcription PCR products from mouse fibroblast cDNA) in the presence of salmon sperm DNA ( Sigma ) for 1 h at 68°C. Final washes (twice for 15 min) were performed in 0.2% SSC, 0.1% SDS at 25°C. RNA loading was controlled by normalization to a β-actin probe. The different mRNA levels were quantified by phosphorimaging. Section title: Flow Cytometric Analysis. Educational score: 4.020908355712891 Domain: biomedical Document type: Study Language: en Cells were treated with LPS and/or cytokines, harvested, and stained with PE-conjugated hamster anti–murine Fas mAb Jo2 for 30 min on ice. Cells were washed twice, fixed in 4% paraformaldehyde, and analyzed using a Becton Dickinson flow cytometer. Section title: Analysis of Fas-mediated Cell Death. Educational score: 4.067697048187256 Domain: biomedical Document type: Study Language: en MEFs were cultured for 12 h in the presence or absence of LPS, TNF-α, and/or IFN-γ. After treatments, cells were washed with fresh medium and anti– mouse Jo2 was added overnight at 1 μg/ml. Cells were collected and counted in the presence of trypan blue. Data are expressed as a percentage of viable cells in the untreated population. Section title: The RelA Component of NF-κB Is Critically Required for Activation of Key Genes Involved in Adaptive Immune Responses. Educational score: 4.424384593963623 Domain: biomedical Document type: Study Language: en The gene encoding for the MHC class I H-2 molecule can be activated by TNF-α and IFN-γ ( 19 ). Although NF-κB sites have been found within transcriptional control regions of this gene ( 20 , 21 ), the role of NF-κB proteins in its activation is not known. Furthermore, transcription factors that do not belong to the NF-κB family have also been shown to bind specifically to the MHC κB site ( 22 ). Therefore, we wished to determine whether RelA participates in activation of this gene. To this end, primary MEFs derived from RelA +/− or RelA −/− mice were first treated with TNF-α or LPS. We have previously shown that LPS is a potent inducer of NF-κB in MEFs, although the effect of such treatment on gene induction was not determined ( 23 ). Treatments were carried out for 6 h, since this time period is sufficient for maximal activation of the genes we have studied and does not result in significant death of RelA −/− MEFs by TNF-α. Northern blot analysis of RNA obtained from TNF-α– or LPS-treated RelA +/− MEFs showed a moderate increase in H-2 expression (two- to threefold). However, this increase was significantly potentiated in the presence of IFN-γ (10.5-fold with TNF-α). In contrast, no induction of expression was seen in the RelA −/− cells after TNF-α or LPS treatment, whereas IFN-γ–mediated activation of H-2 expression (threefold) was unaltered in these cells . These results demonstrate that RelA is required for activation of the H-2 gene after TNF-α or LPS and for potentiation of expression in the presence of IFN-γ. Section title: The RelA Component of NF-κB Is Critically Required for Activation of Key Genes Involved in Adaptive Immune Responses. Educational score: 4.12592887878418 Domain: biomedical Document type: Study Language: en MHC class I is critical for the initial interaction between T lymphocytes and target cells. We next tested whether other genes functionally important in interactions between T lymphocytes and target cells or APCs are also under RelA control. The CD40 gene can be inducibly expressed on APCs and is important for activation of both T cells which express the CD40 ligand and the APCs ( 24 ). Once again, TNF-α or LPS treatment increased expression of CD40 mRNA in RelA +/− MEFs, and the presence of IFN-γ synergistically increased the amount of CD40 mRNA. In contrast, RelA −/− MEFs showed virtually no increase in the amount of CD40 mRNA in the presence of TNF-α or LPS alone or in combination with IFN-γ . Section title: The RelA Component of NF-κB Is Critically Required for Activation of Key Genes Involved in Adaptive Immune Responses. Educational score: 4.329408645629883 Domain: biomedical Document type: Study Language: en Next we tested the effect of these inducers on expression of the Fas death receptor. Coupling of Fas expressed on target cells with Fas ligand expressed on T cells generally results in apoptotic demise of the target cell ( 25 ). Fas expression is generally low in most cells, although certain cells such as hepatocytes constitutively express high levels of Fas ( 25 ). As shown in Fig. 1 , Fas expression was dramatically induced by TNF-α plus IFN-γ or LPS plus IFN-γ (∼10-fold), but less strongly by these inducers alone (2–3-fold). Importantly, RelA −/− MEFs showed greatly diminished induction of Fas mRNA (approximately twofold) under the same conditions . These results provide the first direct proof for a role of NF-κB in regulation of the MHC class I, CD40, and Fas death receptor genes, and suggest that NF-κB may play a key role in activation of genes involved in specific immune responses by nonspecific stimuli such as LPS. Activation of NF-κB by such stimuli may thus allow synchronous expression of multiple genes that carry out similar or complementary functions. Section title: IFN-γ–dependent and –independent Gene Activation by RelA. Educational score: 4.387818336486816 Domain: biomedical Document type: Study Language: en IFN-γ is a potent activator of macrophages, especially in combination with LPS. Two genes critically involved in macrophage function that can also be activated in fibroblasts are those encoding the inducible form of nitric oxide synthase (iNOS) and the proinflammatory cytokine IL-6. Therefore, we tested whether activation of these genes was dependent on RelA. As above, both iNOS and IL-6 genes were potently induced by the combined effects of LPS and IFN-γ in RelA +/− MEFs . Interestingly, activation of these genes was considerably less after TNF-α or TNF-α and IFN-γ treatment . Induction was again significantly reduced in RelA −/− MEFs , demonstrating that RelA is specifically required for activation of these genes. Next we tested the potential involvement of RelA in regulation of chemokine gene expression. Chemokines produced at sites of inflammation, often by fibroblasts, play an important role in both initiation and potentiation of an inflammatory response. Thus IFN-inducible protein (IP)-10, a chemokine specific for macrophages and T lymphocytes ( 26 ), was also synergistically induced by LPS or TNF-α and IFN-γ in RelA +/− MEFs, and induction was reduced in RelA −/− MEFs . Taken together, our results demonstrate that the RelA component of NF-κB is important for gene induction by LPS and TNF-α alone and for synergistic activation in the presence of IFN-γ. Section title: IFN-γ–dependent and –independent Gene Activation by RelA. Educational score: 4.525117874145508 Domain: biomedical Document type: Study Language: en An inflammatory response can occur without the involvement of antigen-specific lymphocytes, often providing the first line of defense against invading pathogens. Key leukocytes involved in such “immediate” responses are neutrophils, which are attracted to infected or inflamed sites by chemokines. Two such neutrophil-specific chemokines, KC ( 27 ) and macrophage-inflammatory protein (MIP)-2 ( 28 ), were found to be potently induced by LPS in RelA +/− MEFs (and to a lesser degree by TNF-α), whereas induction was significantly reduced in RelA −/− MEFs . Importantly, and in contrast to the examples described above, IFN-γ neither induced expression of these chemokines nor potentiated expression by LPS or TNF-α treatments, in both RelA +/− and RelA −/− MEFs. These results indicate a potentially dual role for RelA in target gene regulation. In the absence of specific immune effectors such as IFN-γ produced by T cells, RelA alone can function as a potent inducer of innate response genes, e.g., neutrophil chemokines. However, IFN-γ is required for optimal induction by RelA of genes involved in an adaptive immune response. Section title: IFN-γ–dependent and –independent Gene Activation by RelA. Educational score: 4.21457576751709 Domain: biomedical Document type: Study Language: en Interestingly, significant differences in induction of specific genes in response to LPS or TNF-α and in synergy with IFN-γ were noticed. For example, although MHC class I, CD40, and Fas were activated to a comparable extent by TNF-α or LPS, the induction of IP-10, KC, and MIP-2 is dramatically more by LPS than by TNF-α. The basis for such differences is presently unclear, but they indicate that different NF-κB inducers may activate distinct heterodimers of RelA-containing complexes, and κB sites in different genes may preferentially bind different heterodimers. Induction of iNOS expression in macrophages requires c-Rel ( 29 ), suggesting that both RelA and c-Rel are important for activation of this gene. Section title: Increased Resistance of RelA-deficient MEFs to Fas-induced Cell Death. Educational score: 4.258386611938477 Domain: biomedical Document type: Study Language: en We next wished to determine the consequence of RelA-dependent activation of Fas expression on induction of cell death by this receptor. First, we determined whether the observed induction of Fas mRNA correlated with increased surface expression of this molecule after LPS, IFN-γ, or LPS plus IFN-γ treatment of RelA +/− or RelA −/− MEFs. Only LPS was used as the NF-κB activator, since it results in potent induction of Fas mRNA in RelA +/− cells in the presence of IFN-γ and is not cytotoxic to RelA −/− cells. Combined treatment of RelA +/− MEFs with LPS and IFN-γ resulted in an ∼10-fold increase in Fas surface expression, as measured by FACS ® analysis, whereas LPS and IFN-γ treatments alone resulted in less significant increases . In contrast, Fas expression was increased only twofold in RelA −/− cells after LPS plus IFN-γ treatment . Thus, potent mRNA induction of Fas results in increased cell surface expression of Fas in RelA +/− but not in RelA −/− MEFs. We have also found that IFN-γ treatment is moderately cytostatic to both RelA +/− and RelA −/− cells ; in RelA −/− cells, some cytotoxicity through likely production of TNF-α or TNF-β was also noticed after LPS and IFN-γ treatment. Section title: Increased Resistance of RelA-deficient MEFs to Fas-induced Cell Death. Educational score: 4.273456573486328 Domain: biomedical Document type: Study Language: en We then tested whether cross-linking with the Fas-specific Jo2 antibody ( 30 ) resulted in death of RelA +/− or RelA −/− fibroblasts. Significant cell death was observed in RelA +/− cells by trypan blue staining when Jo2 was added after LPS or IFN-γ treatment, and combined treatment resulted in even greater killing . No cell death was observed in the absence of LPS or IFN-γ treatment. In contrast, Fas-induced cell death was significantly reduced in RelA −/− cells after LPS or combined treatment with LPS and IFN-γ . Our results indicate that the level of Fas expression may be critical in determining whether a cell will undergo apoptosis after ligand binding, and that the RelA component of NF-κB is required for activation of Fas expression and thus for induction of cell death by this receptor. Furthermore, activation of Fas expression appears to be directly mediated by RelA, since consensus NF-κB binding sites have been found in the human Fas promoter ( 31 ) and induction of Fas expression was found to be independent of new protein synthesis (data not shown). Section title: Increased Resistance of RelA-deficient MEFs to Fas-induced Cell Death. Educational score: 4.23900032043457 Domain: biomedical Document type: Study Language: en Previous studies have demonstrated an antiapoptotic function for RelA in TNF-α and DNA damage–induced cell death pathways ( 15 , 32 – 34 ). However, the results presented here indicate that RelA is involved in regulation of both proapoptotic and antiapoptotic genes. Recent studies suggest that Fas expression may be responsible for tissue destruction in mouse autoimmune disease models for experimental autoimmune encephalomyelitis (EAE) and insulin-dependent diabetes (IDD) ( 35 – 38 ). Indeed, elevated Fas expression has been found in β cells of the pancreas ( 36 ). Our results indicate that Fas expression may be elevated by cytokines such as IFN-γ, TNF-α, TNF-β, and IL-1 produced by infiltrating T cells and macrophages. A role for NF-κB in regulation of Fas expression suggests that inhibition of this transcription factor may have therapeutic potential for the treatment of autoimmune diseases. | Study | biomedical | en | 0.999999 |
10075984 | Section title: Mice. Educational score: 4.090842247009277 Domain: biomedical Document type: Study Language: en SJL/J and CSJLF1/J mice were obtained from The Jackson Laboratory . BALB/cAnN mice were obtained from Taconic Farms, Inc. hIL-10Tg mice were constructed using a hIL-10 cDNA sequence regulated by a class II MHC Eα promoter sequence ( 13 ). For this study, male hemizygous BALB/c IL-10Tg mice were backcrossed with female BALB/cAnN mice to generate hIL-10Tg and nonTg littermates. SJL × BALB/c F1 transgene–positive and nonTg littermates were generated by crossing male hemizygous hIL-10Tg BALB/cAnN mice with female SJL/J mice. To generate mice expressing the hIL-10 transgene but lacking endogenous murine IL-10, the hIL-10 transgene was backcrossed onto the BALB/cAnN IL-10KO background. Male BALB/cAnN hemizygous hIL-10Tg IL-10KO mice were bred with female BALB/cAnN IL-10KO mice to produce transgene-positive and nonTg littermate mice on the IL-10KO background. Section title: Induction of EAE. Educational score: 4.094536781311035 Domain: biomedical Document type: Study Language: en Mouse spinal cord homogenate (MSCH) was prepared from 8–12-wk-old BALB/cAnN mice as previously described ( 14 ). Bovine MBP was obtained from Sigma Chemical Co. For active induction of EAE, mice were immunized intradermally with 2.5 mg of MSCH and 200 μg of Mycobacterium tuberculosis (strain H37RA; Difco) at days 0 and 7 as described ( 14 ). Mice were examined and scored for clinical signs of EAE, and routine histopathological analyses of hematoxylin and eosin– or Luxol fast blue–stained paraffin sections were performed in a masked fashion as described ( 14 ). Section title: Antibodies. Educational score: 4.114916801452637 Domain: biomedical Document type: Study Language: en All mAb were purified by column chromatography from either ascites fluid or tissue culture supernatants and contained <4 EU endotoxin/mg protein. 1B1.2 is a blocking mAb reactive with mouse IL-10 receptor used in in vitro cultures ( 15 ). The following mAbs were used in vivo: JES3-9D7 (rat IgG1), a hIL-10–specific mAb that does not cross-react with mouse IL-10 ( 16 ); and GL113 (rat IgG1), an isotype control mAb reactive with β-galactosidase. For in vivo mAb treatment, mice were injected i.p. with 1 mg mAb/dose in 100 μl of PBS. Each animal received three injections, once per week. Section title: Cell Purification. Educational score: 4.1687211990356445 Domain: biomedical Document type: Study Language: en CD4+ draining lymph node (DLN) cells were purified by positive selection using MACS ® L3T4 microbeads and MiniMACS ® columns (Miltenyi Biotec). The microbead-labeled cell suspensions were processed through the magnetic column twice, and the purity was routinely >95% CD4+ cells. To prepare T cell–depleted APC, spleen cells from naive CSJLF1/J mice were depleted of CD4 (GK1.5, 20 μg/ml)- and CD8 (2–43, 20 μg/ml)-staining cells by negative selection using anti–rat Ig-coated Dynabeads ( Dynal ). Section title: Cell Culture and Cytokine Detection. Educational score: 4.085501194000244 Domain: biomedical Document type: Study Language: en Mice were immunized with MSCH following the same procedure as for active induction of EAE. 10 d after immunization, DLN cells (4 × 10 6 /ml) and purified CD4+ DLN cells (10 6 /ml) were stimulated with 50 μg/ml of MBP as described ( 14 ). Anti–IL-10 receptor mAb (1B1.2, 10 μg/ml) was added to cultures where indicated. Culture supernatants were harvested after 60 h and levels of IFN-γ and IL-4 were determined using a sandwich ELISA technique as described ( 14 ). Section title: Transgenic IL-10 Prevents Induction of EAE. Educational score: 4.1554107666015625 Domain: biomedical Document type: Study Language: en For the initial study, hIL-10Tg mice and nonTg littermate controls on the BALB/cAnN genetic background were compared for EAE susceptibility. Although BALB/c mice are generally considered to be EAE resistant, it has been reported that BALB/cAnN and BALB/cByJ substrains are susceptible to actively induced EAE ( 17 ). Consistent with these results, EAE was reproducibly induced in these strains of mice by intradermal immunization on days 0 and 7 with MSCH in CFA (84%, n = 86; average EAE grade = 3.2; day of disease onset = 17–30 d after immunization). With this immunization, hIL-10Tg mice were completely EAE resistant (0/23 mice), whereas control littermate mice were highly EAE susceptible and exhibited the same EAE severity as wild-type BALB/cAnN and SJL/J mice . No inflammatory infiltrates were found in the spinal cords of EAE-resistant Tg mice, whereas intense inflammatory infiltrates and demyelination were found in the white matter of EAE-susceptible nonTg littermate mice . Section title: Transgenic IL-10 Prevents Induction of EAE. Educational score: 4.118837356567383 Domain: biomedical Document type: Study Language: en To confirm these findings in a more conventional model of EAE, hemizygous male BALB/c hIL-10Tg mice were crossed with SJL/J mice to generate transgene-positive and nonTg littermates. These F1 mice were compared to CSJLF1/J, which have a highly predictable disease onset at 14 d after immunization with MSCH and a relatively strong in vitro recall response to MBP. Transgene-positive SJL/J × BALB/cAnN F1 mice had a low EAE incidence (4/55 mice) and disease severity compared to nonTg littermate control mice (43/46 mice), which were highly susceptible to EAE. The nonTg littermates exhibited an EAE severity and disease course similar to wild-type CSJLF1/J mice . No difference in EAE severity or day of disease onset was observed when comparing CSJLF1/J with SJL/J mice (data not shown). Section title: Transgenic IL-10 Must Be Present during the Induction of Disease to Prevent EAE. Educational score: 4.180398941040039 Domain: biomedical Document type: Study Language: en To show that disease resistance was due to the direct effects of the transgenic IL-10, immunized transgenic SJL/J × BALB/cAnN F1 mice were injected with either JES3-9D7 mAb (hIL-10–specific mAb that does not cross-react with mouse IL-10) or GL113 mAb (an isotype-matched mAb control). Tg mice receiving the first anti–hIL-10 mAb injection on the day of immunization were susceptible to EAE, whereas the isotype control mAb–injected mice were resistant to EAE . This result suggests that EAE resistance of hIL-10Tg mice was the consequence of hIL-10 present during immunization, rather than of developmental effects of the transgene. A consistent delay in the day of disease onset was found in the anti–hIL-10 mAb–treated mice compared to control littermates . To determine the cause of this delay, anti–hIL-10 mAb treatment was initiated 8 d before MSCH immunization. With this treatment regimen, the EAE clinical grade and day of disease onset for hIL-10Tg and control littermate mice were indistinguishable . Similar results were obtained when hIL-10Tg mice on the BALB/cAnN genetic background were treated with the anti–hIL-10 mAb (data not shown). Section title: MBP-specific Th1 Cells Were Generated Normally in IL-10Tg Mice. Educational score: 4.446398735046387 Domain: biomedical Document type: Study Language: en Three possible mechanisms for the regulatory effect of IL-10Tg in EAE are as follows: inhibition of the initial development of autoreactive Th1 cells, active inhibition of Th1 cells by IL-10, or immune deviation toward a Th2-type response. To test these, the cytokine secretion profiles of DLN cells from MSCH-immunized SJL × BALB/c F1 hIL-10Tg and control littermate mice were analyzed. 10 d after immunization, DLN cells were prepared for culture in the presence of MBP or MBP plus anti–IL-10 receptor mAb. After 3 d, the culture supernatants were tested for secreted IFN-γ and IL-4. Cells from nonTg littermate mice stimulated with MBP secreted higher levels of IFN-γ than cells from Tg mice, a result consistent with the association of EAE pathogenesis with IFN-γ production . However, when stimulated with MBP in the presence of anti–IL-10 receptor mAb, cells from both hIL-10Tg mice and littermate controls produced similar levels of IFN-γ. To eliminate APC differences between hIL-10Tg and control littermate mice, CD4+ T cells were purified and cultured with irradiated, T-depleted splenocytes from CSJLF1/J donor mice. In the absence of APC-derived hIL-10Tg, T cells from both hIL-10Tg and control littermate mice produced equivalent levels of IFN-γ . No MBP-specific IL-4 production was detected in cultures of DLN cells or CD4+ T cells from hIL-10Tg or control littermate mice, suggesting that EAE resistance in the Tg mice was not associated with a Th2-type response (data not shown). These results support the interpretation that myelin-reactive Th1 cells were induced in the hIL-10Tg mice and suggest that their ability to exert an effector function was actively suppressed by IL-10 in vivo. Section title: hIL-10Tg in the Absence of Endogenous Mouse IL-10 Can Prevent EAE. Educational score: 4.297788619995117 Domain: biomedical Document type: Study Language: en To assess whether T cell–derived endogenous mouse IL-10 was required for EAE resistance, the IL-10Tg mice were backcrossed onto an IL-10 null (IL-10KO) background to generate mice that expressed hIL-10Tg but lacked endogenous mouse IL-10. Because murine T cells do not express MHC class II molecules, no T cell–derived IL-10 is present in these mice. The hIL-10Tg IL-10KO mice and nonTg IL-10KO littermates were tested for susceptibility to EAE. Transgene-positive IL-10KO mice, which only have MHC class II–positive cell–derived IL-10, were completely resistant to EAE, whereas nonTg IL-10KO littermates were highly susceptible to EAE . No difference in EAE susceptibility was observed when nonTg IL-10KO littermates were compared with BALB/cAnN IL-10KO mice (data not shown). Treatment with anti– hIL-10 mAb reversed this protection in the Tg IL-10KO mice, which became as susceptible to EAE as nonTg IL-10KO littermates . To determine the extent of the protection provided by the IL-10Tg in the absence of endogenous murine IL-10, the CNS of the three groups of mice from Fig. 5 were examined. Intense inflammatory infiltrates and extensive demyelination as determined by the loss of Luxol fast blue staining were found in the white matter of nonTg IL-10KO littermates, consistent with the clinical disease . In contrast, the CNS of the EAE-resistant mice expressing the IL-10 transgene but lacking endogenous murine IL-10 were completely free of inflammatory infiltrates and demyelination . Examination of the CNS of hIL-10 transgene–positive IL-10KO mice treated with anti–hIL-10 mAb showed extensive inflammation and demyelination similar to the pathological changes found in the nonTg littermates (data not shown). These data suggest that APC production of IL-10 alone, despite the complete absence of T cell–produced IL-10, is sufficient to protect mice from EAE. Section title: Discussion Educational score: 4.3298797607421875 Domain: biomedical Document type: Study Language: en In this study, we have demonstrated that Tg mice expressing hIL-10 under the control of the MHC class II promoter are completely resistant to induction of EAE and that this resistance is independent of T cell–derived IL-10. The use of human IL-10, which has similar specific activity to murine IL-10 ( 1 , 2 ), allowed for the specific measurement and inhibition of Tg and endogenous IL-10. Treatment of MSCH-immunized mice with neutralizing anti–hIL-10 at the time of immunization rendered transgene-positive mice fully susceptible to EAE, although the disease onset was delayed by several days . Treatment of hIL-10Tg mice with anti–hIL-10 mAb 8 d before MSCH immunization resulted in kinetics of disease onset identical to that of nonTg littermates, suggesting that the elevated IL-10 level before immunization can also influence the response to antigen, consistent with our previous study showing that IL-10 has significant effects on the function of APC prior to antigen-induced activation ( 18 ). Furthermore, APC-derived IL-10Tg is sufficient for EAE prevention, as shown by the resistance of transgene-positive BALB/c mice homozygous for a mutated mouse IL-10 gene . Therefore, IL-10 need not be provided by T cells to prevent EAE. Section title: Discussion Educational score: 4.24210262298584 Domain: biomedical Document type: Study Language: en Several observations suggest that myelin-reactive Th1 cells are induced in hIL-10Tg mice despite the absence of disease. Similar levels of IFN-γ were produced by purified, MBP-specific CD4+ cells from EAE-resistant hIL-10Tg mice and EAE-susceptible nonTg littermates when stimulated in vitro with wild-type APC and MBP . No MBP-induced IL-4 was produced by T cells from Tg or nonTg mice, suggesting that EAE resistance was not associated with a Th2 response. These results are consistent with a report showing that, although systemic treatment with rIL-10 decreased the severity of EAE in SJL mice, PLP-specific Th1 cells were induced and no PLP-specific Th2 cells were found ( 9 ). These data are also in agreement with the original finding that IL-10 does not substantially inhibit the development of Th1 cells but does inhibit their effector function ( 1 , 19 ). Section title: Discussion Educational score: 4.224281311035156 Domain: biomedical Document type: Study Language: en EAE in BALB/c and SJL × BALB/c F1 mice is characterized by an intense inflammatory infiltrate found predominantly in the white matter of the CNS and occasionally extending into the gray matter region, causing neuronal destruction and death (not shown). The absence of an inflammatory infiltrate in the CNS of EAE-resistant Tg mice suggests that either autoreactive T cells did not enter the CNS or the few T cells entering the CNS were not able to recruit additional inflammatory cells. In vitro, IL-10 has been shown to inhibit antigen presentation and production of IL-1, IL-6, TNF-α, CD80, and CD86 by LPS- and/or IFN-γ–activated microglia ( 20 , 21 ). Therefore, it is possible that IL-10Tg in the CNS may inhibit microglia antigen presentation and activation of myelin-reactive Th1 cells. Section title: Discussion Educational score: 4.112170696258545 Domain: biomedical Document type: Study Language: en Several parameters, including the amount of IL-10 and the mode of gene regulation in specific local regions, may account for the effectiveness of IL-10Tg in inhibition of EAE. hIL-10 was detectable in the serum of the hIL-10Tg mice at levels of 400–700 pg/ml ( 13 ). In vitro, hIL-10 and mouse IL-10 were detected in the lymph node cell cultures at similar levels of 2–6 ng/ml (data not shown). Thus, IL-10Tg was not present systemically at high levels even though it significantly protected mice from EAE. Section title: Discussion Educational score: 4.316431999206543 Domain: biomedical Document type: Study Language: en Two separate studies have shown that IL-10 regulated by lymphocyte-specific promoters can inhibit EAE. Adoptive transfer of PLP-specific memory T cells expressing IL-2 promoter–regulated IL-10 partially reduced or reversed disease in PLP-immunized recipient mice ( 12 ). FVB × SJL F1 mice expressing Tg murine IL-10 under the lymphocyte-specific CD2 promoter are resistant to EAE induced by PLP immunization ( 22 ). These results and the findings in the present study suggest that, in addition to lymphocyte-derived IL-10, MHC class II–positive cell-derived IL-10Tg, which may be upregulated early in an inflammatory response, can dramatically protect mice from EAE. Furthermore, our preliminary results show that adoptive transfer of MBP-specific encephalitogenic Th1 cells can not induce EAE in hIL-10Tg mice, suggesting a role for local inhibition of Th1 cells by MHC class II promoter-regulated IL-10Tg within the CNS (data not shown). These studies suggest that IL-10, applied with appropriate localization, in appropriate amounts, and at the appropriate time, can completely protect animals from EAE, despite their generation of potentially pathogenic Th1-like cells. This model should permit a more detailed understanding of the conditions necessary for IL-10 inhibition of autoimmune-mediated CNS inflammation, as this insight will provide information about how to use IL-10 in therapeutic situations. | Study | biomedical | en | 0.999997 |
10075985 | Section title: Animals and Treatments. Educational score: 4.040351867675781 Domain: biomedical Document type: Study Language: en Homozygous CD7-deficient mice ( 12 ) were backcrossed five generations onto the C57BL/6 background. C57BL/6 mice were obtained from The Jackson Laboratory . 10–12-wk-old sex-matched mice were studied. Phenol extracted LPS from Escherichia coli ( Sigma Chemical Co. ) was administered intraperitoneally at 100 mg/kg for the high-dose LPS shock model. For the low-dose LPS plus D-gal ( Sigma Chemical Co. ) shock model, animals received 1 μg of LPS and 8 mg D-gal intraperitoneally in 0.5 ml saline. Animals were killed and livers and spleens were excised and studied as indicated below. Mouse handling and experimental procedures were conducted in accordance with the American Association of Accreditation of Laboratory Animal Care guidelines for animal care and use. Section title: Cytokine Measurements. Educational score: 3.955613851547241 Domain: biomedical Document type: Study Language: en Quantification of murine IFN-γ and TNF-α present in sera and culture supernatants was determined using Duoset cytokine-specific ELISA kits as per the manufacturer's protocols ( Genzyme Corp. ). Section title: In Vitro Splenocyte Cultures. Educational score: 4.111346244812012 Domain: biomedical Document type: Study Language: en Splenocytes were stimulated with murine recombinant (r)IL-12 ( Genzyme Corp. ) and murine rIL-18 (Chemicon) in RPMI 1640 with l -glutamine ( GIBCO BRL ) supplemented with 10% FCS, 5.5 × 10 −5 M 2-ME, and 10 μg/ml gentamicin (BioWhittaker) for 3 d at 10 6 cells/ml in tissue culture plates (Costar) at 37°C in a 5% CO 2 humidified incubator. Section title: RNA Isolation and RNase Protection Assays. Educational score: 4.037942886352539 Domain: biomedical Document type: Study Language: en Total RNA was isolated from spleen and liver tissue using Trizol ( GIBCO BRL ) as per the manufacturer's protocol. Steady-state levels of specific cytokine messenger RNA in tissues were determined using the multiprobe RiboQuant RNase Protection Assay ( PharMingen ). Section title: Isolation of Liver Lymphocytes and Flow Cytometry. Educational score: 4.0445661544799805 Domain: biomedical Document type: Study Language: en Liver lymphocytes were prepared as previously described ( 23 ). Phenotypic analysis of liver lymphocytes (10 6 cells in 50 μl) was performed at 4°C after an initial blocking step with 1 μg of unlabeled anti-FcγR Ab ( PharMingen ). mAbs used included CD3 (Caltag), NK1.1 ( PharMingen ), and B220 (Caltag). Section title: Statistical Analysis. Educational score: 3.0200021266937256 Domain: biomedical Document type: Study Language: en Student's t test was used to determine significance of cytokine mRNA and protein levels. Chi-square tests were used to determine P values for mouse survival data. Section title: CD7-deficient Mice Are Resistant to LPS-Induced Shock Syndromes. Educational score: 4.093533039093018 Domain: biomedical Document type: Study Language: en To determine the effect of high-dose LPS treatment in CD7-deficient mice, CD7-deficient mice ( n = 30) and C57BL/6 control ( n = 16) mice were injected intraperitoneally with LPS (100 mg/kg) and survival was assessed daily for 7 d . C57BL/6 mice succumbed to shock between days 1 and 2 after high-dose LPS injection, with only 19% of the animals surviving on day 7. In contrast, 67% of CD7-deficient animals were alive on day 7 ( P < 0.001), and demonstrated partial resistance to high-dose LPS shock. As additional controls, saline injected CD7-deficient ( n = 3) and C57BL/6 control mice ( n = 3), remained alive and healthy throughout the 7-d study . Section title: CD7-deficient Mice Are Resistant to LPS-Induced Shock Syndromes. Educational score: 4.068265914916992 Domain: biomedical Document type: Study Language: en Within 12 h of intraperitoneal injection of low-dose LPS (1 μg) and D-gal (8 mg), only 20% of control C57BL/6 animals ( n = 10) survived , consistent with previously reported data for this model ( 13 ). In contrast, no deaths were observed in CD7-deficient mice ( n = 10) up to 72 h after injection with LPS ( P < 0.001). Because of the total resistance of CD7-deficient mice to death in the low-dose LPS shock syndrome model, we studied this model further. Section title: Elevated Serum Levels of TNF-α and IFN-γ in the Low-Dose LPS Shock Model Are Dependent on CD7 Expression. Educational score: 4.154316425323486 Domain: biomedical Document type: Study Language: en Studies using IFN-γR–deficient ( 15 ) and TNFRI-deficient ( 16 , 18 ) mice have clearly demonstrated the importance of IFN-γ and TNF-α as dominant cytokines that induce hepatitis and death in the low-dose LPS shock model ( 14 ). Therefore, serum levels of TNF-α and IFN-γ were measured in control C57BL/6 and CD7-deficient mice at multiple time points after injection of LPS and D-gal . There was a sharp increase in serum TNF-α levels in C57BL/6 animals 1 h after LPS plus D-gal treatment, that fell to baseline after 4 h of treatment . In contrast, a blunted peak in TNF-α secretion was observed in CD7-deficient mice 1 h after LPS plus D-gal treatment, with a rapid return to baseline 2 h after treatment. Peak TNF-α serum levels in CD7-deficient animals were 55 ± 7% of that of control animals ( P < 0.001) with a decrease in serum TNF-α levels 2 h after LPS plus D-gal treatment. Section title: Elevated Serum Levels of TNF-α and IFN-γ in the Low-Dose LPS Shock Model Are Dependent on CD7 Expression. Educational score: 4.160186290740967 Domain: biomedical Document type: Study Language: en In contrast to the early 2 h rise in TNF-α, serum IFN-γ levels in C57BL/6 mice peaked 6 h after injection of LPS plus D-gal . Importantly, a near complete absence of serum IFN-γ was observed in CD7-deficient mice at the same time point ( P < 0.001), and throughout the entire 12-h study period. Thus, disruption of the CD7 gene resulted in a partial block in serum TNF-α production, and in a complete block in serum IFN-γ production in the low-dose LPS-induced shock model. Section title: IFN-γ and TNF-α Cytokine mRNA Expression Was Decreased in Liver from LPS-Treated CD7-deficient Mice. Educational score: 4.067575931549072 Domain: biomedical Document type: Study Language: en Next, cytokine gene expression in liver and spleen tissue from C57BL/6 and CD7-deficient mice was quantified by RNase protection assays. C57BL/6 ( n = 3) and CD7-deficient ( n = 3) animals were treated with low-dose LPS plus D-gal for either 0 or 6 h. The expression of various proinflammatory cytokine genes in liver and spleen are shown in Fig. 3 . Section title: IFN-γ and TNF-α Cytokine mRNA Expression Was Decreased in Liver from LPS-Treated CD7-deficient Mice. Educational score: 4.128008842468262 Domain: biomedical Document type: Study Language: en Marked increases in TNF-α, IFN-γ, and IL-6 steady-state mRNA levels were observed in liver tissues from C57BL/6 control mice treated with LPS plus D-gal for 6 h . As seen in the control mice, expression of TNF-α and IL-6 mRNA were also significantly elevated in liver tissue from CD7-deficient mice exposed to LPS plus D-gal for 6 h, compared with pretreatment levels . However, there was no significant increase in the steady-state level of IFN-γ in 6-h–treated CD7-deficient mice versus the 0 h mice. In addition, liver from LPS plus D-gal–treated CD7-deficient animals had lower steady-state mRNA levels of TNF-α and IL-6 mRNA compared with those seen in C57BL/6 liver tissues ( P < 0.05). In contrast, spleen of both C57BL/6 and CD7-deficient mice had no significant differences in TNF-α, IFN-γ, or IL-6 steady-state mRNA levels after treatment with LPS plus D-gal for 6 h . Section title: IFN-γ and TNF-α Cytokine mRNA Expression Was Decreased in Liver from LPS-Treated CD7-deficient Mice. Educational score: 4.135867118835449 Domain: biomedical Document type: Study Language: en Deficient IFN-γ production in the liver could be due to a lack of induction of the IFN-γ inducing factors IL-12 and IL-18 or ICE. To address these possibilities, IL-12 (p35, p40), IL-18, and ICE steady-state mRNA expression in liver was determined by RNase protection assays. IL-18 and ICE mRNA expression in liver was determined after 6 h of exposure to LPS plus D-gal. There was no difference between C57BL/6 control mice ( n = 3) and CD7-deficient mice ( n = 3) in constitutive or LPS-induced steady-state levels of IL-18 and ICE mRNA expression. At the 6-h time point no significant induction in p35 or p40 IL-12 mRNA was detected in CD7-deficient or control mice. In addition, no difference in constitutive or LPS-induced steady-state levels of either IL-12 mRNA was observed between C57BL/6 control mice and CD7-deficient mice. Section title: CD7-deficient Splenocytes Responded Normally to IL-18 with IFN-γ Production. Educational score: 4.099061489105225 Domain: biomedical Document type: Study Language: en Splenocytes from C57BL/6 control mice ( n = 3) and CD7-deficient mice ( n = 3) were cultured in vitro for 72 h with a wide dose range of IL-12, IL-18, and IL-12 plus IL-18 . As shown in Fig. 4 A, IL-12 alone did not induce IFN-γ production by splenocytes from either C57BL/6 control or CD7-deficient mice. IL-18 alone induced a low level of IFN-γ production in splenocyte cultures from both C57BL/6 control and CD7-deficient mice ( P = NS) . When IL-12 and IL-18 were combined to stimulate IFN-γ production , an enhanced induction in IFN-γ production was observed for both C57BL/6 splenocytes and CD7-deficient splenocytes. There was no significant difference in the level of IFN-γ stimulated by both cytokines in splenocyte cultures from C57BL/6 versus CD7-deficient mice. Section title: CD7-deficient Mice Have Reduced Numbers of Liver NK1.1 + / CD3 + T Cells. Educational score: 4.148812294006348 Domain: biomedical Document type: Study Language: en NK1.1 + /CD3 + T cells have been recently reported to be major producers of IFN-γ and key effector cells in the pathogenesis of lethal shock syndromes ( 23 , 24 ). Thus, we isolated liver mononuclear cells from C57BL/6 control and CD7-deficient mice and performed phenotypic analysis with respect to CD3 and NK1.1 expression. Three independent experiments comparing C57BL/6 and CD7-deficient mice were performed by pooling of liver mononuclear cells from two to five animals per experiment. There was no difference in the absolute number of liver lymphocytes isolated from C57BL/6 versus CD7-deficient livers . We observed a significant reduction in the percentage and number of NK1.1 + /CD3 + cells in livers from untreated CD7-deficient mice ( P < 0.05). In contrast, the percentage and absolute number of traditional NK cells (NK1.1 + /CD3 − ) and T cells (NK1.1 − /CD3 + ) per liver in C57BL/6 control and CD7-deficient mice were similar. CD7-deficient livers also had an increase in the number of B220 + B cells compared with C57BL/6 controls . Section title: Discussion Educational score: 4.172906398773193 Domain: biomedical Document type: Study Language: en In this study we have shown that CD7-deficient mice are resistant to death in both the high-dose and the low-dose LPS-induced models of shock. Using the low-dose LPS model we demonstrated decreased induction of serum TNF-α and IFN-γ, decreased liver IL-6, TNF-α and IFN-γ steady-state mRNA levels, and normal liver IL-12, IL-18, and ICE mRNA levels in CD7-deficient mice compared with control mice. Moreover, we showed that CD7-deficient lymphocytes responded normally to IL-12 and IL-18 with regard to IFN-γ production. Finally, resistance of CD7-deficient mice to LPS-induced shock was associated with a low number of resident effector NK1.1 + /CD3 + T cells in liver of CD7-deficient mice. Section title: Discussion Educational score: 4.13409948348999 Domain: biomedical Document type: Study Language: en The CD7-deficient mouse is unique among homologous recombinant mouse strains with respect to its responses in low- and high-dose LPS-induced shock syndromes. Similar to ICAM-1 deficient, ICE-deficient, and TNFRII-deficient mice ( 19 , 20 , 22 ), CD7-deficient mice are partially resistant to LPS-induced death in the high-dose shock model. Moreover, CD7-deficient mice are fully resistant to LPS-induced death in the low-dose shock model, similar to IFN-γR–deficient and TNFRI-deficient mice ( 14 , 18 ) (Table I ). Section title: Discussion Educational score: 4.284890174865723 Domain: biomedical Document type: Study Language: en Recent studies have reported that murine liver contains a unique population of α/β T cells that have intermediate TCR expression and are positive for the NK1.1 antigen ( 23 ). These NK1.1 + /CD3 + T cells are phenotypically and functionally distinct from NK 1.1 − T cells, which are high in TCR expression, and from CD3 − NK cells, which are TCR negative ( 23 ). This population of NK1.1 + /CD3 + liver T cells has been suggested to be autoreactive ( 25 , 26 ), a major source of IFN-γ production, and key effector cells in the pathogenesis of lethal shock syndromes ( 23 , 24 , 27 ). These studies further revealed that LPS activates NK1.1 + /CD3 + T cells via IL-12 production by Kupffer cells, resulting in the hepatotoxicity observed in septic shock ( 27 ). Moreover, depletion of these cells significantly decreased mortality in the IL-12–primed LPS-induced shock syndrome ( 24 ). Section title: Discussion Educational score: 4.308193683624268 Domain: biomedical Document type: Study Language: en We have found a significant reduction in the percentage and absolute number of NK1.1 + /CD3 + cells in livers from untreated CD7-deficient mice versus age-matched control mice . These data suggested that the CD7 molecule may play a critical role in development, function, and/or migration of liver NK1.1 + /CD3 + T cells. The ontogeny of NK1.1 + /CD3 + T cells is currently unknown; however, it is clear that they are a functionally and phenotypically distinct subset of cells ( 23 ). That CD7-deficient animals have selectively diminished numbers of CD3 + /NK1.1 + T cells with normal numbers of CD3 − /NK1.1 + NK cells and CD3 + / NK1.1 − T cells, also suggests that the CD3 + /NK1.1 + T cell subset may be a distinct cell lineage. We hypothesize that CD7, which is expressed early in T and NK cell ontogeny ( 7 ), may play a role in development and/or migration of NK1.1 + T cells in the liver. Decreased numbers of these T cells in the livers of CD7-deficient mice may result in blunted cytokine production and be sufficient to protect mice from both high- and low-dose LPS-induced shock. Section title: Discussion Educational score: 4.192601203918457 Domain: biomedical Document type: Study Language: en Thus, CD7-deficient mice are unique in their resistance to both high-dose and low-dose LPS-induced shock models. Our data suggest that CD7 is an essential molecule that is involved in the lethal cellular and molecular events leading to low-dose LPS-induced hepatocyte necrosis/apoptosis, and to ischemia/reperfusion injury in high-dose LPS-induced shock. Understanding the role of CD7 in LPS shock syndromes and in NK1.1 + T cell maturation and function may spur the development of new treatments for endotoxic shock syndrome in humans. | Study | biomedical | en | 0.999997 |
10085282 | Section title: Bni1p, a Formin, Functions in Kip3p-dependent Spindle Positioning and Movement Educational score: 3.8220813274383545 Domain: biomedical Document type: Study Language: en Both groups found that bni1 mutants had defects in spindle positioning, using different approaches. The nucleus did not migrate to the neck efficiently, and the spindle was not aligned along the mother-bud axis, based in part on movies of live cells . The spindle then moved into the neck . Section title: Bni1p, a Formin, Functions in Kip3p-dependent Spindle Positioning and Movement Educational score: 4.181778907775879 Domain: biomedical Document type: Study Language: en These phenotypes are similar to ones observed previously in kip3 and kar9 mutants . Previous genetic analyses suggested that Kip3p and Kar9p act together to position the spindle before the action of dynein . Genetic analyses in the new reports indicate that Bni1p functions in the same process as Kip3p and Kar9p . Bud6p, a protein that physically interacts with Bni1p, has a similar but less important role, based on milder phenotypes and a weaker genetic interaction with dynein . Section title: Bni1p, a Formin, Functions in Kip3p-dependent Spindle Positioning and Movement Educational score: 3.388474941253662 Domain: biomedical Document type: Study Language: en Interestingly, bni1 mutants do display movements of the pre-anaphase spindle, including exaggerated transits back-and-forth through the neck. Therefore, alternative mechanisms for movement may exist, and Bni1p may act as a governor to focus or restrict the action of these other mechanisms. Section title: Bni1p Participates in Kar9p Localization Educational score: 4.219952583312988 Domain: biomedical Document type: Study Language: en Bni1p forms a cap at the incipient bud site and remains at the bud tip, suggesting that Bni1p interacts with microtubules to pull the spindle toward the bud. Kar9p is present as a spot at the bud tip, presumably overlapping the cap of Bni1p. In kar9 null mutants, astral microtubules do not orient into the bud, and, consequently, Kip3p-dependent spindle movements are impaired . In the current work, Kar9p was mislocalized in cells lacking Bni1p, Bud6p, or filamentous actin . Mislocalization of Kar9p correlated with defects in astral microtubule orientation and spindle positioning. Section title: Bni1p Participates in Kar9p Localization Educational score: 4.188068389892578 Domain: biomedical Document type: Study Language: en These results suggest that Bni1p and Bud6p localize Kar9p to the cortex, and that the mislocalization of Kar9p in bni1 and bud6 mutants is responsible for the nuclear positioning and pre-anaphase spindle orientation defects in these mutants. However, microtubules were appropriately oriented in the bni1 mutant, and the spindle positioning defects in kar9 mutants were more severe than the defects in bni1 mutants. Therefore, some Kar9p function appears to be retained despite its mislocalization in bni1 mutants. Section title: A Role for Filamentous Actin Educational score: 4.154476165771484 Domain: biomedical Document type: Study Language: en Studies with a conditional actin mutant have implicated actin in pre-anaphase spindle orientation . To examine the role of filamentous actin more directly, both groups used the actin-depolymerizing drug latrunculin A. Spindle orientation was lost with latrunculin treatment, as seen in kip3 mutants . Kar9p localization also was lost in latrunculin . These results confirm that actin is necessary for Kip3p-dependent spindle movements. Section title: A Role for Filamentous Actin Educational score: 4.308363437652588 Domain: biomedical Document type: Study Language: en What element of the actin cytoskeleton provides this function? Cortical actin patches have been widely assumed to be the attachment site for microtubules because the patches cluster at the bud tip. However, clustering of actin patches may not be necessary for pre-anaphase spindle orientation. An actin cytoskeleton mutant with largely delocalized patches, sla1 Δ SH3#3 , showed normal spindle orientation and positioning . Also, Kar9p localized normally in a sla1 null mutant . In a similar analysis, the bipolar pattern of bud site selection in diploid yeast depended on actin but not patches . Furthermore, several proteins are involved in both bipolar bud site selection and spindle orientation. Thus, both processes may involve some as yet undefined form of filamentous actin; alternatively, a small amount of actin patch clustering may be sufficient. Section title: Nuclear Positioning during Mating Educational score: 4.283841609954834 Domain: biomedical Document type: Study Language: en The nucleus moves during mating, and some of the molecular mechanisms are shared with the Kip3-dependent movements of the nucleus and spindle in dividing cells. During mating, haploid cells undergo polarized cell growth toward each other, forming a projection that makes cells resemble shmoos. Nuclei migrate into projections via astral microtubules that interact with the cortex at projection tips. Upon cell fusion, astral microtubules from each nucleus contact each other, permitting the nuclei to move together and fuse. Section title: Nuclear Positioning during Mating Educational score: 4.170836925506592 Domain: biomedical Document type: Study Language: en In shmoos, bni1 and bud6 mutations impaired Kar9p localization, astral microtubule orientation and nuclear movement into the projection. The extent of Kar9p mislocalization correlated with the severity of the defects in microtubule orientation and nuclear movement. However, the phenotypes in shmoos were more severe than those in dividing cells. Therefore, mating may provide a simpler model for cortical capture of astral microtubules. Section title: Conclusions Educational score: 3.3967621326446533 Domain: biomedical Document type: Other Language: en These papers provide important new information about how microtubules interact with the cell cortex in yeast. Astral microtubules are presumed to connect the mitotic spindle to the cell cortex and thereby dictate the position and movement of the spindle. This work should represent another case where discoveries in yeast influence research on related processes in other systems. Section title: Conclusions Educational score: 3.624558687210083 Domain: biomedical Document type: Other Language: en Bni1p, actin and Kar9p are all necessary for the early phases of spindle positioning and orientation, which depend on astral microtubules and Kip3p, a kinesin. Bni1p and actin function together to localize Kar9p. Section title: Future Directions Educational score: 3.416391134262085 Domain: biomedical Document type: Other Language: en In yeast, much remains to be learned about how these proteins interact with each other and how they function to mediate the attachment between microtubules and the cortex. Additional proteins will surely be identified as necessary for the attachment, and biochemical studies will be needed to define the activities. Section title: Future Directions Educational score: 2.9884438514709473 Domain: biomedical Document type: Other Language: en The mechanism of force production to move the spindle is unknown. The kinesin Kip3p is presumably involved, but whether Kip3 functions as a microtubule motor or causes microtubule shortening by destabilizing ends is an important open question. Section title: Future Directions Educational score: 4.027182579040527 Domain: biomedical Document type: Study Language: en Whether this microtubule/cortex attachment mechanism operates outside of yeast is also unknown. Formins, such as Bni1p, are found in many different organisms. Formins appear to influence the actin cytoskeleton but have not yet been implicated in interactions between actin and microtubules or been shown to have primary effects on microtubules. Kar9p has no obvious homologues in the sequence databases. Studies of formins and associated proteins, including perhaps functional equivalents of Kar9p, in other systems will be important. Section title: Future Directions Educational score: 2.829291582107544 Domain: biomedical Document type: Other Language: en In addition, little is known about how microtubules attach to the cell cortex during the dynein-mediated movement of the spindle into the neck in yeast. Dynein-dependent spindle movements are known to occur in organisms other than yeast . | Study | biomedical | en | 0.999997 |
10085283 | Section title: Cell Culture Educational score: 4.16635799407959 Domain: biomedical Document type: Study Language: en Indian muntjac (DM) cells were grown in DME + 10% FCS ( GIBCO BRL ) and plated on glass-bottom microwell dishes coated with poly- d -lysine (Mattek). Cells were bead loaded using 100-μm glass beads ( Sigma Chemical Co. ) as described by McNeil with 10 μl 0.1 mM Cy5-10-dUTP (made as described below), fluorescein-12-dUTP, or Bodipy-TR-14-dUTP (both from Molecular Probes, Inc.) in L15 medium ( GIBCO BRL ). After loading, cells were grown in medium + 0.1 mM Trolox (a free-radical scavenger; Fluka AG) during imaging. Cy5-10-dUTP was made by coupling 5-(3-aminoallyl)-2′-deoxyuridine-5′-phosphate ( Sigma Chemical Co. ) with Cy5 monofunctional dye (Nycomed Amersham ). Cy5-10-dUTP is now supplied by Amersham . Unsynchronized cells were used for Figs. 1 , E and F, 2, 3, and 5. For Fig. 1 , E and F, cells were loaded with fluorescein-dUTP, grown for 45 min in bromodeoxyuridine, fixed (10 min) in 4% paraformaldehyde, refixed (20 min) in 8% paraformaldehyde, and Br-DNA indirectly immunolabeled with Cy3 . For Fig. 2 A, cells were loaded with a mixture of fluorescein-dUTP and Bodipy-TR-dUTP, and grown for 4 h before the live cells were imaged. For Fig. 2 B, cells were loaded with fluorescein-dUTP, grown for 3 h, loaded with Bodipy-TR-dUTP, and grown for 4 h before imaging. Section title: Cell Culture Educational score: 4.102880954742432 Domain: biomedical Document type: Study Language: en S-phase cells were enriched to >90% for experiments in Figs. 1 , A–D, and 4, B–D. Mitotic cells were collected by shake-off, grown for 13–15 h in 2.5 mM thymidine, washed, regrown for 2–4 h in 5 μM deoxycytidine to reverse the block, and loaded. G2 cells were enriched to >50% for the experiment in Fig. 4 A. Mitotic cells were grown for 8 h, loaded, grown for 5 h, regrown for 12 h in thymidine, washed, and regrown for 11 h in deoxycytidine. Section title: Microscopy and Image Collection Educational score: 4.307819366455078 Domain: biomedical Document type: Study Language: en Phase-contrast images were captured with a Hamamatsu CCD attached to a Nikon Diaphot-200 microscope, fitted with a heated (Bioptech) 60× PlanApo objective and stage ( Zeiss ). The stage was surrounded by an insulated box. Stacks of green, red, or far-red confocal images were collected using a Bio-Rad MRC1024 (12 single scans of 170 pixels square, axial steps of 0.5 μm for interphase, and 1 μm for mitotic nuclei). Cy5 was generally used for live cell work using a 647-nm excitation beam, an intensity of 50 nW (<1/100 intensity commonly used with fixed cells), and an open pinhole. Note that doubling the excitation intensity often slowed progression around the cycle. The bottom four to six sections from each stack were projected onto a plane, converted to 510 pixels square, and contrast-stretched to fill the 256-level gray scale, the same settings were used for all images of one movie. Finally, noise was reduced by Gaussian filtering using Adobe Photoshop. For Fig. 5 , positions of 48 foci were located manually using SIS EasiVision software, and exported into Excel. Images were aligned first by superimposing centers of gravity of 48 foci, and then by rotation (using a least-squares fit). Time series were displayed using Confocal Assistant (T.C. Brelje). The inaccuracy of position measurement, due to instrument movement, noise, and positioning focal centers, was determined as 0.1 μm by measuring and remeasuring the positions of foci in fixed cells under identical conditions. The length of mitotic chromosomes in two living cells (loaded with fluorescein-dUTP in early S phase) was determined using positions obtained from stacks of 37 and 66 sections. Section title: Visualizing Sites of DNA Synthesis in Living Cells Educational score: 4.221513748168945 Domain: biomedical Document type: Study Language: en Fig. 1 illustrates the approach applied to Indian muntjac cells . These cells were chosen because they have only nine chromosomes, which facilitates analysis. They also grow rapidly with a doubling time of ∼18 h. At time zero, Cy5-dUTP was introduced into S-phase cells by bead-loading . Addition of glass beads transiently permeabilizes the plasma membrane, allowing the analogue to enter. The petri dish containing living cells was placed on the heated stage of a microscope equipped with both phase-contrast and confocal fluorescence optics. Within 6 min, most of the precursor has been transported into the nucleus , where it becomes concentrated in discrete nuclear foci . These foci result because active DNA polymerases are concentrated at this stage of the cell cycle in discrete factories which duplicate the genome . Essentially all analogue is incorporated into DNA in <30 min, so only DNA made in this short period fluoresces. Therefore, roughly the same pattern of foci is seen after 90 min . Section title: Visualizing Sites of DNA Synthesis in Living Cells Educational score: 4.25693941116333 Domain: biomedical Document type: Study Language: en Various features indicate that these foci result from S-phase synthesis. First, they have diagnostic patterns, both in interphase and mitosis . After loading in G1 phase, nuclear foci only appear when S phase is reached, then, they have the pattern typical of early-S phase (not shown). If Cy5-dUTP, or another analogue, fluorescein-dUTP, is loaded late during S phase, foci appear immediately, arranged in the pattern typical of this stage . Second, fluorescein-dUTP is incorporated into the same foci as bromodeoxyuridine (Br-dU), a precursor routinely used to mark sites of S-phase replication . Third, these patterns are different from those due to nucleotide-excision repair, which takes place in thousands of tiny foci in all cells in the population . Fourth, unincorporated analogues can be extracted with 0.1% Triton X-100, and the nuclear foci by an additional treatment with DNase (0.5 U/ml, 10 min, 20°C, not shown). Section title: Labeling Regions That Replicate at Different Times Educational score: 4.091243743896484 Domain: biomedical Document type: Study Language: en Regions replicating at different times during S phase can be labeled after successive loadings with fluorescein-dUTP and Bodipy-TR-dUTP. For example, simultaneous loading with both precursors yields yellow foci , as the two labels become intermingled in DNA. However, loading one followed by the other 3 h later yields discrete green or red foci , as the two labels are incorporated into different parts of the genome. These results confirm others obtained earlier with fixed cells . Section title: Cy5-DNA Strands in Diploid Cells Segregate Normally Educational score: 4.20528507232666 Domain: biomedical Document type: Study Language: en All cells, including the line of muntjac cells used here , possess sophisticated mechanisms for repairing DNA damage . These might be expected to recognize fluorescent adducts, and to arrest the cell cycle at checkpoints in S and G2 phases. Laser illumination would also be expected to generate additional damage that would compound the arrest. Only when most adducts and any light-induced damage had been removed did we expect the nonfluorescent cells to pass through mitosis. Surprisingly, muntjac cells containing Cy5-DNA divided normally (see below). As these cells have been grown continuously in culture for many generations and might have accumulated mutations in the repair pathway, we also investigated whether diploid cells behaved similarly. Therefore, a secondary culture of human fibroblasts which had been grown for only 15 passages was loaded with Cy5-dUTP, and the cells regrown for 30 h. Imaging then showed that the fluorescent DNA was divided among daughters, which were easily recognized because they shared similar fluorescent patterns . All cells containing Cy5-DNA tested to date (muntjac cells, diploid human cells, HeLa cells) proved equally sensitive to illumination (not shown), although we have not yet examined this systematically. Cy5-DNA is detected after illumination with laser light of 647 nm, and, as expected, use of shorter wavelengths (568 nm for Bodipy-Texas red-DNA, and 488 nm for fluorescein-DNA) proved more toxic (not shown). We have not yet examined this systematically. Section title: Passage through Mitosis Educational score: 3.686267852783203 Domain: biomedical Document type: Study Language: en Unfortunately, high doses of illumination did prevent passage through mitosis (not shown). Therefore, we could only make movies of dividing cells using low exposures, with consequent reduction in image quality and length. Fig. 4 illustrates movies of four different muntjac cells as they traverse different parts of the cell cycle. Each movie also contains a set of phase-contrast images, although none are shown in Fig. 4 , B–D. Note that each fluorescent image shown is derived from a complete stack of 12 images, so that three-dimensional information is also available. Section title: Passage through Mitosis Educational score: 4.189186096191406 Domain: biomedical Document type: Study Language: en Fig. 4 A illustrates passage through mitosis. The cell was loaded with Cy5-dUTP during S phase so that late replicating DNA became fluorescent. Then, the cell was allowed to divide once (to confirm that it was well) before a stack of 12 optical sections was collected as one daughter was about to enter mitosis again (28:00 h after loading). Inspection of this stack gives a three-dimensional view of foci arranged in a pattern typical of late-S phase. Projecting the 12 images onto one plane gives a complex pattern that is difficult to analyze, so a simplified projection of only the bottom four sections is shown as the first frame in Fig. 4 A. As most late-S phase foci are peripheral, ∼50% foci are seen. Other projections were made similarly, as the daughter passed from G2 phase (28:01), through mitosis (28:22–28:40), to give two granddaughters (only one is shown after 31:35). Section title: Passage through Mitosis Educational score: 4.307187080383301 Domain: biomedical Document type: Study Language: en Fig. 4 , B and C, illustrates entry into and out of mitosis in more detail. In Fig. 4 B, the cell was loaded early in S phase, so the pattern of fluorescent foci is more complex than in Fig. 4 A. The movie begins 7 h later when the cell has reached the end of G2 phase, and during filming the cell crawls to the right; by the last frame the cell is in mitosis. Between 7:35 and 7:45 h after loading, phase-contrast shows that the nuclear membrane breaks down; however, the fluorescent images reveal that chromosomes form earlier. Individual foci (three bright foci in the middle) can be traced throughout the movie. Surprisingly, their position changes little relative to neighboring foci, even when chromosomes are forming. In Fig. 4 C, the cell was loaded early during S phase, to give many foci. By 9:00 h, it has reached mitosis, and chromosomes reorient extensively on the metaphase plate (9:07–9:28), before segregating (9:44). By 10:00 h, nuclear membranes reform as chromosomes decondense, nuclei flatten (10:22), and the bottom daughter continues to grow (10:11–21:00). Section title: Passage through Mitosis Educational score: 4.144920349121094 Domain: biomedical Document type: Study Language: en Inspection of many movies, like those shown in Fig. 4 , underlies our operational definition of when a cell should be considered alive. For example, cells are often able to crawl across the surface of the culture dish without being alive enough to pass through mitosis (not shown). Similarly, they may contain individual foci that move considerably relative to neighboring foci, even though they cannot form those foci into recognizable mitotic chromosomes (not shown). However, we find that if a cell is able to transform its interphase nucleus into visible chromosomes, it is usually able to segregate those chromosomes to two daughter cells. This is in accord with earlier results showing that chromosomes, once formed, become inert passengers during mitosis, as they can even be substituted or removed . Section title: The Segregation of Individual DNA Strands to Daughters and Granddaughters Educational score: 4.301939964294434 Domain: biomedical Document type: Study Language: en Individual DNA strands can also be followed as they segregate to great-granddaughters. In Fig. 4 D, the parent cell was loaded early during S phase, grown for 6:00 h, and photographed. It was then allowed to divide to give daughters (26:00) and granddaughters (54:00). As a result of semiconservative replication and random segregation, ∼50% of the chromosomes in a granddaughter contain a fluorescent strand. During interphase, these are seen as fluorescent domains against a dark background (54:00). Fluorescent domains condense into fluorescent chromosomes (54:25), dark domains into dark chromosomes (54: 36). After aligning on the metaphase plate (54:43), fluorescent chromosomes (and other dark chromosomes) segregate to each of the two great-granddaughters (54:48–55: 40). If Cy5-DNA strands obey the rules of semiconservative replication and random chromosome segregation, we would expect two to three of the nine chromosomes great-granddaughters receive to be fluorescent, as is the case. Eventually descendants with only one fluorescent chromosome are born, and subsequently this chromosome is inherited unilinearly . Section title: The Dynamics of Chromosome Formation: Similarity of Interphase and Prometaphase Architectures Educational score: 4.218599319458008 Domain: biomedical Document type: Study Language: en Because information on how chromosomes form is limited, we analyzed the transition from mid-G2 phase to early prophase in one cell in detail . The cell was loaded late in S phase and filmed as it progressed from G2 phase into prophase. We collected 21 phase-contrast images and 252 fluorescence images (21 complete stacks of 12 confocal sections through the cell) of the living cell. The phase-contrast images allowed us to monitor breakdown of the nuclear membrane and chromosome formation, while the fluorescence images provided three-dimensional information on the distribution and movement of (heterochromatic) DNA replicated late in S phase. To simplify the fluorescence images for presentational purposes, the bottom four to six sections in each of the 21 stacks were projected onto a plane . After collecting the last stack of images, the cell was fixed and total DNA stained, enabling individual prophase chromosomes to be identified . A final stack of 12 images was also collected to reveal the distribution of the fluorescent foci in the fixed cell. During filming, the cell crawled 20 μm across the field, as the nucleus rotated ±20° around the z axis, such translation and rotation are common . A film incorporating corrections for these global cellular movements can be viewed on http://www.path.ox.ac.uk/prc/images/emm/emmov.htm . It contains 21 frames, each showing fluorescent foci in the bottom half of the cell. Section title: The Dynamics of Chromosome Formation: Similarity of Interphase and Prometaphase Architectures Educational score: 4.045202255249023 Domain: biomedical Document type: Study Language: en After inspection of the complete set of three-dimensional fluorescence images, we identified 48 foci that could be tracked unambiguously from the first (interphase) image to their final positions in a chromosome. Others that could not be tracked unambiguously (mainly because they fused and/or split) were not analyzed further. Simplified tracks have been superimposed on the final image of the fixed and stained cell to illustrate the movement of the selected foci, with tracks of the same color marking foci from the same chromosomal segment . Note that it is sometimes difficult to trace some of the selected foci unambiguously from frame to frame in the movie, which contains only two-dimensional information. Section title: The Dynamics of Chromosome Formation: Similarity of Interphase and Prometaphase Architectures Educational score: 4.304869174957275 Domain: biomedical Document type: Study Language: en Quantitative analysis of the movie indicates that a typical focus tracks ∼2 μm from first to last frame (after correction for cellular movements). The rate of movement progressively increases from 0.2 μm/h in mid-G2 phase, reaching 0.8 μm/h when the nuclear envelope breaks down, and 2.5 μm/h during the final leg. Foci in one segment often move together , sometimes sliding past adjacent segments moving in a different direction (e.g., foci in segments 2 and 3). This movement could result indirectly from cellular movement, as individual chromosomal segments are elastically deformed by external forces. It also occurs in dead cells that have been exposed to so much illumination that they cannot enter prophase (not shown). The net result of such movement is an average translocation of the center of mass of foci in a typical segment by 0.7 μm (range 0.2–1.6 μm). As a segment moves, individual foci move within that segment. The net movement of foci is 0.8 μm (range 0.7–0.9 μm) relative to the center of mass of the segment. Section title: The Dynamics of Chromosome Formation: Similarity of Interphase and Prometaphase Architectures Educational score: 4.0850324630737305 Domain: biomedical Document type: Study Language: en Foci could have been distributed randomly in a domain to move to their appropriate places in the chromosome . However, our results suggest foci are so prealigned that only subtle movements are required to generate recognizable chromosomes . These subtle movements are reflected by the shortening of axis 1 by 12% from first to last frame. Once formed, a prophase chromosome roughly halves its end-to-end length to give a metaphase chromosome . We believe the results obtained with this cell are representative, because similar chromosome dynamics were seen in five others that were treated similarly. Moreover, another 10 cells, which were not fixed at the end of the experiment, were seen to pass through mitosis after exposure to similar levels of illumination. Section title: Imaging Sites of DNA Synthesis and Individual DNA Strands in Living Cells Educational score: 4.350804328918457 Domain: biomedical Document type: Study Language: en We describe a method that allows sites of DNA synthesis, and individual DNA strands, to be imaged directly in living cells. Fluorescent analogues (fluorescein-dUTP, Bodipy-TR-dUTP, Cy5-dUTP) of the natural precursor, thymidine triphosphate, are introduced into cells. Analogues are incorporated by endogenous enzymes into DNA so that it becomes fluorescent. Active DNA polymerases are concentrated in discrete factories , and as the fluorescent precursor is exhausted, the newly made (fluorescent) DNA becomes locally concentrated in discrete foci . These foci persist for many generations . Surprisingly, attaching fluorescent tags to DNA has remarkably little effect on growth; the mechanisms for recognizing and repairing such unnatural DNA residues do not prevent subsequent progress around the cell cycle. Therefore, the fluorescent DNA can be imaged in the light microscope. This enabled us to use a confocal microscope to collect images of DNA strands as they condensed into chromosomes and segregated to daughters and granddaughters . These cells remain both biochemically and genetically alive during imaging, as they can make DNA and pass through mitosis. Section title: Imaging Sites of DNA Synthesis and Individual DNA Strands in Living Cells Educational score: 4.146549701690674 Domain: biomedical Document type: Study Language: en Unfortunately, the quality and length of such movies is limited by the effects of the laser light used to excite fluorescence. These effects include a lengthening of the cell cycle, delayed segregation, and the induction of strand exchanges which leads to the generation of subchromosomal domains in great-granddaughters and their descendants (not shown). Irradiation with high doses generally arrested cells in G2 phase, presumably at the major checkpoint . If cells passed this checkpoint, they generally divided (not shown). As expected , illuminating Cy5-DNA with far-red light of 647 nm proved less toxic than the use of fluorescein or Bodipy-TR and shorter wavelengths (488 and 568 nm, respectively). Although Bodipy-TR-dUTP and fluorescein-dUTP were incorporated into DNA, and the resulting fluorescent DNA strands segregated to daughters, we generally used Cy5-dUTP when following chromosomes through mitosis. We also had to use a low intensity of the laser (5 nW measured at the position of the specimen). In practice, the length of each movie represents a compromise between minimizing exposure (to reduce toxicity) and maximizing the intensity (to increase resolution within individual frames and the total number of frames). Even so, we were able to collect >240 images of one cell , sufficient to provide four-dimensional information. Section title: Imaging Sites of DNA Synthesis and Individual DNA Strands in Living Cells Educational score: 4.348837852478027 Domain: biomedical Document type: Study Language: en Inspection of the movies directly confirms important conclusions inferred earlier using indirect approaches. First, they confirm that the replication foci or factories seen in fixed or permeabilized cells have their counterparts in vivo, and are not preparative artifacts. Second, DNA foci move little during interphase compared with the rapid movements of mitosis . These interphase movements are within the range seen by others . Third, individual chromosomal territories seen in granddaughters and great-granddaughters are compact , again in agreement with earlier findings . Fourth, characteristic distributions of foci are inherited through interphase and mitosis. Thus, in Fig. 4 D, a distinctive pattern (many small foci spread throughout the nucleoplasm) is established early in S phase, and is passed to daughters (26:00) and the appropriate chromosomal domains in granddaughters (55:40). In Fig. 4 A, a different (late replication) pattern is passed down the generations. This suggests that whatever underlying structure maintains the distinctive pattern, the structure can either pass through mitosis intact, or, if disassembled, can reform accurately afterwards . Fifth, Cy5-DNA strands obey the rules of semiconservative replication and random chromosome segregation. For example, two to three of the nine chromosomes a great-granddaughter receives would be expected to be fluorescent, as in Fig. 4 D. Section title: Chromosome Formation Educational score: 4.209434509277344 Domain: biomedical Document type: Study Language: en We also used this method to visualize DNA strands during the critical phase of the cell cycle when interphase chromatin fiber forms into a recognizable chromosome . This phase was inaccessible to classical cytologists, as individual fibers in the interphase nucleus cannot be resolved. Cells were loaded with Cy5-dUTP late in S phase so that heterochromatic foci became labeled. We expected all the foci on one chromosome to be distributed randomly throughout that chromosome's territory, and those foci to move to their appropriate places in the chromosome . However, our results suggest the foci are so prealigned that only subtle movements are required to generate recognizable chromosomes . They show that axis 2 shortens little as the chromosome forms. Most shortening (and considerable movement) occurs after prometaphase, during the stages visible to classical cytologists. It remains to be seen to what extent early replicating foci, which are arranged in complex patterns difficult to analyze, are similarly prealigned during interphase. Just as identifiable chromosomes form without much movement, they disappear after mitosis without much movement , although we have not yet analyzed this in detail. Section title: Chromosome Formation Educational score: 4.499251365661621 Domain: biomedical Document type: Study Language: en These results, and those obtained earlier , are consistent with the model illustrated in Fig. 6 B. Here, late replicating (heterochromatic) foci are prealigned during interphase. They are also relatively immobile, perhaps because they are attached to the lamina. Much of their movement seems to result indirectly from cellular movement, as individual chromosomal segments are elastically deformed as the cell crawls across the surface of the culture vessel. On entry into prophase, it is easy to imagine that these (fixed) heterochromatic foci nucleate the condensation of neighboring euchromatin. Once the lamina has dispersed, the foci become free to collapse both laterally and axially into the mitotic chromosome. Rapid chromosome movements of mitosis occur, and sister chromatids separate. After segregation to daughters, the (heterochromatic) foci could now nucleate lamina reassembly, so that they become fixed in new positions. They can now remain in those positions as the euchromatin decondenses to form a G1 domain. It remains to be established what path the chromatin fiber follows within such a domain, and how much individual foci move during the rest of interphase . Nevertheless, our results suggest the architecture of the G2 nucleus is directly related to that of the prophase chromosome. Section title: Future Prospects Educational score: 3.9049220085144043 Domain: biomedical Document type: Study Language: en This approach can be extended to follow early, mid, and late replicating regions in one cell (e.g., after loading successively fluorescein-dUTP, Bodipy-TR-dUTP, and Cy5-dUTP; not shown). We also anticipate that further technical advances in imaging should facilitate the production of longer movies with higher resolution. This method also opens up the prospect of using fluorescence resonance energy transfer to monitor the docking onto DNA of DNA-binding proteins tagged with GFP or FLASH. | Study | biomedical | en | 0.999998 |
10085284 | Section title: Antibodies and Cell Lines Educational score: 4.208955764770508 Domain: biomedical Document type: Study Language: en The rabbit polyclonal antiserum against the COOH-terminal peptide of p27 BBP/eIF6 (NH 2 -CTIATSMRDSLIDSLT-COOH) was tested for its specificity by Western blotting and immunoprecipitation both on the recombinant protein and on cellular lysates . Integrin β4 was detected with the rat mAb3E1 (10 μg/ml; Chemicon International, Inc.), or with the mouse mAb AA3 at 10 μg/ml (gift of Vito Quaranta, Scripps Research Institute, La Jolla, CA). The human autoantibodies against fibrillarin were a generous gift of Robert Ochs (Scripps Research Institute) and were diluted 1:300. Cytokeratins were detected either with mouse monoclonal anticytokeratin 8:18, IgG2a (Diagnostika) at 1:200, or with mouse monoclonal anticytokeratin 7/17 IgG1, according to the manufacturer's protocol (C46; Euro-Diagnostica). Secondary antibodies were rhodamine- and fluorescein-tagged swine anti–rabbit IgGs (1:50; DAKO Corp.), rhodamine-tagged goat anti–human IgGs (10 μg/ml; Chemicon International, Inc.), rhodamine-tagged goat anti–mouse IgGs (7.5 μg/ml; Molecular Probes Europe) and fluorescein-tagged goat anti–mouse IgGs (1:50; Antibodies Inc.). In control experiments, primary antibodies were replaced by preimmune sera or irrelevant mAbs. In addition, the p27 BBP/eIF6 antiserum was preadsorbed with the peptide used for its generation (1 μM, overnight, 4°C), or with the bacterially produced human recombinant full length protein (at 10 μg/ml, 2 h at 4°C) purified by ion exchange chromatography. Section title: Antibodies and Cell Lines Educational score: 3.6863787174224854 Domain: biomedical Document type: Study Language: en The cell lines and primary cells used in this study, as well as the conditions for their propagation, are described in the American Type Culture Collection cell line catalogue or in the references between parentheses. They are as follows: mouse NIH/3T3 fibroblasts, human A431 epidermoid carcinoma, human HeLa epitheloid carcinoma, human pancreatic carcinoma FG2 , human Jurkat T cells, transformed human keratinocytes HaCat , human insulinoma cells Rin2A , and human neuroblastoma SK-N-MC. The 804G rat epithelial cell line clone A was a gift of F. Giancotti (Memorial Sloan-Kettering Cancer Center, New York) and has been described in Spinardi et al. . Section title: Antibodies and Cell Lines Educational score: 1.4541229009628296 Domain: biomedical Document type: Other Language: en Mouse resting splenocytes, human fibroblasts from the umbilical cord, and Xenopus oocytes were gifts of A. Cabibbo, E. Bianchi, and E. Pannese (all at DIBIT, Milano, Italy) and were obtained by standard procedures. Section title: Actinomycin Treatment Educational score: 3.7962863445281982 Domain: biomedical Document type: Study Language: en Cells were treated with actinomycin D ( Boehringer Mannheim GmbH ) at the final concentration of 5 μg/ml for 1, 4, and 12 h, washed, and fixed as described. In some experiments cells were allowed to recover after treatment by switching them to their normal medium. Section title: Electron Microscopy on Human Amnion Educational score: 4.177465915679932 Domain: biomedical Document type: Study Language: en Human fresh amniotic membrane (obtained immediately upon delivery from the Department of Obstetrics, San Raffaele Hospital, Milano, Italy) was dissected and pieces of tissue were fixed with 4% paraformaldehyde and 0.25% glutaraldehyde in 125 mM sodium phosphate buffer, pH 7.4, for 45 min at 4°C. The samples were infiltrated with polyvinylpyrrolidone and frozen in a 3:1 (vol/vol) mixture of propane and isopentane cooled with liquid nitrogen. Ultrathin cryosections (50–100 nm thick) were obtained using an Ultracut ultramicrotome equipped with a Reichert FC4 cryosectioning apparatus. The cryosections were processed as described in Villa et al. using the rabbit anti-p27 BBP/eIF6 antiserum and the mouse monoclonal anticytokeratin 7/17 IgG1. Cryosections were examined in an electron microscope . Section title: Western Blot Analysis Educational score: 4.070823669433594 Domain: biomedical Document type: Study Language: en All samples were denatured before loading in Laemmli buffer and run on denaturing 12% SDS–acrylamide gel, transferred to Immobilon P membranes ( Millipore Corp. ), and blotted with the rabbit p27 BBP/eIF6 antiserum at 1:1,000 dilution as previously described . In the control of the fractionation experiment, a mouse monoclonal anticytokeratin 8/18 IgG2a at 1:200 was used. Detection was always performed by the commercially available chemiluminescence detection system (ECL) technique (Nycomed Amersham ). Section title: Extraction of Nuclear Matrix and Ribosomal Proteins Educational score: 4.140950679779053 Domain: biomedical Document type: Study Language: en Intermediate filaments/nuclear matrix filaments fractions were prepared exactly according to He et al. . Briefly, all the soluble proteins, the nonintermediate filament cytoskeleton, DNA associated proteins, and proteins loosely associated with the nuclear matrix proteins were removed by sequential washes in buffers (Triton X-100, 250 mM ammonium sulphate, DNase I, and 2 M NaCl). At the end of this procedure, a cytoplasmic and nuclear intermediate filament network containing keratins, lamins, and intermediate filament-associated proteins was left. The efficiency of the extraction was routinely controlled by DNA staining, or by immunostaining for keratins. Section title: Extraction of Nuclear Matrix and Ribosomal Proteins Educational score: 4.2245073318481445 Domain: biomedical Document type: Study Language: en Preparation of ribosomes was performed through established procedures and exactly as described in Madjar . 804G clone A cell line monolayer was washed and scraped with cold PBS. The pellet was resuspended in cold buffer A (0.25 M sucrose, 25 mM KCl, 5 mM MgCl 2 , 50 mM Tris-HCl, pH 7.4), stirred slowly with a vortex, while adding NP-40 to a final concentration of 0.7%, and kept on ice for 10 min. The suspension was centrifuged at 750 g for 10 min at 4°C and the resulting pellet containing nuclei and insoluble proteins was resuspended in Laemmli buffer for biochemical analysis. The supernatant was centrifuged at 12,500 g , 10 min at 4°C and the resulting pellet containing mitochondria was resuspended in Laemmli buffer. The low-speed supernatant was added to 0.32 vol of buffer C (0.25 M sucrose, 2 M KCl, 5 mM MgCl 2 , 50 mM Tris-HCl, pH 7.4), layered on top of a 1 M sucrose cushion, and ultracentrifuged (TL100; Beckman Instruments, Inc. ) at 245,000 g , 2 h at 4°C. The high-speed pellet containing ribosomal proteins was resuspended in Laemmli buffer. The high-speed supernatant was precipitated with cold 10% TCA, for 45 min on ice, centrifuged at 14,000 rpm at 4°C, and resuspended in Laemmli buffer for biochemical analysis. Section title: Indirect Immunofluorescence and Immunocytochemistry Educational score: 4.226294040679932 Domain: biomedical Document type: Study Language: en Immunofluorescence was performed as previously reported . In brief, the following was performed: cell monolayers were fixed in 3% paraformaldehyde in PBS, pH 7.6, containing 2% sucrose for 10 min at room temperature; permeabilized in Hepes–Triton X-100 buffer for 5 min at 4°C (20 mM Hepes, 300 mM sucrose, 50 mM NaCl, 3 mM MgCl 2 , 0.5% Triton X-100, pH 7.4); and blocked with 5% BSA in PBS for 30 min at room temperature. Next, the cells were incubated in primary antibodies (diluted in 5% BSA in PBS) for 2 h at room temperature, washed in 0.2% BSA in PBS, and treated with secondary antibodies diluted in PBS. Staining for F-actin was performed with 200 nM fluorescein-labeled phalloidin ( Sigma Chemical Co. ) for 20 min at 37°C in the dark and with 2 μg/ml DNA counterstaining . Once mounted in Mowiol 4-88 (Hoechst AG), coverslips were analyzed with a confocal microscope equipped with a krypton/argon laser. To reduce bleed through, double-label confocal images (XY and XZ sections) were acquired sequentially. Micrographs were taken using either a Focus Imagecorder Plus (Focus Graphics Inc.) on Kodak film or a Professional color Point II dye sublimation printer (Seiko). Stained cells were observed in parallel with a Zeiss Axiophot microscope equipped for epifluorescence and a 63× planapochromatic lens; pictures were taken on Kodak T-MAX 400 films exposed at 1000 ISO and developed at 1600 ISO in T-MAX developer for 10 min at 20°C. Section title: Indirect Immunofluorescence and Immunocytochemistry Educational score: 4.102736473083496 Domain: biomedical Document type: Study Language: en In some experiments, p27 BBP/eIF6 was revealed by immunoperoxidase labeling using the avidin–biotin amplification method (ABC Kit Vectastain; Vector Labs Inc.). Briefly, after incubation with the primary antiserum cell monolayers were washed and treated with a goat anti–rabbit biotin-conjugated antibody for 30 min at room temperature, followed by the preformed avidin–biotin complex (ABC). The staining was revealed by horseradish peroxidase and 3,3′-diaminobenzidine as chromogen (BioGenex Labs). Section title: Electron Microscopy on Extracted Cells Educational score: 4.224691867828369 Domain: biomedical Document type: Study Language: en FG2 cells were extracted to reveal the nuclear matrix as described above and fixed with fresh 3.7% paraformaldehyde in digestion buffer for 30 min at 4°C. They were washed once in digestion buffer, once in TBS-1 (10 mM Tris-HCl, pH 7.7, 150 mM NaCl, 3 mM KCl, 1.5 mM MgCl 2 , 0.05% Tween 20, 0.1% BSA, 0.2% glycine), and blocked in 5% BSA in TBS-1 for 30 min at room temperature. The cells were incubated in rabbit p27 BBP/eIF6 antiserum, 1:200 in 5% BSA in TBS-1 rocking overnight at 4°C. After this incubation, they were sequentially washed in TBS-1, blocked with 5% BSA in TBS-1, 10 min at room temperature, and incubated with 5-nm gold bead-conjugated goat anti–rabbit antibody (Jackson ImmunoResearch Laboratories, Inc.), 1:40 in TBS-2 (20 mM Tris-HCl, pH 8.2, 140 mM NaCl, 0.1% BSA) rocking for 1 h at room temperature. Cells were washed in TBS-1, postfixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, and centrifuged at low speed (600 g for 5 min). The cell pellets were included in diethylene glycol distearate as described by Nickerson et al. . Resinless sections were examined in a Hitachi H-7000 electron microscope. Section title: Database Searches Educational score: 4.138057708740234 Domain: biomedical Document type: Study Language: en Homology searches were performed with the Blast programs available through http://www.ncbi.nlm.nih.gov , or by the alerting system of EMBL ( http://www.bork.embl-heidelberg.de/Alerting ). The accession number of the sequences retrieved are: Homo sapiens , Y11435 ; S. cerevisiae , Z49919 ; Caenorhabditis elegans , Z99709 ; Arabidopsis thaliana , AC003000 ; Methanococcus jannaschii , U67463 ; S. acidocaldarius , P38619 ; Methanobacterium thermoautotrophicum , AE000920 ; P. bomkoshii , AB009481 ; and Archaeoglobus fulgidus , AE000961 . The alignment was created using the CLUSTAL W algorithm . The phylogram of the aligned proteins were produced with the GROWTREE program in GCG with the Jukes-Cantor distance matrix and neighbor-joining method. Section title: Yeast Strains and Media Educational score: 4.096193313598633 Domain: biomedical Document type: Study Language: en All strains used are derivatives of W303 ( MATa , ade2-1 , trp1-1 , leu2-3 , 112 , his3-11,15 , ura3 , can1–100 ). Cells were grown in YEP medium (1% yeast extract, 2% bactopeptone, 50 μg/liter adenine) supplemented with either 2% glucose (YEPD) or 2% raffinose and 1% galactose (YEPRG). Transformants carrying the kanMX4 cassette were selected on YEPD plates containing 400 μg/ml G418 (US Biological). Section title: Plasmid Construction and Genetic Manipulations of Yeast Strains Educational score: 4.262840270996094 Domain: biomedical Document type: Study Language: en Standard techniques were used for genetic crosses and DNA manipulations . The yeast integrin interacting homologue ( IIH1 ) gene was cloned by PCR using as a template the genomic DNA of strain W303 and oligonucleotides oSP44 (5′CAG-AATAGTCGGAGAAGCGGAC3′) and oSP45 (5′GTAAGGTGCAAGATCAGACAAAG 3′). To construct a IIH1 chromosomal deletion ( iih1::kanMX4 ), the heterologous kanMX4 cassette was amplified by PCR using plasmid pFA6a- kanMX4 as a template and oligonucleotides oSP43 (5′ CGCATACAACTGTAAACAGACTTGA - GGAAGGAGGGGAATCCCCTCAGGAG ATC GATGAAT TC GAGCTC-G3′) and oSP46 (5′ GCCTCATCCCTCGTTCTTATAGTATAA TTACAAGAAGCAATACGACAG CGTACGCTGCAGGTCGAC3′) as primers. The amplification product contained the kanMX4 cassette flanked by IIH1 sequences (underlined in the oligonucleotide sequences) and was used to transform the diploid strain W303. G418-resistant transformants were shown by PCR analysis to be heterozygous for the replacement of most of the IIH1 chromosomal ORF with the kanMX4 cassette. By sporulation and tetrad analysis of one of these transformants (ySP478), iih1::kanMX4 segregants were shown to be inviable, since all tetrads contained only two viable spores that were always G418 sensitive. Section title: Plasmid Construction and Genetic Manipulations of Yeast Strains Educational score: 4.3173346519470215 Domain: biomedical Document type: Study Language: en To construct the GAL–IIH1 fusion (pSP43), the IIH1 XbaI/SspI fragment was cloned in XbaI (SalI) of a Yiplac211-derived plasmid that carried the BamHI–EcoRI GAL1-10 promoter fragment . To construct the GAL-Hsp27 BBP/eIF6 fusion (pSP40), a PCR fragment containing a hemagglutinin (HA)-tagged version of Hsp27 BBP/eIF6 was amplified using as a template the pSG5-p27 expression plasmid in which the entire open reading frame of human p27 BBP/eIF6 gene was cloned in frame with a 10–amino acid HA tag in the NH 2 terminus . The oligonucleotides YSTAG (5′CGG-AATTCAACAATA ATGTACCCATACGAGCTTCCA 3′) and YAS-TAG2 (5′CGGAATTCCTA GGTGAGGCTGTCAATGAGGGA 3′), where the sequences of the human gene and of the HA tag are underlined were used as primers. The obtained PCR product was cut with EcoRI and cloned in the EcoRI site of c2139. Both GAL-IIH1 and GAL-Hsp27 BBP/eIF6 fusions were integrated at the ura3 locus of ySP478 by cutting pSP43 and pSP40 with ApaI before transformation. As a result, strains ySP650 ( GAL-IIH1 single copy), ySP653 ( GAL-Hsp27 BBP/eIF6 single copy), and ySP652 ( GAL-Hsp27 BBP/eIF6 multiple copies), respectively were generated. The copy number of the integrated plasmids was checked by Southern analysis. Strain ySP661 ( MATa , iih1::kanMX4 , and ura3::URA3:: GAL-IIH1 ) was obtained after sporulation and tetrad dissection of ySP650, whereas ySP664 ( MATa , iih1::kanMX4 , and ura3::URA3::GAL-Hsp27 BBP/eIF6 ) was obtained after sporulation and tetrad dissection of ySP653. Section title: Polysome and Western Blot Analysis of Ribosomal Fractions Educational score: 4.13582181930542 Domain: biomedical Document type: Study Language: en Polyribosome preparation and polysome analysis were done exactly according to Foiani et al. . Briefly, cell cultures of W303 (wt), ySP661 ( iih1 Δ, GAL-IIH1 ), and ySP664 ( iih1 Δ, GAL-HSp27 BBP/eIF6 ) were grown in YEPRG medium and shifted to YEPD at time 0 to repress the GAL promoter. Yeast extracts were prepared from 300 ml of cell culture at OD = 0.5–1 , layered on a 7–47% sucrose gradient in 50 mM Tris-acetate, pH 7.0, 50 mM NH 4 Cl, 12 mM MgCl 2 , and 1 mM dithiothreitol and centrifuged at 4°C in a SW41 Beckman rotor for 2 h at 39,000 rpm. Gradient analysis was performed with a gradient collector with continuous monitoring at A 254 . Section title: Polysome and Western Blot Analysis of Ribosomal Fractions Educational score: 4.0367655754089355 Domain: biomedical Document type: Study Language: en For protein analysis, the collected fractions were precipitated with TCA to a final concentration of 10% and left on ice for 30 min. Fractions were centrifuged at 15,000 g , for 15 min at 4°C, and resuspended in Laemmli buffer. Equal amounts of extracts were run on denaturing 12% acrylamide gels and blotted as described above. Section title: p27 BBP/eIF6 Is Present in all Cell Lines, at Early Developmental Stages and Is Associated with the Cytoskeleton Educational score: 4.170144081115723 Domain: biomedical Document type: Study Language: en It was previously observed that p27 BBP/eIF6 mRNA was highly expressed during mouse embryonic development and that highly conserved homologues were present in the unicellular organism S. cerevisiae . These data suggested that p27 BBP/eIF6 might have a general role in cellular processes that is not limited to epithelial cells expressing β4 integrin only. To test this hypothesis, the expression of p27 BBP/eIF6 was first measured by Western blot analysis with a polyclonal antiserum directed against the COOH terminus of p27 BBP/eIF6 on total protein lysates from immortalized cell lines of various origin (see Materials and Methods for original references). So far, p27 BBP/eIF6 has been detected in all cell lines analyzed. Fig. 1 (left) shows the levels of p27 BBP/eIF6 in the immortalized cell lines NIH/3T3 (nontransformed mouse fibroblasts), Jurkat (human T cells), SK-N-MC (human neuroblastoma), A431, HeLa, HaCaT, and FG2 (transformed human epithelial cell lines), and Rin2A (human insulinoma). Constitutive expression of p27 BBP/eIF6 was also detected in two out of two primary cultures tested, respectively, human primary fibroblasts, and mouse resting splenocytes. Section title: p27 BBP/eIF6 Is Present in all Cell Lines, at Early Developmental Stages and Is Associated with the Cytoskeleton Educational score: 4.137823581695557 Domain: biomedical Document type: Study Language: en It was previously observed that p27 BBP/eIF6 mRNA was abundant during embryonic development and declined in the adult, where it was mainly retained in epithelial tissues . To test the hypothesis that p27 BBP/eIF6 protein may be present already at early phases of development, its onset was studied in embryos. The protein was found to be expressed from the earliest developmental stage and later on. Fig. 1 (right) shows p27 BBP/eIF6 in the Xenopus egg, between fertilization and the beginning of segmentation. Comparable results were obtained in mice. As previously observed for p27 BBP/eIF6 mRNA, in the adult, high levels of p27 BBP/eIF6 protein were retained mostly in epithelial tissues, and testis (not shown). Section title: p27 BBP/eIF6 Is Present in all Cell Lines, at Early Developmental Stages and Is Associated with the Cytoskeleton Educational score: 4.117536544799805 Domain: biomedical Document type: Study Language: en In epithelial cells containing β4 integrin, ∼50% of p27 BBP/eIF6 was associated with the intermediate filament cytoskeleton . The association of p27 BBP/eIF6 with the cytoskeleton was analyzed in cell lines not containing β4 integrin. For this purpose, cells were first extracted with detergent containing buffers (Materials and Methods) and the various fractions were analyzed by Western blot. Part of p27 BBP/eIF6 was always found in the cytoskeletal fraction. However, the extent of the association varied according to the cell line (not shown). Fig. 1 (right) shows the results of the fractionation experiments in Xenopus eggs, where at least half of p27 BBP/eIF6 was found to be resistant to detergent extraction and associated with the cytoskeleton. Section title: Nucleolar Localization of p27 BBP/eIF6 Educational score: 4.166591644287109 Domain: biomedical Document type: Study Language: en The topographical distribution of p27 BBP/eIF6 was studied in detail by immunofluorescence, immunocytochemistry, and electron microscopy. To summarize our findings, as shown in Figs. 2 – 4 , p27 BBP/eIF6 was present in the nucleus, with a clear nucleolar pattern. The nucleolar staining of p27 BBP/eIF6 was present in all the organisms analyzed so far (from worms to humans) and in all cell lines. In addition, in some cell lines containing β4 integrin, p27 BBP/eIF6 was clearly evident in the cytoplasm . All the immunoreactivity described is specific, since both the nuclear and the cytoplasmic staining could be routinely abolished by preincubating the antiserum with either the peptide used for immunization or with the recombinant protein . In addition, a similar nucleolus-enriched staining pattern could be seen on NIH/3T3 fibroblasts transfected with a HA-tagged version of p27 BBP/eIF6 , followed by immunofluorescence with a mouse anti-HA mAb (not shown). Section title: Nucleolar Localization of p27 BBP/eIF6 Educational score: 4.07370662689209 Domain: biomedical Document type: Study Language: en The nuclear staining of p27 BBP/eIF6 and its dynamic features will be described using the FG2 cell line as a model. In the interphase nucleus, p27 BBP/eIF6 was clearly concentrated in the nucleolus . This pattern was similar to the one obtained with an antiserum recognizing the nucleolar protein, fibrillarin . The nucleolar colocalization was supported by double immunofluorescence studies with fibrillarin and p27 BBP/eIF6 (not shown). Section title: Nucleolar Localization of p27 BBP/eIF6 Educational score: 4.153329372406006 Domain: biomedical Document type: Study Language: en To establish whether p27 BBP/eIF6 was dynamically associated with the nucleolus, epithelial cells were treated with low doses of actinomycin D and p27 BBP/eIF6 localization was analyzed after 1, 4, and 12 h. This treatment caused the collapse of the nucleolus and the redistribution of nucleolar-associated proteins . In actinomycin D–treated cells, both p27 BBP/eIF6 and the nucleolar antigen, fibrillarin , reversibly weakened their association with the nucleolus and became mostly diffuse in the cell's nucleus. Importantly, no effect on p27 BBP/eIF6 localization was seen when cells were treated with the protein synthesis inhibitors, cycloheximide and puromycin (not shown). Nucleolar localization of p27 BBP/eIF6 was confirmed by immunoelectron microscopy, using the anti-p27 BBP/eIF6 antiserum, followed by 5-nm gold-labeled secondary antibodies. 5-nm gold beads were strongly concentrated within the nucleolus . Section title: Nucleolar Localization of p27 BBP/eIF6 Educational score: 4.114928722381592 Domain: biomedical Document type: Study Language: en Next, we tested whether p27 BBP/eIF6 was stably associated with ribosomal proteins in the cytoplasm of the 804G-clone A epithelial cell line. For this purpose, ribosomes and ribosomal proteins were separated from all of the following: mitochondria, nuclear matrix/intermediate filaments, and soluble proteins. Afterwards, the different fractions were tested for the presence of p27 BBP/eIF6 by Western blot analysis. As shown in Fig. 2 H, most of the protein was present in the nuclear matrix/intermediate filament cytoskeleton fraction. A faint band was associated with the ribosomal fraction. Section title: p27 BBP/eIF6 Redistributes during Mitosis Educational score: 4.403353214263916 Domain: biomedical Document type: Study Language: en The strong nucleolar-associated pattern of p27 BBP/eIF6 was visible in all cell lines during interphase, as well as in various normal and neoplastic tissues (Sanvito, F., manuscript in preparation). Therefore, it was expected that during mitosis, when the nucleolus disappears, the protein would be redistributed. Indeed, during cell division p27 BBP/eIF6 dramatically changed its topographical pattern. At prophase, the immunoreactivity tended to become more dispersed at first. Later, it became associated with the periphery of condensed chromosomes . At metaphase, p27 BBP/eIF6 was enriched in the central mass of chromatin formed by the condensed chromosomes of the metaphasic plate and this pattern was even more noted at anaphase . With the onset of telophase and the reappearance of the nucleolar organization, p27 BBP/eIF6 first scattered and then regained its association with the nucleolus . The redistribution of p27 BBP/eIF6 during the mitotic phases was not associated with its proteolytic degradation. Furthermore, no obvious physical association of p27 BBP/eIF6 with tubulin was observed (not shown). A similar redistribution was observed for some nucleolar antigens, chromosome passengers, which redistribute around chromosomes during mitosis , as well as for some nuclear matrix-associated antigens, whose immunoreactivity become more dispersed during mitosis . Section title: p27 BBP/eIF6 Is Associated with the Nuclear Matrix Educational score: 4.168627738952637 Domain: biomedical Document type: Study Language: en To investigate whether the nucleolar p27 BBP/eIF6 was associated with the nuclear matrix, FG2 cells were extracted with a sequential treatment by means of detergents, DNase, RNase, and high salts , and then analyzed by immunofluorescence and electron microscopy. This treatment removed >90% of the proteins, and 95% of the DNA. In addition, the treatment uncovered a nuclear matrix consisting of a nuclear lamina connected to the cytoplasmic intermediate filaments and of an internal meshwork of polymorphic fibers connecting the lamina to masses within the nucleus. In conditions that lead to the complete loss of DNA , the p27 BBP/eIF6 staining, associated with the nucleolus was clearly retained . Also, the nuclear staining of p27 BBP/eIF6 was unaffected after digestion of residual RNA with RNase (not shown). Section title: p27 BBP/eIF6 Is Associated with the Nuclear Matrix Educational score: 4.124835968017578 Domain: biomedical Document type: Study Language: en To establish whether the residual staining of p27 BBP/eIF6 was present in specific structures, extracted cells were examined by immunoelectron microscopy. By this analysis, immunoreactivity of p27 BBP/eIF6 was always found to be associated with the residual thick filaments of the nuclear matrix . Taken together these data show that in the nucleolus and in the nucleus a relevant part of p27 BBP/eIF6 is tightly associated with the nuclear matrix. Section title: Topographical Relationships of p27 BBP/eIF6 and β4 at Hemidesmosomes in Epithelial Cells Educational score: 4.369216442108154 Domain: biomedical Document type: Study Language: en In epithelial cells containing the β4 integrin, the pattern of immunoreactivity of p27 BBP/eIF6 was slightly different and is briefly described using the epithelial cell line 804G clone A. This cell line contains human β4 integrin, clustered in rosettes of hemidesmosomes . As a result, when stained with antibodies against β4, these cells exhibit a typical Swiss cheeselike pattern in which intense β4 staining surrounds cytoplasmic areas devoid of integrin . Confocal laser scanning microscopy analysis in the horizontal section (x, y) of p27 BBP/eIF6 immunolocalization in these cells clearly showed a cytoplasmic staining partially superimposable to the one for β4 integrin . Most importantly, in the vertical (x, z) and horizontal (x, y) sections, both β4 and p27 BBP/eIF6 stainings were excluded from the small circular areas forming the holes of the Swiss cheeselike pattern . However, staining with the labeled actin-binding drug, phalloidin, showed that these holes contained other cytoskeletal components such as actin and actin-binding proteins (data not shown; Spinardi, L., manuscript in preparation). These data suggest that in epithelial cells that require β4 to form hemidesmosomes, p27 BBP/eIF6 can be specifically recruited in the intermediate filament's cytoskeleton converging on these adhesion structures. Section title: Topographical Relationships of p27 BBP/eIF6 and β4 at Hemidesmosomes in Epithelial Cells Educational score: 4.221792221069336 Domain: biomedical Document type: Study Language: en To extend these observations, the presence of p27 BBP/eIF6 was analyzed by immunoelectron microscopy on cryosections of human amnion, a tissue that contains hemidesmosomes clustered at the basal cell surface. Consistent with the pattern observed in the 804G clone A cells, p27 BBP/eIF6 was detected at the level of inner plaque of the hemidesmosome, where it seemed associated with a thin filament network running between the intermediate filaments and the hemidesmosomal dense plaque . A specific immunolabeling was also noticed in the cytoplasm associated with filamentous structures , and also at the inner face of desmosomes . In agreement with the association with the intermediate filament cytoskeleton, p27 BBP/eIF6 immunolocalization was resistant to high salt extraction (not shown). However, the p27 BBP/eIF6 positive structures (5-nm gold beads) were within intermediate filament bundles, as shown by a double staining with antikeratin antibodies . Section title: p27 BBP/eIF6 Is Essential for Yeast Cell Viability Educational score: 4.29540491104126 Domain: biomedical Document type: Study Language: en To gain more insights into p27 BBP/eIF6 function, several approaches were taken, but our efforts to manipulate the levels of p27 BBP/eIF6 in mammalian cell lines were not successful. Briefly, the expression of p27 BBP/eIF6 antisense mRNA in NIH/3T3 cells led only to a small decrease of protein levels and established clones could not be derived (Sanvito, F., unpublished observations). Furthermore, transient expression of several mutated constructs in COS cells led in some cases to accumulation of p27 BBP/eIF6 either in the nucleus or in the cytoplasm, and was toxic to the cells (Sanvito, F., unpublished observations). These observations, together with the nucleolar localization and the fact that the protein is conserved from yeast to humans , might suggest a conserved function for this protein, which should be independent of β4 integrin ( S. cerevisiae does not have β4 homologues). The possibility that p27 BBP/eIF6 has an ancestral function is further supported by the finding that putative genes encoding peptides homologous to human p27 BBP/eIF6 are present in the genome of different Archibacteria and are also found in plants . Section title: p27 BBP/eIF6 Is Essential for Yeast Cell Viability Educational score: 4.154456615447998 Domain: biomedical Document type: Study Language: en The analysis of the conserved amino acid sequences does not provide any insight into p27 BBP/eIF6 function. However, the fact that S. cerevisiae contains a p27 BBP/eIF6 homologue, 80% identical to the human protein, allowed us to analyze the functional role of the protein in the yeast model. The yeast protein is encoded by a single copy gene, which we called IIH1. We disrupted one chromosomal copy of the IIH1 gene in a diploid strain (see Materials and Methods), followed by sporulation of the obtained IIH1/iih1 Δ heterozygous strain. Tetrad dissection and analysis showed that all tetrads contained only two viable spores , none of which carried the disruption marker KanMX4 , indicating that deletion of IIH1 was lethal. Spores carrying the iih1 Δ allele were able to germinate, but arrested cell division either in the first or the second cell cycle. Section title: p27 BBP/eIF6 Is Essential for Yeast Cell Viability Educational score: 4.249650001525879 Domain: biomedical Document type: Study Language: en We asked whether the human protein could rescue the lethality caused by deletion of the IIH1 gene. For this purpose, we constructed fusion genes where the yeast or the human p27 BBP/eIF6 coding sequences were expressed under control of the yeast galactose inducible GAL1-10 promoter. These fusions were integrated in either single or multiple copies at the yeast URA3 locus of IIH1/iih1 Δ heterozygous diploid strains. Subsequently, these integrated fusions underwent induced sporulation to analyze viability of their meiotic segregants under galactose-induced conditions. As shown in Fig. 7 , most tetrads derived from any of these diploid strains contained either three or four viable spores, as expected if expression of human p27 BBP/eIF6 (Hsp27 BBP/eIF6 ) was able to rescue the lethality caused by the iih1 Δ allele. These data indicate that human and yeast p27 BBP/eIF6 share a common function. However, expression of human p27 BBP/eIF6 seems to complement the defect less efficiently than its yeast counterpart; as indicated by the slower growth of the clones derived from spores expressing a single copy of the human gene and the iih1 Δ allele . This might be due to inefficient translation of the human mRNA gene in yeast (CAI-S.c. = 0.076); consistently with this hypothesis, the slow growth phenotype was substantially abolished when multiple copies of the GAL- Hsp27 BBP/eIF6 fusion were integrated at the ura3 locus . Section title: Depletion of p27 BBP/eIF6 Causes Accumulation of G1 Cells Educational score: 4.2082109451293945 Domain: biomedical Document type: Study Language: en To study the function of p27 BBP/eIF6 in yeast cells, we characterized the phenotype caused by its depletion. For this purpose, wild-type and iih1 Δ cells, carrying either the GAL-IIH1 or the GAL-Hsp27 BBP/eIF6 fusion and logarithmically growing in galactose, were transferred to glucose-containing medium, to switch off the GAL promoter. The switch to a glucose-containing medium resulted in the progressive loss of the p27 BBP/eIF6 protein (not shown). Since the shut-off of the GAL-Hsp27 BBP/eIF6 fusion caused a much quicker arrest of cell division than that of the GAL-IIH1 fusion, we used the GAL-Hsp27 BBP/eIF6 fusion-expressing strain for all the described depletion experiments. As shown by the FACS ® profiles in Fig. 8 , yeast cells depleted of p27 BBP/eIF6 , progressively stopped growing and accumulated as G1 cells with 1C DNA content. This phenotype is consistent with a role of p27 BBP/eIF6 in protein synthesis since yeast cells need to grow in cell mass and reach a critical size before they can enter the S phase. Section title: p27 BBP/eIF6 Depletion Correlates with the Loss of Free 60S Ribosomal Subunit Educational score: 4.1481499671936035 Domain: biomedical Document type: Study Language: en The arrest of p27 BBP/eIF6 depleted cells in G1, the fact that p27 BBP/eIF6 has been independently identified as a putative translation initiation factor and our observation that p27 BBP/eIF6 is detected in the nucleolus of all cell lines, suggested that this protein might be involved in protein synthesis and/or ribosome assembly. To understand the relevance of p27 BBP/eIF6 in one of these processes in yeast, the polysome profiles of wild-type– and p27 BBP/eIF6 -depleted cells were analyzed. For this purpose, wild-type and iih Δ strains carrying the GAL-Hsp27 BBP/eIF6 were grown in galactose-containing medium, and then shifted to glucose-containing medium to switch off the GAL promoter. As a control, a strain where the iih1 Δ allele lethality was rescued by the GAL-IIH1 fusion was also used. Section title: p27 BBP/eIF6 Depletion Correlates with the Loss of Free 60S Ribosomal Subunit Educational score: 4.2439775466918945 Domain: biomedical Document type: Study Language: en As shown in Fig. 9 , the ribosomal profiles of wt and iih1 Δ GAL-IIH1 strains were very similar at time 0, whereas iih1 Δ GAL-Hsp27 BBP/eIF6 cells, consistently with their slow growth phenotype, already showed a marked decrease in the amount of both the 60S subunit and the polysome fraction at the same time point. In contrast, the levels of the free 40S subunit seemed unaffected or slightly increased. This phenotype was even more dramatic 6 h after shifting to the glucose-containing medium of iih1 Δ GAL-Hsp27 BBP/eIF6 cells . Furthermore, an accumulation of half-mer polysomes (i.e., 80S + 60S) was detectable under these conditions. These data suggest that p27 BBP/eIF6 might have a primary function in the correct assembly of the 60S ribosomal subunit in yeast. Section title: p27 BBP/eIF6 Depletion Correlates with the Loss of Free 60S Ribosomal Subunit Educational score: 4.114828109741211 Domain: biomedical Document type: Study Language: en Cofractionation of human p27 BBP/eIF6 in yeast cells was analyzed in parallel. For this end, fractions from the ribosomal gradients were precipitated with TCA and analyzed by Western blot using antibodies against the human protein. As shown in Fig. 10 , p27 BBP/eIF6 was detected in the 80S and in the free 60S fractions, but absent from polysomes. Section title: Discussion Educational score: 4.1807475090026855 Domain: biomedical Document type: Study Language: en p27 BBP/eIF6 was simultaneously identified by two laboratories using two different approaches. It was isolated in our laboratory as a cytoplasmic interactor of the β4 integrin subunit, and we have shown that it can specifically bind the cytodomain of β4 in vitro . However, the discovery of p27 BBP/eIF6 homologues in organisms that do not contain β4 indicated that this protein might have a function independent of β4. Along this line, p27 BBP/eIF6 was independently identified by Si et al. as a putative translation initiation factor, able to inhibit the association between the 60S and the 40S ribosomal subunits. Section title: Discussion Educational score: 4.287449359893799 Domain: biomedical Document type: Study Language: en In this study, we have shown that although in epithelial cells p27 BBP/eIF6 is coherent with β4 at hemidesmosomes, its association with the cytoskeleton is not a unique feature of epithelial cells. Indeed the protein is in the nuclear matrix of all growing cells. Consistently with its conserved nucleolar expression pattern and sequence, p27 BBP/eIF6 is necessary for growth in yeast cells where its loss correlates with a reduced level of the free 60S ribosomal subunit. The in vivo findings were unexpected because they were consistent with a role of p27 BBP/eIF6 in ribosomal biogenesis rather than in mRNA translation. In addition, the association of p27 BBP/eIF6 with the nuclear matrix suggested that this process was linked to the nuclear cytoskeleton. The ability of the human protein to complement yeast mutation further suggested a conserved function for p27 BBP/eIF6 . Section title: An Evolutionarily Conserved Function for p27 BBP/eIF6 in 60S Metabolism Educational score: 4.384406089782715 Domain: biomedical Document type: Study Language: en Database analysis indicates that p27 BBP/eIF6 is a very ancient, evolutionarily conserved protein. It is striking to note that the homology is not restricted to a particular domain of the protein, and that even the length of the protein is constant among different species (246 amino acids in C. elegans ; 245 in humans, fly, yeast, and A. thaliana ; and 215–222 in different Archibacteria ). These data suggest that p27 BBP/eIF6 may have a critical and conserved function. Indeed, we have shown that the deletion of the S. cerevisiae IIH1 gene, encoding the p27 BBP/eIF6 homologue, is lethal to yeast cells, and that the human protein can complement the yeast-null mutation. Some lines of evidence suggest that also in mammalian cells, p27 BBP/eIF6 may be required for growth because of the following: (a) the inability to produce stable p27 BBP/eIF6 mRNA antisense expressing mammalian cells (not shown); (b) the ubiquitous p27 BBP/eIF6 expression in all immortalized cell lines so far analyzed; (c) and the presence of a single p27 BBP/eIF6 gene in the human genome . The generation of p27 BBP/eIF6 -null mice will help to understand whether p27 BBP/eIF6 is also necessary for growth in higher vertebrates. Unfortunately, extensive sequence analysis did not yield significant clues to understand p27 BBP/eIF6 function. Section title: An Evolutionarily Conserved Function for p27 BBP/eIF6 in 60S Metabolism Educational score: 4.471742630004883 Domain: biomedical Document type: Study Language: en To gain some insights into this problem, we used two complementary approaches: the depletion of p27 BBP/eIF6 in the genetically manipulable yeast model, and the study of its topographical localization and biochemical properties in mammalian cell lines and tissues. Yeast cells depleted of p27 BBP/eIF6 are progressively arrested in G1, a phenotype consistent with a defect in either protein synthesis or ribosomal biogenesis. This fact, and the localization of p27 BBP/eIF6 in nucleoli prompted us to analyze the effect of its depletion on the polysome profile. These experiments provide useful information about how p27 BBP/eIF6 , based on its in vitro ribosomal anti-association activity, could be a translation initiation factor . Polysome profiles of p27 BBP/eIF6 -depleted yeast cells showed a dramatic reduction in the peak of free 60S subunits and the appearance of half-mer polysomes. Similar polysome profiles have been observed for mutants defective in ribosomal proteins of the 60S ribosomal subunit , or for components involved in pre-rRNA processing and 60S ribosomal subunit assembly . Thus, the primary function of p27 BBP/eIF6 in yeast is likely related to the 60S ribosomal subunit metabolism. Section title: An Evolutionarily Conserved Function for p27 BBP/eIF6 in 60S Metabolism Educational score: 4.267489433288574 Domain: biomedical Document type: Study Language: en Polysome profiles of yeast cells, defective in translation initiation factor proteins, are generally characterized by the reduction of the rate of polysomes accompanied by the gradual accumulation of both the free 40S and 60S subunits. Therefore, the polysome profile of p27 BBP/eIF6 -depleted yeast cells does not support its primary function as a translation initiation factor. However, on the basis of the in vitro data of Si et al. , and in view of the presence of p27 BBP/eIF6 also in the cytoplasm of some human cells, the possibility that this protein might have a function also as a cytosolic initiation factor cannot be ruled out, as such activity could be masked by the predominant defect in 60S metabolism. Section title: An Evolutionarily Conserved Function for p27 BBP/eIF6 in 60S Metabolism Educational score: 4.065515518188477 Domain: biomedical Document type: Study Language: en The polysome profile does not enlighten the precise role played by p27 BBP/eIF6 in 60S metabolism. The protein may be necessary for ribosome assembly/transport, or may act as a structural ribosomal protein. On the basis of the available data, this last possibility is less likely to be true. In fact, the amount of p27 BBP/eIF6 sedimenting with the ribosomal fraction in several cell lines represents only a minor fraction of the total p27 BBP/eIF6 content. Furthermore, no p27 BBP/eIF6 was detected in the polysome fraction. Section title: An Evolutionarily Conserved Function for p27 BBP/eIF6 in 60S Metabolism Educational score: 4.244480133056641 Domain: biomedical Document type: Study Language: en p27 BBP/eIF6 accumulates in the nucleolus of all the analyzed cell lines, where its pattern follows nucleolar evolution (redistribution at mitosis, when the nucleolar organizing region disappears, and redistribution after actinomycin D treatment). Since the nucleolus is the site where ribosomal subunits are assembled, it seems plausible to speculate that p27 BBP/eIF6 might be involved in 60S ribosomal biogenesis. The process of ribosome biogenesis is complex and involves several factors including proteins with diverse functions such as RNA helicases, transcription factors, and nucleases. Further studies will address the precise role that p27 BBP/eIF6 might play in this process. Section title: An Evolutionarily Conserved Function for p27 BBP/eIF6 in 60S Metabolism Educational score: 4.224125862121582 Domain: biomedical Document type: Study Language: en Finally, it is possible that p27 BBP/eIF6 may be involved in the transport of the 60S subunit from the nucleus to the cytoplasm. To date, very little is known about this process , and only a few nucleolar proteins have been found to shuttle between the nucleolus and the cytoplasm. In this context, three observations are particularly intriguing: (a) the presence of p27 BBP/eIF6 both in a soluble pool and in a cytoskeletal bound compartment; (b) the existence of trace amounts of soluble cytoplasmic p27 BBP/eIF6 in all cells; and (c) the ability of p27 BBP/eIF6 to bind also the mature 60S subunit . Section title: An Evolutionarily Conserved Function for p27 BBP/eIF6 in 60S Metabolism Educational score: 4.182278156280518 Domain: biomedical Document type: Study Language: en It is also worth noting that the nucleolar localization of p27 BBP/eIF6 is observed in the absence of a consensus nuclear localization signal. Therefore, either p27 BBP/eIF6 carries an unknown sequence for nuclear targeting or it is targeted into the nucleus by binding an additional factor in the cytoplasm. The second hypothesis is supported by the fact that even in its most soluble form, p27 BBP/eIF6 partitions in gel filtration as a high molecular weight complex (unpublished observation). The molecular dissection of this high molecular weight complex may shed light on the mechanism by which p27 BBP/eIF6 is transported to the nucleus. Section title: p27 BBP/eIF6 in the Nuclear Matrix/Intermediate Filaments Fraction Educational score: 4.259835720062256 Domain: biomedical Document type: Study Language: en Our study shows that a relevant fraction of p27 BBP/eIF6 is highly insoluble in vivo and is associated both with the nuclear matrix and with the intermediate filament pool. In the cytoplasm, electron microscopy studies have detected p27 BBP/eIF6 on thin cytoplasmic filaments of unknown composition that are spatially separated from the classical keratin intermediate filaments, and converge both upon hemidesmosomes and desmosomes. To our knowledge, beside keratins, only another intermediate filament associated protein, IFAP300, has been described both in hemidesmosomes and desmosomes . In this context, it is interesting to note that a recent thorough electron microscopy analysis of human hemidesmosomes has shown the presence of a novel filamentous structure in the proximity of the inner plaque of the hemidesmosome . Section title: p27 BBP/eIF6 in the Nuclear Matrix/Intermediate Filaments Fraction Educational score: 4.315181732177734 Domain: biomedical Document type: Study Language: en Nuclear matrix consists of both thick polymorphous filaments and of thin filaments known as core filaments . In the nucleus, p27 BBP/eIF6 is associated with polymorphous thick filaments, and is absent from the core filaments. This observation is fully consistent with the notion that core filaments may be formed by nuclear RNA, and that p27 BBP/eIF6 distribution is resistant to RNase digestion . The localization of p27 BBP/eIF6 in the nuclear matrix is of extreme interest in the context of ribosome biogenesis. Our data provide an intriguing link between the nuclear cytoskeleton and the process of ribosome assembly. Section title: p27 BBP/eIF6 in the Nuclear Matrix/Intermediate Filaments Fraction Educational score: 4.197432518005371 Domain: biomedical Document type: Study Language: en In recent years growing evidence has indicated that most nuclear and cytoplasmic processes including transcription, DNA replication, and protein synthesis are spatially organized in association with the cytoskeleton. The combined roles of p27 BBP/eIF6 protein in 60S assembly, its association with the cytoskeleton, and its ability to bind β4 integrin and the mature 60S ribosome subunit belong to an integrated view of cell regulation that encompasses structure as well as biochemical processes . Section title: p27 BBP/eIF6 and β4 Integrin Educational score: 4.536279678344727 Domain: biomedical Document type: Study Language: en We have previously shown that p27 BBP/eIF6 binds specifically to the cytodomain of β4 integrin in vitro and in yeast . Our previous data, and specifically the association of p27 BBP/eIF6 with keratin intermediate filaments, strongly suggested that this interaction could occur also in vivo and be necessary for targeting β4 to hemidesmosomes and intermediate filaments. Since intermediate filament-associated proteins can be solubilized only upon SDS treatment, rendering the maintenance of biochemical interactions impossible, an association between β4 and p27 BBP/eIF6 in tissues could not be proved. We now provide two further elements suggesting that p27 BBP/eIF6 is functionally associated to the β4 integrin in vivo: (a) its peculiar Swiss cheese distribution is superimposable to that of β4 in cells that form hemidesmosomes; and (b) the presence of the protein, in vivo, in hemidesmosomes of the human amnion. Further experiments are needed to clarify the functional significance of β4–p27 BBP/eIF6 interaction, and specifically whether p27 BBP/eIF6 may direct β4 to hemidesmosomes. Alternatively, as it has been recently suggested, on the basis of in vitro evidence and yeast two-hybrid assays, the crucial step in targeting β4 to hemidesmosomes is the interaction with the large intermediate filament-associated protein, HD-1 . If this is the case also in vivo, then the role of p27 BBP/eIF6 binding to β4 may be related to a nonstructural function of β4 integrin, similar to that shown in the case of the recruitment of shc and grb2 or of PI3 kinase . Section title: p27 BBP/eIF6 and β4 Integrin Educational score: 4.270535945892334 Domain: biomedical Document type: Study Language: en In the absence of further evidence, we may reasonably suggest that p27 BBP/eIF6 has an evolutionarily conserved function linked to 60S ribosome biogenesis, and one acquired during evolution in epithelial cells containing β4 integrin. At least one precedent of a protein with a dual function acquired during evolution, i.e., β-catenin/armadillo, has already been reported. This remarkable protein can be found both at sites of cell–cell adhesion in connection to cadherins and in the nucleus where it can signal in conjunction with LEF-1 . | Study | biomedical | en | 0.999995 |
10085285 | Section title: Subcellular Fractionation Educational score: 3.8811216354370117 Domain: biomedical Document type: Study Language: en The yeast strains Saccharomyces uvarum , considered a strain of Saccharomyces cerevisiae , or S . cerevisiae were used throughout the procedure. Enriched nuclei, NEs and heparin-extracted NEs were prepared as previously described . Enriched NPCs were prepared from nuclei (fraction 7) exactly as described in Rout and Blobel and Rout and Strambio-de-Castillia . Section title: Subcellular Fractionation Educational score: 4.280483722686768 Domain: biomedical Document type: Study Language: en Proteins contained in 30 ml of the highly enriched NE fraction described above (fraction 10), were precipitated by mixing the sample with 9 vol of methanol and harvested by centrifugation. The methanol pellet was solubilized in 4 ml of 10 mM MES, pH 6.5, 100 mM DTT, 1% SDS at 90°C for 10 min. The resuspended proteins were mixed with 36 ml of 20 mM MES, pH 6.5, 7 M Urea, 1% (vol/vol) Triton X-100, 0.1% SDS, 1 mM DTT (buffer 7). 16 ml of a 1:1 suspension of the cation-exchange S–Sepharose resin (8 ml of resin bed) were loaded on a broad base, 50-ml column and washed three times with 20 ml of buffer 7. The NE sample was loaded onto the column and was allowed to absorb onto the resin by incubating for 1 h at 25°C with gentle rocking. After the binding step, the flow-through from the column was harvested and pooled with a 20-ml wash in buffer 7 (this pooled material was termed unbound fraction). The column was eluted two times with 30 ml each of 1 M NaCl in buffer 7. Proteins from both bound and unbound fractions were harvested by methanol precipitation. Aliquots were separated on SDS-PAGE. After electrophoresis, the fractionation pattern of known NPC components was analyzed by immunoblotting using mAb414 . The unbound fraction (termed S-NE), was found to be selectively depleted of most nucleoporins recognized by mAb414. Proteins from this fraction were harvested by methanol precipitation, resuspended in PBS, and used to immunize mice. Section title: Mice Immunization and Production of mAbs Educational score: 3.9649906158447266 Domain: biomedical Document type: Study Language: en The production of hybridomas from B-lymphocytes derived from mice spleens was as previously described . Supernatants were screened by indirect IF microscopy of whole yeast cells . Positive supernatants were also screened by immunoblotting of enriched NEs. Cells from positive lines were cloned up to four times by limiting dilution using a standard protocol . Section title: Molecular Cloning and Sequence Analysis Educational score: 4.204051494598389 Domain: biomedical Document type: Study Language: en A λgt11 S . cerevisiae genomic expression library ( Clontech Laboratories Inc. , Palo Alto, CA) was screened by immunoblotting using mAb148G11 following the specifications of the manufacturer. Three positive λ clones containing an identical insert of ∼1.8 kb were obtained and purified to homogeneity by four consecutive rounds of screening. The insert from one of the positive λ clones was subcloned into pBluescript SK(+/−), sequenced from both ends using the T3 and the T7 standard primers and found to contain a sequence identical to the coding region for AA 1,105– 1,700 of MLP1 . DNA sequence comparisons were performed using the BLAST algorithm . Deducted amino acid sequences were compared with sequences in the SGD ( Saccharomyces Genome Database), GenBank and EMBL databases using the FASTA algorithm . Amino acid sequence alignments were performed using FASTA and CLUSTAL W v. 1.6 . Amino acid sequences were analyzed using Protean v. 1.08 (DNAStar Inc.) and MacStripe 1.3.1 . Section title: Ultrastructural Studies Educational score: 4.1942572593688965 Domain: biomedical Document type: Study Language: en IEM analysis of isolated NEs was performed using a modification of a published procedure . For the IEM of isolated whole nuclei, the nuclear preparations were subjected to mild osmotic shock by diluting them with 9 vol of PVP solution (8% polyvinylpyrrolidone [PVP]; 20 mM potassium phosphate, pH 6.5; 0.75 mM MgCl 2 ). These broken nuclei were then transferred (100 μl of diluted sample per well) to microtiter wells pretreated as described for the IEM of isolated NEs, and centrifuged at 23,500 g for 30 min at 4°C. Nuclei pellets were washed twice with PVP solution at 25°C followed by one wash each for 5 min with the following three solutions: (a) 25% (vol/vol) M buffer (5% dried milk in bt-DMSO [10 mM Bis-Tris-Cl, pH 6.50, 0.1 mM MgCl 2 , 20% DMSO]), 75% (vol/vol) PVP solution; (b) 50% (vol/vol) M buffer, 50% (vol/vol) PVP solution; and (c) 75% (vol/ vol) M buffer, and 25% (vol/vol) PVP solution. The nuclei pellets were washed once in M buffer for 5 min at 25°C. Subsequent steps were also as described above with the following exceptions: (a) 0.5× PBS-K, 1 mM MgCl 2 was substituted with bt-DMSO; and (b) after treatment with osmic acid, samples were postfixed with 1% tannic acid in 50 mM potassium phosphate, pH 7.0, for 30 min at 25°C. Section title: Localization of Green Fluorescent Protein Fusion Proteins Educational score: 4.171038627624512 Domain: biomedical Document type: Study Language: en To generate green fluorescent protein (GFP)-tagged proteins we used the plasmid pGFP-N-FUS encoding the GFP gene under the control of the MET25 promoter . DNA sequences encoding the following Mlp1p fragments: (a) NT1 (AA 1–667; pGFPNT1); (b) NT2 ; (c) CT (AA 1,447–1,875; pGFPCT); and (d) putative nuclear localization sequence (NLS), pNLS (AA 1,486–1,545; pGFPpNLS), were PCR-amplified and cloned in-frame downstream of the GFP gene using the unique cloning sites XbaI and XmaI. pGFPNT1, pGFPNT2, pGFPCT, pGFPpNLS, and the negative control pGFP-N-FUS were transformed into W303 cells. To induce the expression of the GFP-tagged Mlp1p fragments, cells were grown to mid-log phase in selective medium without methionine. GFP fusion proteins were visualized in living cells after mounting on microscope slides with 2% methyl cellulose, using a Zeiss Axiophot microscope. ( Carl Zeiss ) equipped with a Chroma no. 41014 GFP Filter (Chroma Technology Corp.). Photomicrographic recording was performed using a Sony DKC5000 digital photo camera (Morrel Instruments Inc.) interfaced with Adobe Photoshop v. 4.01 (Adobe Systems, Inc.) Section title: Gene Disruption and Protein A Tagging of MLP1 and MLP2 Educational score: 4.215219974517822 Domain: biomedical Document type: Study Language: en All yeast strains were derived from W303 . Gene replacement of MLP1 and MLP2 was accomplished using published methods . MLP1 was replaced with URA3 by generating a PCR product containing the entire URA3 gene flanked by 75 nucleotides directly upstream and 75 directly downstream of the MLP1 coding region. A similar procedure was followed to disrupt MLP2 with HIS3 . A haploid strain carrying disrupted copies of both MLP1 and MLP2 (CSDC09α) was constructed by mating CSDC03α and CSDC05a and subsequent sporulation. Haploid strains of opposite mating types carrying the double deletion of MLP1 and MLP2 were mated to generate a homozygous diploid strain (CSDC13a/α; mlp1&mlp2 Δ hd ). In all cases correct integration and segregation of each individual disruptions were verified by PCR analysis of the genomic DNA. The expression of Mlp1p in wild-type and mutant strains was analyzed by immunoblotting of whole yeast cell lysates and by indirect IF microscopy using mAb148G11. Section title: Gene Disruption and Protein A Tagging of MLP1 and MLP2 Educational score: 4.101806640625 Domain: biomedical Document type: Study Language: en For protein A tagging of Mlp1p and Mlp2p, PCR products were generated that contained the coding region for four and a half IgG-binding repeats of protein A followed by a HIS3, URA3 cassette flanked by 75 nucleotides immediately upstream and 75 nucleotides immediately downstream of the stop codon (i.e., excluding the stop codon itself) of each gene . Section title: In Vivo Import and Diffusion Assays Educational score: 4.15733528137207 Domain: biomedical Document type: Study Language: en The in vivo import assay was performed as described . In brief, wild-type W303 and mlp1&mlp2 Δ hd cells expressing GFP fused to the SV40 large T antigen NLS were incubated in dextrose-free medium containing 10 mM each of 2-deoxy- d -glucose and sodium azide at 30°C for 45 min. At the end of the incubation, cells were washed once and incubated in dextrose-containing medium at 30°C to allow reimport of the substrate into the nucleus. The number of normal cells showing a clear accumulation of GFP-NLS in the nucleus (nuclear cells) and the number of cells in which the reporter was cytoplasmic were counted at each time point. At least 40 independent cells were scored per time point. At least four independent sets of cells were counted to construct the graph presented in Fig. 8 . The results are presented as the percentage of cells presenting nuclear signal as a function of time. Linear regression lines were drawn through the linear portion of each curve using KaleidaGraph and the slope of these straight lines were used to estimate the relative import rates. Section title: In Vivo Import and Diffusion Assays Educational score: 4.102484226226807 Domain: biomedical Document type: Study Language: en Passive diffusion assays were performed as follows. pGFP-LEU transformed wild-type and mutant cells were grown as described . Cells were harvested by centrifugation and resuspended in one-fifth of the initial volume of selective medium. Resuspended cells were held at 4°C until use. The diffusion assay was started by centrifuging the cells, washing them once in sterile water, resuspending them in 1 vol of 10 mM sodium azide and 10 mM deoxyglucose in dextrose-free selective medium and finally placing them at 30°C. Aliquots were taken at each time point and scored as described above. At least 3 independent sets of 40 cells were scored at each time point. The relative passive equilibration rates were estimated as described for the import rates. Section title: Overexpression of MLP1 Educational score: 4.294463157653809 Domain: biomedical Document type: Study Language: en To achieve the overexpression of Mlp1p in yeast cells, the MLP1 gene was inserted into the pYES2 yeast expression plasmid (Invitrogen Corp.) downstream of the GAL1/10 inducible promoter. The unique BspHI site located at nucleotide position 4958 of pYES2 was disrupted using the Klenow fragment of DNA polymerase I to generate pYES2-no BspHI. DNA from the λ clone λPM-5620 containing a yeast genomic fragment of ∼16,800 bp from chromosome XI , was used as the template to generate a ∼650 bp PCR product containing the 5′ region of MLP1 from nucleotide position −12 (relative to the first bp of the coding region) to nucleotide position +634 flanked by a BamHI site at the 5′ end. This PCR product was cut with BamHI and EcoRI (site located at nucleotide position +572 of the MLP1 coding region) and inserted into the BamHI and EcoRI sites of pYES2-no BspHI, to generate pYES2-570MLP1. Finally, an ∼5,900-bp BspHI fragment of λPM-5620 that contains the entire MLP1 coding region except the first 8 bp, was inserted into pYES2-570MLP1 that had been linearized with BspHI (site located at nucleotide position +8 of the MLP1 coding region) to generate pGALMLP1. Section title: Overexpression of MLP1 Educational score: 4.114249229431152 Domain: biomedical Document type: Study Language: en pGALMLP1 was transformed into W303 cells by electroporation and transformants were selected taking advantage of the URA3 selectable marker present on the plasmid. Only freshly transformed cells were used for each experiment. To induce the expression of MLP1 , W303/ pGALMLP1 cells were grown to mid-logarithmic phase in selective medium containing 2% D(+)-raffinose (referred to as raffinose throughout the text), transferred to selective medium containing 1% raffinose and 2% galactose and incubated at 30°C for up to 4 h. For repression of MLP1 expression, W303/pGALMLP1 cells were grown in raffinose as above, transferred to selective medium containing 1% raffinose and 2% dextrose and incubated for 4 h at 30°C. Section title: Miscellaneous Educational score: 3.909540891647339 Domain: biomedical Document type: Study Language: en SDS-PAGE and immunoblotting were performed essentially as described . The intensity of bands on immunoblots was quantified using the ImageQuant v.1.1 software in the PhosphorImager system (Molecular Dynamics), when a Cy5-conjugated anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories) was used. Occasionally, computer images of enhanced chemiluminescence signals on photographic films were quantified using the gel plotting macro of NIHImage v.1.60 (Research Services Branch, National Institutes of Health, Bethesda, MD). Section title: Miscellaneous Educational score: 4.14246940612793 Domain: biomedical Document type: Study Language: en Cells were prepared for indirect IF microscopy using the procedure of Kilmartin and Adams with the modifications of Wente et al. and Kilmartin et al. . Double labeling with mAb148G11 and a polyclonal rabbit anti-Nup159p antibody was visualized using Cy3-labeled polyclonal donkey anti–rabbit IgG (cross absorbed against rabbit IgG) and DTAF-labeled polyclonal donkey anti–mouse IgG (cross absorbed against mouse IgG; Jackson ImmunoResearch Laboratories). In all single labeling experiments, the bound antibody was visualized using Cy3-labeled polyclonal donkey anti–mouse or anti–rabbit IgG (Jackson ImmunoResearch Laboratories). The staining conditions were as described . Photomicrographic recording was performed using a Zeiss Axioplan2 microscope ( Carl Zeiss ) equipped with a Photometrics SenSys A2S digital photo camera (Photometrics) interfaced with IPLab Spectrum p v. 3.1.1c (Signal Analytics). Section title: Miscellaneous Educational score: 2.2489688396453857 Domain: biomedical Document type: Study Language: en The growth competition experiment was performed following published procedures . Section title: A Screen for Non-NPC Proteins Associated with the NE Educational score: 4.254807472229004 Domain: biomedical Document type: Study Language: en Mutations or deletions of the genes that encode for certain nucleoporins (for example, Nup133p, Nup120p, Nup145p, and Nup159p) can cause the NPCs to accumulate at one side of the NE, giving rise to tight clusters that are easily identified as spots or patches when cells from such strains are stained with a nucleoporin specific antibody . It was reasoned that proteins only partially associated with the NPC, or localized to areas of the NE that are not in close contact with the NPC, would either fail to cluster or would only partially cluster with the NPCs in these strains. Isolated yeast NEs prepared as previously described were used to produce a panel of 114 anti-NE mAbs that were screened by an assay based on this NPC clustering phenomenon. The indirect IF pattern generated by each of the individual mAbs on fixed whole wild-type yeast cells was compared with the staining pattern obtained on a yeast strain carrying a gene disruption in the NUP133 gene . The anti-NE antibody, mAb148G11, was identified by this screen as recognizing an antigen that appeared to only partially cluster in the NUP133 disrupted strain. This mAb recognized a protein that runs as a single band of ∼220 kD (apparent molecular mass; p220) on immunoblots . Section title: A Screen for Non-NPC Proteins Associated with the NE Educational score: 4.312445640563965 Domain: biomedical Document type: Study Language: en Both wild-type yeast and NUP133 deleted cells were double stained with mAb148G11 and a rabbit polyclonal antibody against the nucleoporin Nup159p . In wild-type cells Nup159p and p220 colocalize to a great extent although this colocalization is not absolute. In particular, some areas of the NE are devoid of one or the other signals and some cells show some nucleoplasmic staining with mAb148G11 . This raises the possibility that p220 may also be found in the nuclear interior. In the clustering strain the difference in the localization of Nup159p and p220 is striking . In these cells Nup159p clearly forms tight clusters localized at the nuclear periphery consistent with its localization at the NPC, while p220 appears to be localized in large patches or even in continuous rims at the nuclear periphery that only partially overlap with the NPC clusters. Furthermore, Nup159p signal is closer to the cytoplasm than the p220 signal . This is consistent with the localization of p220 by IEM (see below) and is in accord with results recently obtained in Xenopus oocytes . In addition, similarly to data obtained with different spindle associated markers , these findings demonstrate that it is possible to obtain IF localization to subregions of the NE even in yeast. Similar results were obtained when the localization of Mlp1p and Nup159p were compared in a NUP120 knock-out strain . Section title: Isolation of the Gene Encoding p220 Educational score: 4.703520774841309 Domain: biomedical Document type: Study Language: en The gene encoding p220, mAb148G11, was identified as MLP1 . MLP1 encodes for a protein of 1875 AA with a predicted molecular mass of 218 kD cloned on the basis of its cross-reactivity with a mAb recognizing human platelet myosin . This nonessential protein was hypothesized to have a nuclear function on the basis of its subcellular localization (see Discussion). The major structural features of Mlp1p, the position of the cloned fragment of the gene and of the epitope of mAb148G11, are shown in Fig. 2 A. The NH 2 -terminal ∼80% of the protein is predicted to have a high α-helical content and contains the heptad-repeats pattern characteristic of coiled-coil proteins. The COOH-terminal ∼400 amino acid residues of the protein are predicted to form a globular tail rich in phenylalanine and proline residues . The sequence with the highest degree of similarity to MLP1 based on FASTA analysis (28% identical and 66% similar) was the uncharacterized yeast open reading frame (ORF), YIL149c . YIL149c is expected to encode a protein of 1,680 AA with a predicted molecular mass of 195 kD. MLP1 and YIL149c belong to a duplicated chromosomal region of the yeast genome present both on Chromosome XI and on Chromosome IX. The similarity between Mlp1p and Yil149p extends over the whole amino acid sequence and is underscored by the similarity of the overall predicted secondary structure of the proteins. Yil149p also contains a phenylalanine and proline rich COOH terminus. Based on its similarity to Mlp1p and the fact that MLP1 and YIL149c appear to have arisen from a genome duplication event, we propose the name MLP2 for YIL149c (see Discussion). The complete yeast genomic database contains no other putative homologues of MLP1 . Section title: Isolation of the Gene Encoding p220 Educational score: 4.329401969909668 Domain: biomedical Document type: Study Language: en Mlp1p and Mlp2p are also similar to the vertebrate and Drosophila Tpr proteins . These proteins are associated with structures found on the nuclear side of the NPC and may be involved in facilitating nucleocytoplasmic transport . The main difference between Tpr and Mlp1p is that the COOH-terminal tail (excluding the predicted coiled-coil region) of Tpr is relatively much longer, spanning the final 30% of the protein, and has a much more marked acidic character . A recently identified S. pombe uncharacterized ORF (SPC162.08c) also shows significant similarities to MLP1, MLP2, and Tpr. Section title: Mlp1p Is Associated with Intranuclear Filaments that Are Localized at the Interface between the NPCs and the Nuclear Interior Educational score: 4.254875659942627 Domain: biomedical Document type: Study Language: en Results from the indirect IF analysis shown in Fig. 1 demonstrated that Mlp1p is localized at the NE in areas that are only partly occupied by NPCs, and that it cannot be only a NPC constituent, even a peripheral one (e.g., a nuclear basket component). To better understand its localization, the fractionation pattern of Mlp1p was followed during the preparation of NEs and NPCs . As expected, the majority of the Mlp1p fractionated with the nuclei and highly enriched NE fraction, in agreement with the indirect IF data . After heparin-extraction of NEs, nearly all of the NE-associated Mlp1p pool (68% of the total) was found in the heparin supernatant , demonstrating it is not strongly associated with the membrane. Only some of Mlp1p cofractionated with the enriched NPC preparation , suggesting a partial or weak association of this protein with NPCs, again consistent with the indirect IF results . It is important to note at this point that Mlp1p represents the first NE-associated protein to display such a subcellular fractionation pattern (see Discussion). Section title: Mlp1p Is Associated with Intranuclear Filaments that Are Localized at the Interface between the NPCs and the Nuclear Interior Educational score: 4.3645758628845215 Domain: biomedical Document type: Study Language: en The ultrastructural localization of Mlp1p was investigated by pre-embedding labeling IEM using mAb148G11 on both isolated NEs and isolated whole nuclei that had been subjected to mild osmotic shock to expose the nuclear interior . It should be remembered that using this procedure only the position of the epitope of the antigen is being localized rather then the position of the protein as a whole. On the scale of the immunostained structures here this may not actually reflect the extent of the localization of the entire protein. A second important caveat of many immunolocalization techniques is that the dimensions of the antibody-gold conjugate have to be taken into account in determining the precise localization of the epitope. Nonetheless, on isolated NEs labeled with mAb148G11, gold particles were found almost exclusively on the nuclear side of the NE often though not always in the vicinities of NPCs. The average of the distance between gold particles and the nearest NPC was 66 ± 19 nm on the y axis and 41 ± 34 nm on the x axis . On numerous occasions the gold appeared to be associated with fibrillar structures stretching from the nuclear side of the NE towards the nucleoplasm . This was in marked contrast to the labeling of Nup159p, a nucleoporin known to be associated with the cytoplasmic fibrils attached to the outer ring of the NPC . In this case, and consistent with published results , the gold particles were found significantly closer to the NPC . The results obtained with the Nup159p control demonstrate that the absence of Mlp1p signal on the cytoplasmic side of the NE can not be due either to a lack of accessibility or to gross alterations or damage of the NE. Section title: Mlp1p Is Associated with Intranuclear Filaments that Are Localized at the Interface between the NPCs and the Nuclear Interior Educational score: 4.337636947631836 Domain: biomedical Document type: Study Language: en The distribution of the Nup159p control in the isolated whole nuclei was unchanged with respect to the NEs . While the majority of the Mlp1p signal in whole nuclei was found in the immediate vicinity of the NE, a significant fraction was found at a considerable distance into the nuclear interior, sometimes as much as ∼300 nm from the mid-plane of the NE . This resulted in the average distribution of Mlp1p being further from the mid-plane of the NE than in isolated NEs (Y = 84 ± 56 nm; X = 33 ± 16 nm; n = 50; using the nearest NPC as a referent). The shorter distance in the case of isolated NEs could be due to the collapse of filaments during the NE isolation procedure (see Discussion). Gold was found even further into the nucleoplasm but it was difficult to establish whether this signal was significantly above background . No obvious structure was observed in association with gold localized in the interior of the nucleus, although fibrils could again be seen associated with gold particles found near the NE. Interestingly, in both isolated NEs and isolated whole nuclei the Mlp1p signal appeared to extend a maximum of ∼120 nm from the cylindrical axis of the NPCs, which corresponds to the proposed minimum in vivo inter-NPC distance . The results of the IEM localization studies are consistent with the indirect IF and immunoblot results presented above , and suggest that Mlp1p is associated with filaments localized at an interface between the nuclear interior and the NPC. Section title: The COOH Terminus of Mlp1p Is Responsible for Its Nuclear Localization Educational score: 4.28913688659668 Domain: biomedical Document type: Study Language: en To determine which region of Mlp1p is responsible for its nuclear localization, the coding sequence of the protein was roughly divided in three thirds and each third was GFP-tagged. In addition, a short sequence localized at the non-coiled-coil COOH terminus was selected because of its high lysine and arginine content and hence its similarities to known NLSs, and was also GFP-tagged . The intracellular localization of GFP was determined in living cells expressing either GFP alone or the GFP-tagged fragments of Mlp1p . As expected, untagged GFP had a predominantly cytoplasmic distribution even though it was not excluded from the nucleus. A similar distribution was observed with cells expressing GFP fused to the both of the NH 2 -terminal thirds of Mlp1p and to the putative NLS. Interestingly however, the COOH terminus of Mlp1p was able to direct the targeting of GFP to the nucleus but not to the NPCs. This suggests that it contains a bona fide NLS that has no obvious homologies to other NLSs, and is not sufficient for NPC association. Section title: Mlp2p Resembles Mlp1p in Its Fractionation Behavior and Cellular Localization Educational score: 4.303515434265137 Domain: biomedical Document type: Study Language: en To determine the subcellular localization of Mlp2p, the gene was genomically tagged with an in-frame COOH-terminal fusion of the IgG binding domains of protein A . As a control, MLP1 was similarly tagged. Highly enriched NEs fractions were prepared from the Mlp1p- and Mlp2p-tagged strains and the fractionation pattern of the proteins was assessed on immunoblots. As expected, the fractionation pattern of Mlp1p-pA was indistinguishable from the one observed in Fig. 3 (data not shown). Similarly, Mlp2p-pA cofractionated with the highly enriched NE fraction but was almost entirely stripped off by the heparin treatment . The tagged strains were used to determine the subcellular localization of the proteins by indirect IF microscopy . Both proteins were found localized predominantly at patches found at the nuclear periphery similar to that previously observed for Mlp1p . The localization of Mlp1p and Mlp2p in tagged strains was also determined by preembedding IEM on isolated NEs. Again these two proteins displayed a very similar localization pattern to that observed for Mlp1p (data not shown). These observations are consistent with the hypothesis that Mlp1p and Mlp2p are functional homologues in accordance with their structural similarities and their genetic interaction (see below). Section title: Double Deletions of MLP1 and MLP2 Cause a Marked Decrease in the Yeast Comparative Fitness Educational score: 4.237033843994141 Domain: biomedical Document type: Study Language: en The entire coding regions of both MLP1 and MLP2 were individually disrupted in the diploid yeast strain W303 by integrative transformation of the URA3 and HIS3 genes respectively. Each heterozygous diploid strain ( mlp1:: URA3/ + and mlp2::HIS3/ +) was sporulated and tetrads were dissected. In both cases four viable spores from most tetrads were observed, demonstrating that neither of these genes is essential and confirming and extending published results . Immunostaining with mAb148G11 revealed that the punctate IF staining pattern and the ∼220-kD immunoblot band recognized by both antibodies was absent in mlp1::URA3 cells but was present in mlp2::HIS3 cells (data not shown). This confirmed that mAb148G11 binds specifically to Mlp1p and demonstrated that mAb148G11 does not cross-react with Mlp2p. Segregants of opposite mating types carrying the individual disruptions as confirmed by both phenotypic and genotypic analyses were mated and sporulated. Spores were isolated that carried both selectable markers demonstrating that the double knock-out of MLP1 and MLP2 is viable. Section title: Double Deletions of MLP1 and MLP2 Cause a Marked Decrease in the Yeast Comparative Fitness Educational score: 4.114840507507324 Domain: biomedical Document type: Study Language: en To assess the degree of selective disadvantage conferred by individual and double disruptions in the MLP1 and MLP2 genes, the mlp1 Δ, mlp2 Δ, and mlp1&mlp2 Δ strains were each grown competitively with their wild-type counterpart in rich medium . While mlp1 Δ and mlp2 Δ competed successfully with wild-type, the strain harboring the double deletion lost ground rapidly even though it was initially added in twofold excess and appeared to be eliminated from the population after 30 generations (data not shown). These results demonstrated that mlp1&mlp2 Δ had a fitness defect relative to the parental stock equal to 24% and was effectively non-viable outside the protected laboratory environment. These results also indicated that MLP1 and MLP2 are homologous. Section title: Deletion of MLP1 and MLP2 Affects the Efficiency of Nuclear Import Educational score: 4.421191215515137 Domain: biomedical Document type: Study Language: en To investigate the possibility that Mlp1p and Mlp2p may be involved in transport of molecules in and out of the nucleus, an in vivo import assay was performed as described by Shulga et al. . This assay allows the detection of kinetic defects in the import rates of a NLS-GFP reporter . In this assay logarithmically growing yeast cells constitutively expressing NLS-GFP are harvested and poisoned in order to block the production of energy. Under these conditions the active import of the NLS-GFP reporter into the nucleus is dramatically reduced resulting in the equilibration of the GFP signal between the nucleus and the cytoplasm by passive diffusion across the NPC. When the metabolic inhibitors are removed and the cells are allowed to recover, NLS-GFP is once again rapidly imported in the nucleus. The relative rates of accumulation of the mutant strain are compared with the ones found with wild-type, revealing any defect in the mutant's efficiency of nuclear import. The steady state distribution of NLS-GFP was indistinguishable in homozygous diploid cells carrying a double deletion of MLP1 and MLP2 as compared with wild-type (data not shown). Nevertheless, mlp1&mlp2 Δ hd (homozygous diploid) cells displayed a markedly slower relative accumulation rate of NLS-GFP into the nucleus with respect to their wild-type counterpart, 13.5 ± 0.2%/min (wild-type) versus 8.9 ± 0.1%/min ( mlp1&mlp2 Δ hd ). Significantly, when double mutant cells were allowed to recover for extended periods of time (up to double the time required for wild-type), the initial equilibrium distribution of reporter protein was regained, again indicating that the efficiency of import and not its steady state balance was affected by the absence of Mlp1p and Mlp2p. The rates of passive equilibration of the NLS-GFP reporter during the incubation with the metabolic inhibitors were also measured in both wild-type and mutant cells . In this case, mlp1&mlp2 Δ hd displayed a significant increase in the relative passive nuclear egress rates of the NLS-GFP reporter as compared with wild-type, −8.75 ± 1.0%/min (wild-type) versus −13.1 ± 0.6%/ min ( mlp1&mlp2 Δ hd ). A model that could explain both the import and the diffusion assay results is presented in Fig. 8 C. The basic assumption of this model is that the rates of passive diffusion of the reporter across the NPC are constant, both in the presence and in the absence of metabolic inhibitors (i.e., they do not require NTP hydrolysis). Consequently, during recovery, after the removal of the metabolic inhibitors (− Inhibitor), the rate of active import becomes greater than the diffusion rate and the net effect is accumulation of the transport substrate into the nucleus. If the rate of import is lower in mlp1&mlp2 Δ hd cells with respect to wild-type the prediction is that mutant cells will take a longer period of time to reach the steady state levels of nuclear accumulation of the substrate, and this is exactly what is observed. On the other hand, during the incubation with the inhibitors (+ Inhibitor), the rate of active import is drastically reduced in both wild-type and mutant cells. However, in the mutant cells this residual import is even further compromised as compared with the wild-type cells. As the diffusion rate remains constant this will appear as an overall faster egress rate from the nucleus in the mutant cells. Section title: Deletion of MLP1 and MLP2 Affects the Efficiency of Nuclear Import Educational score: 4.103917121887207 Domain: biomedical Document type: Study Language: en The involvement of Mlp1p and Mlp2p in active nuclear export was investigated using two steady state assays (data not shown). In the first assay, the subcellular distribution of poly(A) + RNA was analyzed by in situ hybridization using digoxigenin-labeled oligo(dT) 30 as a probe . In the second assay, the steady state distribution of a nuclear export sequence (NES)-GFP was studied by direct fluorescent microscopy . In both cases no effect on export was detected at steady state and the double mutant cells appeared indistinguishable from wild-type. Section title: Overexpression of Mlp1p Educational score: 4.421881675720215 Domain: biomedical Document type: Study Language: en When Mlp1p was expressed from a high copy number 2 μm plasmid, the anti-Mlp1p antibody used by Botstein and coworkers recognized intensely staining dots and sometimes rings localized adjacent to the nucleus . This localization does not correspond to the native localization of Mlp1p discussed in this manuscript (see above). Mlp1p was overexpressed in W303 cells in order to investigate whether this discrepancy could be accounted for by different levels of expression of this protein. The entire coding region of MLP1 was subcloned in a 2 μm–based yeast expression vector under the control of the GAL inducible promoter (see Materials and Methods). Using this plasmid (pGALMLP1), it was possible to overexpress Mlp1p at least ∼100-fold over wild-type levels as demonstrated by semi-quantitative immunoblotting performed with mAb148G11 . Strikingly, overexpression of Mlp1p at these levels was not lethal as demonstrated by the ability of cells carrying pGALMLP1 to grow for days on galactose (data not shown). Cells containing this construct were either induced with galactose for various periods of time or repressed with dextrose for 4 h and analyzed by indirect IF microscopy using mAb148G11 to reveal the localization of Mlp1p . As expected, cells in which the expression of the exogenous copy of MLP1 was repressed with dextrose showed a staining pattern very similar to the one observed in wild-type cells. In contrast, induced cells showed an increase of the Mlp1p-specific signal as a function of the induction time. Initially, small dots (1–4 per cell) could be seen at the nuclear periphery and a clear punctate rim pattern could still be distinguished in most cells . Subsequently, these dots appeared to coalesce and generally gave rise to one prominent circular patch per cell and the nuclear rim pattern became increasingly more diffuse . Finally, the large patch grew to occupy most of the nuclear interior . This staining pattern closely resembled the one described by Kolling et al. , thus reconciling the apparent difference. Careful examination of the overexpressing cells showed that the intense nuclear DAPI signal was either much reduced of excluded from the overexpression spots. It often appeared that the growing Mlp1p spheroids had pushed the chromatin aside, leaving clear indentations in the chromatin or in some cases even compressing it between two spheroids . Cells overexpressing Mlp1p were observed by thin-section transmission EM to reveal whether any novel structure could be detected (data not shown). As a comparison, cells that had been grown in dextrose to repress the expression of the non-chromosomal copy Mlp1p were also analyzed using the same technique. Extensive electron-dense fibrillogranular networks that extended from different areas of the NE towards the nuclear interior were observed in cells grown in galactose but were absent in cells grown in dextrose. To determine if the electron-dense networks present in induced cells were indeed formed of large accumulations of Mlp1p, isolated nuclei from cells grown in both galactose and dextrose (data not shown) were immunostained with mAb148G11 and prepared for IEM using the same pre-embedding labeling technique used above. As expected, the dense fibrillogranular networks present in induced cells specifically stained with mAb148G11 demonstrating that they contain large quantities of Mlp1p and that the Mlp1p spheroids are highly permeable to large macromolecules such as the antibody-gold conjugate. In cells grown in dextrose, Mlp1p appeared to have a localization that was indistinguishable from wild-type. Section title: Discussion Educational score: 4.537554740905762 Domain: biomedical Document type: Study Language: en The clustering of yeast NPCs within characteristic patches in the NE induced by the deletion of certain nucleoporins has previously been used to confirm the association of various proteins with NPCs . Here we used this clustering assay to screen a bank of monoclonal antibodies raised against NE proteins, and isolate those which recognize NE components not associated with the NPC from those recognizing NPC components and nuclear transport factors. We expected to find mainly integral nuclear membrane proteins, analogous to the LAPs or LBR proteins in vertebrate NEs . Instead, a monoclonal antibody was found which identified a protein, Mlp1p, associated with the nuclear periphery of the NPC and NE and extending into the nuclear interior. Sequence comparisons of the predicted amino acid sequence of Mlp1p with the entire GenBank database revealed numerous proteins with strong similarities. The most significant was a second yeast ORF predicted to have arisen as a chromosomal duplication event . We showed this to encode a redundant homologue of Mlp1p which we therefore named Mlp2p. Next, the vertebrate protein Tpr, its probable Drosophila homologue, and an uncharacterized S . pombe ORF showed strong similarity to Mlp1p and Mlp2p along their entire lengths. Though sequence similarity alone is not necessarily definitive, given the similarities in their localization and possible roles in nuclear transport, we conclude that Mlp1p and Mlp2p are the yeast homologues of Tpr . This represents the first identification and characterization of Tpr homologues in yeast, setting the stage for studies that will hopefully elucidate the functions of this protein in a genetically and molecularly tractable system. Section title: Discussion Educational score: 4.35286808013916 Domain: biomedical Document type: Study Language: en The Tpr homologues are predicted to consist of a coiled-coil NH 2 terminus occupying most of the primary sequence. It seems likely that the putative coiled-coil domain forms an extended structure that may in turn be involved in organizing higher order homopolymers (for example, filaments). In contrast, the COOH terminus may interact with heterologous factors and anchor these polymeric structures to the NPCs or to the nuclear interior (see also below). Despite its name, based on its cross reactivity with a mAb against myosin , Mlp1p does not belong to the myosin family due to the different organization of the structural domains along the primary sequence of the protein. Section title: Discussion Educational score: 4.6804518699646 Domain: biomedical Document type: Study Language: en IF microscopy of the NPC clustering strains shows a partial coclustering of Mlp1p with nucleoporin markers, indicating a significant fraction of Mlp1p is associated with the NPCs. Our IEM experiments localizing Mlp1p and Mlp2p to the nuclear face of the nuclear envelope in yeast agree with the recent results obtained with vertebrate Tpr . Our IEM localization also suggests that, like Tpr, Mlp1p and Mlp2p form extensive filamentous structures radiating into the interior of the nucleus from foci at the NPC periphery, perhaps attached at the distal ends of the NPC baskets or fishtraps . Also like Tpr, Mlp1p penetrates deep into the yeast nucleus, as far as half of the nuclear radius, and is thus potentially in contact with at least 80% of the chromatin. Interestingly, a significant fraction of Mlp1p failed to cocluster with NPCs, remaining instead as numerous punctate foci distributed around the nuclear periphery. However, virtually all of Mlp1p cofractionated with the NE fraction, and the majority of Mlp1p signal was in close proximity to the NE inner membrane by IEM. Taken together, this suggests that there is also a portion of Mlp1p not associated with NPCs but distributed around the interporous regions of the NE inner face. This may differ from Tpr, which was reported to be absent from these interporous regions, although other studies have found structures believed to be composed of Tpr interconnecting between NPCs over the inner NE surface . The punctate IF staining pattern of Mlp1p in regions of NE devoid of nucleoporin signal (and hence NPCs) may indicate that it can also organize from non- NPC-associated foci distributed around the nuclear face of the inner nuclear membrane. In an alternative scenario, structures formed of Mlp1p would absolutely depend on NPCs for their nucleation at the NE but would be subsequently able to spread all around the nuclear rim in a non-NPC-dependent manner. Section title: Discussion Educational score: 4.364886283874512 Domain: biomedical Document type: Study Language: en It has long been observed that the NPCs are structurally continuous with the nuclear interior . Interconnecting open channels have been observed radiating from the nuclear interior towards NPCs , and the movement of proteins and RNAs along distinct intranuclear pathways can be studied both in vivo and in vitro . These and other similar observations have led to speculations that efficient exchange of material between the nuclear periphery and the nuclear interior could occur along a filamentous network of tracks. . Indeed, one model proposes the existence of a nucleoskeleton (defined as a nonchromatin intranuclear structural framework) composed of a filamentous network organized from the nuclear periphery and playing a major role in the direction of nuclear trafficking to and from the NPCs . Until recently, the existence of tracks had remained unproven, as no clear candidates for track components had emerged. Section title: Discussion Educational score: 4.721327781677246 Domain: biomedical Document type: Study Language: en A case can be made for the Mlp1p/Tpr family of proteins meeting some of the characteristics expected of nucleoskeletal track components. As one would expect for track proteins they are localized further away from the NPCs than the most peripherally described structures of the NPCs (the nuclear baskets and cytoplasmic filaments) and penetrate most of the volume occupied by chromatin . Their peripheral association with the NPC is emphasized by the partial fractionation of Mlp1p with isolated NPCs, though an independent tight association with the nuclear periphery is suggested by its cofractionation with enriched NEs and continuous distribution around the NE even in strains where the NPCs have clustered. Furthermore, such components would not necessarily be stoichiometric with respect to NPC proteins, and not coassemble with them during NPC assembly. Instead, they could carry an NLS to be imported separately into the nucleus. Thus, the domain necessary for NPC association could be separated from that for nuclear localization, as indeed we showed for Mlp1p and was also recently shown for vertebrate Tpr . The Tpr family seems to form filamentous structures of considerable length (unsurprising considering their predicted coiled-coil structure) that are likely polymeric . Interestingly, the most distal of the intranuclear signal appeared to collapse towards the inner nuclear membrane during the preparation of isolated NEs. Preliminary data indicate an extreme version of this collapse occurs when NEs are isolated from cells overexpressing Mlp1p (data not shown; see below). Thus it would seem, as expected of a track component, that interaction with intact chromatin is required to maintain the normal distribution of Mlp1p within the nucleus and even opens the possibility of a role for this protein in the maintenance of the nuclear architecture. As lamins are absent from yeast, it may even substitute in part for their function. The ability of Mlp1p to self-assemble and form regular polymers that could account for such structural functions remains to be demonstrated and will be the subject of future studies. Section title: Discussion Educational score: 4.660946369171143 Domain: biomedical Document type: Study Language: en As with Tpr, the copious labeling of the COOH-terminal epitope recognized by our antibody indicates that this part of the protein is free, perhaps to interact with chromatin or transport factors . A possible interaction has been found between Tpr and the vertebrate karyopherin β1 nuclear import factor . Here we show that Mlp1p and Mlp2p are required for the efficient nuclear import of a substrate of the homologous yeast karyopherin β1 plus karyopherin α. Although this represents the first in vivo evidence for a role of this protein family in nuclear transport, it must still be regarded with caution given the potential for pleiotropic effects in this experiment (see Results). Nonetheless, together such data support the idea of a direct role for the Tpr family in nuclear transport by active transport of substrates between binding sites positioned opportunely along the filaments. However, Tpr also coincides with clear channels extending from the NPC into the nuclear interior . Overexpression of Mlp1p forms spheroidal fibrillogranular structures which can occupy large portions of the nucleus. Overexpression aggregates such as these usually only display some of the characteristics of the protein under normal conditions, and other characteristics may be anomalous. However, contrary to many other proteinaceous aggregates the overexpression spheroids of Mlp1p are surprisingly open to large molecules, as indicated by the apparently unhindered access and accumulation of antibody conjugated ∼10 nm gold (added before fixation, embedding and sectioning for IEM) throughout them. In addition, it is interesting that the Mlp1p spheroids displaced chromatin as they grew to form large chromatin free regions within the nucleus. One could imagine a polymeric cylinder of Mlp1p with the properties of these spheroids projecting from the NPC into the nucleus. Such a structure would maintain a chromatin free channel that remains highly permeable to even large proteinaceous particles. These data are consistent with binding tracks for transport factors, but also raise another possible and less direct role for this protein family; they may maintain efficient nucleocytoplasmic transport by holding open diffusion channels, for the unhindered intranuclear movement of transport substrates. Section title: Discussion Educational score: 4.444684028625488 Domain: biomedical Document type: Study Language: en It came as a considerable surprise that the deletion of both yeast Tpr homologues was not lethal. The function of both proteins must therefore not be essential in yeast, or other functionally redundant proteins must exist. Either way, this result holds important consequences for the increasing number of studies underway on the vertebrate Tpr proteins. Thus despite a reported association with transport factors and a deleterious effect of vertebrate Tpr overexpression on mRNA export , Tpr homologues are not necessary to support basic nuclear transport. In vivo experiments should be conducted to determine if vertebrate cells have a greater requirement than yeast for Tpr, and what factors (such as cell size) might contribute to any differences. Further work is also required on the Mlp1p/Mlp2p deficient strain to find conditions where at least one of these proteins is required for viability. Such experiments may lead to interacting proteins and processes; yeast studies have been key in showing that many nucleocytoplasmic transport operatives are redundant with numerous subtly overlapping functions, and the Tpr family are proving no exception . The behavior of these proteins should also be studied in living yeast cells (such as by using GFP-tagged Mlp1p), taking advantage of available conditional mutant strains, to avoid some of the problems previously associated with studying potential nucleoskeletal proteins biochemically . Section title: Discussion Educational score: 4.185712814331055 Domain: biomedical Document type: Study Language: en In conclusion, we believe that Mlp1p and Mlp2p, together with their multicellular eukaryotic counterparts, satisfy all the criteria expected of non-nucleoporin nucleoskeletal components. We further suggest they represent the strongest candidates to date for the molecular components of the long hypothesized nuclear tracks connecting the NPCs with the nuclear interior. The characterization of the homologues of Tpr in a genetically tractable organism complements the studies of these proteins in vertebrate systems and opens new avenues in the study of nuclear cell biology. | Study | biomedical | en | 0.999997 |
10085286 | Section title: Construction of the Calreticulin Knockout Vector Educational score: 4.289483547210693 Domain: biomedical Document type: Study Language: en The plasmid pNTK (containing the PGK neomycin cassette and PGK thymidine kinase) was used to generate the knockout vector. The calreticulin promoter was cloned previously from a mouse liver genomic library and digested by SalI and HindIII restriction endonucleases. Afterwards, the 1.5-kb fragment was blunt ended and ligated into the pNTK plasmid to generate the pNTC1 plasmid. The plasmid pCM101 containing the full-length mouse calreticulin gene was cut with EcoRV. Furthermore, an 11-kb fragment, containing part of exon 4 and exons 5–9 and 3-kb of 3′ flanking region, was ligated with the NotI/SalI cut and blunted pNTC1 plasmid that resulted in the calreticulin gene knockout construct pNTC6 . In the pNTC6 plasmid the PGK NEO cassette replaced the first four exons of the calreticulin gene, thus removing the proposed transcription initiation site, the ATG start codon, and interrupting translation of the protein. Section title: Electroporation, Selection, and Screening of ES Cell Clones Educational score: 4.289858818054199 Domain: biomedical Document type: Study Language: en J1 129/Sv embryonic stem (ES) cells (5 × 10 7 ) were electroporated with 100 μg of linearized pNTC6 vector using a Bio-Rad Gene Pulser at 400 V/cm and 25 μF. Cells were plated on mitomycin C–treated G-418–resistant mouse embryonic fibroblast feeder cells. Recombinant clones were selected with G418 (0.2 mg/ml) and gancyclovir (2 μM). 200 colonies were picked after 10 d in selection medium and expanded. 100 clones were screened for a homologous recombination event by PCR using the Expand Long Template PCR System ( Boehringer Mannheim ). The efficiency of homologous recombination was 1 in 30 clones. The heterozygote calreticulin knockout ES cells were identified by PCR using one 5′ primer (5′-GCTGGTCAAGTGTGATTCTCATGTTCCTGCCTG-3′) and two different 3′ primers (5′-CTCTGACCTTCACACTAGACACCCTTCATC-3′ for the wild-type gene and 5′-CTCTGACCTTCACACTAGACACCCTTCATC-3′ for the knockout allele) and confirmed by Southern blot analysis. These ES cells were microinjected into 3.5-d-old C57BL/6J blastocysts to generate chimeric mice. Chimeric males were analyzed for germline transmission by mating with C57BL/6J females, and the progeny were analyzed by PCR and Southern blot. For PCR amplification of the genomic DNA of knockout mice, two sets of primers were utilized . One resulted in detection of the wild-type gene (the primer sequences were: 5′-GAAGATCTAAACCAGTCAAAAGGACC-3′ and 5′-CTCCAGGTCCCCGTAAAATTTGCC-3′) and the second resulted in detection of the targeted knockout construct (the primer sequences were: 5′-CAGAGATCTCAGCAGCAAGGGC-3′ and 5′-CTCTGACC TTCACACTAGACACCCTTCATC-3′). EcoRI-digested genomic DNA was used for Southern blot detection of the calreticulin gene and for detection of the targeted mutant allele using the 5′ DNA probe indicated in Fig. 1 A. Section title: Generation of the GFP Reporter Gene Vector Educational score: 4.177191734313965 Domain: biomedical Document type: Study Language: en The plasmid pS65T-C1 containing cDNA encoding GFP was purchased from CLONTECH Laboratories, Inc. The nucleotide sequence corresponding to the CMV promoter of this construct was removed using AseI and NheI restriction sites and the ends were blunted using Klenow polymerase. The CMV promoter was replaced with a 2.3-kb mouse calreticulin promoter , and cut with SmaI and StuI to generate blunt ends. The 3.58-kb vector containing the calreticulin promoter, GFP cDNA, and the SV-40 polyA site is referred to as the pCPGF construct. To generate transgenic mice, pCPGF was linearized with NaeI/SpeI , purified, and injected into the fertilized oocytes from the FVB/N mice . Afterwards cells were transferred into pseudopregnant FVB/N mice. Genomic DNA was isolated from tail biopsies of each of the transgenic mouse litters and the presence of the GFP reporter targeting vector was detected by PCR using a 5′ primer in the CRT promoter region (5′-GATTCCTTCTGGGCAGTTCATAGTC-3′) and a 3′ primer in the GFP protein cDNA (5′-ATCTAATTCAACAAGAATTGGGACAA-3′). The locations of these primers are indicated in Fig. 3 A. Transgenic animals and calreticulin-deficient mice were generated in the Transgenic Facility (University of Alberta Health Sciences Laboratory Animal Services, Edmonton, Alberta, Canada). Section title: Histological Analysis Educational score: 4.192193984985352 Domain: biomedical Document type: Study Language: en Mouse embryos, at different gestational age, were dissected out of the uterus and fixed in 4% paraformaldehyde for 30–90 min. The embryos were embedded in 30% sucrose overnight at 4°C, washed once in PBS for 1 h, placed in 50% Tissue Tek OCT compound (mounting media) in PBS saline for 8 h at room temperature, and incubated in 100% OCT overnight at 4°C. The embryos were frozen in 2-methylbutane cooled in liquid nitrogen. Cryostat sections, 8–10 μm thick, were prepared in transverse and sagittal sections. The sections were either mounted in mounting media containing 0.2% DABCO (for fluorescence analysis) or processed for immunohistochemistry and standard hematoxylin and eosin staining. A confocal microscope (MRC600; Bio-Rad Laboratories) was used to obtain images at 10× or 60× and images were reconstructed using the Adobe Photoshop program. For better visualization, simulated phase-contrast images of each fluorescent image were generated using the Adobe Photoshop program. Section title: Immunohistochemistry Educational score: 3.98091721534729 Domain: biomedical Document type: Study Language: en Sections from wild-type and transgenic mouse embryos were stained with appropriate antibodies followed by staining using Vectastain Elite ABC kit and Vector DAB substrate kit (Vector Labs Inc.). Primary antibodies were polyclonal rabbit antibodies to GFP (1:150 dilution; CLONTECH Laboratories, Inc.) and affinity purified anticalreticulin (1:10 dilution) . The sections were counterstained with hematoxylin. Section title: Activation of NF-AT3 in Mouse Embryonic Fibroblasts Educational score: 4.159059524536133 Domain: biomedical Document type: Study Language: en Embryos from two litters of heterozygous crosses were used to derive mouse embryonic fibroblasts. Embryos crt −/− and wild-type genotype were dissociated, washed, trypsinized for 30 min, and cultured in 6-well tissue culture plates. Cells were maintained in DME containing 20% FCS. For transfection experiments, plasmid DNA was purified by column chromatography (Qiagen Inc.). Cells were transfected transiently with NF-AT3 expression vector alone, or cotransfected with NF-AT3 and calreticulin expression vectors as described by Burns et al. . After 1 d, cells were stimulated with 200 nM bradykinin followed by indirect immunofluorescence with monoclonal anti–NF-AT (mAb 7A6, a gift of G.R. Crabtree, Stanford University) and polyclonal goat anticalreticulin antibodies . The secondary antibodies were: Texas red–conjugated sheep anti–mouse (diluted 1:50 in PBS) and FITC-conjugated donkey anti–goat (used at 1:50 dilution). For double labeling, all incubations were carried out sequentially. A confocal fluorescence microscope (MRC-600; Bio-Rad Laboratories) equipped with a krypton/argon laser light source was used. Section title: Ca 2+ Measurements Educational score: 4.10957145690918 Domain: biomedical Document type: Study Language: en Wild-type and calreticulin-deficient mouse embryonic fibroblasts (1.5 × 10 6 cells/ml) were loaded with fura-2/AM (Molecular Probes, Inc.). Fluorescence measurements were carried out as described by Mery et al. . Ca 2+ release from internal stores was induced with either 1 μM thapsigargin ( Sigma Chemical Co. ), or 200 nM bradykinin ( Sigma Chemical Co. ). Changes in the cytoplasmic Ca 2+ concentration were monitored in Ca 2+ -free media . Section title: Miscellaneous Educational score: 3.96091890335083 Domain: biomedical Document type: Study Language: en Proteins were separated by SDS-PAGE on 10% polyacrylamide gels as described by Laemmli , transferred to nitrocellulose membranes, and stained by immunoblotting with affinity purified rabbit anticalreticulin antibody . Section title: Miscellaneous Educational score: 4.0394134521484375 Domain: biomedical Document type: Study Language: en Genomic DNA was isolated from mouse tissue as described by Ausubel et al. and digested with EcoRI. The DNA was separated by electrophoresis on 0.8% agarose gel and transferred to Hybond-N membranes ( Amersham Pharmacia Biotech ). The disruption of the calreticulin gene was characterized by Southern blotting . Section title: Calreticulin Knockout Educational score: 4.2842254638671875 Domain: biomedical Document type: Study Language: en Fig. 1 A summarizes the gene targeting strategy used to generate the calreticulin gene knockout mice. During the process of homologous recombination, the PGK NEO cassette was inserted into the calreticulin gene, replacing the first four exons, thus removing the initiator ATG and interrupting the expression of calreticulin protein . PCR analysis of genomic DNA using the specific sets of primers depicted in Fig. 1 A helped to identify the genotype of the mice. Analysis of genomic DNA by Southern blotting showed two hybridizing bands in genomic DNA from crt +/− mice corresponding to the wild-type allele (5.7 kb) and the targeted knockout allele (3.8 kb), whereas Southern blotting of genomic DNA isolated from crt −/− mice showed only one DNA band of 3.8 kb corresponding to the size of the targeted knockout gene. Western blot analysis revealed that the interruption of a single allele in crt +/− mice resulted in a significant decrease in calreticulin protein level, whereas in the calreticulin gene knockout animals ( crt −/− ) there was no detectable expression of the protein . crt +/− mice contained ∼50% lower level of calreticulin protein than wild-type mice, as estimated by densitometry. Identical results were obtained with three different anticalreticulin antibodies (not shown). Section title: Phenotype of Calreticulin Knockout Mice Educational score: 4.136192798614502 Domain: biomedical Document type: Study Language: en Chimeric male mice were crossed with wild-type females to generate first generation heterozygotes. The crt +/− mice had normal phenotype, being viable and fertile. Intercrossing of the crt +/− males with crt +/− females was carried out to generate homozygote ( crt −/− ) gene knockout mice. We were unable to obtain any viable crt −/− pups from this cross. Living crt −/− embryos were obtained at 18 d and earlier. In addition, a number of 12.5–16.5-d-old embryos were dead. Analysis of embryos at or after day 14.5 showed a deficit number of crt −/− embryos (15% instead of 25%), indicating that a significant fraction of crt −/− embryos died earlier. We concluded that the homozygote ( crt −/− ) gene knockout was embryonic lethal and that calreticulin is essential for survival. Section title: Phenotype of Calreticulin Knockout Mice Educational score: 4.308045387268066 Domain: biomedical Document type: Study Language: en Accordingly, serial timed matings were set up to follow embryonic development in an attempt to find the cause of death of the crt −/− mice. Fig. 2 , A and B, shows photographs of 18-d-old crt +/− (left) and crt −/− (right) mouse embryos. At this level of analysis the most visible difference between the crt +/− and crt −/− embryos was the failure of absorption of the umbilical hernia (omphalocele) in the crt −/− embryos . Histological analysis of crt −/− mice confirmed the omphalocele and the presence of midgut in the umbilical hernia . The other significant defect was observed in morphology of the hearts of the crt −/− mice . Fig. 2 B shows that there was a marked decrease in the thickness of the ventricular wall in crt −/− as compared to crt +/− mice. Histological analysis of 12.5-d-old crt −/− embryo also revealed defects in morphology of the heart , indicating that calreticulin is essential for cardiac development at relatively early stages of cardiogenesis. No other gross morphological changes were detected in crt −/− mice . Higher magnification analysis of hearts from 12.5-, 14.5-, and 18-d-old embryos revealed deep intertrabecular recesses and increased fenestration that were associated with the thinner ventricular wall . However, no significant changes in the histology of the atrial wall was observed . At high magnification, pictures of the ventricular wall of 18-d-old crt −/− embryo further demonstrate all of the following: increased fenestration, thinner ventricular wall, and the impaired growth of the compact layer of the ventricles as compared to the crt +/− embryos . Section title: Developmental Activation of the Calreticulin Promoter Educational score: 4.170873641967773 Domain: biomedical Document type: Study Language: en The mature heart contains a very low level of calreticulin but histological analysis of calreticulin-deficient mice suggests an essential role for the protein in cardiac development. To answer the question of whether the calreticulin gene is activated during cardiac development we studied the activation of its promoter in transgenic mice expressing the GFP reporter gene under control of the calreticulin promoter. Fig. 5 A illustrates the strategy used to construct the reporter transgene. The calreticulin promoter (2.3 kb) was introduced upstream of the GFP reporter gene. Genomic DNA from the transgenic mice was analyzed by PCR for the presence of the GFP transgene . Three separate transgenic founder mice (L9, L13, and L17) were generated and all three gave identical results. Activation of the calreticulin promoter was monitored by detection of the fluorescent signal obtained from calreticulin promoter-driven expression of the GFP reporter gene. The first generation (F1) male transgenic mice were used in serial timed matings with wild-type FVB/N females. Embryos were harvested at different gestation times, fixed, and frozen. Every embryo was tested for the presence of the GFP transgene by PCR, followed by analysis of sections prepared from the transgenic and wild-type littermates. Section title: Developmental Activation of the Calreticulin Promoter Educational score: 4.293349742889404 Domain: biomedical Document type: Study Language: en Fig. 6 , A and B, shows sagittal sections of a 9.5-d-old transgenic mouse embryo. The highest fluorescent signal, indicative of high expression of GFP, was found in the cardiovascular system including: ventricular walls, atrial walls, aortic sac, sinus venosus, dorsal aorta, and some of the smaller arteries (e.g., branchial arch arteries). This indicates that the highest activity of the calreticulin promoter occurs in these tissues at day 9.5 of embryonic development. The optic vesicle and the lining of brain ventricles also exhibited high calreticulin promoter activity . At 10.5 d of embryonic development the calreticulin promoter remained highly active in cardiovascular and brain tissue, but its activity was also detected in the midgut and in intersomitic vessels . In older embryos (13.5-d-old) high activity of the calreticulin promoter was maintained in the heart and arteries . In addition, activation of the calreticulin promoter was also seen in the liver, midgut, and the umbilical hernia. Section title: Developmental Activation of the Calreticulin Promoter Educational score: 4.265065670013428 Domain: biomedical Document type: Study Language: en A high level of activation of the calreticulin promoter continued in the cardiovascular system and umbilical hernia in the 14.5-d-old mouse embryo . The expression of GFP reporter protein was localized exclusively to the cytosol of the myocytes of atria and ventricles . There was no fluorescent signal in the thoracic wall and blood cells, indicating differential expression of GFP in these tissues. At late stages of development (18-d-old embryos) a relatively low fluorescent signal, indicative of a lower expression of GFP, was found in the heart . A negligible level of fluorescence was found in the heart of 3-wk-old transgenic mice . This indicates that the activity of the calreticulin promoter is downregulated at late stages of development and after birth. These findings are in agreement with earlier observations that mature hearts express a low level of calreticulin . Section title: Developmental Activation of the Calreticulin Promoter Educational score: 4.18338680267334 Domain: biomedical Document type: Study Language: en To confirm the expression of GFP protein was under the control of the calreticulin promoter in the cardiovascular system, we carried out immunohistological analysis of GFP transgenic embryos. Fig. 8 shows immunohistological staining of sagittal section of hearts from transgenic mice with antibodies to GFP. In agreement with observed fluorescent signals of GFP the protein was highly expressed in the embryonic heart . There was no staining of the wild-type embryonic heart with anti-GFP antibodies . To determine if activation of the calreticulin promoter correlated with expression of calreticulin protein we carried out immunohistological analysis of mouse embryos with anticalreticulin antibodies. Fig. 9 shows low (A–D) and high (A′–D′) magnification of immunohistological staining of 9.5- (A, A′), 13.5- (B, B′), and 18- (C, C′) d-old embryos and mature (D) hearts. Calreticulin protein was highly expressed in myocytes during early stages of embryonic development . The highest expression of calreticulin was observed in the 13.5-d-old embryonic heart . In agreement with our earlier biochemical studies , virtually no staining for calreticulin was detected in the mature heart . This is in full agreement with the levels of GFP expressed in GFP transgenic mice . Section title: Nuclear Translocation of NF-AT3 Is Impaired in Calreticulin Knockout Cells Educational score: 4.3057146072387695 Domain: biomedical Document type: Study Language: en Recent observations indicate that the NF-AT3 transcription factor plays an important role in cardiac hypertrophy and development . The activation of NF-AT is controlled by calcineurin, a Ca 2+ calmodulin-dependent phosphatase . Dephosphorylation of NF-AT by activated calcineurin triggers its nuclear translocation . To determine whether the role of calreticulin in cardiac development might be associated with NF-AT nuclear translocation, we isolated mouse embryonic fibroblasts from crt −/− and wild-type embryos and tested them for nuclear import of NF-AT3 transcription factor. Cells were stimulated with bradykinin to induce depletion of the intracellular Ca 2+ stores and activation of calcineurin . Fig. 10 A shows that wild-type cells expressed calreticulin and the protein is localized to an ER-like network. As expected, there was no expression of calreticulin in crt −/− cells . In wild-type cells stimulated with bradykinin, NF-AT3 was efficiently translocated to the nucleus . However, nuclear import of NF-AT3 in crt −/− cells was impaired and the majority of the transcription factor was found in the cytoplasm indicating that NF-AT3 transcription factor was not efficiently translocated into the nucleus. Nuclear translocation of NF-AT3 was reestablished in the crt −/− cells transiently transfected with calreticulin expression vector . Section title: InsP 3 -dependent Ca 2+ Release Is Inhibited in Calreticulin-deficient Cells Educational score: 4.279153823852539 Domain: biomedical Document type: Study Language: en It is well established that Ca 2+ release by the InsP 3 -dependent pathway is required to activate calcineurin, leading to nuclear import of NF-AT transcription factor . To assess whether calreticulin-deficient cells exhibit any alteration in Ca 2+ homeostasis, we used the fluorescent Ca 2+ indicator fura-2. Wild-type and crt −/− mouse embryonic fibroblasts were stimulated with either thapsigargin, an inhibitor of SERCA type Ca 2+ pumps , or with bradykinin, a potent activator of the InsP 3 -dependent Ca 2+ release channel located in the ER . When cells were stimulated with thapsigargin, the peak amplitude and the duration of enhanced cytoplasmic Ca 2+ concentration were comparable in wild-type and in crt −/− mouse embryonic fibroblasts . Next we investigated the effects of bradykinin, a receptor agonist known to activate phospholipase C and InsP 3 -dependent Ca 2+ release from the ER . Fig. 11 B (solid line) shows that bradykinin caused a rapid and transient increase in the cytoplasmic Ca 2+ concentration in wild-type cells, but not in crt −/− mouse embryonic fibroblasts . Ca 2+ release by InsP 3 -dependent pathway was clearly impaired in calreticulin-deficient cells. Section title: Discussion Educational score: 4.421061038970947 Domain: biomedical Document type: Study Language: en In this study we demonstrated that disruption of the calreticulin gene results in embryonic lethality. Analysis of the crt +/− and crt −/− embryos showed defects in cardiac morphology. The crt −/− mice show a marked decrease in ventricular wall thickness and deep intertrabecular recesses in the ventricular walls. Investigation of the activation pattern of the calreticulin gene during embryonic development revealed that at early stages of embryogenesis the highest level of activation of the calreticulin gene was in the cardiovascular system: ventricular walls, atrial walls, aortic sac, sinus venosus, dorsal aorta, and some of the smaller arteries. In later stages of embryogenesis a high activity of the promoter in the cardiovascular system was maintained, but activation of the gene was also seen in the brain, midgut, intersomitic vessels, and, finally, in the liver and the umbilical hernia. Analysis of InsP 3 -dependent Ca 2+ release and of nuclear import of NF-AT3 transcription factor in cells isolated from crt −/− and wild-type mice revealed that calreticulin is a component of the Ca 2+ regulatory system in these cells. Therefore, calreticulin influences the Ca 2+ and calcineurin-dependent pathway in developing heart. Section title: Discussion Educational score: 4.15955114364624 Domain: biomedical Document type: Study Language: en In the adult, calreticulin is expressed mainly in nonmuscle and smooth muscle cells, and is only a minor component of the skeletal muscle and cardiac sarcoplasmic reticulum . However, data presented in Figs. 6 – 9 show that calreticulin is highly expressed in the cardiovascular system during early embryogenesis. Thus, it was not surprising to find that crt −/− mice have defects in cardiac development and function. The only other gross morphological change in crt −/− embryos was the failure to absorb the umbilical hernia. It is unlikely that embryonic lethality of calreticulin-deficient mice occurs because of failure to absorb the umbilical hernia since this pathology is not embryonic lethal in humans . Embryonic lethality of crt −/− mice most likely resulted from a lesion in cardiac development. Section title: Discussion Educational score: 4.114301681518555 Domain: biomedical Document type: Study Language: en Numerous functions have been postulated for calreticulin including: modulation of steroid-mediated gene expression , chaperone activity , regulation of cell adhesion , and regulation of Ca 2+ homeostasis . A loss of any of these potential functions of calreticulin could lead to a lesion in cardiac development. Because there were no obvious histological abnormalities in any other tissues during embryogenesis, it is unlikely that this is directly due to chaperone function of calreticulin, or its role in the regulation of cell adhesion. Section title: Discussion Educational score: 4.487585544586182 Domain: biomedical Document type: Study Language: en Cardiac development is an extremely complex process under strict transcriptional control . The first morphogenetic event in mouse cardiac development is the formation of a primordial heart tube by cells that segregate from the splanchnic mesoderm in 7-d-old embryos . Between 8 and 9.5 d, the primitive heart tube begins to exhibit rhythmic contractions and undergoes asymmetrical elongation, creating an S-shape loop . Later in embryogenesis, the growing heart progresses through several additional morphological stages including all of the following: the formation of endocardial cushion tissue, septation, trabeculation, compaction (expansion of the ventricular wall), and envelopment of the epicardial mantle . Based on analogies to skeletal myogenesis, it is presumed that cardiomyocyte lineage commitment is directed by a group of tissue-specific transcription factors . Section title: Discussion Educational score: 4.445284843444824 Domain: biomedical Document type: Study Language: en However, the factors essential for cardiomyocyte differentiation in vertebrates remain poorly characterized . How can calreticulin, an ER lumenal protein, affect cardiac development? In this work we have shown that nuclear import of NF-AT3 transcription factor is impaired in crt −/− cells indicating that calreticulin plays a role in the Ca 2+ /calcineurin/NF-AT/GATA-4 transcription pathway . Cardiomyogenesis depends on activation of the GATA family of transcription factors . GATA-4 null mice display a severe defect in the formation of the cardiac tube, which is required for the migration and folding of the precardiogenic splanchnic mesoderm . Furthermore, inhibition of GATA-4 expression affects terminal cardiomyocyte differentiation . During cardiomyogenesis NF-AT, in the nucleus, forms active heterodimer complexes with GATA-4 and significantly enhances its transcriptional activity . The ability of NF-AT to translocate to the nucleus depends on the activation of calcineurin phosphatase activity and efficient dephosphorylation of the transcription factor . Therefore, it is not surprising that NF-AT–deficient mice are not viable and the major defect in these animals relates to cardiac development . Section title: Discussion Educational score: 4.538572788238525 Domain: biomedical Document type: Study Language: en Activation of calcineurin and nuclear import of NF-AT requires Ca 2+ release by the InsP 3 -dependent pathway . In this study we show that calreticulin-deficient mouse embryonic fibroblasts do not have a measurable InsP 3 -dependent Ca 2+ release when stimulated with bradykinin. This is in agreement with a previous report in which the Ca 2+ response to bradykinin was diminished as a result of antisense oligodeoxynucleotide downregulation of calreticulin expression . Calreticulin is a Ca 2+ -binding protein that affects Ca 2+ storage in the ER lumen . It may regulate function of the ER Ca 2+ -release channel (InsP 3 receptor), Ca 2+ -ATPase (SERCA2b) , and store-operated Ca 2+ influx . Camacho and Lechleiter proposed that calreticulin may be responsible for modulation of the Ca 2+ release function of the InsP 3 receptor and/or of the Ca 2+ transport function of SERCA. Recently, John et al. reported that calreticulin may interact with SERCA2b, resulting in a lower transport capacity by the Ca 2+ -ATPase. Here we show that loss of calreticulin reduces Ca 2+ release through the InsP 3 receptor. Despite inhibition of the InsP 3 -dependent Ca 2+ release pathway, cardiomyocytes cultured from 14.5-d-old crt −/− embryos undergo spontaneous contraction indicating that their excitation-contraction coupling is functional in these cells (Mesaeli, N., and M. Michalak, unpublished observations). This implies that Ca 2+ handling by the ER, but not by the cardiac sarcoplasmic reticulum, is affected in calreticulin knockout cardiomyocytes. ER and sarcoplasmic reticulum membranes may form separate functional entities in muscle cells . Section title: Discussion Educational score: 4.0802764892578125 Domain: biomedical Document type: Study Language: en An intriguing finding of this work is that calreticulin-deficient mice develop omphalocele (umbilical hernia). At the early stages of embryogenesis the small intestine develops outside the body to form a physiological umbilical hernia. However, in 15.5-d-old embryos the intestine enters the body cavity and the body wall closes (only the umbilical cord and its contents remain in this region) . Failure of the intestine to return to the coelom results in an omphalocele . Section title: Discussion Educational score: 4.480544090270996 Domain: biomedical Document type: Study Language: en Recent studies indicate that umbilical hernia is associated with the function of a cyclin-dependent kinase inhibitory protein p57 KIP2 , a regulator of cell proliferation . IGF-II receptor mutation and overexpression of IGF-II also results in omphalocele . Disruption of the MARCKS gene that encodes a substrate of protein kinase C also leads to an omphalocele . Protein kinase C is activated by Ca 2+ , suggesting that Ca 2+ release from the ER may play an important role in the closure of umbilical hernia. GATA-4 transcription factor is also expressed during gut development by playing a role in the regulation of intestine epithelial cell differentiation . It is conceivable that a similar Ca 2+ /calcineurin/NF-AT/GATA-4 transcription pathway may be activated during cardiac development and closure of umbilical hernia. This may explain why NF-AT3–deficient mice also show signs of abdominal necrosis . Section title: Discussion Educational score: 4.317529201507568 Domain: biomedical Document type: Study Language: en In Fig. 12 we present a model in which calreticulin is linked to NF-AT function and cardiac development. Ca 2+ release through the InsP 3 -dependent pathway is required to activate calcineurin and to maintain NF-AT transcription factor in the nucleus . We propose that calreticulin regulates calcineurin activity indirectly by affecting Ca 2+ release from the ER and the ability of NF-AT to translocate to the nucleus . This model is supported by our demonstration that InsP 3 -dependent Ca 2+ release is inhibited in calreticulin-deficient cells, concomitant with the inhibition of the NF-AT nuclear import. Section title: Discussion Educational score: 4.538939952850342 Domain: biomedical Document type: Study Language: en The role of the InsP 3 receptor and the InsP 3 -dependent pathway in cardiac development is further supported by studies on another regulator of the InsP 3 receptor and Ca 2+ release, the immunophilin protein FKBP12 (FK506 binding protein) . Knockout of the FKBP12 gene is also embryonic lethal and the animals show cardiac defects . FKBP12 could be considered as a cytosolic regulator of the ER Ca 2+ release channel, whereas calreticulin could be a lumenal regulator of Ca 2+ homeostasis. The calreticulin/Ca 2+ /calcineurin/NF-AT/GATA-4 regulatory pathway is critical for the developing heart , but it is likely to be switched off in the adult myocardium and many components are absent or present at very low levels (NF-AT, GATA-4, and calreticulin) in the mature cardiomyocytes . In mature organisms NF-AT and calreticulin may play a more important role in the immune response . Our results demonstrate an essential role for calreticulin during embryogenesis. Although calreticulin is not a transcription factor, this work shows that the protein is a regulator of Ca 2+ homeostasis and of transcriptional pathways involved in cardiac development. | Study | biomedical | en | 0.999994 |
10085287 | Section title: Immunofluorescence Microscopy of Fixed Normal Rat Kidney (NRK) Cells Educational score: 4.31304931640625 Domain: biomedical Document type: Study Language: en NRK cells were maintained in DME containing 5 g glucose per liter, 10% FCS, and antibiotics in a humidified 5% CO 2 incubator. For transfection with ts-G-GFP all of the following occurred: one barely confluent 10-cm dish of NRK cells, ∼10 7 cells, was removed from the plate with trypsin-EDTA, washed once with PBS, resuspended in 0.6 ml of PBS, and incubated for 10 min on ice with 10 μg of plasmid DNA in a 0.4-cm gap electroporation cell (Bio-Rad Laboratories). The cells were electroporated using 960 μF and 0.25 kV in an electroporator (Bio-Rad Laboratories) with capacitance extender, and plated on 2.2-cm 2 glass coverslips. After 5 h at 37°C, sodium butyrate was added to 5 mM and the cells were shifted to 40°C for 12 h. For microscopy, coverslips (from control nontransfected cells at 37°C or transfected cells at 40°C) were either dropped directly into a well containing 4% paraformaldehyde, 0.1 M sodium phosphate, pH 7.0, or dropped into wells of medium preequilibrated at 32°C, and incubated for various lengths of time before fixation. After 30 min of fixation, coverslips were moved to wells containing 0.1 M glycine in PBS for >10 min, and equilibrated in permeabilization buffer (PBS containing 0.4% saponin, 1% BSA, and 2% normal goat serum). Staining was carried out at room temperature for 1 h in a permeabilization solution containing combinations of a calnexin polyclonal antiserum , a commercial rabbit polyclonal antibody to Bip (Stressgen), the affinity-purified rabbit anti–rat membrin 2-125 or rbet1 mAb 16G6 . Coverslips were washed three times for 15 min each in permeabilization buffer and incubated for 30 min with anti–rabbit or anti–mouse secondary antibodies conjugated to FITC or Texas red. For staining with one of these antisera relative to GFP-G protein, Texas red secondary was used for staining so that it could be detected relative to the inherent green fluorescence of the GFP-G protein. After washing with permeabilization solution as above, coverslips were mounted in mounting medium (Vectashield; Vector Laboratories) and viewed using a fluorescence microscope ( Olympus IX70; Olympus Optical Co.) with a 100× oil immersion lens, an image acquisition system (DeltaVision; Applied Precision), and a computer (model O2; Silicon Graphics). Images were captured to disk using filter sets appropriate for FITC (for FITC or GFP fluorescence) or Texas red (for Texas red fluorescence). Images were cropped, adjusted, and arranged using Adobe Photoshop and printed on a photodigital printer (Fujix Pictography; Fuji Photo Film Co.). Colocalization was quantitated blindly by placing puncta into three categories: SNARE alone, ts-G-VSV alone, or SNARE and ts-G-VSV. From each time point at least 50 puncta were categorized. Section title: Immunogold Labeling of Ultrathin Cryosections Educational score: 4.038313865661621 Domain: biomedical Document type: Study Language: en HepG2 cells were fixed in a mixture of 2% formaldehyde and 0.2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, and prepared for cryosectioning and double-immunogold labeling according to the protein A gold method as described . Section title: Construction of Fluorescent Protein Fusions Educational score: 4.1827850341796875 Domain: biomedical Document type: Study Language: en Coding regions of SNAREs were amplified using PCR and cloned in frame into pEGFP-N3 or pEGFP-C1 ( CLONTECH Laboratories, Inc.). EYFP, ECFP, and EBFP ( CLONTECH Laboratories, Inc.) coding regions were amplified using PCR and cloned into pEGFP-N3 and pEGFP-C1 lacking GFP to produce pEYFP-N3, pEYFP-C1, pECFP-N3, pECFP-C1, pEBFP-N3, and pEBFP-C1 mammalian expression vectors (collectively called pEFP). Coding regions from each SNARE were cut from pEGFP-N3-SNARE and pEGFP-C1-SNARE and ligated into various pEFP vectors. All PCR-amplified inserts were sequenced to ensure no mutations were introduced. VSV-G-ts045 coding region was cut from VSV-G-ts-045-GFP and ligated into various pEFP-N3 constructs. Section title: Time-Lapse Imaging Educational score: 4.1520819664001465 Domain: biomedical Document type: Study Language: en NRK cells were electroporated with 7 μg plasmid DNA for single-transfected or 5 μg of each plasmid for double-transfected cells and plated onto glass coverslips one day before imaging as previously described. Coverslips with transfected cells were transferred to an imaging chamber (Warner Instruments) containing DME without phenol red supplemented with 25 mM Hepes, pH 7.4, 10% FCS, 1× penicillin/streptomycin. Cells were visualized with an inverted microscope ( Olympus IX70; Olympus America Inc.) and a 60× or 100× oil immersion objective. The entire microscope was enclosed in a plastic chamber and prewarmed to either 32°C for VSV-FP movies, or 37°C for movies not involving VSV. Images were acquired using a CCD camera and DeltaVision software every 1.5–4 s with 0.3–1.0-s exposure times for up to 100 exposures for single- and 200 exposures for double-transfected cells. An FITC filter set was used for GFP movies and a custom made CFP/YFP excitation/emission filter set and polychroic was used for two-color CFP and YFP movies (Chroma Technologies Corp.). Resulting movies were analyzed on a computer (octane; Silicon Graphics) and DeltaVision image analysis software. Section title: Punctuate Structures Containing rbet1 and Membrin Are ER Exit Sites Educational score: 4.209394454956055 Domain: biomedical Document type: Study Language: en Previous immunofluorescence studies localized SNAREs (syntaxin 5, membrin, rsec22b, and rbet1) to the ER, Golgi, and numerous punctate structures throughout the cytoplasm. Immuno-EM established the identity of the punctate structures as VTCs of the peripheral and Golgi-adjacent ER–Golgi intermediate compartment (IC). To further characterize these elements and investigate their role as intermediates in secretory cargo transport, we employed double-label immunofluorescence microscopy experiments using ts-G-GFP and antibodies that specifically recognize SNAREs . Section title: Punctuate Structures Containing rbet1 and Membrin Are ER Exit Sites Educational score: 4.146819114685059 Domain: biomedical Document type: Study Language: en First, we examined the relative distributions of endogenous rbet1 and membrin in fixed NRK cells. As shown in Fig. 2 (top), both proteins heavily labeled the juxtanuclear Golgi area as well as small punctate structures throughout the cytoplasm. These steady-state, 37°C structures are similar to those that were observed to contain rbet1, rsec22b, and membrin after incubations at 15°C. The difference is that at 15°C, these peripheral structures become markedly more intense relative to juxtanuclear staining and their size increases (not shown). As documented previously , rbet1 and rsec22b are present at higher levels in the ER than membrin. Although many bright cytoplasmic structures containing rbet1 also contained membrin , VTCs were also visible that contained one but not the other of the two, suggesting some differentiation in their functions (see arrowheads). Section title: Punctuate Structures Containing rbet1 and Membrin Are ER Exit Sites Educational score: 4.24241828918457 Domain: biomedical Document type: Study Language: en Next we investigated whether the VTCs containing rbet1 and membrin were closely associated with ER elements by costaining with antibodies against resident ER proteins. We focused on peripheral VTCs because the density of membranes in the central Golgi area of the cell is too high to observe distinct VTCs. As shown in Fig. 2 (middle) many rbet1-containing elements were closely associated with ER tubules marked with calnexin, an integral membrane ER chaperone. Some of the rbet1 foci actually exclude or were de-enriched for calnexin (see arrows). Despite their apparent distinctness, the exclusive rbet1 elements and ER tubules appeared conspicuously associated, since the rbet1 spots were usually arranged in very close proximity to ER tubules. This is consistent with at least a portion of the rbet1-containing spots representing structures situated downstream of the sorting of secretory cargo away from ER resident proteins. These VTCs may originate adjacent to the ER by local fusion of ER-derived vesicles. We occasionally observed rbet1 spots that were not immediately adjacent to ER tubules , perhaps representing more mature VTCs en route to the Golgi area. Another ER marker, Bip, displayed a similar overall staining pattern as calnexin; it also shared only limited overlap with rbet1 in the peripheral VTCs . Section title: Punctuate Structures Containing rbet1 and Membrin Are ER Exit Sites Educational score: 4.269379138946533 Domain: biomedical Document type: Study Language: en The organization of these SNARE-enriched foci was investigated at higher resolution using immuno-EM and examples are shown of the labeling observed with rsec22b and COPII antibodies . COPII coat proteins are present on anterograde-directed transport vesicles that bud from the ER. Several conclusions are drawn from these micrographs. First, no difference is observed in the organization of these sites when they are labeled with antibodies that recognize rbet1, rsec22b, or membrin, consistent with the colocalization of the spotty immunoreactivity at the light level. Second, sites close to the nucleus and those located close to the plasma membrane have the same general organization. Third, the structures are comprised of an intricate network of vesicles and/or tubules, and many are immunoreactive for both the SNAREs and COPII. It is not possible to discern whether long tubules emanate from the ER traveling in and out of the plane of section, or, alternatively, if the observed elements represent a myriad of individual vesicles. Finally, as is noted at the light level, these structures are adjacent to clearly recognizable ER cisternae. Section title: Punctuate Structures Containing rbet1 and Membrin Are ER Exit Sites Educational score: 4.344184875488281 Domain: biomedical Document type: Study Language: en To help clarify the relationship of the SNARE-containing structures to secretory cargo, we used a GFP-tagged version of VSV-G-ts045 (ts-G-GFP) as a model ER-to-Golgi cargo protein . The temperature-sensitive nature of ts045 allows abrupt release of the cargo G protein from the ER, enabling morphological characterization of sequential transport intermediates . NRK cells were transfected with a ts-G-GFP protein construct and held at 40°C to accumulate the cargo in the ER. At 40°C, ts-G-GFP was found to largely colocalize with calnexin in the ER . It often appeared that calnexin was enriched in different sections of ER tubules, whereas the ts-G-GFP was more homogeneously distributed. However, both of these proteins clearly resided in contiguous regions of the same ER tubules. As expected, rbet1 peripheral structures only partially overlapped with the ER-retained cargo. Fig. 4 (middle) demonstrates that although many rbet1 spots fall along ER tubules and contain the green cargo, many also lie just adjacent to (see arrows) or even some distance (arrowheads) from the ER structures and lack ts-G-GFP. Hence, it appears that cargo in the ER does not have access to a portion of the rbet1-containing structures, indicating that sorting and/or vesicle transport is required to move from the ER into these structures. A similar relationship is observed between VSV-G and membrin under these conditions . Before releasing ts-G-VSV from the ER 18% of the rbet1 puncta and 4% of the membrin puncta colocalize with ts-G-VSV. Section title: Punctuate Structures Containing rbet1 and Membrin Are ER Exit Sites Educational score: 4.302036285400391 Domain: biomedical Document type: Study Language: en When released from the ER by shifting to 32°C, the ts-G-GFP cargo quickly began entering peripheral structures distinct from the ER. As seen in Fig. 5 (top), after 5 min at the permissive temperature, the cargo was still very apparent in the ER, but was also present in calnexin-negative puncta (see arrows). This is similar to those that contained the ER/Golgi SNAREs. 5 min after release of cargo from the ER, 57% of rbet1 and 27% of membrin puncta colocalized with ts-G-GFP. The cargo became more concentrated in these puncta as transport continued. By 10 min after release from the ER, the ER staining was weak and the peripheral puncta and Golgi area staining was intense. At this time 42% of rbet1 and 52% of membrin puncta colocalized with ts-G-GFP , The data indicate that these SNARE-containing structures represent bona fide intermediates in cargo transport and are suggestive of a sequential localization, first in rbet1 and later in membrin puncta. Concentrated cargo colocalized with SNAREs both in foci in close proximity to and some distance away from ER tubules. Section title: Punctuate Structures Containing rbet1 and Membrin Are ER Exit Sites Educational score: 4.030200481414795 Domain: biomedical Document type: Study Language: en By 60 min after release from the ER, cargo was visible predominantly in the Golgi and plasma membrane, with little ER or peripheral IC staining (not shown). Overall the data suggest that the cargo moves from broadly dispersed locations within the ER into the SNARE-containing peripheral sites where it exits these domains and travels to the Golgi apparatus. Section title: SNARE-containing Organelles Revealed by Fluorescent Protein Fusions Educational score: 4.084232807159424 Domain: biomedical Document type: Study Language: en To better understand the dynamics of transport intermediates containing SNARE proteins we transfected cells with various fluorescent proteins (FPs) fused to either the NH 2 - or COOH-terminal ends of the SNAREs . Either stable or transient expression in NRK cells was used to observe the localization of the SNARE-FP fusion proteins. Consistent with our previous experience, the transfected SNARE-FPs localized in very similar, if not identical, patterns to those of the endogenous proteins . Section title: SNARE-containing Organelles Revealed by Fluorescent Protein Fusions Educational score: 4.25407075881958 Domain: biomedical Document type: Study Language: en In transient expression experiments we examined the SNARE-FP localization in cells expressing widely varying levels of the transfected product based on the fluorescence intensity. At all intermediate levels of expression the pattern matched closely with that of the endogenous protein. To further investigate the dynamics of the GFP-tagged SNAREs compared to the native protein we treated transfected cells with Brefeldin A (BFA) or lowered the temperature to 15°C. Again the GFP-tagged SNARE proteins behaved as the wild-type proteins . For example, GOS-28 is little effected at 15°C, while membrin, rbet1, and Sec22 become dispersed within the cytoplasm into a punctate distribution. Treatment of syntaxin 13–transfected cells with BFA resulted in redistribution into a compact spot whereas the Golgi region staining of sec22b and the other IC SNAREs dispersed into a punctate fluorescence pattern. These data support the notion that the trafficking pathways of the transfected GFP-tagged SNAREs are very similar if not identical to the endogenous proteins. Section title: SNARE-containing Organelles Revealed by Fluorescent Protein Fusions Educational score: 4.103999137878418 Domain: biomedical Document type: Study Language: en To see if SNARE-FP incorporates into complexes we used antibodies against syntaxin 5 to immunoprecipitate solubilized membranes from cells transfected with ER/ Golgi SNARE-FPs. SNARE-FPs were found to be present in complexes with endogenous proteins. For example, immunoprecipitation of endogenous syntaxin 5 resulted in coimmunoprecipitation of membrin-GFP (not shown). Also, GFP mAbs ( CLONTECH Laboratories) were able to precipitate endogenous syntaxin 5 in membrin-GFP– transfected cells but not GFP-transfected cells. Finally, the presence of GFP-tagged proteins did alter the localization of other endogenous SNAREs (data not shown). Taken together, these data support the hypothesis that the transfected SNARE-FPs have many, if not all, of the properties of the endogenous molecules. Section title: SNARE-containing Organelles Revealed by Fluorescent Protein Fusions Educational score: 4.283907413482666 Domain: biomedical Document type: Study Language: en To directly test the hypothesis that cargo moves into rsec22b/rbet1 peripheral sites and exits these sites, we performed time-lapse imaging of cells transfected with spectrally distinct fluorescent proteins . We used various fluorescent protein combinations and found that although GFP and BFP are spectrally distinct, photodamage of BFP led to rapid diminution of the signal making this combination impossible for imaging studies. YFP and CFP are spectrally distinct and also photostable, allowing imaging of both proteins in a single cell. We did not observe any bleedthrough into the CFP channel in YFP-transfected cells and vice versa. Thus, the CFP-YFP combination of fluorescent proteins is ideal for double-labeled imaging studies. After release of ts-G-YFP from the temperature block we observed accumulation of the cargo protein in a preexisting rsec22b-CFP–labeled site . Subsequently, the colabeled sites lose the ts-G-YFP, leaving behind the rsec22b-CFP . The ts-G-YFP dynamics observed are similar to those previously described . These data confirm our hypothesis suggesting that the peripheral rsec22b/rbet1-enriched sites are indeed ER exit sites. We interpret our data as the direct observation of the filling and exit of cargo from ER exit sites which remain behind at a fixed position within the cell. Section title: The Dynamics of SNARE-FP–labeled Organelles in Living Cells Educational score: 4.206540107727051 Domain: biomedical Document type: Study Language: en Many types of organelle movements were observed in the video images of living cells transfected with FP-tagged SNAREs. A summary of movements observed at 3-s intervals with a 1-s exposure time is shown in Fig. 8 . Each type of event is depicted by a particular combination of symbols so that summaries of organelle movements can be presented in later figures. The first type of movement involves a fluorescent organelle moving in the field without significant morphological changes . Between 0 and 4 s the organelle has moved 3.9 μm and between 4 and 8 s the organelle has moved in the same general direction another 2.4 μm. This movement is summarized by a closed circle with an arrow pointing in the direction that the organelle is moving. Since the organelle is observed in the second frame it is again depicted by a circle followed by another arrow that ends at the position of the organelle in the third frame. We interpret this movement to be a membrane element or cluster of elements maintaining its structure and simply moving from one position to the next. The rate of this type of movement is ∼0.75–1.0 μm/s. Section title: The Dynamics of SNARE-FP–labeled Organelles in Living Cells Educational score: 4.143120288848877 Domain: biomedical Document type: Study Language: en A similar movement is observed in Fig. 8 B; however, this time a streak of fluorescence is observed emanating from an organelle in frame one and ending at an organelle that appears in frame three. Since the two types of movements may be mechanistically different we illustrate them slightly differently; in the latter case an open circle is connected to a closed circle by an arrow pointing in the direction of the movement. A streak of fluorescence may originate from either a structure moving while the shutter is open, in effect blurring across the CCD array, or from an elongated, tubular organelle. Here we favor the former case since the structure in the original position is not present in the final image and the streak is the same width as the original organelle. Section title: The Dynamics of SNARE-FP–labeled Organelles in Living Cells Educational score: 4.202333450317383 Domain: biomedical Document type: Study Language: en We also observe streaks emanating from round, ∼0.75-μm organelles. However, in this case the streaks either loose their fluorescence or are lost from our field of view in some other fashion . These events are illustrated by a closed circle at the base of an arrow. In many cases we are able to follow the fluorescent streaks emanating from a sphere through several frames . We depict these events by a closed circle with an arrow emanating in the direction of the streak, as in Fig. 8 C. The subsequent fluorescent streaks are illustrated as arrows (without a circle at the base or the end) pointing in the direction of the movement and proportional to the length of the streak . Rates of movements seen with this type of event are 1.3 ± 0.7 μm/s. These dynamics we interpret as extending tubules or vesicle streams because the streaks are thinner than the organelle from which they originate and at least a fraction of the original organelle remains behind at the initial position. In addition, in images collected with 0.5-s exposure times with no dark time between exposures we see partial overlap in different exposures along the length of the tubule (not shown). In other words, the end of a tubule in one exposure may have moved to the midpoint of the tubule in the next image. Section title: The Dynamics of SNARE-FP–labeled Organelles in Living Cells Educational score: 3.8814964294433594 Domain: biomedical Document type: Study Language: en Another type of movement observed is a streak that ends in a spherical organelle, depicted by an arrow with a closed circle only at the arrow head . Fluorescent spheres are also observed to emit a streak that appears to consume the fluorescence of the sphere. This is depicted by an arrow pointing in the direction of the streak with an open circle at the origin . These movements could either be tubules emanating from the sphere or the sphere itself moving while losing fluorescence. Lengths of the arrows are proportional to the distance of the migration during the time interval or the length of the fluorescent streak. Section title: The Dynamics of SNARE-FP–labeled Organelles in Living Cells Educational score: 4.184439659118652 Domain: biomedical Document type: Study Language: en Post-Golgi SNARE proteins exhibited additional events as illustrated in Fig. 8 , G and H. Organelles are observed approaching the plasma membrane followed by an increase in the fluorescence along the membrane, suggesting an exocytotic membrane fusion event. Conversely, we observe bright fluorescent regions of the membrane followed by the appearance of an intracellular organelle, which suggests an endocytic event. These events are depicted by a line along the plasma membrane followed by an arrow pointing either toward (exocytosis), or away (endocytosis) from the plasma membrane. At this level of resolution we cannot rule out the possibility that the organelles are only in close proximity to the membrane and that we are not observing actual exo- or endocytic events. Finally, using several labeled SNAREs we have observed two organelles that appear to become connected by a tubule, suggesting the transfer of contents between the structures. To illustrate these events we connected two circles with a line . Section title: ER and Golgi SNARE Dynamics Educational score: 3.8246004581451416 Domain: biomedical Document type: Study Language: en To follow the dynamics of individual organelles and/or transport intermediates labeled with SNARE-FP we imaged cells and plotted the movements according to the key outlined in Fig. 8 . Movements of SNARE-GFP organelles are shown in Fig. 9 , where each color represents the pathway of a separate element plotted at up to nine positions. Section title: ER and Golgi SNARE Dynamics Educational score: 4.3509626388549805 Domain: biomedical Document type: Study Language: en rsec22b-GFP and rbet1-GFP are observed at peripheral sites, consistent with our previous observations of a significant contingent of these SNAREs in the ER . Peripheral fluorescent sites are most easily observed with rsec22b-GFP and are largely stationary with the exception of some small movements that might be due to Brownian motion. Far fewer back and forth movements are observed with rsec22b than with membrin (see below). Importantly, these relatively stationary structures are the points of origin of streaks of fluorescence that often travel across the cytoplasm toward the Golgi region . 29% of the movements observed were of this type and 40% of the movements were migrating fluorescent streaks . We hypothesize that these streaks represent tubules or closely aligned rows of vesicles carrying cargo from ER exit sites to the Golgi area. Since distinct streaks emanating from a particular site at different times follow a similar pathway, the organelles are likely traveling on the same or parallel microtubule tracks. Other types of rsec22b-GFP movements were as follows: 16% , 10% , and 4% . Relatively little motion is seen in the retrograde direction, suggesting that the recycling of rsec22b occurs via a process that is rare or difficult for us to observe. In the example of a retrograde movement in Fig. 9 A (dashed white and purple arrows pointing away from the Golgi region), the site is consumed. While quite similar motions are recorded for rsec22b-GFP and rbet1-GFP, it was more difficult to follow the rbet1-GFP particles for long distances, perhaps because the fluorescence intensity is fainter. We observed a few rbet1-GFP sites near the Golgi region that appear to connect to each other via tubules . Section title: ER and Golgi SNARE Dynamics Educational score: 4.270697116851807 Domain: biomedical Document type: Study Language: en Syntaxin 5-GFP displays less frequent movement events in our recordings. The events observed are similar in character to the other ER and Golgi SNAREs although fewer long distance movements from peripheral sites were recorded . The cell in Fig. 9 D expresses the transfected membrin-GFP fusion protein at a moderate to low level. Hence, the fainter more peripheral spotty structures distal to the intermediate compartment are not readily observed. Interestingly, 100% of the observed membrin-FP– labeled organelles transit away from and back to the Golgi region . The most intricate of these is illustrated in yellow where a membrin-FP particle originates in a region that may be within the Golgi complex, transits out and away from the Golgi complex, wanders, and then returns to the Golgi region. Other organelles (blue, violet, pink) display similar patterns, moving back and forth between the Golgi region and more peripheral sites. The average rate of movement is 0.7 ± 0.3 μm/s. Organelles can be observed to intersect and form a pair, or to dissociate from a previously existing pair. Section title: ER and Golgi SNARE Dynamics Educational score: 4.169574737548828 Domain: biomedical Document type: Study Language: en We speculate that these movements are antero- and retrograde movements of vesicles, clusters of vesicles, or small tubules between the ER, IC, and the Golgi apparatus. Interestingly, as a cell enters mitosis the membrin-GFP becomes fragmented, appearing more evenly dispersed about the cell (data not shown). Furthermore, organelle movements cease or are dramatically reduced, which is consistent with the previously described change in secretory events associated with mitotic cells . GOS28-GFP shows few if any detectable events on the time scale we recorded from the transfected cells (data not shown). This behavior of GOS28 is consistent with our previous observations that the protein changes little upon a temperature shift to 15°C and that GOS28 appears to be in distinct complexes from the other ER to Golgi SNAREs . Section title: ER and Golgi SNARE Dynamics Educational score: 4.1461639404296875 Domain: biomedical Document type: Study Language: en We also studied the dynamics of two post-TGN SNARE proteins, syntaxin 6-GFP and syntaxin 13-GFP . The movements recorded for syntaxin 6-GFP are very unique as they are all of the type illustrated in Fig. 8 A, that is, spherical organelles migrating within the cell. A single organelle was traced through 11 frames illustrating movements towards, away from, and parallel to the TGN . Syntaxin 6-GFP–labeled organelles can also be seen to reverse direction abruptly. In contrast to syntaxin 6-GFP, syntaxin 13-GFP–labeled organelles exhibit a wider variety of movements. Movements of spherical organelles as well as tubules are documented in Fig. 9 F. In addition, we observe syntaxin 13-GFP organelles that approach the plasma membrane, possibly fusing, and events that have the appearance of endocytosis . Directions of the syntaxin 6 and 13-GFP movements are much more random in orientation than those of rsec22b-GFP and membrin-GFP. Section title: ER and Golgi SNARE Dynamics Educational score: 4.158087730407715 Domain: biomedical Document type: Study Language: en To follow the movements of multiple SNAREs in a single cell we again used the YFP, CFP combination of fluorescent proteins. rsec22b-CFP coexpressed with membrin-YFP exhibits a higher degree of colocalization when compared to rsec22b-CFP coexpressed with syntaxin 13-YFP. However in the rsec22b-CFP/membrin-YFP cotransfected cells, the ratios of the two SNARE-FPs are not always equal because some organelles appear to contain more of one SNARE while other organelles contain more of the other . This observation is consistent with the different overall dynamics observed for the two SNAREs. With this technique we are further able to observe the following: some membrin and rsec22b movements are distinct, and in some cases the colabeled rsec22b and membrin sites move synchronously. Section title: Discussion Educational score: 4.297901153564453 Domain: biomedical Document type: Study Language: en We have studied the organization of the secretory pathway by fusing fluorescent proteins to VSV-G, a widely utilized cargo protein, and SNAREs, molecules important for membrane fusion. By following the dynamic movements of organelles labeled with these reagents we are able to better understand the mechanisms whereby proteins are translocated through the secretory pathway. Since SNARE proteins are critical determinants of an organelle's identity, these proteins serve as specific labels of functional compartments. Beginning at the ER, several interesting observations emerged from this work. At 40°C the ts-G-GFP is retained in the ER where the staining is similar to both calnexin and Bip. Many of the SNARE-labeled structures are closely juxtaposed to the ER, as if they lie alongside the tubules of the organelle. When the temperature of the transfected cells is lowered, allowing the VSV-G to flow through the secretory pathway, the protein accumulates in the 0.75–1-μm structures that precisely overlap with SNAREs at sites we believe to be the position of exit from the ER. Section title: Discussion Educational score: 4.270740509033203 Domain: biomedical Document type: Study Language: en Double labeling with SNAREs and cargo reveals that rsec22b precedes cargo at these ER sites. The theory that these structures are ER exit sites is further confirmed by the dynamics of fluorescent SNARE proteins, particularly rsec22b-GFP. We observe streaks of fluorescence emanating from these sites that we interpret to be tubules or rows of closely spaced vesicles. Multiple streaks are commonly observed to emanate sequentially from a single exit site that suggests the ER exit sites are used multiple times. Their direction of transport is most often toward the Golgi region (suggesting anterograde trafficking) and sequential streaks follow similar paths along what are likely to be the same or closely aligned microtubules. This picture is somewhat different than that observed for the real-time dynamics of cargo, where the peripheral structures were noted to move toward the Golgi complex . Section title: Discussion Educational score: 4.345983505249023 Domain: biomedical Document type: Study Language: en In contrast, our data support the view of a fixed ER exit site that serves to load transport organelles (vesicles or emerging tubules) with cargo while sorting ER proteins in a retrograde fashion back to the ER. When the transport vesicles or nascent tubules have matured, we propose that they translocate to the Golgi apparatus along microtubules and a new round of cargo loading and protein sorting initiates at the same site. Nevertheless, the regulation of membrane dynamics at these ER exit sites needs to be further investigated. For example, it would be important to learn what accounts for the apparently sporadic nature of the fluorescent streaks; perhaps they signify cargo export. In addition, it is of interest to know whether there is a functional difference between the SNARE-labeled sites that appear to colocalize with ER markers (perhaps in segments of ER tubules) and those puncta that exclude ER markers and lie directly alongside or some distance from ER tubules. Also, it is not yet known how SNAREs concentrate at certain sites while excluding resident ER proteins. Section title: Discussion Educational score: 4.480545997619629 Domain: biomedical Document type: Study Language: en Interestingly, membrin, a SNARE proposed to function in concert with rsec22b through the formation of complexes and whose still-life localization significantly overlaps that of rsec22b, appears to display significantly different dynamics than rsec22b. Membrin appears to reside on mobile ∼1-μm organelles that themselves move between the cis-Golgi region and more peripheral sites. On the other hand, while rsec22b is more apparent on stationary, peripheral, ∼1-μm structures from which steaks of fluorescence emanate, generally toward the Golgi region. One possibility is that rsec22b and rbet1 are more enriched in small transport vesicles/tubules that stream outward from larger foci. This would be consistent with the EM study that found a larger percentage of rsec22b and rbet1 vesicles also contained COPII when compared to syntaxin 5– or membrin-containing vesicles . The mobile 1-μm structures containing membrin may contain rsec22b and rbet1 as well, but these SNAREs may be more vigorously sorted away when these structures are translocated. This hypothesis might explain why the movements shown in Fig. 8 A appear rarer than those shown in Fig. 8 F, which are more common in the movies of rsec22b- and rbet1-FPs. What is the function of the mobile 1-μm structures? Perhaps they function as anterograde and/or retrograde transport organelles in conjunction with small ER-derived vesicles. The small vesicles/tubules may serve to fill the membrin-enriched larger structures which then act as carriers in and out of the Golgi area. These organelles could then be thought of as mobile extensions of the cis-Golgi network. Section title: Discussion Educational score: 4.134737014770508 Domain: biomedical Document type: Study Language: en Since the fluorescence of syntaxin 5–transfected cells was not as bright as rsec22b, we may not have detected particular classes of movements. Given this caveat, syntaxin 5 dynamics were generally the same types as the other mobile ER and Golgi SNAREs; however, movements were less frequent, perhaps reflecting a less dynamic nature of this SNARE. The tubular extensions observed between organelles may be conduits for the transfer of cargo between organelles. Alternatively, these structures may represent transport tubules that fuse with the acceptor compartment before fission from the donor compartment. Section title: Discussion Educational score: 4.3215718269348145 Domain: biomedical Document type: Study Language: en The dynamics of the two post-Golgi SNAREs, syntaxin 6 and syntaxin 13, were very different from the ER-to-Golgi proteins. Syntaxin 6 localizes largely to the TGN, and less so to endosomes. Syntaxin 6-FP appears on organelles that meander through the cytoplasm, some of which travel away from the Golgi region, reverse direction, and travel back toward this site again, perhaps reflecting cycling between the TGN and endosomes. The precise nature of any cargo contained in these organelles remains unknown. However, the localization and dynamics are consistent with a role in TGN to endosome trafficking. Syntaxin 13 is proposed to represent a SNARE in the plasma membrane/recycling endosome pathway . Consistent with this proposal, syntaxin 13-FP labels were the only organelles we were able to observe in potential plasma membrane fusion and recycling events. In addition, the dynamics appear to reveal tubules that extend from a central organelle. Perhaps these tubules, formed as proteins, are sorted within the early endosome. Section title: Discussion Educational score: 4.318171977996826 Domain: biomedical Document type: Study Language: en The most remarkable feature of cellular dynamics reinforced by these studies is the specificity of the multitude of trafficking events. The direction and mode of movements, the size and shape of the organelles, and the destinations of the organelles are all precisely regulated. These events are likely to be the critical determinants of the specificity of vesicle trafficking and the membrane organization of cells. Clearly when an organelle arrives at its appropriate destination within the cell an adequate machinery, likely comprised of SNAREs, must catalyze the membrane fusion events. But how cargo and SNAREs are specifically coupled to the organelle translocation machinery, likely comprised of motors and cytoskeletal tracks, remains unclear. Recent reports of motor proteins binding specific Rab proteins may be part of the solution to the molecular recognition issues that underlie this problem. Perhaps purification of the SNARE-FP–tagged organelles will help lead to a solution to this problem. | Study | biomedical | en | 0.999997 |
10085288 | Section title: Culture of Sympathetic Neurons Educational score: 4.058577537536621 Domain: biomedical Document type: Study Language: en Sympathetic neurons from newborn rat cervical superior ganglia were cultured as previously described . For electron microscopy studies, neurons were fixed with 1% glutaraldehyde in PBS for 48 h at 4°C and washed in PBS before pre-embedding. Section title: Subcellular Fractionation Educational score: 4.124692916870117 Domain: biomedical Document type: Study Language: en Sympathetic neurons (2 × 10 5 in 3.5-cm-diam Petri dish) were harvested in 100 μl of isotonic buffer (210 mM mannitol, 70 mM sucrose, 1 mM EDTA, and 10 mM Hepes, pH 7.5) supplemented with protease inhibitors cocktail Complete ( Boehringer Mannheim ) and homogenized with a Dounce homogenizer. Samples were transferred to Eppendorf centrifuge tubes, centrifuged at 900 g for 5 min to remove nuclei, and then followed by centrifugation at 10,000 g for 30 min at 4°C to obtain the heavy membrane pellet (HM) enriched in mitochondria. The HM material was resuspended in 20 μl PBS, 0.2% Triton X-100. The protein concentration was determined by the method of Bradford in both the HM and soluble fractions. 5 μg (HM fraction) and 8 μg (soluble fraction) were used for Western blotting. Section title: Isolation of Mouse Liver Mitochondria and Incubation with Bax Educational score: 4.129192352294922 Domain: biomedical Document type: Study Language: en Mitochondria were isolated by sucrose density gradient centrifugation as previously described . Mitochondria were incubated with 5 μM BaxΔTm for 30 min at 30°C in a buffer containing 125 mM KCl, 4 mM MgCl 2 , 5 mM Na 2 HPO 4 , 5 mM succinate, 5 μM rotenone, 0.5 mM EGTA, 15 mM Hepes-KOH, pH 7.4. For electron microscopy, pellets of mitochondria were fixed in 1.5% glutaraldehyde in Sorensen phosphate buffer for 1 h at 4°C and processed as indicated. Section title: Electron Microscopy Studies Educational score: 4.186622619628906 Domain: biomedical Document type: Study Language: en Pellets of glutaraldehyde-fixed neurons and isolated mitochondria were pre-embedded into low viscosity agarose, washed with Sorensen phosphate buffer, and then postfixed in 2% OsO 4 in phosphate buffer for 1 h at room temperature. Then the samples were washed again in phosphate buffer, dehydrated in alcohol and propylene oxide, and then embedded in Epon. Ultrathin sections of comparable thickness were prepared with a Leica Ultracut ultramicrotome and placed on formvar carbon-coated copper grids. The grids were stained with uranyl acetate and lead citrate and observed with a Philips CM10 transmission electron microscope at 80 kV using a 30–40-μm objective aperture. Section title: Immunocytochemistry Educational score: 4.079407691955566 Domain: biomedical Document type: Study Language: en Neurons were fixed with 4% paraformaldehyde in PBS, permeabilized for 10 min with PBS containing 0.2% Triton X-100, incubated for 2 h with an anti–cytochrome c monoclonal antibody (dilution 1:15 in PBS with 5% normal goat serum; PharMingen ), and then revealed with a fluorescein-labeled goat anti–mouse antibody. Section title: Cytochrome C Release from NGF-deprived Sympathetic Neurons Educational score: 4.178157806396484 Domain: biomedical Document type: Study Language: en We have studied the distribution of cytochrome c in cultured sympathetic neurons from rat superior cervical ganglia (SCG) undergoing apoptosis induced by NGF deprivation. Cytochrome c was analyzed by Western blotting in both soluble cytosolic and the mitochondria enriched heavy membrane (HM) fractions obtained from SCG neurons at 8, 15, and 24 h after NGF. Fig. 1 , a and b, highlights a significant decrease of cytochrome c in the HM fraction of neurons deprived of NGF for 8, 15, and 24 h , accompanied by an increase of cytochrome c in the cytosolic fraction . This result was confirmed by immunostaining studies . Although all neurons cultured in the presence of NGF displayed a punctate staining , ∼50–75% of those deprived of NGF for 15 h displayed a diffuse cytosolic pattern and >95% of the latter had a condensed apoptotic nucleus (data not shown). Immunocytochemistry studies revealed that mitochondria present in neurites preserved their cytochrome c content longer than mitochondria present in soma (data not shown). Section title: Cytochrome C Release from NGF-deprived Sympathetic Neurons Educational score: 4.161341667175293 Domain: biomedical Document type: Study Language: en We also analyzed the cytochrome c distribution in sympathetic neurons rescued from NGF-deprivation with the caspase peptide inhibitor BAF. In the presence of 100 μM BAF, >80% neurons were able to survive after NGF deprivation for 5 d, although they had markedly smaller somas and atrophic neurites . In agreement with previous data , we found that BAF was able to inhibit caspase activity as assessed by immunoblot analysis of poly-(ADP ribose) polymerase (data not shown). A major decrease in cytochrome c levels was detected in the HM fraction of those neurons , a finding confirmed by cytochrome c immunostaining . These data indicate that, during apoptosis, the release of cytochrome c from mitochondria of sympathetic neurons does not require caspase activity. Section title: Cytochrome C Recovery by Mitochondria of NGF-rescued Neurons Re-exposed to NGF Educational score: 4.339616775512695 Domain: biomedical Document type: Study Language: en The major objective of this article was to test whether mitochondria from BAF-rescued neurons could recover their cytochrome c content following re-exposure of neurons to NGF. Cytochrome c analysis in HM fraction of neurons deprived of NGF in the presence of BAF for 2 d and then re-exposed to NGF for 3 d, revealed a significant increase in the level of mitochondrial cytochrome c compared with neurons cultured in the absence of NGF and the presence of BAF alone for 5 d . A kinetic analysis revealed a progressive recovery of cytochrome c by mitochondria which was almost complete after a 4-d NGF treatment . Consistent with this finding, immunocytochemistry studies showed reappearance of a punctate mitochondrial cytochrome c staining in almost all neurons (>80%, n = 3) re-exposed to NGF for 24 h . The effect of re-exposure to NGF was blocked by cycloheximide , suggesting the requirement of de novo protein synthesis for its action. It is possible that synthesis of cytochrome c precursor apocytochrome c was a prerequisite for cytochrome c recovery by mitochondria as only apocytochrome c has been shown to be imported into mitochondria . In contrast, the recovery of normal cytochrome c levels by mitochondria was not prevented by the microtubule disrupting agent colchicine , therefore excluding the possibility that reappearance of a normal cytochrome c staining pattern in the cell body was due to microtubule associated migration of still intact mitochondria from neurites to the soma. Section title: Cytochrome C Recovery by Mitochondria of NGF-rescued Neurons Re-exposed to NGF Educational score: 4.224786758422852 Domain: biomedical Document type: Study Language: en In agreement with previous data , we observed that addition of NGF back to BAF-protected SCG neurons caused an increase in soma diameter and neurite extension . Altogether, these results indicate that the NGF receptors TRKA (tyrosine kinase receptor) and their signaling components remained functional in neurons deprived of NGF for at least 5 d. Interestingly, addition of NGF alone (without BAF) back to BAF-rescued neurons was sufficient to promote mitochondria recovery, regrowth of neurons, and long-term survival, suggesting that the caspases which had been activated during apoptosis had been irreversibly inhibited by BAF. Section title: Ultrastructure of Mitochondria Educational score: 4.199755668640137 Domain: biomedical Document type: Study Language: en The reappearance of cytochrome c in mitochondria of BAF-rescued neurons re-exposed to NGF suggested that the ultrastructure of mitochondria had been preserved. This hypothesis was tested by electron microscopic studies. Fig. 4 a shows that mitochondria from neurons cultured continuously in the presence of NGF appeared elongated or oval-shaped, with sparse cristae . 24 h after NGF deprivation most mitochondria were round in shape, smaller than normal with a hyperdense matrix. No obvious rupture of the outer mitochondrial membrane has been observed. Mitochondria from BAF-rescued neurons were even smaller , often forming aggregates surrounded by lysosomes (data not shown). After addition of NGF back to BAF-rescued neurons , mitochondria recovered the shape and size typical of neurons continuously cultured in the presence of NGF . Lysosomes containing myelin figures, lipid droplets, as well as images of autophagy, have also been observed as previously described by Martin et al. . Section title: Bax Induces Mitochondrial Shrinking and Triggers Cytochrome C Efflux from Mitochondria Educational score: 4.223189353942871 Domain: biomedical Document type: Study Language: en Bax is known to play an essential role in neuronal apoptosis. Sympathetic neurons from Bax-deficient mice can survive more than 20 d in the absence of NGF . Moreover, Bax is known to trigger cytochrome c release when added directly to isolated mitochondria . We therefore wondered whether Bax could also be responsible for the mitochondrial morphological change observed in neurons after NGF deprivation. This hypothesis was tested by electron microscopy using isolated mitochondria that had been incubated for 15 min in the presence of 5 μM BaxΔTm or, for comparison, with 100 μM calcium which stimulates PTP opening . Although, as previously reported , opening of the PTP with calcium led to mitochondrial swelling, Bax, in contrast, triggered mitochondrial shrinkage resulting in a morphology similar to that observed in apoptotic neurons . Section title: Discussion Educational score: 4.536986827850342 Domain: biomedical Document type: Study Language: en We report that in sympathetic neurons undergoing apoptosis triggered by NGF deprivation, mitochondria are reduced in size, display a hyperdense matrix, and are depleted of cytochrome c. A similar morphology of mitochondria lacking cytochrome c was observed in neurons protected from NGF deprivation by the caspase peptide inhibitor BAF. Interestingly, upon re-exposure to NGF, the mitochondria from BAF-rescued neurons recovered both a normal size and cytochrome c content. Altogether, these data do not support the hypothesis according to which cytochrome c release is the result of opening of the PTP and subsequent mitochondrial swelling and rupture of the outer mitochondrial membrane since neither of these two features were observed in apoptotic neurons. Instead, our data agree with previous morphological studies of various cell types undergoing apoptosis showing preservation of mitochondrial membrane ultrastructure and a reduction in total mitochondrial volume . More recently, ultracondensation of mitochondria has also been observed in apoptotic nodal myocytes , in lymphoblastic leukemic cells undergoing tumor necrosis factor-induced apoptosis , in a colon carcinoma cell line treated with herbimycin A and in etoposide-treated THP.1 cells . In some of these studies, a condensed conformation of mitochondria associated with hyperdensity of the matrix, as described here, has been postulated to be the result of water and ion loss from the matrix and to correspond to low-energy states of mitochondria . Section title: Discussion Educational score: 4.331386089324951 Domain: biomedical Document type: Study Language: en Bax, a pro-apoptotic member of the Bcl-2 family essential for neuronal apoptosis , can form ion channels in synthetic lipid membranes and therefore could be a likely candidate responsible for these mitochondrial changes during apoptosis. In support of this hypothesis, addition of Bax directly to isolated mitochondria triggers the release of cytochrome c by a mechanism that may involve pore formation. We now show that this effect is accompanied by a reduction in mitochondrial volume. However, the mechanisms of action of Bax are still unclear. It has been recently reported that Bax can interact with the adenine nucleotide translocator (ANT), a component of the mitochondrial PTP . Moreover, in yeast, the ANT appears to be required for the Bax killing function . However, the importance of the ANT in apoptosis of mammalian cells has not yet been demonstrated. Section title: Discussion Educational score: 4.141657829284668 Domain: biomedical Document type: Study Language: en One of the most striking observations reported here is the ability of mitochondria from BAF-rescued neurons to recover a normal size and cytochrome c content after re-exposure of neurons to NGF. The transition from small to large mitochondria induced by NGF deserves particular attention. This may be the result of mitochondrial fusion, an hypothesis that we are currently testing. We cannot exclude the possibility that proliferation of mitochondria may also take part in the complete recovery of neurons associated with the ability of neurons to grow in size, to extend neurites and to survive over long periods after re-exposure to NGF. Section title: Discussion Educational score: 4.589282989501953 Domain: biomedical Document type: Study Language: en Recovery of function after protection by caspase inhibitors does not apply to all cell types. Indeed, it has previously been reported that inhibition of caspases in diverse types of apoptosis is incompatible with long-term survival, suggesting that in those cells caspases are activated after the cells become committed to apoptosis . Moreover, it has been shown that overexpression of Bax in Jurkat cells leads to mitochondrial dysfunction and caspase-independent apoptosis . In the case of sympathetic neurons undergoing apoptosis induced by NGF deprivation, the situation is different as caspase activation appears to represent the point at which cells become committed to die . Consistent with this, caspase inhibitors can inhibit apoptosis induced by overexpression of Bax or Bak in these neurons. The difference between sympathetic neurons and other cell types may reside in intrinsic specificities of their apoptotic pathway, in specific properties of their mitochondria or could be related, at least in vitro, to their ability to produce ATP through glycolysis rather than through oxidative phosphorylation. The ability of mitochondria to recover fully their function when homeostatic conditions are restored may be specific for mitochondria from neurons. This could explain why in both caspase 3– and caspase 9–deficient mice, only neurons are protected from apoptosis during development . | Study | biomedical | en | 0.999997 |
10085289 | Section title: Antibodies Educational score: 3.917039632797241 Domain: biomedical Document type: Study Language: en Rabbit pAbs were raised against amino acids 1–21 of human Bax , amino acids 11–30 of human Bax (Bax N-20 sc-493; Santa Cruz Biotechnology ), amino acids 43–61 of murine Bax . Note that the antibodies raised against human Bax also recognize murine Bax. A mouse mAb raised against amino acids 3–16 of human Bax was also used for in vitro binding assays. Rabbit pAb raised against amino acids 4–21 of human Bcl-2 was from Santa Cruz Biotechnology (Bcl-2 N-19 sc-492). Mouse mAb raised against amino acids 41–54 of Bcl-2 was obtained from Genosys (OM-11-925A). Rabbit pAb against amino acids 18–233 of human Bcl-x S/L was from Transduction Laboratories . Rabbit pAb against the NH 2 terminus of human Bcl-x was from Santa Cruz Biotechnology (Bcl-x S/L S-18 sc-634). Mouse mAb against mitochondrial heat shock protein 70 (mt-hsp-70) was from Affinity Bioreagents , Inc. (MA3-028). Anti–human Bak mAb raised against amino acids 1–52 was obtained from Calbiochem (AM03). Mouse mAb against bovine cytochrome oxidase subunit IV (COX-IV) which reacts specifically with the rat and human homologue of the protein was from Molecular Probes . The mouse monoclonal anti–cytochrome c antibody that recognizes the native form of rat, mouse, and human cytochrome c was from PharMingen and the rabbit polyclonal anti–cytochrome c antibody was generated against bovine cytochrome c. A rabbit pAb was generated against recombinant full-length Bid. Section title: Cell Cultures Educational score: 4.159863471984863 Domain: biomedical Document type: Study Language: en Primary cultures of cerebellar granule cells (CGC) were prepared from 8-d-old rat pups according to Villalba et al. with slight modifications. In brief, freshly dissected cerebella were incubated with 0.25 mg/ml trypsin for 15 min at 37°C and trypsin inhibitor (0.5 mg/ml) was added to stop the reaction. Then, digested cerebella were mechanically dissociated with a flame-narrowed Pasteur pipette in HBSS in the presence of DNase I (0.1 mg/ml) and trypsin inhibitor. Cells were seeded at a density of 0.25 × 10 6 cells/cm 2 in Falcon dishes previously coated with poly- d -lysine hydrobromide (10 μg/ml) in basal medium Eagle (BME; GIBCO BRL ) supplemented with 10% FCS, 20 mM KCl, 50 IU penicillin, 50 μg/ml streptomycin, 10 mM Hepes, 2 mM l -glutamine, and 1 mM sodium pyruvate. After 24 h, 10 μM cytosine β- d -arabinofuranoside was added to the culture medium. Cultures were incubated at 37°C in a humidified atmosphere of 5% CO 2 . Neurons were used after 7 d in culture. Section title: Cell Cultures Educational score: 4.1031951904296875 Domain: biomedical Document type: Study Language: en HeLa cells and the stable HeLa cell line that constitutively overexpresses Bcl-2 (HeLa-Bcl-2) were cultured in a 1:1 mixture of basal Iscove's medium and Ham's F12 medium (Seromed) supplemented with 10% FCS and 2 mM l -glutamine. HEK cells were cultured in DME-F12 medium ( GIBCO BRL ) supplemented with 10% FCS and 2 mM l -glutamine. LoVo (colon, adenocarcinoma, human), DU145 (prostate, carcinoma, human), and LS180 (colon, adenocarcinoma, human) were from the American Type Culture Collection and cultured according to the instructions provided. Section title: Immunocytochemistry Educational score: 4.203446865081787 Domain: biomedical Document type: Study Language: en For immunocytochemistry analysis, cells were seeded onto glass coverslips. Apoptosis was induced in primary cultures of CGC by washing twice and placing the neurons in a culture medium containing a normal concentration of KCl (5 mM) and lacking serum (basal medium Eagle– Glutamax-I supplemented with 50 IU penicillin, 50 μg/ml streptomycin, 10 mM Hepes, 2 mM l -glutamine, and 1 mM sodium pyruvate). Apoptosis in HeLa cells was induced by addition of 1 μM staurosporine in the presence or absence of 100 μM z-VAD-fmk. Stock solutions of the drugs (×1,000) were made in DMSO. Control cultures received solvent alone. Cells were fixed with 4% paraformaldehyde in PBS for 15 min and permeabilized with 0.2% Triton X-100 in PBS at room temperature. After washing, the cells were incubated for 2 h with anti-Bax pAbs (all diluted 1:100 in PBS + 5% normal goat serum) and anti–cytochrome c mAb (dilution 1:15 in PBS + 5% normal goat serum) or anti–mt-hsp-70 (dilution 1:500 in PBS + 5% normal goat serum), washed twice in PBS, and developed with fluorescein and Texas red–labeled goat anti–rabbit and goat anti–mouse antibodies, respectively. In the last wash, 1 μg/ml of Hoechst 33258 was added to the cells. Coverslips were placed on a glass slide in Vectashield mounting medium and observed by conventional or confocal fluorescence microscopy. Section title: Subcellular Fractionation Educational score: 4.208943843841553 Domain: biomedical Document type: Study Language: en At different times after induction of apoptosis, HeLa cells and CGC were harvested in isotonic mitochondrial buffer (MB: 210 mM mannitol, 70 mM sucrose, 1 mM EDTA, and 10 mM Hepes, pH 7.5) supplemented with protease inhibitor cocktail Complete ( Boehringer Mannheim ), and homogenized for 30–40 strokes with a Dounce homogenizer. Samples were transferred to Eppendorf centrifuge tubes and centrifuged at 500 g for 5 min at 4°C to eliminate nuclei and unbroken cells. The resulting supernatant was centrifuged at 10,000 g for 30 min at 4°C to obtain the heavy membrane pellet (HM) enriched for mitochondria. This supernatant was further centrifuged at 100,000 g for 1 h at 4°C to yield the light membrane pellet (not analyzed) and the final soluble fraction (S). The HM material was resuspended in MB supplemented with 1% Triton X-100. Soluble and HM fractions (30 and 15 μg, respectively) were separated by SDS-PAGE (4–20% Tris-Glycine gels; NOVEX) and transferred to a nitrocellulose membrane (NOVEX). After blocking nonspecific sites for 1 h at room temperature with 5% nonfat milk in PBS supplemented with 0.2% Tween 20, the membrane was incubated overnight at 4°C with the antibody raised against amino acids 1–21 of human Bax (Upstate Biotechnology) diluted 1:500 in PBS supplemented with 2.5% nonfat milk. To confirm equal loading and transfer, the membrane was subsequently stripped and reprobed for COX-IV. The immunoreactive proteins were visualized using horseradish peroxidase–linked goat anti–mouse antibody (Jackson ImmunoResearch Laboratories Inc.) and enhanced chemiluminescence ( Amersham Life Sciences ). Section title: cDNA Cloning and Site-directed Mutagenesis of Bid and Bcl-x L Educational score: 4.181785583496094 Domain: biomedical Document type: Study Language: en Single-stranded random-primed cDNA prepared from mouse thymus total RNA was used as a template for PCR amplification of Bid cDNA using sense primer 5′ ATGGATCCCCATGGACTCTGAGGT 3′ and antisense primer 5′ CACCTCGAGCCTCAGTCCATCTCGTTTC 3′. The PCR product was digested with BamH1/Xho1 and subcloned in the BamH1/Xho1 site of pBluescript II KS (Stratagene). The sequence of Bid was confirmed by sequencing. This construct was used as template to prepare NH 2 -terminal His-tagged Bid using as sense primer 5′ ATACCATGGCTCACCACCACCACCACCACATGGACTCTGAGGTCAGC 3′ and the same antisense primer as above. The PCR product was digested with Nco1/Xho1 and subcloned in the Nco1/Xho1 site of pET23d (Novagen). Section title: cDNA Cloning and Site-directed Mutagenesis of Bid and Bcl-x L Educational score: 4.194637298583984 Domain: biomedical Document type: Study Language: en BidmIII-1 and BidmIII-3 were generated in three steps. First, the 5′ portion of Bid was amplified using the 6-His sense primer, and mutant antisense primers 5′ CTGGATGTTGTGGGCGGCCTCATCG 3′ for BidmIII-1 and 5′ GTCCATCTCATCGGCTATTTGGGCGA 3′ for BidmIII-3. The 3′ portion of Bid was amplified using the sense 5′ CGATGAGGCCGCCCACAACATCCAG 3′ for BidmIII-1, 5′ TCGCCCAAATAGCCGATGAGATGGA 3′ for BidmIII-3, together with the antisense Xho1 containing primer as above. Second, the PCR products of each corresponding mutant were mixed and reamplified by PCR with the sense His primer and the antisense Xho1 primer. Third, the PCR products were digested with Nco1/Xho1 and subcloned in the Nco1/Xho1 site of pET23d. Mutations were confirmed by DNA sequencing. Section title: cDNA Cloning and Site-directed Mutagenesis of Bid and Bcl-x L Educational score: 3.9011542797088623 Domain: biomedical Document type: Study Language: en The constructs pET23dHisBid, pET23dHisBidmIII-1, and pET23dHisBidmIII-3 were transformed in BL21 (DE3) Escherichia coli and protein expression was induced by 100 μM of isopropyl β- d -thiogalactopyranoside (IPTG). Section title: cDNA Cloning and Site-directed Mutagenesis of Bid and Bcl-x L Educational score: 4.200826644897461 Domain: biomedical Document type: Study Language: en Bcl-x L m: G 138 → A was generated by PCR site-directed mutagenesis on the template pET23dHisBcl-x L , using Pwo DNA polymerase, sense primer 5′ GGGGTAAACTGGGCCCGCATTGTGGCC 3′, and antisense primer 5′ GGCCACAATGCGGGCCCAGTTTACCCC 3′. The PCR product was then digested with Dpn, purified using QIAquick (QIAquick PCR purification kit; Qiagen), and 2.5 μl purified PCR DNA transformed in competent E . coli XL1-Blue. Mutation was confirmed by DNA sequencing. Section title: Production of Recombinant Proteins Educational score: 4.09233283996582 Domain: biomedical Document type: Study Language: en His-tagged Bid and Bid mutants were expressed in the pET23d vector in E . coli . The recombinant proteins were recovered in the soluble bacteria fraction and purified by chromatography on Ni-NTA-Agarose followed by Q-Sepharose. The purified proteins which were >95% pure were stored in 25 mM Tris-HCl, 0.1 mM DTT, 30% glycerol, pH 7.5, at −80°C. Section title: Production of Recombinant Proteins Educational score: 4.130738258361816 Domain: biomedical Document type: Study Language: en His-tagged human Bcl-x L and His-tagged human mutant Bcl-x L (Bcl-x L m: G 138 → A) both lacking 24 amino acids at the COOH terminus were expressed in the pET23d vector in E . coli . The recombinant proteins were recovered in the soluble bacteria fraction and purified by chromatography on Ni-NTA-Agarose followed by Q-Sepharose. The purified proteins which were >95% pure were stored in 25 mM Tris-HCl, 0.2 mM DTT, 30% glycerol, pH 7.5, at −80°C. Section title: Production of Recombinant Proteins Educational score: 3.6559979915618896 Domain: biomedical Document type: Study Language: en Human Bax-α lacking 20 amino acids at the COOH terminus and human Bcl-2 lacking 34 amino acids at the COOH terminus were produced as described previously . Section title: In Vitro Binding Assay Educational score: 4.162564277648926 Domain: biomedical Document type: Study Language: en To characterize the interactions of Bid and the BH3 mutants BidmIII-1 and BidmIII-3 with Bax, Bcl-2, and Bcl-x L , recombinant proteins were mixed in 50 μl PBS (100 nM each, or 1 μM Bcl-2 or Bcl-x L with 100 nM Bid or BidmIII-1). After 30 min of incubation on ice, 450 μl of NP-40 buffer (142.5 mM KCl, 5 mM MgCl 2 , 1 mM EDTA, 0.25% NP-40, and 10 mM Hepes, pH 7.5) supplemented with protease inhibitor cocktail Complete ( Boehringer Mannheim ) was added to each binding mixture. Samples were rotated overnight at 4°C in the presence of 4 μg antibody. Then, 50 μl protein A–Agarose (for pAbs) or protein G–Agarose (for mAbs) (Boeh– ringer Mannheim) was added to each sample, rotated for 3 h at 4°C, recovered by centrifugation at 10,000 g for 1 min, and washed three times in 1 ml of NP-40 buffer. Materials bound to the beads were eluted by the addition of 50 μl of 3× SDS-PAGE sample buffer, thorough vortexing, and boiling for 5 min. Immunoprecipitated proteins were analyzed by SDS-PAGE and Western blotting, as described above. Section title: Isolation of Mitochondria Educational score: 4.207898139953613 Domain: biomedical Document type: Study Language: en Mitochondria were isolated from HeLa, HEK, HeLa-Bcl-2, LoVo, LS180, and DU145 cell lines by sucrose density gradient centrifugation. In brief, cells were harvested with PBS containing 1 mM EDTA, centrifuged at 750 g for 10 min, washed, and resuspended in isotonic MB supplemented with protease inhibitors. Cells were broken by five passages through a 25G1 0.5 × 25 needle fitted on a 2-ml syringe and the suspension was centrifuged at 2,000 g in an Eppendorf centrifuge at 4°C. This procedure was repeated until almost all of the cells were broken. Supernatants from each step were pooled before centrifugation at 13,000 g at 4°C for 10 min. The pellet was resuspended in 1 ml of MB and layered on top of a discontinuous sucrose gradient consisting of 20 ml of 1.2 M sucrose, 10 mM Hepes, pH 7.5, 1 mM EDTA, 0.1% BSA on top of 17 ml of 1.6 M sucrose, 10 mM Hepes, pH 7.5, 1 mM EDTA, 0.1% BSA. The samples were centrifuged at 27,000 rpm for 2 h at 4°C in a Beckman SW28 rotor. Mitochondria were recovered at the 1.6–1.2 M sucrose interface, washed, and resuspended in MB. Protein concentration was estimated by the method of Bradford with BSA as the standard. Electron microscopy studies revealed that most of the organelles recovered by this procedure are mitochondria with negligible contamination by endoplasmic reticulum membranes (not shown). Section title: Analysis of Mitochondrial Bax Immunostaining by Flow Cytometry Educational score: 4.155661582946777 Domain: biomedical Document type: Study Language: en Mitochondria (50–70 μg of proteins) were incubated with recombinant Bid proteins for 15 min at 30°C in 200 μl of KCl buffer (125 mM KCl, 4 mM MgCl 2 , 5 mM NaHPO 4 , 5 mM succinate, 0.5 mM EGTA, 15 mM Hepes-KOH, pH 7.4, 5 μM rotenone). Then mitochondria were fixed with PBS 4% paraformaldehyde, washed twice in PBS, incubated for 1 h at room temperature with the anti-Bax (amino acids 1–21) antibody or with the anti–Bcl-2 (amino acids 4–21) antibody both diluted 1:100 in PBS + 5% normal goat serum, washed twice in PBS, and incubated for 30 min at room temperature with fluorescein-labeled goat anti–rabbit IgG antibody (dilution 1:100 in PBS + 5% normal goat serum). After the final wash, fluorescence was analyzed by flow cytometry using a Facscalibur ® flow cytometer ( Becton Dickinson ). Only mitochondria exhibiting a light scatter profile typical of intact mitochondria were analyzed and debris or nonmitochondrial membranes were excluded from the study by electronic gating. Section title: In Vitro Assay for Cytochrome c Release Educational score: 4.168375015258789 Domain: biomedical Document type: Study Language: en Mitochondria (30 μg) were incubated in the presence or absence of various recombinant proteins in 200 μl of KCl buffer for 15 min at 30°C and then centrifuged for 5 min at 13,000 g at 4°C. Mitochondrial pellets corresponding to 1.5-μg proteins and the corresponding volume of the supernatant fractions were separated by SDS-PAGE using 4–20% Tris-Gly gels (NOVEX) and their respective contents of cytochrome c were estimated by Western blotting using polyclonal anti–cytochrome c antibody (dilution 1:2,500). Equal loading of the mitochondrial pellet was verified using either an antibody against COX-IV or an antibody against mt-hsp-70. Antigen–antibody complexes were detected using a horseradish peroxidase– conjugated goat anti–rabbit IgG and enhanced chemiluminescence detection reagents. Section title: Bax Immunostaining in Cells Undergoing Apoptosis Educational score: 4.106806755065918 Domain: biomedical Document type: Study Language: en We have investigated the subcellular distribution of Bax during apoptosis in HeLa cells treated with 1 μM staurosporine. Immunocytochemical studies with a pAb raised to amino acids 1–21 of Bax are shown in Fig. 1 . Untreated HeLa cells displayed staining of the nucleolus but no cytosolic immunoreactivity . The nucleolar staining does not appear to involve Bax since the same pattern was observed in two Bax-deficient tumor cell lines, LoVo and LS180 (not shown). Section title: Bax Immunostaining in Cells Undergoing Apoptosis Educational score: 4.170221328735352 Domain: biomedical Document type: Study Language: en In contrast to untreated cells, in cultures exposed to staurosporine for 4 h, ∼30% of the cells displayed a punctate cytosolic pattern of Bax immunostaining suggestive of an association with the mitochondria . No mitochondrial staining was observed with this Bax antibody in staurosporine-treated LoVo and LS180 cell lines, confirming the specificity of the Bax immunostaining in these experiments (not shown). Similar results were also obtained with another pAb raised against amino acids 11–30 of Bax . The mitochondrial localization of Bax in these cells was confirmed by confocal microscopy and double immunostaining with an antibody to mt-hsp-70 . This protein, closely related to but distinct from the cytosolic hsp-70, is a 75-kD protein which resides constitutively in the mitochondrial matrix . Section title: Bax Immunostaining in Cells Undergoing Apoptosis Educational score: 3.528041124343872 Domain: biomedical Document type: Study Language: en It is interesting to note that the mitochondria revealed by Bax immunostaining displayed a small round shape and had a tendency to aggregate , a finding previously observed in cells overexpressing Bax or Bid . Section title: Bax Immunostaining in Cells Undergoing Apoptosis Educational score: 4.263966083526611 Domain: biomedical Document type: Study Language: en During apoptosis induced by diverse stimuli, cytochrome c is released from mitochondria into the cytosol , and this is thought to be a crucial event in the apoptotic pathway. Recent evidence suggests that Bax may be involved in the pathway leading to cytochrome c release . Therefore, the appearance of Bax in mitochondria after staurosporine-induced apoptosis prompted us to study the cytochrome c distribution in these cells. A double immunostaining showed that while normal HeLa cells displayed a reticulate staining for cytochrome c , the Bax positive cells almost invariably (>95%) displayed a diffuse cytosolic pattern of cytochrome c staining . In all cases, Bax positive cells displayed a fragmented nucleus as assessed by Hoechst 33258 staining . Therefore, detection of mitochondrial Bax is closely correlated with both depletion of cytochrome c from mitochondria and cell death as revealed by apoptotic nuclei. Section title: Bax Immunostaining in Cells Undergoing Apoptosis Educational score: 4.204664707183838 Domain: biomedical Document type: Study Language: en We next decided to test whether blocking cell death, either by Bcl-2 or by using inhibitors of caspase activity, could influence the appearance of Bax staining during apoptosis. In HeLa cells constitutively overexpressing Bcl-2 (HeLa-Bcl-2) which we have shown previously to be resistant to staurosporine-induced apoptosis , we found that <0.1% of the cells became Bax positive after a 4-h treatment with 1 μM staurosporine and no release of cytochrome c was detected. The results of immunofluorescence in the Bcl-2 overexpressing strain were essentially indistinguishable from those obtained using untreated cells . In contrast, the presence of 100 μM of the caspase peptide inhibitor z-VAD-fmk did not prevent the appearance of Bax immunoreactivity and cytochrome c release from mitochondria, despite total blockade of nuclear fragmentation . This is consistent with recent observations that cytochrome c release is an upstream event in the pathway leading to caspase activation . Section title: Bax Immunostaining in Cells Undergoing Apoptosis Educational score: 4.185604095458984 Domain: biomedical Document type: Study Language: en We next tested whether the appearance of a mitochondrial Bax staining during apoptosis also occurs in other cell types. We used primary cultures of CGC undergoing apoptosis after KCl and serum deprivation . We show that, as with HeLa cells exposed to staurosporine, these neurons displayed a mitochondrial staining with the antibody raised against amino acids 1–21 of Bax, cytochrome c–depleted mitochondria, and a condensed nucleus . Similar results were obtained with another pAb raised against amino acids 43–61 of murine Bax. Interestingly, in CGC, unlike HeLa cells, only the neurons with a marginalized chromatin, representing an early stage in apoptosis, displayed a Bax staining, whereas those in the terminal phase of apoptosis, with a highly condensed nucleus and a shrunken cell body, appeared Bax negative . This suggests that in these neurons the Bax immunoreactivity is a transient event in the apoptotic pathway. Section title: Bax Mitochondrial Levels Do Not Increase during Apoptosis Educational score: 4.382511138916016 Domain: biomedical Document type: Study Language: en Two hypotheses could explain the appearance of Bax mitochondrial staining during apoptosis. Either Bax could translocate to mitochondria as recently reported or alternatively, Bax may be present continuously in mitochondria but may undergo a conformational change rendering it immunoreactive towards the NH 2 -terminal antibodies. To distinguish between these two possibilities, we compared Bax levels by Western blot analysis in mitochondria from control and apoptotic HeLa cells and CGC . These studies showed that in control cultures of both cell types Bax is distributed in both the soluble fraction (S) and HM fraction enriched in mitochondria and that the level of mitochondrial Bax does not increase during apoptosis . Therefore, Bax translocation to the mitochondria is unlikely to account for the appearance of Bax immunoreactivity. It is interesting to note that the level of Bax detected by the antibody raised against amino acids 1–21 of Bax decreases in the soluble fraction during apoptosis as noted earlier . However, the concomitant appearance of smaller immunoreactive fragments, at least as seen in CGC, suggests that this may be the result of Bax proteolysis. Therefore, the results in Fig. 3 suggest that the punctate pattern of Bax immunostaining observed in apoptotic cells is not due to an increased level of mitochondrial Bax but rather to a structural change of the protein which leads to exposure of the NH 2 -terminal domain. Section title: Translocation of Bid to Mitochondria during Apoptosis Educational score: 4.349373817443848 Domain: biomedical Document type: Study Language: en Resolution of the three-dimensional structure of Bcl-x L in its non–membrane-integrated conformation revealed the existence of a hydrophobic cleft on the surface of the protein formed by the BH1, BH2, and BH3 domains which provides a likely binding site for BH3 containing peptides . This suggested to us that the binding of a BH3 domain protein to Bax could induce the structural changes which we postulate to explain the appearance of Bax immunostaining during apoptosis. A likely candidate for such a protein was Bid, because this protein had been shown previously to strongly interact with Bax . As Bid is expected to be mainly cytosolic , we reasoned that in order to induce a change in the Bax structure, Bid might translocate to mitochondria during apoptosis. This hypothesis was tested using HeLa cells undergoing apoptosis. Mitochondria from these cells were purified on a sucrose gradient and the respective levels of Bid and Bax were determined by Western blotting at 5 and 12 h after addition of 1 μM staurosporine . These results first of all confirmed our earlier observation that the levels of Bax in mitochondria do not change during apoptosis and in addition showed a significant increase in the level of Bid. The increased level of Bid in these mitochondria was confirmed by flow cytometry . Thus, the observed translocation of Bid to mitochondria during apoptosis is consistent with the idea that Bid could be responsible for the change in Bax conformation, and this hypothesis was tested using isolated mitochondria from HeLa cells. Section title: Bid Triggers Bax Conformational Change Educational score: 4.156364440917969 Domain: biomedical Document type: Study Language: en In a first series of experiments, mitochondria from HeLa cells were isolated on a sucrose gradient and incubated for 15 min with or without 1 μM Bid. Bax immunostaining was then assessed by flow cytometry . When an antibody to the NH 2 -terminal sequence (amino acids 1–21) of Bax was used , the mean fluorescence intensity of mitochondria from HeLa cells exposed to 1 μM Bid was about fivefold higher than that of untreated mitochondria . A similar effect was observed with mitochondria from HEK cells . Dose–response analysis using Bid concentrations in the range of 1 nM to 10 μM indicated that half the maximal increase in Bax-immunofluorescence intensity (EC 50 ) in mitochondria from HeLa cells was obtained at a concentration of 5 nM Bid, with saturation reached at 1 μM. In contrast, using mitochondria from the three Bax-deficient tumor cell lines LoVo, DU145, and LS180, no increase in fluorescence was detected after addition of 8 μM Bid . Section title: Bid Triggers Bax Conformational Change Educational score: 4.056345462799072 Domain: biomedical Document type: Study Language: en Using mitochondria isolated from HeLa-Bcl-2 cells and an antibody raised against the NH 2 terminus of Bcl-2 (amino acids 4–21), we found that, in contrast to Bax, the Bcl-2 fluorescence intensity did not change after incubation with 8 μM Bid . Section title: Bid Triggers Bax Conformational Change Educational score: 4.279290199279785 Domain: biomedical Document type: Study Language: en To investigate the role of the BH3 domain of Bid in the stimulation of Bax immunoreactivity, we produced two different recombinant BH3 mutants of Bid, BidmIII-1 (M 97 ,D 98 → A,A) and BidmIII-3 (G 94 → A) described previously by Wang et al. . First, we tested these mutants for binding to Bax, Bcl-2, and Bcl-x L in vitro . As shown by Wang et al. , the mutant BidmIII-3 had a lowered affinity for Bax but still bound Bcl-2 or Bcl-x L , whereas BidmIII-1 showed a lowered affinity for Bcl-2 or Bcl-x L but was still able to interact strongly with Bax . When tested for their ability to stimulate Bax immunoreactivity, BidmIII-3 was 10-fold less efficient (EC 50 < 50 nM) than wild-type Bid, whereas BidmIII-1 was as effective as wild-type Bid . Importantly, the effect of Bid on Bax immunoreactivity was dramatically reduced by at least two orders of magnitude when 1 μM Bcl-2 or Bcl-x L was added to the mitochondrial preparation . Section title: Bax Conformational Change Is Accompanied by a Release of Cytochrome c from Mitochondria Educational score: 4.224616050720215 Domain: biomedical Document type: Study Language: en Overexpression of Bax in cells or addition of recombinant Bax directly to mitochondria results in the release of cytochrome c . This Bax-induced cytochrome c release from mitochondria could be related to a change in conformation of the protein triggered by Bid. To test this hypothesis, mitochondria were incubated with Bid (1–100 nM) for 15 min and levels of cytochrome c were assessed by Western blotting in both mitochondrial pellets and supernatants . As with Bax , we found that Bid induced the release of cytochrome c in a concentration-dependent manner. A maximal effect was observed at 10 nM Bid which corresponds to >90% loss of mitochondrial cytochrome c. BidmIII-1 was as active as wild-type Bid, whereas BidmIII-3 was ∼10-fold less active . Section title: Bax Conformational Change Is Accompanied by a Release of Cytochrome c from Mitochondria Educational score: 4.134212970733643 Domain: biomedical Document type: Study Language: en Altogether, these results demonstrate a tight correlation between the effect of Bid on mitochondrial cytochrome c release and on Bax NH 2 -terminal immunoreactivity . Section title: Bcl-x L and Bcl-2 Inhibit Bid-induced Cytochrome c Release Educational score: 4.169697284698486 Domain: biomedical Document type: Study Language: en To test whether Bcl-x L and Bcl-2 were able to inhibit Bid-induced cytochrome c release, mitochondria were incubated with 100 nM Bid together with 1 μM recombinant Bcl-x L or Bcl-2 . Consistent with the reduced Bax immunofluorescence reported in Fig. 7 B, we found that both proteins were able to inhibit Bid-induced cytochrome c release. Furthermore, Bcl-x L and Bcl-2 were also able to abrogate the effect of BidmIII-1 . However, even under these conditions in which they are in excess (1 μM), Bcl-x L and Bcl-2 do not bind BidmIII-1 (100 nM) sufficiently to explain their inhibitory effect on cytochrome c release . Therefore, these data indicate that the inhibition of cytochrome c release from mitochondria by Bcl-2 and Bcl-x L does not require interaction with Bid. Section title: Bcl-x L and Bcl-2 Inhibit Bid-induced Cytochrome c Release Educational score: 4.117244720458984 Domain: biomedical Document type: Study Language: en The inhibitory effect of Bcl-2 on Bid-induced cytochrome c release was further confirmed using mitochondria isolated from Bcl-2–overexpressing HeLa cells. Fig. 9 A shows that Bid was 10-fold less efficient in triggering the release of cytochrome c from HeLa-Bcl-2 mitochondria compared with mitochondria from control HeLa cells. Section title: Bcl-x L and Bcl-2 Inhibit Bid-induced Cytochrome c Release Educational score: 4.237666130065918 Domain: biomedical Document type: Study Language: en To test whether the inhibition of Bid-induced cytochrome c release by Bcl-x L required its binding to Bax, we used a Bcl-x L mutant in which Gly 138 was replaced by an alanine (Bcl-x L m: G 138 → A). This mutant does not bind to Bax and is unable to rescue cells from apoptosis . In contrast to wild-type Bcl-x L , we found that this Bcl-x L mutant was unable to inhibit the Bid effect . However, in vitro binding experiments showed that Bcl-x L m was still able to bind Bid . Altogether, these results indicate that Bcl-x L prevents Bid-induced cytochrome c release through its interaction with Bax and not through its ability to bind Bid. Section title: Bid-induced Release of Cytochrome c from Mitochondria from Bax-deficient Cells Educational score: 4.165963172912598 Domain: biomedical Document type: Study Language: en To demonstrate that Bid-induced cytochrome c release was mediated by Bax, we tested the ability of Bid to trigger cytochrome c release from mitochondria isolated from the Bax-deficient tumor cell line LS180. The results of Western blot analysis showed that, as expected, Bax was not detectable in this cell line, whereas the levels of Bcl-x L and Bak found associated with mitochondria were similar to levels in mitochondria from HeLa cells (not shown). The results in Fig. 9 A show that Bid was at least 10-fold less efficient in triggering cytochrome c release from mitochondria from LS180 cells, compared with mitochondria from HeLa cells. In contrast, if mitochondria from LS180 cells were incubated with both Bid and Bax together, the mitochondria were completely depleted of cytochrome c within 15 min , confirming that the major effect of Bid in these mitochondria was indeed mediated by Bax. Section title: Bid-induced Release of Cytochrome c from Mitochondria from Bax-deficient Cells Educational score: 4.190707206726074 Domain: biomedical Document type: Study Language: en However, it should also be noted that even in LS180 mitochondria without exogenous Bax, Bid was nevertheless able to induce release of cytochrome c at higher concentrations , suggesting that there may be other proapoptotic targets for Bid in the mitochondrial membrane. Since we showed that Bak is present at about the same level in mitochondria from HeLa cells and LS180 cells, we therefore decided to test whether Bid was also able to induce a conformational change in Bak. Results in Fig. 9 C show that exposure of mitochondria from both HeLa cells and LS180 cells to 1 μM Bid, followed by immunostaining with an NH 2 -terminal antibody to Bak (amino acids 1–52), resulted in a threefold increase in Bak immunoreactivity in both cell types as judged by subsequent FACS ® analysis. Thus, a component of the Bid-induced cytochrome c release from these mitochondria is also likely to be mediated via Bak. Section title: Discussion Educational score: 4.29525899887085 Domain: biomedical Document type: Study Language: en Recent reports have shown that Bid is responsible for the release of cytochrome c from mitochondria during Fas- and TNF-mediated apoptosis . However, the mechanisms by which Bid triggers this event are unclear. We now report, in a different system, that Bax mediates Bid-induced cytochrome c release from mitochondria. During staurosporine-induced apoptosis of HeLa cells, Bid translocates from the cytosol to mitochondria, an event associated with a change in conformation of Bax and a release of cytochrome c. Using isolated mitochondria, we demonstrate that direct binding of Bid to Bax is a prerequisite for Bax structural change and cytochrome c release. Section title: Bid but Not Bax Translocates from the Cytosol to Mitochondria during Staurosporine-induced Apoptosis Educational score: 4.203211307525635 Domain: biomedical Document type: Study Language: en It has been reported recently that, during Fas- and TNF-mediated apoptosis, Bid becomes cleaved by caspase 8 and its COOH-terminal domain translocates to mitochondria . Using staurosporine-treated HeLa cells, we found that full-length Bid can also translocate to mitochondria during apoptosis. However, we have been unable to detect the cleaved form of Bid attached to mitochondria. Our findings suggest that caspase-induced Bid cleavage is not an essential requirement for its movement to mitochondria. Translocation of full-length Bid may thus occur in types of apoptosis in which activation of caspases is not a primary event in the apoptotic pathway. Section title: Bid but Not Bax Translocates from the Cytosol to Mitochondria during Staurosporine-induced Apoptosis Educational score: 4.166064262390137 Domain: biomedical Document type: Study Language: en In contrast to Bid, we did not detect any significant translocation of Bax to mitochondria, although we did observe a decrease in Bax cytosolic levels in both HeLa cells and CGC undergoing apoptosis. At least in the case of CGC, the appearance of several smaller Bax immunoreactive bands by Western blot suggests that this decrease in the level of Bax in the cytosol may be the result of proteolytic degradation. The characterization of these lower molecular weight species and the involvement of caspases or, as reported recently, calpain in Bax proteolysis is currently ongoing. Section title: Bid but Not Bax Translocates from the Cytosol to Mitochondria during Staurosporine-induced Apoptosis Educational score: 4.386880874633789 Domain: biomedical Document type: Study Language: en The absence of Bax translocation to mitochondria during apoptosis has also been observed by Goping et al. in some of their experiments with FL5.12 cells undergoing apoptosis after IL-3 deprivation. In contrast, others have reported a translocation of Bax to mitochondria in IL-3–deprived FL5.12 cells in staurosporine-treated HL-60 and in GFP-Bax–overexpressing Cos-7 kidney epithelial cells . These apparent conflicting results may be due to the type of cells or cell death stimuli used in these various studies. In all cultured cells that we examined so far, including HeLa, Cos-7, and HEK cell lines as well as cultured primary neurons from superior cervical ganglia and cerebellar granule neurons (data presented here; Nichols, A., and J.-C. Martinou, unpublished results), we have observed high levels of Bax associated with mitochondria in addition to cytosolic Bax. In clear contrast, in cells from freshly dissected tissues but not exposed to culture conditions, including thymus, spleen, or brain, Bax was practically cytosolic and hardly detectable in mitochondria (Antonsson, B., unpublished data), a finding which confirms previous results from Hsu et al. . Thus, it seems that simply maintaining cells in culture leads to redistribution of a significant part of cytosolic Bax to mitochondria. The important question which remains to be understood is why these cells, despite significant levels of mitochondrial-associated Bax, do not spontaneously undergo apoptosis. Does Bax remain inactive in cultured cells because of possible association with antiapoptotic proteins such as Bcl-x L or Bcl-2? or another protein? and/ or because of an inert configuration? The data presented here provide a possible explanation to this question. Section title: Bax Undergoes a Bid-dependent Conformational Change Educational score: 4.38946008682251 Domain: biomedical Document type: Study Language: en During apoptosis of HeLa cells and cerebellar granule neurons, Bax immunoreactivity was found to increase despite the apparent absence of Bax translocation to mitochondria. Moreover, addition of Bid directly to isolated mitochondria was sufficient to stimulate the appearance of Bax immunoreactivity towards NH 2 -terminal antibodies. Altogether, these results suggest that Bax immunoreactivity increases because the NH 2 -terminal region of the protein undergoes a conformational change making it accessible to antibodies. We presume that this domain, which may include the BH3 domain, must be localized on the external face of the outer mitochondrial membrane since it was accessible to antibodies without the need for membrane permeabilization. Alteration of the structure of Bax was inhibited by Bcl-2 or Bcl-x L , but not by the caspase inhibitor z-VAD-fmk, suggesting that, at least in the models of apoptosis described here, exposure of this domain is not a caspase-dependent event. Section title: Bax Undergoes a Bid-dependent Conformational Change Educational score: 4.548766613006592 Domain: biomedical Document type: Study Language: en Bid-induced exposure of the NH 2 -terminal domain of Bax could reflect either a conformational change in Bax or a displacement of another protein serving to mask critical immunoreactive residues. For example, interaction of Bid with antiapoptotic proteins may be responsible for the dissociation of Bax heterodimers and the unmasking of Bax epitopes. However, BidmIII-3, with reduced affinity for Bax but which retained a high affinity for Bcl-2 and Bcl-x L , was much less active in enhancing Bax immunoreactivity and resulted in a 10-fold lower release of cytochrome c than wild-type Bid. On the other hand, BidmIII-1, with a reduced affinity for Bcl-2 and Bcl-x L but which retained high affinity for Bax, behaved as wild-type Bid. In addition, in ELISA competitive binding studies, Bid was found unable to dissociate Bax/Bcl-2 and Bax/Bcl-x L complexes (not shown), confirming previous data demonstrating that Bid failed to bind Bax/Bcl-2 heterodimers . Altogether, these data suggest that Bid-induced unmasking of the NH 2 -terminal domain of Bax results from a direct binding of Bid to Bax and not from a loss of Bax interaction with Bcl-2 or Bcl-x L . Section title: Bax Undergoes a Bid-dependent Conformational Change Educational score: 4.259332656860352 Domain: biomedical Document type: Study Language: en Our data also suggest that Bcl-x L and Bcl-2 inhibit the effect of Bid by interacting directly with Bax and not by preventing the interaction between Bid and Bax. First, Bcl-x L and Bcl-2 were able to inhibit Bid as well as BidmIII-1, which has a low affinity for Bcl-x L and Bcl-2, indicating that their mechanism of action was independent of their affinity for Bid. Second, a mutant of Bcl-x L (Bcl-x L m: G 138 → A) which has lost its ability to interact with Bax but which is still able to bind Bid was unable to block Bid effect. This finding shows that Bcl-x L must bind Bax to prevent its activation but that its inhibitory effect is independent of its binding to Bid. Section title: Bax Undergoes a Bid-dependent Conformational Change Educational score: 4.22550106048584 Domain: biomedical Document type: Study Language: en On the other hand, it has been reported that heterodimerization of Bcl-x L with Bax was not always essential for its death-repressing action since several mutations that disrupted the ability of Bcl-x L to interact with Bax still retained an antiapoptotic activity . Therefore, our data suggest that these Bcl-x L mutants exerted their activity either upstream of Bax conformational change or downstream of Bax activation and cytochrome c release, as recently reported for Bcl-2 . Section title: Bax Plays a Key Role in the Bid-induced Release of Cytochrome c from Mitochondria Educational score: 4.432570457458496 Domain: biomedical Document type: Study Language: en Addition of Bid to mitochondria is sufficient to trigger cytochrome c release . Here, we extend this observation and provide evidence for a mechanism by which Bax may mediate the Bid effect. Consistent with this, we have shown that Bid was at least 10-fold less efficient in triggering cytochrome c release from mitochondria from LS180 tumor cells which possess no detectable Bax compared with mitochondria from HeLa cells. This difference seems to be due to the deficiency in Bax and not to an intrinsic property of mitochondria from LS180 cells acquired during tumorigenicity, since in the presence of recombinant Bax, mitochondria from these cells respond to Bid in the same way as mitochondria from HeLa cells. Importantly, in these complementation studies, addition of Bid was found to potentiate Bax effect. This potentiation may be explained by a change in conformation of Bax induced by Bid. However, Bax does not seem to be acting alone in this process since a low level of cytochrome c was released when mitochondria from LS180 cells were exposed to high concentrations of Bid. This may be due to another Bax-like protein present on their mitochondria. Bak (which is present in similar amounts in the mitochondria of LS180 and HeLa cells) may represent such an alternative protein and was also found to undergo a conformational change in Bid-treated mitochondria. Section title: Bax Plays a Key Role in the Bid-induced Release of Cytochrome c from Mitochondria Educational score: 4.532475471496582 Domain: biomedical Document type: Study Language: en In conclusion, we have shown that upon binding to Bid, mitochondrially associated Bax undergoes a conformational change, leading to release of cytochrome c in the cytosol. A change in Bax conformation leading to exposure of the NH 2 -terminal domain had been observed already by Hsu and Youle when the protein was in a solution containing nonionic detergents. This change in conformation may expose the BH3 domain of Bax which, as proposed for Bak , may normally be oriented toward the interior of the protein and thus be unavailable for homo- or heterodimerization. As a dimer, Bax may then be able to insert within mitochondrial membranes leading to the observed changes in mitochondrial activity . A recent report indicates that, during apoptosis, the association of Bax with mitochondrial membranes changes from a weak to a strong insertion , a process which is regulated by the NH 2 -terminal domain of Bax. As a working hypothesis, we propose that Bax remains weakly attached to mitochondria in the absence of apoptotic stimulus, and that BH3 only proteins such as Bid may promote apoptosis by modifying the structure of Bax leading to its insertion into mitochondrial membranes. | Study | biomedical | en | 0.999996 |
10085290 | Section title: Cell Culture, Drug Treatment, and Analysis of Apoptosis Educational score: 4.1758341789245605 Domain: biomedical Document type: Study Language: en The human Jurkat T-leukemic cell line was grown in RPMI 1640 medium supplemented with 10% fetal bovine serum and 2 mM l -glutamine ( GIBCO BRL ). The human CEM-C7A T-leukemic cell line was grown in Optimem 1 medium ( GIBCO BRL ) supplemented with 5% FBS as were the CEM-C7A cells stably transfected with Bcl-2 (CEM-Bcl-2) or an empty control vector (CEM-Neo; reference 4 ). Cultures were incubated at 37°C in a humidified atmosphere of 5% CO 2 . Cells in a logarithmic phase of growth were resuspended at a density of 3 × 10 5 cells/ml in fresh medium before drug treatment. Etoposide, staurosporine, and dexamethasone were purchased from Sigma Chemical Co. Stock solutions of drugs in DMSO were stored at −20°C. Control cells received solvent alone. The final concentration of DMSO solvent in the culture medium never exceeded 1% (vol/vol), which was nontoxic to the cells. Mouse IgM anti-CD95 monoclonal antibody (mAb; clone CH-11) was purchased from Coulter Electronics Ltd. An irrelevant mouse IgM antibody (Coulter Electronics Ltd.) was used as negative control. The morphological changes of chromatin condensation, typical of apoptosis, were assessed by fluorescence microscopy after staining of cells with acridine orange (10 μg/ml) and the % apoptosis was scored after scoring at least 200 cells. Section title: Analysis of Protein Expression by Western Blotting Educational score: 4.17410135269165 Domain: biomedical Document type: Study Language: en After treatment, cells were washed twice in PBS, lysed in lysis buffer (50 mM Tris, pH 7.4, 1 mM EDTA, 150 mM NaCl, 1 mM Na orthovanadate, 0.5% NP-40, and protease inhibitors (0.1 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 10 μg/ml trypsin inhibitor). Cell lysates (30 μg protein) were separated by SDS-PAGE (12% acrylamide) and transferred to a PVDF membrane (hybond-PVDF; Amersham Life Sciences ). After blocking nonspecific binding sites overnight with 5% nonfat milk in TPBS (PBS, 0.1% Tween-20), the membrane was incubated for 2 h at room temperature with a 1/1,000 dilution of a murine anti– human Bak monoclonal antibody designated as Ab-1 and raised against the peptide sequence amino-acids 1–52 (AM03; Calbiochem-Novabiochem Ltd.), or Ab-2 (AM04; Calbiochem-Novabiochem Ltd.) also made to the same peptide, or murine anti–human Bcl-2 mAb, made to a peptide sequence of amino acids 41–54 (Dako Ltd.). To ensure equal loading and transfer, membranes were also probed for actin using the anti-actin mouse monoclonal AC-40 ( Sigma Chemical Co. ). The immunoreactive proteins were visualized using rabbit horseradish peroxidase-linked goat anti– mouse antibody (Dako Ltd.) and enhanced chemiluminescence (ECL; Amersham Life Sciences ). Section title: Flow Cytometric Analysis of Bak and Bcl-2-associated Immunofluorescence Educational score: 4.157016754150391 Domain: biomedical Document type: Study Language: en At specific times after drug-treatment, cells (10 6 ) were fixed in paraformaldehyde (PFA; 0.25%) for 5 min at room temperature and washed three time in PBS. Cells were kept at 4°C until analysis, then centrifuged and incubated for 30 min with the anti-human Bak monoclonal antibodies Ab-1 or Ab-2, the anti-human Bcl-2 mAb (see above), a rabbit polyclonal anti-human Bak antibody , or a mouse IgG2b isotype-specific mouse antibody raised against Aspergillus niger glucose oxidase, designated as the irrelevant Ab (Dako Ltd.). All antibodies were diluted 1 in 50 in PBS containing digitonin (500 μg/ml). After three washes in PBS, cells were incubated with FITC-labelled goat anti–mouse or anti–rabbit IgG secondary antibody, diluted 1 in 100 in PBS, for 30 min, washed twice in PBS and resuspended in 1 ml of PBS. Analysis was performed on a FACS ® Vantage flow cytometer equipped with an Enterprise laser (Innova Technology, Coherent Inc.) set to excite at 250 mW using the 488-nm laser line. Green fluorescence (FITC, FL-1) was detected at 530 ± 30 nm. Fluorescence was acquired using logarithmic amplifiers. 10,000 cells were analyzed per sample at a flow rate of 300 cells/s. Section title: Flow Cytometric Analysis of Bak and Bcl-2-associated Immunofluorescence Educational score: 4.081977367401123 Domain: biomedical Document type: Study Language: en The effect of coincubation with the broad spectrum caspase inhibitor zVAD-fmk (40 μM) on Bak Ab-1 (NH 2 -terminal) immunofluorescence was analyzed by flow cytometric (FCM) in Jurkat cells before and after exposure to etoposide and in CEM cells before and after treatment with dexamethasone, both for 4 h. Section title: Flow Cytometric Analysis of Bak and Bcl-2-associated Immunofluorescence Educational score: 3.9639744758605957 Domain: biomedical Document type: Study Language: en CEM-Bcl-2 and CEM-Neo cells ( 4 ) were analyzed for immunofluorescence of Bak Ab-1 before and after exposure to etoposide for 4 h. Section title: Flow Cytometric Analysis of Bak and Bcl-2-associated Immunofluorescence Educational score: 4.124631881713867 Domain: biomedical Document type: Study Language: en In order to quantitate the flow cytometric results obtained using Bak Ab-1, the raw data obtained were manipulated in the following way: (a) cells exhibiting a light scatter profile typical of apoptotic cells or cell debris were excluded from the analysis by electronic gating; (b) the median specific Bak-associated fluorescence ( f Bak ) was determined by subtraction of the median fluorescence of the parallel irrelevant antibody control sample ( f IgX ; the fluorescence of samples containing secondary antibody alone was closely similar to, and always less than f IgX ); (c) f Bak was multiplied by the percentage of cells with fluorescence above f IgX to generate a figure designated S ; (d) S C for control untreated samples at a particular timepoint was subtracted from S toxin for toxin-treated samples to generate a final fluorescence value designated θ. The mean value of θ was determined in two or more separate repeat experiments. Section title: Analysis of Cellular Bak and Bcl-x L Immunofluorescence by Microscopy Educational score: 4.198931694030762 Domain: biomedical Document type: Study Language: en Control and etoposide-treated cells (10 μM) were fixed in PFA 0.25% for 5 min at room temperature and washed three times in PBS. Cells were incubated overnight at 4°C with anti-human Bak mAb (Ab-1, specific to the NH 2 terminus) and/or anti-human Bak polyclonal antibody (pAb-Bak; for details see above) and/or Bcl-x L using the rabbit anti–human Bcl-x L polyclonal antibody raised to residues 18–233 (pAb Bcl-x L ; Transduction Laboratories) diluted 1 in 50 in PBS-digitonin (500 mg/ml), washed in PBS and then incubated with either a FITC-conjugated goat anti–mouse IgG antibody diluted 1 in 100 in PBS or a goat anti–rabbit conjugated with indocarbocyanine (Cy3; Jackson Immunochemicals). Nuclei were stained with a solution of Hoechst 33342 (1 μg/ml). Cells were then suspended in Vectashield mounting medium (Vector Laboratories), pipetted dropwise onto a glass slide and observed by fluorescence microscopy using a Zeiss Axioskop microscope equipped with an epiilluminator and appropriate filters. Section title: Subcellular Localization of Bak and Other Proteins Educational score: 4.3304057121276855 Domain: biomedical Document type: Study Language: en Cells were washed twice in ice-cold PBS and then resuspended at 5 × 10 7 /ml in ice-cold lysis buffer (20 mM Hepes-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl 2 , 1 mM EDTA, 1 mM EGTA, 250 mM sucrose, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 10 mg/ml leupeptin, 10 μg/ml aprotinin, and trypsin inhibitor 10 μg/ml). The cell suspension was homogenized in a Dounce homogenizer and centrifuged at 700 g for 7 min at 4°C. The pellet containing any remaining intact cells and nuclei (designated as N) was washed once in lysis buffer and the postnuclear supernatant was centrifuged at 10,000 g for 15 min. The resulting pellet, designated P10, was washed once in lysis buffer and the supernatant was subjected to ultracentrifugation at 100,000 g for 1 h to pellet the remaining membrane, termed P100. The remaining supernatant was the cytosolic fraction (designated S100). The fractions N, P10, and P100 were resuspended in 1 vol of lysis buffer. Each fraction was subjected to SDS-PAGE electrophoresis and analyzed by Western blotting for Bak content. The relative purity of fractions was ascertained by Western blotting using the mouse anti-cytochrome oxidase IV mAb (Molecular Probes) as a marker of the mitochondria, the rabbit anti-calnexin pAb and a rat anti-KDEL peptide (anti-grp78) pAb (both from StressGen) as markers for endoplasmic reticulum, the mouse anti–human poly(ADP-ribose)polymerase mAb (Serotec) as a nuclear marker and the anti-procaspase-3 mAb (Transduction Laboratories) and a rabbit anti-aldolase (a kind gift of Dr. C. Schnarrenberger, University of Manchester, UK) to check the purity of the cytosolic fraction. Section title: Immunoprecipitations Educational score: 4.076863765716553 Domain: biomedical Document type: Study Language: en Immunoglobulins were bound to protein A–Sepharose for 1 h at room temperature in the Hepes buffer described below. Beads were sedimented and washed twice with 10 vol of 0.2 M sodium borate, pH 9, then cross-linked with 20 mM DMP for 30 min. Unreacted cross-linker was removed by washing beads with 0.2 M ethanolamine, pH 8. They were resuspended in PBS with 0.01% thimerosal ( Sigma ). Section title: Immunoprecipitations Educational score: 4.161474704742432 Domain: biomedical Document type: Study Language: en Jurkat cells were grown to a density of 10 6 cells/ml and treated with etoposide (10 μM) for 20 h, to ensure maximum exposure of the NH 2 -terminal epitope of Bak. Control cells received solvent alone. Cells (1.2 × 10 8 ) were washed with PBS and then lysed in 2 ml of 10 mM Hepes, pH 7.4, 150 mM NaCl, 0.1% NP-40, 40 μg/ml PMSF, 1 μg/ml leupeptin, and aprotinin. Lysis was carried out on ice for 30 min, with regular vortexing. A post-nuclear supernatant was prepared by centrifugation at 10,000 g for 10 min at 4°C. The resulting lysate was precleared with 200 μl of neat pig-serum, DMP (dimethylpimelimidate) cross-linked to protein A–Sepharose ( Pharmacia ), rotated at 4°C for 1 h to remove nonspecific IgG binding proteins, and the protein A beads recovered by centrifugation at 10,000 g for 5 min at 4°C. The supernatant was further cleared by the addition of 200 μl of protein A–Sepharose slurry (prepared by equilibrating in Hepes buffer according to the manufacturers protocol, and resuspended in two bed volumes), rotated at 4°C for 30 min, and removed by centrifugation at 10,000 g for 5 min at 4°C. The resulting cleared lysate was then used for immunoprecipitation of Bak and Bcl-x L proteins. Section title: Immunoprecipitations Educational score: 4.059792518615723 Domain: biomedical Document type: Study Language: en Immunoprecipitations were performed at 4°C for 1–3 h. There was no significant difference in the results if these were left overnight. Each of the antibodies used for immunoprecipitation were cross-linked with DMP (as described above) onto the protein A–Sepharose media. Antibodies were as described above. Both the monoclonal and the polyclonal antibodies were used. In addition, mouse irrelevant , and rabbit irrelevant antibodies were used as controls. The antibody/protein A–Sepharose matrix was sedimented before use to remove the storage buffer (PBS/thimerosal); Ab-1 (10 μg) and Bcl-x L (20 μg) antibodies were added to 500 μl of lysate. The protein concentration for the Bak polyclonal was unknown and was used at 30 μl per immunoprecipitation. Section title: Immunoprecipitations Educational score: 4.156973838806152 Domain: biomedical Document type: Study Language: en The immunoprecipitated proteins were recovered from the lysate by centrifugation at 10,000 g for 5 min at 4°C. The pelleted Sepharose was washed 3 times with 1-ml vol of the Hepes buffer described above. The immunoprecipitated proteins were eluted from the antibody–bead complex by the addition of 100 μl of 3× SDS-PAGE sample buffer. The beads were vortexed thoroughly, sedimented at 15,000 g , and the supernatant boiled before SDS-PAGE analysis, loading 30 μl per lane. The beads could be boiled directly and no difference was seen in protein recovery. However, in some instances immunoglobulin heavy chain was also eluted from the beads. Lysates, precleared lysates, and immuno-precipitate-depleted lysates were analyzed by SDS-PAGE and Western blotting, as described above. The signals from the bands were quantitated using a Bio-Rad GS-700 image densitometer with manufacturer's software. Section title: Two-Dimensional Gel Electrophoresis Educational score: 4.154515266418457 Domain: biomedical Document type: Study Language: en Isoelectric focusing tube gels were prepared essentially as in Knowles ( 20 ). Tube gels were prepared by water displacement and allowed to polymerize. These were prefocused for 1 h at 200 V. Treated or untreated cells were lysed in urea sample buffer of 9 M urea, 2% ampholine 3-10, 100 mM DTT, 4% NP-40, and 2% CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate). 500 μg of protein was routinely loaded per tube gel. Samples were then electrophoresed at 700 V for 16 h, then fine focused for 1 h at 1,000 V. Gels were incubated in equilibration buffer (0.5 M Tris-HCl, pH 6.8, 1 M glycerol, 1% SDS, with bromophenol blue to color) for 10–15 min before overlaying onto a 15% polyacrylamide slab gel. Two-dimensional electrophoresis was carried out followed by Western blotting, as detailed above. Section title: Kinetics of Apoptosis Educational score: 4.1855902671813965 Domain: biomedical Document type: Study Language: en The kinetics of apoptosis of Jurkat and CEM-C7A lymphoma cells were characterized after exposure to staurosporine, etoposide, and an agonistic antibody to CD95. Dexamethasone-induced apoptosis in CEM-C7A cells was also measured; the Jurkat cell line used did not respond to dexamethasone. Table I summarizes the apoptotic response of the two cell types to these agents acting at different cellular loci. These were protein kinase inhibition by staurosporine, protein-associated DNA single and double strand breaks induced by etoposide (by inhibition of topoisomerase II), activation of a known death inducing receptor CD95, and activation of glucocorticoid receptors and transcriptional changes by dexamethasone. Timepoints were then chosen for analysis of Bak content and immunofluorescence before and after the appearance of significant numbers of apoptotic cells. Section title: Measurement of Bak Protein Content Educational score: 4.130151748657227 Domain: biomedical Document type: Study Language: en Measurements of the cellular content of Bak were made by Western blotting at various times in Jurkat and CEM-C7A cells before and after the death-inducing stimuli. Bak was constitutively expressed in both lines, as determined by the use of the anti-Bak antibody Ab-1 (NH 2 -terminal). The cellular content of Bak was unaffected by any of the toxic stimuli over the entire time course . Two other antibodies to the Bak protein (another monoclonal antibody to the NH 2 terminus and the polyclonal, see Materials and Methods) similarly showed no change in the amount of Bak protein by Western blotting (data not shown). There was also no evidence for cleavage of Bak in any experiments. There were also no changes in Bcl-2 protein content in either cell line after toxin treatment . Actin was used as a loading control. Section title: Epitope-specific Changes in Immunofluorescence of Bak after Treatment with Toxins Measured by Flow Cytometry Educational score: 4.156627655029297 Domain: biomedical Document type: Study Language: en FCM was used to determine whether there were changes in the amount of immunofluorescence associated with Bak using the two epitope-specific antibodies made to the NH 2 terminus of Bak (Ab-1 and Ab-2, see Materials and Methods). Although no changes were observed by Western blotting , it has been suggested elsewhere ( 2 ) that this may represent the limitations of a methodology which is population averaged and thus masks cellular heterogeneity. Using FCM there can be two potential explanations for changes in immunofluorescence: firstly they may indeed reflect changes in cell protein content, but secondly they may identify changes in epitope availability in intact cells without a change in protein content. The flow cytometric data shown are displayed as frequency histograms of Bak immunofluorescence, detected using a FITC-conjugated secondary antibody. The data are presented on a logarithmic scale and a shift to the right in the histogram indicates an increase in Bak-associated immunofluorescence. Fluorescence associated with Bak Ab-1 or Ab-2 (NH 2 -terminal specific) was undetectable in intact untreated cells by flow cytometry and the histograms are superimposed upon those of an irrelevant antibody control . Bak Ab-1-associated immunofluorescence became detectable 4 h after treatment of both cell lines with staurosporine . Similar results were obtained using Bak Ab-2. There was no change in the immunofluorescence associated with Bcl-2 using an antibody to amino acids 41–54 . Section title: Epitope-specific Changes in Immunofluorescence of Bak after Treatment with Toxins Measured by Flow Cytometry Educational score: 4.282553195953369 Domain: biomedical Document type: Study Language: en Similarly, treatment with etoposide (10 μM) that irreversibly commits >90% of CEM-C7A cells to a loss of clonogenic survival after 30 min of exposure (data not shown), resulted in the appearance of Bak Ab-1 NH 2 -terminal-associated immunofluorescence by 4 h, without any increase in the amount of protein, measured by Western blotting . This was also seen with Jurkat cells . The increase in NH 2 -terminal epitope availability at 4 h was dependent on the concentration of etoposide added to Jurkat cells . Thus increased amounts of etoposide-induced DNA damage, and presumably some DNA damage signal, induced more Bak immunofluorescence associated with an NH 2 -terminal epitope of the protein. Again, analysis of Bcl-2-associated immunofluorescence, showed this to be unchanged after etoposide, as it was after STS treatment . In all FCM experiments, cell debris, the cells that were overtly apoptotic at 4 h and cell aggregates were all electronically excluded (gated out) by virtue of altered light scatter signals, before examination of the Bak immunofluorescence profile. This may not remove all apoptotic cells, particularly those in the early stages. Nevertheless, the changes in Bak-associated immunofluorescence did not appear to be associated with the apoptosis per se, that is, with the execution phase of cell death (see also Table I ). Some type of apoptosis-associated proteolytic cleavage of Bak to change epitope availability is also unlikely as we saw no change in the size of Bak protein on the Western blots . Section title: Dexamethasone also Changes Bak Ab-1 Immunofluorescence but Ligation of CD95 (Fas, APO-1) Does Not Educational score: 4.1064300537109375 Domain: biomedical Document type: Study Language: en The immunofluorescence profiles for Bak were examined after the addition of other death stimuli. Fig. 3 A shows that the non-genotoxic drug dexamethasone revealed the Bak Ab-1 epitope in CEM-C7A cells. In contrast, CEM-C7A cells treated with an agonistic antibody to the CD95/ Fas/APO-1 receptor did not exhibit Bak immunofluorescence despite the induction of 22% apoptosis (Table I ). Similar results were obtained after treatment of Jurkat cells with the anti-CD95 antibody (data not shown). Section title: Exposure of the NH 2 -terminal Epitope of Bak Occurs before Caspase Activation Educational score: 4.089010238647461 Domain: biomedical Document type: Study Language: en In experiments using the broad spectrum caspase inhibitor zVAD-fmk, there was no effect on the increase in Bak-associated immunofluorescence . Data were quantitated using an equation to derive the parameter θ (see Materials and Methods and below). The θ values were 583 ± 123 and 578 ± 120 ( n = 3) for cells treated with etoposide in the presence or absence of zVAD-fmk, respectively. The caspase inhibitor effectively inhibited apoptosis in etoposide-treated cells to 6 ± 3% ( n = 3). Section title: Immunofluorescence Using a Polyclonal Antibody to Bak Is Detectable before and after Cell Perturbation Educational score: 4.170722961425781 Domain: biomedical Document type: Study Language: en Critically, using the polyclonal antibody to Bak (pAb), raised against all but the transmembrane domain of the protein, immunofluorescence was detectable by FCM in both untreated and etoposide-treated cells . A decade increase in fluorescence above that of the irrelevant antibody control was observed, with a slight increase after etoposide treatment. Further comparison of data obtained with the monoclonal and polyclonal antibodies is described below in Fig. 5 . The ability of the polyclonal antibody (pAb) but not Bak Ab-1 antibody to bind Bak in untreated cells supports the idea that a selective change in epitope availability is occurring specifically at the NH 2 terminus of Bak after disparate toxin treatment and that this epitope is concealed in untreated cells. Additionally, the lack of change in immunofluorescence associated with Bcl-2 also supports the idea of a specific damage-induced change at the NH 2 terminus of Bak and not that this was an artefact of FCM. Section title: Quantitation of Changes in Bak-associated Immunofluorescence Educational score: 4.117484092712402 Domain: biomedical Document type: Study Language: en The changes in immunofluorescence measured by flow cytometry were quantified using an equation (see Materials and Methods) that essentially determines the area under curve (histogram) after subtraction of nonspecific fluorescence signals detected using an irrelevant antibody control to generate a value termed θ. The absolute increase in Bak Ab-1 immunofluorescence, calculating a θ value as described above, was different in magnitude for etoposide, STS and dexamethasone , possibly reflecting differences in the kinetics of commitment to and onset of apoptosis, as discussed by us previously ( 40 ). For example, we have found that while STS induces changes in immunofluorescence throughout the cell cycle, etoposide does this only at specific cell cycle phases, presumably reflecting cell cycle checkpoint-associated signals (manuscript in preparation). Section title: Quantitation of Changes in Bak-associated Immunofluorescence Educational score: 4.079030513763428 Domain: biomedical Document type: Study Language: en Bak Ab-1-associated immunofluorescence was detected in both CEM-Neo and CEM-Bcl-2 cells after treatment with etoposide. In two preliminary experiments, four hours after drug treatment the θ values for Bak Ab-1-associated immunofluorescence were 154 and 160 for the CEM-Bcl-2 cells compared with 216 and 210 for the CEM-Neo cells. Section title: Microscopy Confirms that the Exposure of the Bak NH 2 -terminal Epitope Is a Specific Event Occurring before Apoptosis Educational score: 4.302688121795654 Domain: biomedical Document type: Study Language: en In order to test the hypothesis further that the NH 2 -terminal epitope of Bak is exposed after perturbation but before the appearance of an apoptotic morphology, we exposed Jurkat cells to toxins and compared by microscopy the immunofluorescence of Bak Ab-1 and nuclear morphology by costaining with Hoechst 33342 . Untreated cells did not stain positively for Bak using Bak Ab-1 (not shown), confirming the FCM data. Three patterns of staining were observed 4 h after cells had been treated with etoposide : there were some cells with an intact, non-apoptotic nucleus which remained Bak Ab-1 negative (a); others had an intact nucleus but stained positively for Bak Ab-1 (b). The remainder displayed Bak Ab-1 staining and exhibited fragmented and condensed chromatin (c). These data confirm that Bak Ab-1 fluorescence is induced before morphological indications of apoptosis, characterized by a loss of nuclear integrity. Fig. 5 , B and C show Jurkat cells dual stained with both the polyclonal Bak antibody (red) and the Bak Ab-1 (green). B shows an untreated cell: it displays only polyclonal antibody red fluorescence in a punctate distribution. C shows a typical etoposide-treated Jurkat cell with predominantly yellow fluorescence indicating that at 4 h after drug treatment, the majority of Bak protein molecules appear to be dual stained with both antibodies. However, at this time, some Bak molecules remain detectable only by the polyclonal antibody. This may indicate the presence of different pools and/or isoforms of Bak which possess different antibody binding profiles (discussed further below). The staining is no longer punctate, implying the compaction of the mitochondria in a reduced cytoplasm, an established feature of apoptotic cells. Section title: Colocalization of Bak and Bcl-x L by Fluorescence Microscopy Educational score: 4.158914566040039 Domain: biomedical Document type: Study Language: en To determine whether Bak and Bcl-x L were colocalized in etoposide-treated Jurkat cells, cells were dual stained with Bak Ab-1 visualized with a FITC tagged secondary antibody (green) and with Bcl-x L pAb visualized with a Cy3 tagged secondary antibody (red). It was not possible assess colocalization before etoposide treatment since the Bak Ab-1 epitope was unavailable nor was it possible to dual stain with polyclonal antibodies for reasons of secondary antibody overlap. Fig. 6 A shows a typical untreated cell exhibiting Bcl-x L immunofluorescence but no Bak Ab-1 staining. 4 h after etoposide treatment the cell population is heterogeneous, comprised of cells with intact nuclei and cells with fragmented nuclei and condensed chromatin. In Fig. 6 B, in a cell that has not yet undergone apoptosis, Bcl-x L pAb and Bak Ab-1 are predominantly colocalized (observed as yellow fluorescence). In Fig. 6 C, where the cell is clearly apoptotic, the degree of colocalization appears to be decreased. Section title: Subcellular Localization of Bak Protein after STS Educational score: 4.374774932861328 Domain: biomedical Document type: Study Language: en The change in Bak immunofluorescence induced by etoposide and other agents represents the exposure of an otherwise cryptic epitope at the NH 2 terminus. This suggested the possibility that like Bax protein, which moves from cytosol to mitochondrial membranes on exposure of its NH 2 terminus ( 39 ), Bak may also change its cellular location following a damage signal. Therefore, we attempted to investigate the subcellular location of Bak and whether it was translocated in Jurkat cells after STS using cell fractionation methods similar to those used previously ( 36 , 39 ). However, we were concerned about the purity of the mitochondrial (P10) and endoplasmic reticulum (P100) enriched fractions using these methods and consequently examined markers for each fraction. This showed that, using these methods, P10 and P100 both contained calnexin and grp78, both indicative of the presence of endoplasmic reticulum. Additionally, there was also some contamination of the nuclear fraction by the endoplasmic reticulum. However, procaspase 3 and aldolase were only present in the cytoplasmic fraction (S100). It is thus only possible to conclude that Bak protein was absent from the cytosolic fraction, before and after STS treatment, and that it was membrane associated . Importantly, this differs from reports of the location of the congener Bax which is claimed to be cytosolic before signals which initiate apoptosis ( 17 , 39 ). Due to the lack of purity in cellular subfractions generated by this protocol we also examined the cellular location of Bak and Bcl-x L by fluorescence microscopy using organelle markers. Fig. 7 B shows that Bcl-x L and Bak predominantly colocalized with the mitochondrial marker cytochrome oxidase IV and that neither Bak nor Bcl-x L colocalized with the endoplasmic reticulum markers grp78 or calnexin, supporting the idea of mitochondrial association for both molecules. Section title: Assessment of Changes in Binding of Bak to Bcl-x L before and after Toxin Treatment Educational score: 4.182491302490234 Domain: biomedical Document type: Study Language: en We considered that the exposure of the NH 2 -terminal epitope of Bak after cellular perturbation might represent a conformational change and/or the release of a Bak binding partner to change epitope availability. Since Bcl-x L has been shown to bind to Bak ( 7 , 8 , 11 , 35 ) and we have shown that they colocalized in Jurkat cells by immunofluorescence (see above), we examined whether these two proteins were physically bound in untreated cells and whether their association was altered by toxin treatment. Immunoprecipitations were carried out using the Bcl-x L polyclonal antibody, the polyclonal Bak antibody (which binds Bak in the presence and absence of toxin treatment), and Bak Ab-1 (NH 2 terminus, which only binds to Bak after toxin treatment). In initial experiments we attempted to coimmunoprecipitate Bak with Bcl-x L in the absence of any detergent, which would mimic the conditions used for FCM where cells are fixed before detergent is added. Hsu and Youle ( 17 ) cautioned that the use of detergents in protocols for the immunoprecipitation of Bcl-2 family members might initiate conformational changes promoting altered patterns of protein–protein interactions. However, we were unable to immunoprecipitate either Bak or Bcl-x L in the absence of detergent, as the proteins were not solubilized from the membrane fraction. Section title: Assessment of Changes in Binding of Bak to Bcl-x L before and after Toxin Treatment Educational score: 4.2077531814575195 Domain: biomedical Document type: Study Language: en Using a low concentration of detergent (0.1% NP-40) it was possible to immunoprecipitate Bak using the Ab-1 NH 2 -terminal antibody in control untreated cells . Using Bak Ab-1 there was no difference in the ability to immunoprecipitate Bak comparing untreated cells with those treated with etoposide for 20 h to maximize epitope exposure at the NH 2 terminus . These data are in contrast to the inability to detect Bak using this antibody in untreated cells by FCM and microscopy and most likely reflect the ability of detergent to expose the Bak Ab-1 epitope. Immunoprecipitations performed using a monoclonal or polyclonal antibody to Bak, or a polyclonal antibody to Bcl-x L showed that Bak and Bcl-x L were constitutively associated in control, healthy cells . Analysis of the immunoprecipitates and supernatant lysates showed that using the Bak polyclonal antibody 100% of Bak was bound to Bcl-x L . In contrast, analysis of the supernatants after immunoprecipitation with the Bak pAb showed that ∼40% of Bcl-x L remained unbound to Bak and was therefore in excess. When these immunoprecipitations were performed using etoposide-treated cells, the association between Bak and Bcl-x L was clearly reduced . In two independent experiments where we obtained very clearly defined bands after Western blotting, densitometry showed that a 30% and a 41% reduction of binding had occurred. The irrelevant antibody controls indicate that immunoprecipitations were epitope specific and not due to the type of immunoglobulin used (data not shown). Section title: Bak Exists in Multiple Isoforms Identified by Two-Dimensional Gel Electrophoresis and Is Unchanged after STS Treatment Educational score: 4.123371124267578 Domain: biomedical Document type: Study Language: en To establish whether the changes in Bak NH 2 -terminal-associated immunofluorescence were associated with a posttranslational modification of Bak, we performed two-dimensional electrophoresis and Western blotting for Bak after treatment of Jurkat cells with STS, which produced the most substantial change in epitope availability. Fig. 9 shows that Bak exists in multiple forms based on charge differences in untreated cells, without major changes in mass. There were no discernible changes in protein mobility observed after STS treatment. Taken together, protein spots before or after treatment at positions 1 to 6 were very reproducible. However, the more acidic proteins at positions 7 and above were more variable from sample to sample, even in untreated cells, and we cannot exclude the possibility that subtle changes may be occurring which we are unable to resolve sufficiently. This is the subject of further experimentation. Similar data were obtained when cells were treated with etoposide. Section title: Discussion Educational score: 4.343482971191406 Domain: biomedical Document type: Study Language: en We have found that very different types of cellular perturbation, all of which induced apoptosis (Table I ), brought about exposure of a specific epitope at the NH 2 terminus of the Bak protein. The epitope was completely concealed from antibodies raised to the NH 2 terminus in untreated cells . Importantly, the damage-induced exposure of the epitope occurred before detection of both morphological and biochemical changes typical of apoptosis. This is particularly exemplified by the 24 h data for dexamethasone where we showed previously that caspase activity was undetectable until after 30 h of exposure ( 4 ). These alterations in Bak-associated immunofluorescence induced by dexamethasone also preceded the changes in mitochondrial membrane potential, occurring before caspase activation ( 4 ). Thus, the change in the availability of the NH 2 terminus of Bak is not a component of the execution phase per se, but presages it and is an early indicator that the cells had been perturbed or damaged. The specificity of this change in epitope availability was indicated by the findings that while the two different antibodies to the NH 2 terminus showed significant changes in immunofluorescence , this was not observed when a polyclonal antibody was used . The observation that totally disparate types of cellular perturbation initiated this change also suggests that either Bak itself or an associated molecule was acting as an integrator of damage signals arising from different types of disturbance in the cell, generated at different locations. The change in epitope availability observed here in lymphoid cells has been reproduced for Bak in both neuronal and epithelial cell lines and for an NH 2 -terminal epitope of Bax in a B cell lymphoma line (our unpublished data). Section title: Discussion Educational score: 4.244663238525391 Domain: biomedical Document type: Study Language: en In experiments where cells were treated with etoposide, which rapidly commits them to apoptosis, simultaneous probing with both the polyclonal and NH 2 terminus–targeted monoclonal antibodies to Bak showed that the immunofluorescence mostly overlapped . There was a small proportion of Bak, recognized by the polyclonal antibody, that did not show toxin-induced exposure of the Bak NH 2 terminus. Whether this was a temporal phenomenon, arising from the cell cycle specificity of the damage signals generated by etoposide, or whether this represents some subpopulation of the Bak protein remains to be determined. Immunoprecipitation showed that, in the presence of some detergent, all of the Bak was bound to Bcl-x L but this does not preclude heterogeneity. As discussed below, Bak protein is present in multiple, posttranslationally modified forms and it is possible that only some of these represent the active species, with others forming latent or inactive subpopulations, perhaps in the complex with Bcl-x L . Section title: Discussion Educational score: 4.429227352142334 Domain: biomedical Document type: Study Language: en Interestingly, the ligation of CD95, an effective initiator of apoptosis (Table I ) did not bring about a change in the availability of the epitope at the NH 2 terminus of Bak . This suggests that ligation of CD95 (APO-1/Fas) must act in some other way from the other toxins used to stimulate cell death. Whereas CEM-C7A cells transfected to overexpress bcl -2 were afforded significant delay in the onset of apoptosis induced by etoposide, and complete protection of the loss of clonogenicity induced by dexamethasone ( 4 ), over expression of Bcl-2 protein completely failed to inhibit apoptosis induced by the ligation of CD95 (our unpublished data). It has been shown recently that apoptosis induced by CD95 ligation may proceed either via activation of CD95-associated FADD and FLICE (caspase 8), with subsequent caspase activation, a process which is not suppressed by Bcl-2, or via Daxx and the activation of the JNK pathway. The Daxx pathway to apoptosis is Bcl-2 inhibited in certain cells ( 43 ). We are unable to state how CD95 ligation activated apoptosis occurs in these CEM-C7A cells. Whatever the precise pathway used by CD95, unlike the damage signals arising from treatment with etoposide, dexamethasone and staurosporine , it did not bring about a change in the immunofluorescence of Bak. This supports the idea that the signalling cascade for the initiation of apoptosis by CD95 differs from that imposed by the toxins. Section title: Discussion Educational score: 4.228092670440674 Domain: biomedical Document type: Study Language: en When bcl -2 overexpressing CEM-C7A cells ( 4 ) were challenged with etoposide there was only a 30% reduction in the exposure of the NH 2 -terminal epitope of Bak, measured by a fall in the θ value (see Results). Thus overexpression of bcl -2 somehow either attenuates the damage signal that reveals the Bak NH 2 -terminal epitope or it changes the protein–protein interactions between Bak and its partners. Bak preferentially binds to Bcl-x L ( 11 ) and in immunoprecipitations of Bak we found no association with Bcl-2 (Savory, P., unpublished results). However, Bcl-2 overexpression may change the stoichiometry of the protein complex containing Bak, but until the components of that complex have been identified, no mechanistic explanation can be offered to explain the reduction of the exposure of the Bak NH 2 terminus by Bcl-2. Section title: Discussion Educational score: 4.676969051361084 Domain: biomedical Document type: Study Language: en How does the NH 2 -terminal epitope of Bak, recognized by Ab-1, become exposed after damage? This could be the result of direct changes in the conformation of Bak and/or due to the release of a protein(s) that conceals the NH 2 -terminal epitope. Is Bak itself the recipient of damage signals, perhaps becoming posttranslationally modified, so as to change conformation? Or, are damage signals received by accessory proteins, which then bring about the dissociation of protein(s) from the NH 2 -terminal domain of Bak, exposing this epitope? And how do these changes contribute to the irreversible commitment of the cell to subsequent apoptosis? Hsu and Youle ( 17 ) reported changes in epitope availability of murine Bax, again specifically at the NH 2 terminus, after addition of nonionic detergent to cell lysates. They proposed a model whereby the detergent-induced changes in Bax epitope availability, observed in lysates, equated to a change in conformation. This brought about different patterns of protein–protein interactions. An open NH 2 terminus of Bax promoted Bax homodimerization and heterodimerization with Bcl-x L ( 17 ). Additionally, it was suggested that the detergent-induced conformational change in Bax mimics the conformation that Bax adopts when it becomes located in the mitochondrial membrane after translocation from the its monomeric form in cytosol to a membrane-bound, dimeric form. When associated with the mitochondrial membrane, Bax-Bcl-x L heterodimerization may then occur ( 17 ). Elegant studies of murine Bax by Gross et al. ( 14 ) confirm that aspects of this model for Bax translocation from cytosol to mitochondria occur in whole cells. Using protein cross-linking agents they showed that monomeric Bax translocated to mitochondria after an apoptotic stimulus (IL-3 withdrawal) where it then homodimerized. Manipulation of Bax to enforce homodimerization resulted in translocation to mitochondrial membranes and in apoptosis ( 14 ). Section title: Discussion Educational score: 4.302923679351807 Domain: biomedical Document type: Study Language: en Our results, which examine endogenous human Bak in intact cells, show that there are distinct differences between the phenomena observed with Bak compared with those reported for the murine Bax protein either in lysates or murine cells. As discussed above, in cells that were fixed before the use of any detergent, the NH 2 terminus of Bak became available to the antibody Ab-1 after different types of cell damage. We considered that this might promote a change in its subcellular location in a manner similar to that suggested for Bax using cell lysates and, subsequently, a change in its association with Bcl-x L (17). The subcellular fractionation experiments and immunofluorescence showed that Bak was not cytosolic before or after toxin treatment , rather both were more closely associated with mitochondria, before and after perturbation . Taken together our data show that, unlike the scenario for murine Bax, an apoptotic stimulus does not induce a change in subcellular location of Bak. Signals derived from cellular perturbation by toxins are thus presumably integrated at intracellular membrane locations. Section title: Discussion Educational score: 4.3547682762146 Domain: biomedical Document type: Study Language: en When we performed immunoprecipitations, with a low concentration of nonionic detergent present, using either Ab-1 or the polyclonal antibody to Bak or the polyclonal antibody to Bcl-x L , probing the subsequent Western blots showed that Bak-Bcl-x L binding occurred constitutively in healthy cells . Lysates prepared from these cells treated with etoposide to induce maximal unmasking of the NH 2 terminus of Bak, showed a 30–40% decrease in Bak-Bcl-x L binding . Increasing the detergent concentration from 0.1 to 1% did not alter this result (data not shown). Therefore, toxin treatment did not recapitulate for Bak the detergent-mediated promotion of Bax association with Bcl-x L , which had been reported using murine cell lysates ( 17 ). Instead, Bak and Bcl-x L appear to be constitutively bound in healthy cells and a proportion of Bak became dissociated as the cells committed to apoptosis, although it remained membrane bound. The damage induced conformational change in Bak, either at the NH 2 terminus itself, or as other domains then undergo subsequent topological changes, therefore modulates the stoichiometry of binding of these particular pro- and anti-apoptotic molecules. Section title: Discussion Educational score: 4.646790504455566 Domain: biomedical Document type: Study Language: en What changes are taking place in this Bak-Bcl-x L complex as the cells commit to apoptosis? And how are these involved in the commitment of cells to death? Using transient transfections, a mutational analysis of Bak had suggested that its BH3 domain was required both for binding to Bcl-x L and for its pro-apoptotic activity, implying that heterodimerization may be necessary for the induction of apoptosis ( 8 ). Our data are compatible with a model in which death may be suppressed by heterodimerization (Bak and Bcl-x L constitutively bound) and where the dissociation of Bak from Bcl-x L correlates with promotion of apoptosis. Using stably transfected Bak mutants it was found that cytotoxic drug-induced apoptosis of Fl5.12 cells was accelerated even when the BH3 domain of Bak was mutated, an event that abrogated its binding to Bcl-x L ( 35 ). This suggested that Bak works to kill cells independently of its binding to Bcl-x L . In a complementary study, Cheng et al. ( 7 ) showed that mutations of Bcl-x L could be made which retained most of their anti-apoptotic effect yet did not bind Bax or Bak as effectively in immunoprecipitations. These latter studies support growing evidence that heterodimerization of some of the pro- and anti-apoptotic members is not required for function ( 10 , 21 , 38 ). Thus, although the heteromeric binding of these two molecules has been elegantly modeled in vitro ( 34 ) it may be that the Bak responsible for apoptosis is in a cellular pool different from that bound to Bcl-x L and that this is increased after cell perturbation. The questions of Bak population heterogeneity, is all the Bak associated with Bcl-x L and whether the Bak-Bcl-x L interaction actually does occur in vivo, are difficult to resolve. The essential use of detergent to immunoprecipitate these membrane proteins has been shown to induce conformational changes that promote heteromeric associations, at least in lysates containing Bax and Bcl-x L ( 17 ). It might be argued that the results of the immunoprecipitations shown in Fig. 8 , and our finding that all the Bak was constitutively associated with Bcl-x L in untreated cells, may be the consequence of detergent use. Significantly, however, they do show that the association of Bak with Bcl-x L is changed after toxin treatment. And, critically, we observed a dissociation of pro- and anti-apoptotic molecules following damage and the exposure of Bak's NH 2 terminus, in complete contrast with the association observed between Bax and Bcl-x L that followed the exposure of the NH 2 terminus of Bax ( 17 ). The generality of some of the models for changes in heteromerization that presage apoptosis are therefore questionable. Section title: Discussion Educational score: 4.3132171630859375 Domain: biomedical Document type: Study Language: en Two-dimensional electrophoretic analysis of Bak, which surprisingly showed considerable posttranslational modification of Bak, allows speculation that different isoforms may contribute to a heterogeneity of associations in membranes . This remains to be determined. We had originally considered that toxin-induced exposure of an epitope at the NH 2 terminus of Bak may induce or alter some of the posttranslational modifications of the Bak protein. This would represent an event whereby there was an integration of signals arising from different types of cellular damage directly at the Bak molecule itself which would then contribute to changes in conformation and/or protein–protein interactions. However, we could detect no obvious change in the pattern of isoforms after toxin treatment, for example with STS or etoposide (data not shown). This suggests that damage signals are not received directly by Bak but at some associated protein(s) which may promote a change in Bak conformation and/or interactions of Bak with other proteins, including Bcl-x L . The association of accessory protein(s) in a complex with Bak is presumably changed once the NH 2 terminus has been exposed. Section title: Discussion Educational score: 4.216797828674316 Domain: biomedical Document type: Study Language: en In summary, FCM has permitted, for the first time, an analysis of changes in protein conformation and/or of protein–protein binding in intact cells expressing endogenous levels of native Bak protein. Events at the NH 2 terminus of Bak involving changes in protein–protein interactions are suggested to be important for the integration of damage signals and to the subsequent commitment of these human lymphoid cells to apoptotic death. Precise characterization of the protein(s) bound to the NH 2 terminus may clarify both how damage signals become integrated at the locus of Bak and how this subsequently commits a cell to die. | Study | biomedical | en | 0.999998 |
10085291 | Section title: Cell Lines and Cell Production Educational score: 4.140338897705078 Domain: biomedical Document type: Study Language: en 697 human lymphoblastoid cells stably infected with a retroviral expression construct containing bcl-2 cDNA or a control neomycin resistance gene were used in these studies. The cells were maintained in mid-log phase growth in RPMI 1640 medium (Irvine Scientific) supplemented with 10% FBS (Hyclone), 0.2 mg/ml G-418 ( GIBCO BRL ) and 0.1 mg/ml penicillin/streptomycin (Irvine Scientific). Murine dopaminergic MN9D cells (obtained from Dr. A. Heller, University of Chicago) were grown in MEM (Irvine Scientific) supplemented with 10% FBS, 2 mM glutamine and 0.1 mg/ml penicillin/ streptomycin. Mouse brain cortical cells were prepared at E15 of gestation in Hank's buffered saline solution (Irvine Scientific) with 15 mM Hepes. The tissue was briefly dissociated with 0.1% trypsin and washed thoroughly with MEM supplemented with 10% FBS and 0.4 mg/ml DNase I ( Sigma Chemical Co. ), gently triturated and flash frozen. The human breast carcinoma cell line T47D was obtained from American Type Culture Collection and cultured as suggested by the manufacturer. MCF-7 cells stably transfected with an expression plasmid coding for procaspase-3 was kindly supplied by Dr. C. Froelich (Northwestern Healthcare Research Institute, Evanston, IL). Section title: Subcellular Fractionation Educational score: 4.303437232971191 Domain: biomedical Document type: Study Language: en Frozen cell pellets containing ∼10 9 cells were thawed and resuspended in cold hypotonic buffer (10 mM Na-Hepes, 5 mM MgCl 2 , 42 mM KCl, pH 7.4) supplemented with 1 mM PMSF, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, 5 μg/ml aprotinin, 0.1 mM EDTA, 0.1 mM EGTA, and 5 mM DTT ( Sigma Chemical Co. ) to a density of ∼1.5 × 10 8 cells/ml. The samples were incubated on ice for 30 min at which time the cells were lysed using 30–40 strokes with a Dounce homogenizer. The sample was centrifuged twice for 10 min at 500 g , 4°C to separate the nuclei. The nuclear pellets were then washed twice in the same buffer supplemented with 1.6 M sucrose, yielding the nuclear fraction. The supernatant was then centrifuged at 14,000 g for 30 min at 4°C to pellet the heavy membranes. The heavy membranes were washed three times with 1.5 ml cold hypotonic buffer containing protease inhibitors and DTT. The washed membranes were resuspended in hypotonic buffer so that the total protein concentration was ∼2 mg/ml, yielding the heavy membrane fraction, that was either flash frozen or used immediately for enzymatic measurements without freezing. The 14,000 g supernatant was centrifuged at 100,000 g for 30 min at 4°C, yielding a supernatant (cytoplasmic fraction) and a pellet (light membrane fraction). Protein concentrations were measured using Protein Assay Kit II (Bio-Rad Laboratories) with bovine serum albumin as the calibration standard. In some experiments, cell pellets were lysed as above, but without a freezing step. To test effects of cytochrome c on caspase activity, some samples were treated with 10 μg/ml bovine cytochrome c ( Sigma Chemical Co. ) throughout the entire isolation procedure. In some experiments, mitochondrial fractions were prepared from lysed 697-neo and 697-Bcl-2 cells by the rat liver mitochondrial methods of Mancini and collaborators and used without freezing. Section title: Western Immunoblotting Educational score: 4.179834365844727 Domain: biomedical Document type: Study Language: en Subcellular fractions (50 μg protein per lane) were resolved by SDS-PAGE on 12% or 16% gels (Novex) and transferred to Immobilon PVDF membranes ( Millipore ). Membranes were blocked in PBS and 0.1% Tween 20 (PBST) + 0.4% casein (I-block, Tropix). Blots were incubated in 1 μg/ml primary antibody diluted in PBST/casein for 1 h. After three washes in PBST, blots were incubated for one hour in 1:15,000 dilutions of alkaline phosphatase conjugated goat anti–rabbit IgG or goat anti–mouse IgG (Tropix) in PBST/casein. Blots were then washed twice with PBST, twice in assay buffer (10 mM diethanolamine, pH 10.0, 1 mM MgCl 2 ), and then incubated in 250 μM chemiluminescent substrate CSPD (Tropix) in assay buffer and exposed to Biomax film ( Kodak ) overnight. In some cases, after the secondary antibody incubations, the blots were washed with 10 mM Tris, pH 9.5, 1 mM MgCl 2 . The blots were then incubated for 30 min in 1.25 μg/ml DDAO phosphate ( Amersham ) dissolved in the Tris buffer. The blots were scanned using the STORM fluorescence imager (Molecular Dynamics). The antibodies used were against Bcl-2 (clone 7; Transduction Labs), caspase-3 , cytochrome c (clone 7H8.2C12; PharMingen ), cytochrome oxidase, subunit IV (clone 1A12-A12; Molecular Probes), D4-GDP dissociation inhibitor (D4-GDI; a kind gift of Dr. G. Bokoch, Scripps Research Institute, La Jolla, CA) and poly(ADP-ribose) polymerase (PARP) (clone C2-10; Enzyme Systems). Section title: Immunocytochemistry Educational score: 4.226964950561523 Domain: biomedical Document type: Study Language: en T47D human breast carcinoma cells, MCF7 human breast carcinoma cells transduced with a control vector or caspase-3 expression vector were cultured on 8-chamber permanox slides (Nalge Nunc International Corporation). The MCF7/cont and MCF7/casp-3 cells were cultured in separate wells on the same 8-chamber slide. When the cells reached 40–50% confluence, they were fixed in ice-cold 10% formalin for 20 min, washed twice with PBS and immunostained immediately. For immunostaining, fixed cells were incubated for one hour at room temperature in blocking buffer (2% normal goat serum, 2% BSA, 0.2% nonfat milk powder, 0.4% Triton X-100 in PBS). Cells were then incubated with affinity-purified anti-caspase-3 rabbit polyclonal antibody CSP3 or purified rabbit IgG ( PharMingen ; 0.3–1.2 μg/ml), plus anti-cytochrome c mouse monoclonal antibody (clone 6H2.B4; PharMingen , 0.25 μg/ml) diluted in blocking buffer, for 1 h at room temperature. After three 5-min washes in wash buffer (PBS/0.1% Tween 20), cells were incubated for 1 h at room temperature with 0.8 μg/ml each of goat anti–rabbit IgG Alexa 488 conjugate and goat anti–mouse Alexa 594 conjugate (Molecular Probes). Finally, cells were washed three times, 5 min each, in wash buffer. The chamber divisions were removed and the cells were coverslipped under Citiflor mounting fluid (Ted Pella, Inc.). Immunstained cells were visualized by laser scanning confocal microscopy and conventional fluorescence microscopy; procaspase-3 and cytochrome c immunostaining were visualized with FITC and Texas red filters, respectively. The confocal images are single 0.4-μm optical sections. Section title: Enzyme Activity and Inhibition Studies Educational score: 4.2986626625061035 Domain: biomedical Document type: Study Language: en Caspase activity was measured by mixing 50 μl of an enzyme-containing fraction and 200 μl of 25 μM acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin(acDEVD-amc) substrate in ICE buffer (20 mM Hepes, 1 mM EDTA, 0.1% CHAPS, 10% sucrose, 5 mM DTT, pH 7.5) in duplicate 96-well Cytoplate wells (Perseptive Biosystems). Product formation was monitored by the increase in fluorescence (ex = 360 nm, em = 460 nm) over 1–2 h at 30°C using the CytoFluor 4000 plate reader (Perseptive Biosystems). For kinetic studies, the substrate concentration was varied in the range 1–100 μM. For inhibition studies the enzyme was pretreated with 150 μl inhibitor for 30 min at room temperature before the addition of 50 μl of 50 μM substrate solution. Inhibitor IC 50 values were determined using the equation: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\Delta}FL/{\Delta}t=({\Delta}FL/{\Delta}t)_{o}/(1+[I]/IC_{50})\end{equation*}\end{document} Section title: Enzyme Activity and Inhibition Studies Educational score: 4.069092273712158 Domain: biomedical Document type: Study Language: en where ΔFL/Δt is the observed initial rate of fluorescence change at inhibitor concentration [I] and (ΔFL/Δt) o is the initial rate fluorescence change for the uninhibited enzyme. Section title: Caspase Activation Educational score: 4.184632301330566 Domain: biomedical Document type: Study Language: en Heavy membrane samples were diluted to 1 mg/ml in hypotonic buffer or in 0.25 M sucrose, 10 mM MOPS, 2 mM EDTA, pH 7.4 containing 5 mM DTT with or without 1% NP-40. Caspase activation was induced by adding either 60–160 ng/ml recombinant murine caspase-1 (in bacterial lysate), 2 μg/ml of purified human granzyme B (Enzyme Systems Products) or buffer, and incubating the samples for 60 min at 30°C or 37°C. After the activation period, the heavy membrane pellet was removed from the sample by centrifugation for 10 min at 14,000 g at 4°C. The acDEVD-amc cleaving activities in the resulting supernatants were corrected for the activity of the exogenous enzymes. To examine the time course of spontaneous activation of caspase activity from membranes, 50 μl of heavy membrane slurry containing 50–100 μg total protein was mixed with 200 μl hypotonic buffer containing 25 μM acDEVD-amc substrate and 6 mM DTT in 96-well Cytoplates and fluorescence was measured over time. At selected time points, aliquots were removed from some wells, centrifuged for 10 min at 14,000 g to remove the heavy membranes, and then the supernatant was added back into the 96-well plate to measure the soluble acDEVD-amc cleavage activity. In some experiments, subcellular fractions were treated with 1 μg/ml bovine cytochrome c ( Sigma Chemical Co. ) and 50 μM dATP ( New England Biolabs ) for 40 min at 30°C before measurement of caspase activity. Section title: Production of Recombinant Caspase-1 and Caspase-3 Proteins Educational score: 4.215565204620361 Domain: biomedical Document type: Study Language: en BL21 (DE3) cells harboring a plasmid containing the cloned human caspase-3 cDNA ligated into the BamHI/XhoI sites of pET21b (Novagen) were grown in one liter LB medium containing 0.1 mg/ml ampicillin at 37°C. When the culture density reached A 600 = 1, IPTG ( Sigma Chemical Co. ) was added to a concentration of 1 mM and the culture was incubated at 25°C for 3 h. The cells were harvested by centrifugation at 2,000 g for 15 min at 4°C. The cells were lysed using one freeze-thaw cycle in 100 ml binding buffer (20 mM Tris-HCl, 500 mM NaCl, 5 mM imidazole, 0.1% Triton X-100) with 0.1 mg/ml lysozyme. Cell debris was removed from the sample by centrifugation at 20,000 g , for 30 min at 4°C. The lysed cells were treated just before centrifugation with 0.5 mM MgCl 2 and 2 μg/ml DNase I ( Sigma Chemical Co. ) to reduce viscosity. The supernatant was filtered through a 0.45-μm syringe filter and loaded onto a 1 ml Ni 2+ -charged HiTrap chelating column ( Amersham Pharmacia ) at a 1 ml/min flow rate. The column was washed at 1 ml/min with 10 ml binding buffer followed by 10 ml binding buffer containing 60 mM imidazole. The caspase-3 protein was eluted from the column using a 30-ml linear gradient of imidazole (60–500 mM). Section title: Production of Recombinant Caspase-1 and Caspase-3 Proteins Educational score: 4.2094550132751465 Domain: biomedical Document type: Study Language: en Recombinant murine caspase-1 was expressed using BL21 (DE3) pLys S cells harboring pET3ap30mICEFLAG plasmid (a generous gift of Drs. H.R. Horvitz and Ding Xue, Massachusetts Institute of Technology) which contains the p30 caspase-1 cDNA inserted into the NdeI/BamHI sites of the pET3a expression vector (Novagen). A 3-liter culture was grown at 37°C in induction medium (20 g/liter tryptone, 10 g/liter yeast extract, 6 g/liter NaCl, 3 g/liter Na 2 HPO 4 , 1 g/liter KH 2 PO 4 , 1 mM MgCl 2 , 0.1 mM CaCl 2 , pH 7.4) containing 0.1 mg/ml ampicillin and 0.025 mg/ml chloramphenicol. When the culture reached a density of A 600 = 1.0, IPTG was added to 1 mM and the culture was shaken at 25°C for 3 h. The cells were collected by centrifugation at 2,000 g for 15 min at 4°C and resuspended in 100 ml cold buffer containing 25 mM Tris-HCl, pH 8.0, 25 mM KCl, 0.1% Triton X-100, and 0.1 mg/ml lysozyme (InovaTech). The cells were lysed using one freeze/thaw cycle and the lysate was clarified by treating the sample with 2 μg/ml DNase I, 0.5 mM MgCl 2 for 60 min and then centrifuging at 20,000 g for 30 min at 4°C to remove cell debris. Section title: Characterization of Subcellular Fractions from 697 Cells Educational score: 4.203376293182373 Domain: biomedical Document type: Study Language: en Subcellular fractions were prepared from 697 cells stably infected with retroviral constructs expressing either bcl-2 cDNA or a neomycin resistance gene . Nuclear, heavy membrane, light membrane, and cytosolic fractions were isolated from these cells, and were characterized by Western blot analysis with antibodies specific for proteins with distinct known subcellular distributions. Antibodies used were directed against cytochrome oxidase, specific for mitochondrial inner membrane , PARP, specific for nuclei , D4-GDP dissociation inhibitor (D4-GDI), specific for cytoplasm and Bcl-2. As shown in Fig. 1 , the mitochondrial marker was found almost exclusively in the heavy membrane fraction, the nuclear marker only in the nuclear fraction, and the cytoplasmic marker only in the cytoplasmic fraction. Thus, the fractionation methods used generated fractions with the expected subcellular distribution of marker proteins. Importantly, we could not detect cytoplasmic contamination of the nuclear and membrane fractions, and detected only minimal mitochondrial contamination of nuclear fractions . Western analysis of fractions from 697-neo cells with an antibody to human Bcl-2 demonstrated strong reactivity in nuclear and heavy membrane fractions, weaker reactivity in the light membrane fraction, and undetectable signal in cytoplasm, in accord with previous results . Similar analysis of fractions from 697-Bcl-2 cells showed significant overexpression. Section title: Subcellular Distribution of acDEVD-amc Cleavage Activity in 697 Cells Educational score: 4.136667728424072 Domain: biomedical Document type: Study Language: en Preliminary experiments indicated that caspase activity was associated with membranes derived from unstimulated cells. To determine the subcellular distribution of such caspases, we quantitated the caspase activity in the subcellular fractions from 697-neo cells by incubating them with the substrate acDEVD-amc, and measuring the increase in fluorescence over the subsequent 2 h. acDEVD-amc is a useful substrate for all caspases characterized to date, with the exception of caspase-2 . While most of the acDEVD-amc cleavage activity (∼75%) was in the cytoplasmic fraction, a substantial amount of the cleavage activity was found in the nuclear, heavy membrane and light membrane fractions . The major acDEVD-amc cleaving activity in each fraction was indeed caspase activity since it was potently blocked by specific caspase inhibitors (Table I , column 1, and data not shown). Section title: Bcl-2 Suppresses Membrane-derived Caspase Activity Educational score: 4.365788459777832 Domain: biomedical Document type: Study Language: en Next, we examined the effect of Bcl-2 on the caspase activities in the various subcellular fractions. When subcellular fractions derived from 697-Bcl-2 cells were prepared and incubated with acDEVD-amc substrate, substantially reduced caspase activity was observed in the nuclear and heavy membrane fractions compared with 697-neo cells . This Bcl-2 effect was evident when the caspase activity was measured on a per cell basis or per mg protein and resulted in an 80–90% reduction in caspase activity in these fractions . The effect of Bcl-2 expression on caspase activity in these fractions was specific, since little if any suppression was seen in the activities observed in the cytoplasmic or light membrane fractions . These observations suggested that the membrane-associated caspase activity was not simply derived from a small percentage of apoptotic cells in the 697-neo cultures whose numbers were suppressed in the 697-Bcl-2 cultures. If that were the case, we would also have expected to see major differences in caspase activities between cytoplasmic fractions derived from 697-neo vs. 697-Bcl-2 cells. Indeed, control experiments demonstrated that when 697-neo cells were induced to undergo apoptosis by staurosporine treatment, the major increase in caspase activity was found in the cytoplasm (data not shown). The ability of Bcl-2 to suppress membrane-associated caspase activity was not limited to the 697 lymphoblastoid cells, since similar effects were observed in Jurkat T cells and FL5.12 cells (data not shown). Since our data, as well as other published studies, have demonstrated that Bcl-2 protein is found predominantly in nuclear envelope and heavy membrane fractions , our results were compatible with the possibility that Bcl-2 might act locally to regulate this membrane-derived caspase activity. In an effort to begin analyzing such mechanisms, we further characterized this membrane-derived, Bcl-2–suppressible caspase activity and focused our efforts on the heavy membrane fraction. Section title: Membrane-derived Caspase Activity Reflects Spontaneous Activation and Membrane Release Educational score: 4.3487982749938965 Domain: biomedical Document type: Study Language: en It was possible that the membrane associated caspase activity was due either to an active membrane-bound enzyme, or alternatively, to the spontaneous activation and release of a soluble active enzyme. We therefore designed a set of experiments to distinguish between these two possibilities. First, to freshly prepared heavy membranes derived from 697-neo cells (neo-membranes), we immediately added hypotonic buffer and acDEVD-amc substrate at room temperature, and measured the emergence of amc fluorescence over a 90-min period . The data demonstrate that there is little detectable fluorescence change over the first 15 min of incubation, but after this lag period, the rate of amc production increases markedly . These results indicated that the freshly prepared membranes did not contain active caspase, but that activation occurred spontaneously during the incubation period. To assess whether this newly activated caspase was soluble or membrane bound, membranes were incubated for different periods of time, after which the samples were centrifuged and the resulting supernatants were assayed for caspase activity with acDEVD-amc substrate. These data demonstrated that very little caspase activity was present in the supernatant initially, but that soluble caspase activity appeared thereafter . Quantitative analysis of these data demonstrated that for each supernatant, fluorescence increased linearly, indicating that once released from the membranes, no further activation occurred. Furthermore, the slopes of these curves approximate the instantaneous slopes of the corresponding time points in the progress curve for the heavy membrane slurry . Therefore, all of the caspase-3 activity can be accounted for in the supernatant fraction, indicating that all active enzyme had been released from the membranes. In contrast to the neo-membranes, membranes derived from the 697-Bcl-2 cells (Bcl-2-membranes) failed to generate significant acDEVD-amc cleaving activity . Section title: Procaspase-3 Is Present in Heavy Membranes from Both 697-neo and 697-Bcl-2 Cells Educational score: 4.430646896362305 Domain: biomedical Document type: Study Language: en The lack of acDEVD-amc cleaving activity in the Bcl-2-membranes could be due either to the absence of activatable procaspase or suppression of procaspase activation. To distinguish between these alternatives, we first performed Western blot analysis on the membrane fractions with antibodies specific for caspase-3, since the measured acDEVD-amc cleavage activity is in fact due to caspase-3 (see below). The results demonstrate the presence of a caspase-3 reactive band that is of similar intensity in both the neo-membranes and Bcl-2-membranes, and that is approximately the size expected for the procaspase zymogen. Interestingly, the electrophoretic mobility of the membrane-derived bands was slightly slower than that of cytoplasmic procaspase-3. To further demonstrate the presence of procaspase-3 in both neo- and Bcl-2-membranes, we attempted to activate these fractions by treatment with exogenous caspase-1, since procaspases can be activated by proteolytic cleavage at aspartic acid residues between their large and small subunits . As we have shown above, membranes derived from Bcl-2 cells showed almost no caspase activity when measured under our standard conditions. However, treatment of the Bcl-2-membranes with caspase-1 caused a robust induction of enzymatic activity . The neo-membranes were also activated by exogenous caspase-1. But importantly, after activation, the resulting caspase activities from the Bcl-2- and neo-membranes were always similar, within a factor of two . Together with the procaspase-3 immunoblot data, this supports the conclusion that comparable levels of procaspase-3 are present in neo- and Bcl-2-membranes. Section title: Procaspase-3 Is Present in Heavy Membranes from Both 697-neo and 697-Bcl-2 Cells Educational score: 4.201196670532227 Domain: biomedical Document type: Study Language: en Caspase-1-treatment of membranes not only activated the endogenous caspase activity, but also released it from the membranes, since the activity remained in the supernatant when the membranes were removed by centrifugation . This induction and release were due to the proteolytic activity of caspase-1, since the caspase-1 activation could be completely blocked by 200 nM acYVAD-aldehyde which inhibits caspase-1, but not the membrane caspase, at this concentration (data not shown). Our results indicate that both neo- and Bcl-2–expressing cells contain similar amounts of a membrane-associated inactive procaspase that can be activated by caspase-1. However, without exogenous caspase treatment, only membranes derived from the neo-expressing cells demonstrated spontaneous caspase activation. Section title: Procaspase-3 Is Present in Heavy Membranes from Both 697-neo and 697-Bcl-2 Cells Educational score: 4.192389965057373 Domain: biomedical Document type: Study Language: en To further document the presence of procaspase-3 in heavy membrane fractions we performed immunocytochemical studies, using adherent cell lines for ease of experimentation. First, we demonstrated that our affinity-purified antibody CSP3, generated against recombinant caspase-3 and used in our Western blots , was specific when used as an immunocytochemical reagent. Our staining results showed that the antibody did not react with MCF7/cont breast carcinoma cells which lack procaspase-3 due to a genetic deletion . However, the antibody showed intense staining when reacted with MCF7/casp-3 cells overexpressing recombinant procaspase-3 . To analyze the distribution of endogenous procaspase-3 in an untransfected cell line we used T47D breast carcinoma cells. The caspase-3 antibody demonstrated both diffuse and punctate staining . Much of the punctate staining colocalized with mitochondria, as visualized by anti–cytochrome c antibody . Nonspecific purified rabbit IgG did not stain these cells . These results confirm that procaspase-3 immunoreactivity associates with heavy membrane elements in cells, as was also shown using other cell types . Section title: Enzymological Characterization of the Induced and Spontaneous Caspase Activities Educational score: 4.297173500061035 Domain: biomedical Document type: Study Language: en We further characterized the membrane-derived caspase activities by measuring the inhibition of acDEVD-amc cleavage by several peptide aldehyde inhibitors (Table I ). The IC 50 values for the inhibition of acDEVD-amc activity derived from activated Bcl-2 membranes are quite similar to those for the inhibition of the activity derived from neo-membranes, suggesting that caspase-1 activates the same procaspase in both membrane preparations. Furthermore, these IC 50 values are similar to those for the spontaneously activated acDEVD-amc activity derived from neo-membranes, suggesting that the spontaneous and caspase-1–induced activities derive from the same caspase. In all cases, the inhibition data fit well to a simple competitive inhibition curve as described in the Materials and Methods, suggesting that each acDEVD-amc activity arose from a single caspase rather than a mixture of enzymes. The observed IC 50 values for the membrane associated caspases are very similar to those for purified fully processed recombinant human caspase-3. Kinetic measurements also indicate that K m values for hydrolysis of acDEVD-amc by the membrane-derived caspases (10 μM) are similar to that observed with fully processed caspase-3 . NH 2 -terminal microsequence analysis of activated, affinity-purified heavy membrane caspase confirms that this enzyme is indeed human caspase-3 (manuscript in preparation). Section title: Enzymological Characterization of the Induced and Spontaneous Caspase Activities Educational score: 4.135152816772461 Domain: biomedical Document type: Study Language: en To determine if the presence of membrane-associated caspase activity is a general property of mammalian cells, we measured the acDEVD-amc cleavage activity in heavy membranes from two other cell sources: mouse E15 primary brain cortical cells and the mouse dopaminergic MN9D cell line . Heavy membrane fractions were prepared using identical procedures to those used for the 697 cells and were activated with caspase-1. These fractions contained a membrane-associated caspase activity with similar cleavage activities per mg protein as observed in 697 cells (data not shown) and that was blocked by caspase inhibitors with a similar potency to that observed with fractions derived from 697 cells or with recombinant caspase-3 (Table I ). We conclude that the existence of membrane-derived caspase activity is not specific to 697 cells, but appears to be a more general phenomenon. Section title: Addition of Exogenous Cytochrome c Does Not Activate Membrane-associated Procaspase-3 Educational score: 4.544394493103027 Domain: biomedical Document type: Study Language: en Several recent reports have shown that the release of cytochrome c from mitochondria can cause the activation of cytoplasmic caspase-3 . Other reports have demonstrated that cytochrome c is released from mitochondria after apoptotic insults and that Bcl-2 can inhibit that release . Thus it was possible that the difference we observed between caspase activities in heavy membranes from Bcl-2– and neo-expressing cells simply reflected inhibition by Bcl-2 of cytochrome c release during preparation of the heavy membrane fractions or during subsequent incubation of these fractions. To investigate this possibility, we performed cell fractionation in the presence of exogenous cytochrome c and measured whether this influenced caspase activation. If the Bcl-2-membranes had low caspase activity because of a Bcl-2 effect on cytochrome c sequestration, then the addition of exogenous cytochrome c during membrane fractionation should increase the caspase activity derived from those membranes to the levels seen in membranes from neo-cells. Accordingly, during the fractionation procedure for heavy membranes from neo- and Bcl-2–expressing cells, we added 10 μg/ml cytochrome c to the cell lysate immediately after homogenization, and 10 μg/ml to the buffers used to suspend and wash the heavy membranes. This concentration of cytochrome c was chosen since it represents the estimated total amount of cytochrome c present in the starting cell pellets . Finally, these membranes were resuspended in 1 μg/ml cytochrome c plus 50 μM dATP, incubated, and then assayed for acDEVD-amc cleaving activity . This activity was compared with that from our usual membrane preparations prepared without cytochrome c, and incubated without cytochrome c or dATP. The data demonstrate that inclusion of cytochrome c during membrane fractionation and incubation has no effect on membrane-derived caspase activity; the activity in the membranes derived from Bcl-2–expressing cells remained low compared with the activity in the neo-membranes, and furthermore, there was also no effect of cytochrome c on the caspase activity derived from the neo-membranes . Although the cytochrome c treatments did not activate the membrane-associated caspase, the enzyme could still be activated by subsequent treatment with exogenous caspase-1 (data not shown). The lack of a cytochrome c effect on the activation of the membrane caspase was not due to an inactive preparation of cytochrome c, since the acDEVD-amc cleavage activity of the cytoplasmic fractions from both neo and Bcl-2 cells were strongly activated by inclusion of cytochrome c during fractionation and assay . We conclude that Bcl-2 expression suppresses the activation of the membrane-associated procaspase-3, but that this effect is not overcome by addition of exogenous cytochrome c. Furthermore, Bcl-2 overexpression did not affect the ability of cytochrome c to activate caspase-3 in cytoplasmic fractions. Section title: Release of Membrane-associated Caspase Activity Is Not Due to Simple Leakage from Organelles Educational score: 4.144721508026123 Domain: biomedical Document type: Study Language: en A recent report described the presence of procaspase-3 in the intermembrane space within mitochondria . Thus, it was possible that this material could account for the activatable caspase activity that we measured in our mitochondria-containing heavy membrane fractions. Furthermore, it was possible that the spontaneous activity that we measured in membrane fractions from 697-neo cells was due to leakage of active caspase from mitochondria, and that mitochondria isolated from 697-Bcl-2 cells were simply less leaky . However, several experiments suggested that the activity we measured was not due to leakage from mitochondria, and that the activity is distinct from that described by Mancini et al. . Section title: Release of Membrane-associated Caspase Activity Is Not Due to Simple Leakage from Organelles Educational score: 4.281627655029297 Domain: biomedical Document type: Study Language: en First we tested whether the addition of 1% NP-40 to neo-membranes affected the level of either spontaneous activity or the activity induced by caspase-1 or granzyme B. We reasoned that if procaspase and/or active caspase was sequestered within organelles, then enhanced activity would be measured in the presence of NP-40. Treatment with 1% NP-40 was sufficient to release almost all of the cytochrome c present in heavy membrane preparations (data not shown). Furthermore, it was shown by Mancini and colleagues that treatment of their mitochondrial preparations with 1% NP-40 allowed granzyme B to cleave procaspase-3 whereas no cleavage was observed in the absence of detergent . However, our results demonstrate that 1% NP-40 had little effect either on spontaneous activity or the activity induced by treatment with caspase-1 or granzyme B . Next, to analyze whether membrane preparations from 697-Bcl-2 cells may have low spontaneous activity due to enhanced sequestration of a caspase, we added acDEVD-amc to Bcl-2- and neo-membrane preparations, incubated them in buffer alone or buffer plus 1% NP-40, and measured the appearance of fluorescence . The results indicate that 1% NP-40 had only a minor effect on the magnitude or rate of fluorescence increase. Preparations derived from 697-Bcl-2 cells had low activity regardless of whether 1% NP-40 was present, demonstrating that this low level of activity was not due to sequestration of an active caspase. Finally, we prepared mitochondrial fractions from 697-neo and 697-Bcl-2 cells using the methods described by Mancini et al. to more directly assess the relationship between our results and their published data. As shown in Fig. 7 c, fractions from both 697-neo and 697-Bcl-2 made by these methods have granzyme B–activatable caspase activity in the absence of NP-40. However, in the presence of 1% NP-40, granzyme B treatment yielded enhanced caspase activity . Thus, under these conditions, granzyme B generates caspase activity in both NP-40–independent and –dependent manners. Section title: Discussion Educational score: 4.472675323486328 Domain: biomedical Document type: Study Language: en The present work was motivated by genetic and biochemical studies that suggested that the Bcl-2 homologue CED-9 functions by regulating the activity of the caspase CED-3 through protein-protein interactions . Given that Bcl-2 and the related death-inhibiting protein Bcl-x L are both localized to intracellular membranes , we reasoned that these molecules may act locally to regulate a membrane-associated caspase. As an initial step in investigating this hypothesis, we first demonstrated that intracellular membrane fractions do in fact contain an activatable procaspase, which when characterized, was shown to be caspase-3. Immunocytochemical evidence further supported the conclusion that heavy membrane components contain procaspase-3 . Quantitatively, the amount of membrane-derived caspase activity is relatively small compared with that in the cytoplasmic fraction; the total acDEVD-amc cleavage activity in the heavy membrane fraction was generally ∼5–10% of the total cytoplasmic activity in unstimulated cells . In cells stimulated to undergo apoptosis, there are increases in caspase activity in both membrane and cytoplasmic fractions, but the percentage that is membrane-associated remains low (data not shown). However, our results suggest that the pool of membrane-associated caspase is uniquely regulated by Bcl-2. Heavy membrane and nuclear fractions derived from 697-Bcl-2 cells demonstrated only low levels of spontaneous activation of caspase activity compared with similar fractions derived from control 697-neo cells . The heavy membranes from the Bcl-2–expressing cells did, however, contain appreciable amounts of procaspase-3, measured in two ways: directly by Western analysis , and indirectly, by measurement of acDEVD-amc cleaving activity after activation by exogenous caspase-1 . This demonstrated that Bcl-2 was not affecting the ability of procaspase-3 to associate with membranes, but rather, it exerted specific control over enzyme activation. The Western blot analysis demonstrated that the membrane-associated procaspase-3 had an electrophoretic mobility distinct from that of cytoplasmic procaspase-3. However, we do not yet know the biochemical basis for this observation. Section title: Discussion Educational score: 4.3547444343566895 Domain: biomedical Document type: Study Language: en There are many possible mechanisms by which procaspase-3 could become associated with heavy membranes. It has recently been shown that soluble procaspase-3 is present within the mitochondrial intermembrane space , and thus it was possible that this material represented the Bcl-2–regulated activity that we observed in our heavy membrane fractions. However, whereas the procaspase sequestered in the intermitochondrial space is protected from activation by exogenous granzyme B , we observed that heavy membrane caspase activity in our preparations is readily activated by exogenous caspase-1 or granzyme B . In addition, during the course of membrane-associated procaspase activation, active enzyme is continuously released into the medium in accord with the idea that a bound, inactive procaspase is converted to a soluble, active enzyme. Addition of the membrane-permeabilizing detergent NP-40 to our standard heavy membrane fractions had only a minimal effect on either spontaneous or caspase-1 activated caspase activity , suggesting that these activities were not sequestered within organelles. Thus, we favor the hypothesis that the Bcl-2–regulated procaspase-3 is physically associated with membranes and not simply soluble within a sequestered compartment, mitochondrial or other. However, we did observe that addition of NP-40 enhanced the granzyme B–activated caspase activity when fractions were prepared using methods designed to isolate intact mitochondria . Thus, it is possible that mitochondria may contain two pools of procaspase, one that is accessible to activators only with detergent, and one accessible without detergent. Section title: Discussion Educational score: 4.476629257202148 Domain: biomedical Document type: Study Language: en Caspase activity rises dramatically in the cytoplasm of cells induced to undergo apoptosis and the generation of this activity is blocked by various agents, including Bcl-2, that inhibit apoptosis . Thus, we considered whether the inhibition of caspase activation by Bcl-2 in heavy membranes was an indirect consequence of general apoptotic inhibition. For example, the caspase activity in neo-membranes could simply reflect the existence of more apoptotic cells in unstimulated 697-neo cultures vs. 697-Bcl-2 cultures. However, two observations argue against this interpretation. First, the inhibition of caspase activity by Bcl-2 in unstimulated cultures was only seen in heavy membrane and nuclear fractions. However, in 697 cells induced to undergo apoptosis by staurosporine treatment, or in Jurkat cells stimulated with anti-Fas antibody, the largest increase in caspase activity is seen in the cytoplasmic fraction, and this is blocked by Bcl-2 (data not shown). The absence of an effect of Bcl-2 on the cytoplasmic caspase activity in unstimulated cells suggests that the difference between activity in the neo-membranes and Bcl-2-membranes is not simply a passive consequence of more apoptotic cells in the 697-neo cultures. Note, however, that although the number of apoptotic cells in resting 697-neo cultures is low (<5%), this represents generally two to three times more apoptotic cells than in 697-Bcl-2 cultures, as measured by Hoechst dye or annexin V staining (data not shown). Second, the activity that we measured in neo-membranes does not reflect procaspases that were activated during prior apoptotic events since these membranes do not contain pre-existing active caspase; rather, the activity is generated during subsequent incubation . However, it is possible that the ability to autoactivate heavy membrane caspase is a specific property of a subpopulation of pre-apoptotic cells. A recent report suggests that caspases activated in the cytoplasm during apoptosis can subsequently become membrane bound but this phenomenon does not appear to account for our observation of an activatable procaspase in membranes derived from unstimulated cells. Section title: Discussion Educational score: 4.527674198150635 Domain: biomedical Document type: Study Language: en Another striking difference between membrane bound and cytoplasmic caspase-3 relates to activation by cytochrome c. The membrane-associated procaspase showed no activation in the presence of exogenous cytochrome c, even when the cytochrome c was present throughout the membrane isolation procedure and in the caspase assay buffers. It is unlikely that the failure of cytochrome c to activate this caspase was due to an inability of the cytochrome c to obtain access to the procaspase, since activation was readily effected by exogenous granzyme B. It was possible that cytochrome c failed to enhance caspase activation in neo-membranes because endogenous cytochrome c was saturating the activation mechanism. However, this is also unlikely because NP-40 treatment released endogenous cytochrome c but did not inhibit caspase activation . In contrast, cytoplasmic procaspase-3 could be robustly activated by cytochrome c when added either during fractionation, during assay, or both . Note also that cytochrome c effectively activated cytoplasmic caspases regardless of whether the cytoplasm was derived from neo- or Bcl-2–expressing cells, in accord with published data indicating that Bcl-2 functions upstream of cytochrome c–induced caspase activation . Thus, our results imply the existence of two distinct procaspase-3 activation mechanisms: a Bcl-2–regulated pathway specific to membranes and insensitive to exogenous cytochrome c; and a cytoplasmic activation pathway, directly activated by cytochrome c, but which is not directly Bcl-2–regulated. However, the activation of the cytoplasmic procaspase-3 is indirectly controlled by Bcl-2, since Bcl-2 blocks all downstream caspase events associated with apoptotic stimuli . Section title: Discussion Educational score: 4.348752021789551 Domain: biomedical Document type: Study Language: en The fact that the heavy membrane procaspase-3 from control cells becomes active spontaneously implies that it undergoes proteolytic cleavage by some protease also present in the membranes. It is possible that this cleavage occurs through autoactivation without the intervention of a separate activating protease, but this may be unlikely; procaspase-3 in the cytoplasm does not self-activate, but requires a first cleavage by caspase-9 before a second autocatalytic step . Thus, analogous to the caspase-9/procaspase-3 activating mechanism described in the cytoplasm, we speculate that the heavy membrane and nuclear fractions also contain an activating caspase capable of cleaving the membrane-associated procaspase-3. This membrane-associated activating caspase could be a form of caspase-9, or perhaps another caspase, that is regulated by Bcl-2. Section title: Discussion Educational score: 4.274369716644287 Domain: biomedical Document type: Study Language: en The specific effect of Bcl-2 on the activation of the membrane-associated caspase suggests that this caspase may play an important role in controlling apoptosis. We speculate that the membrane-associated caspase might function as a specific trigger to promote downstream apoptotic activity leading ultimately to cytoplasmic caspase activation. If so, this would place Bcl-2 at a critical control point, regulating the trigger, and thereby inhibiting diverse apoptotic events in cells. | Study | biomedical | en | 0.999997 |
10085292 | Section title: Culture Conditions and Strains Educational score: 3.891291856765747 Domain: biomedical Document type: Study Language: en Basic methods of C . elegans culture and handling were as previously described . Strains carrying the following mutations were used: unc-32(e189) , qC1 dpy-19 glp-1(q339) , him-3 , zyg-8(b235) , zyg-11(b2) , cyk-1 (or36) , mel-27 and mel-28 (Ahringer, J., personal communication), let-733 , let-748 and let-771 , emb-30(g53) , par-3(it62) , and emb-8(hc69) . The following deficiencies were used: sDf121 and sDf125 , sDf110 (from the Genetic Toolkit Project), nDf16 , nDf20 , nDf40 , tDf2 , tDf5 , tDf6 , tDf7 , and tDf9 . Information on these strains and deficiencies can be found in the C . elegans database Acedb which is available by anonymous FTP from ncbi.nlm.nih.gov , directory repository/ acedb or can be accessed online at the following Internet address: http://www.sanger.ac.uk/Projects/C_elegans/webace_front_end.shtml Section title: Mutant Collection and Deficiency Mapping Educational score: 4.105427265167236 Domain: biomedical Document type: Study Language: en Mutations were induced on an unc-32 ( e189 ) labeled third chromosome. L4 hermaphrodites of genotype unc-32 ( e189 )/ qC1 III; him-3 IV were mutagenized with 20 mM ethyl methane sulfonate according to standard procedures . F1 progeny were placed on individual plates and several F2 Unc L4 hermaphrodites were picked to a separate plate and placed at 25°C. Strains giving rise to adult Unc hermaphrodites which produced eggs that did not hatch were kept for further analysis. 254 strains carrying parental-effect embryonic lethal mutations were thus recovered from 15,600 mutagenized genomes (Schnabel, H., T. Kaletta, and R. Schnabel, manuscript in preparation). The mutations were outcrossed twice, and a male stock of each was established. This was facilitated by the him-3 mutation which causes 3.5% male production . Section title: Mutant Collection and Deficiency Mapping Educational score: 4.0768022537231445 Domain: biomedical Document type: Study Language: en The 254 strains were subdivided into sets by deficiency mapping . Complementation analysis was performed among mutations within each set, and with loci previously mapped to each of the corresponding regions. Six strains failed to complement more than one deficiency, or members of more than one complementation group, and are thus considered to carry two mutations, although no attempt has been made to separate them. In some cases, this behavior could be due to the presence of a small deficiency, rather than a double mutation. Thus, the collection comprised 260 mutations, which fell into 125 complementation groups, 81 of which had a single allele, and 44 of which had 2 or more alleles (see Tables II and III ; H. Schnabel, T. Kaletta, and R. Schnabel, manuscript in preparation). Section title: Strict and Paternal Tests Educational score: 4.113250255584717 Domain: biomedical Document type: Study Language: en The “strict” test was conducted to determine whether there is a strict requirement for maternal contribution, or, alternatively, whether a wild-type paternal allele is sufficient to rescue lethality. Five homozygous mutant hermaphrodites ( mut unc-32 / mut unc-32 ) were crossed with plg-1 males at 25°C. After mating with plg-1 males, hermaphrodites have a visible plug in the vulva, indicating that mating took place . As a control, five homozygous mutant hermaphrodites were allowed to self fertilize without males. If only nonviable eggs were produced in the cross and at least some animals showed a mating plug, the mutation was called strict (see “s” in Tables II and III ). In contrast, if live cross-progeny was produced, the mutation was deemed nonstrict. Section title: Strict and Paternal Tests Educational score: 4.117764472961426 Domain: biomedical Document type: Study Language: en The “paternal” test was conducted on nonstrict strains to determine whether zygotic expression or, alternatively, sperm cytoplasm can rescue lethality. Five homozygous mutant hermaphrodites ( mut unc-32 / mut unc-32 ) were crossed with heterozygous animals from the same strain ( mut unc-32 / qC1 ). If only non-Unc progeny ( mut unc-32 / qC1 ) were produced, then homozygous mutant progeny ( mut unc-32 / mut unc-32 ) must have died, and zygotic expression is needed to rescue lethality. Such strains are marked “z” for zygotic rescue (see Tables II and III ). In contrast, if both non-Unc progeny ( mut unc-32 / qC1 ) and Unc progeny were produced, then the rescue is independent of the genotype of the embryo, and sperm cytoplasm rescues lethality. Such strains are marked “p” for paternal rescue (see Tables II and III ). Section title: Number of Strains Analyzed Educational score: 4.1399946212768555 Domain: biomedical Document type: Study Language: en We attempted to analyze each single-allele locus of the collection, two independent alleles for loci represented by two or three alleles, and three independent alleles for loci represented by four or more alleles. However, 13 strains representing mutations in 10 loci could not be analyzed for the following reasons. First, in two strains, including a double-mutant chromosome, adult Unc hermaphrodites could no longer be found in the population at the time of analysis, even though they were present when the strains were initially derived. Second, in seven strains, including a double-mutant chromosome, homozygous mutant hermaphrodites gave rise to no or very little progeny, precluding analysis of events in one cell stage embryos. Third, for two double-mutant chromosomes, the phenotype was identical to that observed with single-mutant alleles at one of the two loci and may have precluded scoring the phenotype of the mutation at the other locus. Fourth, for one double-mutant chromosome, other single-mutant alleles at both loci could not be examined, making it impossible to ascribe the phenotype observed in the double-mutant combination to either locus with certainty. Fifth, we found during preparation of the manuscript that one strain carried two mutations; although this double-mutant chromosome had a pronuclear meeting phenotype (see Table III ), it is not included in this study because no attempt has been made as of yet to determine which of the two mutations is responsible for the phenotype. In addition, 10 strains representing 10 loci were not included in the data set because they gave rise to unfertilized embryos (3 strains) or were extremely slow to progress through the one cell stage (7 strains). Section title: Number of Strains Analyzed Educational score: 3.32140851020813 Domain: biomedical Document type: Study Language: en All together the phenotypes in one cell stage embryos were analyzed for 160 mutations in 106 loci. 2 of the 160 mutations were on double-mutant chromosomes (as indicated in Tables II and III ). Section title: Sample Preparation and Recordings Educational score: 3.967956304550171 Domain: biomedical Document type: Study Language: en Worms were grown at 16°C, picked as L4s, and shifted to 25°C for a minimum of 12 h. Gravid hermaphrodites were then dissected in M9 medium . Their embryos were collected with a mouth pipette and mounted on a 2% agarose pad. A coverslip was placed onto the pad and sealed with melted Vaseline . Section title: Sample Preparation and Recordings Educational score: 4.163647651672363 Domain: biomedical Document type: Study Language: en Embryos were analyzed at 25°C ± 1°C with a 100× (1.25 NA) Achrostigmat objective lens and standard DIC optics on a Zeiss Axioskop microscope. Under these conditions, pronuclei, asters, spindles, and nuclei can be observed in living C . elegans embryos. Early embryonic events were recorded with time-lapse video microscopy (one image every 5 s), typically from the time of pronuclear formation to the four cell stage, using a monochrome CCD camera . Section title: In Utero Observations Educational score: 4.0104522705078125 Domain: biomedical Document type: Study Language: en In a few strains, the embryos lost structural integrity upon dissection or mounting on the agarose pad. These strains were dubbed egg-osmotic sensitive (eos). For eos strains, gravid hermaphrodites were anaesthetized for 10 min in M9 medium containing 0.1% tricaine and 0.01% tetramisole, and mounted intact on an agarose pad to observe embryogenesis in utero . Although early events could usually be scored with confidence in this manner, the resolution achieved by viewing through the body wall does not equal that achieved by observing dissected embryos. Therefore, subtle phenotypic manifestations may have been missed in eos strains. Section title: Phenotypic Classes Educational score: 4.161294937133789 Domain: biomedical Document type: Study Language: en One cell stage embryos were scored for the following: number and position of female and male pronuclei, migration of pronuclei, position of pronuclear meeting, centration and rotation of centrosomes and associated pronuclei, spindle assembly, anaphase spindle positioning, and cytokinesis. The number and position of daughter nuclei in AB and P 1 were also determined. Moreover, the size of the embryo, the appearance of its cytoplasm, and the general timing of events were noted. Section title: Phenotypic Classes Educational score: 3.8371689319610596 Domain: biomedical Document type: Study Language: en Five or more embryos were analyzed for each strain, and a given phenotype was ascribed to the allele if it was observed in at least three out of the first five embryos analyzed. The observed phenotypes fell into different classes; mutations that affect one of the six cell division processes which are the focus of this report are found in Table II , mutations that affect other processes are in Table III . Section title: Phenotypic Classes Educational score: 2.878941774368286 Domain: biomedical Document type: Study Language: en The vast majority of strains (157/160) fell into only one phenotypic class. However, four strains displayed characteristics of two phenotypic classes. These strains have been classified according to their phenotype in one of the six cell division processes (see Table II ), with a mention of the other phenotypic characteristic in the comments column. Section title: Phenotypic Classes Educational score: 3.5008432865142822 Domain: biomedical Document type: Study Language: en Loci were named according to phenotypic classes (see abbreviations in Tables II and III ). Multiple allele loci which displayed allele-specific phenotypes were not given name designations. Instead, they are referred to by gene numbers used during generation of the mutant collection. In addition, strains that fell in the “progression through the one cell stage” or “unique phenotypes” classes were not given name designations, as further analysis is required to identify the primary defect in most of these cases. Section title: Other Deviations from Wild-Type Educational score: 2.2045340538024902 Domain: biomedical Document type: Study Language: en Some embryos were significantly smaller than wild-type. Being small, like being eos, was not considered as a phenotypic class per se; both traits are nevertheless indicated for the relevant strains in Tables II and III . Section title: Other Deviations from Wild-Type Educational score: 3.990638494491577 Domain: biomedical Document type: Study Language: en Aspects that were sometimes variable in wild-type and often variable in many mutant strains were not included in the phenotypic classification. These aspects include the position and the strength of the pseudocleavage furrow, the completeness of the rotation of the centrosomes and associated pronuclei, the extent of rocking of the posterior spindle pole during anaphase displacement, or the extent of cortical contractions in the AB blastomere after cytokinesis. Section title: Other Deviations from Wild-Type Educational score: 3.8086049556732178 Domain: biomedical Document type: Study Language: en Other apparent phenotypic deviations from wild-type seemed too subtle to justify a placement in a phenotypic class at this stage of the analysis. These included a minor lack of yolk granules, a slightly smaller size of pronuclei, a hesitant rotation of the centrosomes and associated pronuclei, a delay for the AB or P 1 nucleus to reach their appropriate positions, or an unstable AB nucleus. Section title: Indirect Immunofluorescence Educational score: 4.203278541564941 Domain: biomedical Document type: Study Language: en Indirect immunofluorescence was carried out with slight modifications of standard procedures . Slides were coated with 1% poly- l -lysine in PBS. 2 μl H 2 O was pipetted onto the slide, and five gravid hermaphrodites were placed into the drop and cut open with a scalpel to release embryos. An 18-mm 2 coverslip was placed onto the drop and excess fluid was wicked away with 3MM Whatman paper. The slide was frozen on a block of metal precooled on packed dry ice. After a few minutes, the coverslip was flicked off with a razor blade, and the slide was plunged into −20°C methanol for 15 min or more. Slides were rehydrated in PBS for 5 min and incubated with 30 μl of primary antibody in PBS for 45 min at room temperature in a wet chamber. The following primary antibodies were used: mouse anti-tubulin (clone DM1A, used at 1:400; Sigma ) and rabbit anti–ZYG-9 . Slides were washed for 5 min in PBT (PBS-0.05% Tween 20), 5 min in PBS, and incubated as described above for 45 min with the following secondary antibodies: 1:800 goat anti–mouse Alexa™ 488 (Molecular Probes) and 1:1,000 donkey anti–rabbit Cy3 (Dianova). Slides were placed for 5 min in PBT containing 1 μg/ml Hoechst 33258 ( Sigma ), washed 5 min in PBT, 5 min in PBS, and mounted in 6 μl of 90% glycerol containing 4% n -propyl-gallate in PBS. An 18-mm 2 coverslip was placed onto the sample and sealed with nail polish. Immunofluorescence microscopy was performed with a Zeiss Confocal Microscope (LSM 510), and images were processed with Adobe Photoshop 4.0. Section title: Screening of an Extensive Mutant Collection Leads to the Identification of 34 Loci Required for Cell Division Processes Educational score: 4.099804401397705 Domain: biomedical Document type: Study Language: en We set out to identify novel components required for cell division processes in a complex eukaryote by mutational analysis, using the one cell stage C . elegans embryo as a model system. To this end, we screened an extensive collection of maternal-effect embryonic lethal mutations on chromosome III with time-lapse DIC video microscopy (see Materials and Methods). 160 mutations in 106 loci were analyzed; an overview of the screen is given in Table I . Section title: Screening of an Extensive Mutant Collection Leads to the Identification of 34 Loci Required for Cell Division Processes Educational score: 4.083869457244873 Domain: biomedical Document type: Study Language: en We identified 48 mutations in 34 loci affecting one of six major processes which normally contribute to proper distribution of chromosomes and cytoplasmic material at cell division. These 48 strains are listed by phenotypic class in Table II . Each of the six phenotypic classes is described in the main text and is illustrated by a figure , with the top three panels (A–C) representing the relevant wild-type sequence, and the bottom three panels (D–F) the corresponding mutant sequence. In addition, Quicktime movies corresponding to these figures can be viewed on the Hyman lab web site ( http://embl-heidelberg.de/ ExternalInfo/ hyman/Data.htm ). We also identified 47 mutations in 34 loci which had other phenotypic manifestations detectable by DIC microscopy in the one cell stage embryo. These 47 strains are listed by phenotypic class in Table III . Finally, 65 mutations in 51 loci had either no phenotype or no penetrant phenotype in the one cell stage embryo (data not shown). Section title: Screening of an Extensive Mutant Collection Leads to the Identification of 34 Loci Required for Cell Division Processes Educational score: 3.461139440536499 Domain: biomedical Document type: Study Language: en All 106 loci analyzed were mapped by deficiencies to distinct regions of chromosome III (see Materials and Methods). The location of the loci reported in Tables II and III is given in Fig. 1 . Section title: Time-Lapse DIC Video Microscopy Reveals Six Major Classes of Mutants in Cell Division Processes Educational score: 4.086790561676025 Domain: biomedical Document type: Study Language: en Analysis of one cell stage embryos typically started ∼20 min after fertilization. In wild-type at this stage, the male and female pronuclei are clearly visible by DIC microscopy at opposite poles of the embryo . The male pronucleus is tightly apposed to the cortex , while the female pronucleus lies slightly off the cortex . Since the position of the male pronucleus defines the future posterior of the embryo , the location of the male and female pronuclei serve to orient the embryo, which is shown with anterior to the left and posterior to the right in all figures. Section title: Time-Lapse DIC Video Microscopy Reveals Six Major Classes of Mutants in Cell Division Processes Educational score: 4.249151706695557 Domain: biomedical Document type: Study Language: en The sperm contributes the single centrosome of the one cell stage embryo . This centrosome duplicates and the two resulting centrosomes then separate, while remaining associated with the male pronucleus . The cell division that follows entails a sequence of six major processes which can be monitored by DIC microscopy: pronuclear migration, rotation of centrosomes and associated pronuclei, spindle assembly, anaphase spindle positioning, chromosome segregation, and cytokinesis . Screening of the mutant collection with time-lapse DIC video microscopy led to the identification of the first point in time when a given mutant deviates from the wild-type sequence. Mutant strains affecting cell division processes were thus classified into six phenotypic classes, corresponding to defects in one of the six major processes which take place in wild-type. For each of these processes, we first describe the events in wild-type and then those in the corresponding mutant strains. Section title: Pronuclear Migration Mutants Educational score: 3.924842119216919 Domain: biomedical Document type: Study Language: en The fiirst process to be considered is pronuclear migration. In wild-type, the female pronucleus migrates away from the anterior region, slowly at first and faster thereafter; meanwhile, the male pronucleus migrates slightly away from the posterior cortex . As a result of these migrations, the female and male pronuclei meet at 70% egg length (0%: anterior-most; 100%: posterior-most). Section title: Pronuclear Migration Mutants Educational score: 4.302588939666748 Domain: biomedical Document type: Study Language: en Pronuclear migration was defective in seven mutants representing six loci in the collection. We define as pronuclear migration mutants those strains in which the female and male pronuclei are initially positioned similarly to wild-type , but fail to migrate towards each other . In two pronuclear migration mutants (see Table II ), a spindle which appears smaller than wild-type assembles around the male pronucleus at the very posterior of the embryo and perpendicular to the longitudinal axis . This spindle moves off the posterior cortex by the end of mitosis, and is bisected by a cleavage furrow ingressing from the posterior and along the longitudinal axis. In the remaining five pronuclear migration mutants (see Table II ), the spindle is usually barely detectable or not visible at all after breakdown of the male pronucleus. Consistent with the absence of a functional spindle, there is usually no cleavage furrow ingression from the posterior of the embryo. In occasional pronuclear migration mutant embryos from either set, the female pronucleus undergoes part of its migration towards the posterior. In others, there is more than one female pronucleus, indicating that the female meiotic divisions can be affected as well. Section title: Pronuclear Migration Mutants Educational score: 4.068514823913574 Domain: biomedical Document type: Study Language: en Interestingly, in all pronuclear migration mutants, the nuclear envelope of the male pronucleus breaks down ∼1 min before that of the female pronucleus . This asynchrony in pronuclear envelope breakdown may reflect a wave in cell cycle progression along the longitudinal axis of the embryo (see Discussion). Section title: Rotation Mutants Educational score: 3.9186766147613525 Domain: biomedical Document type: Other Language: en After pronuclear migration in wild-type, the centrosome pair and associated pronuclei move to the embryo center, while undergoing a 90° rotation that aligns the centrosomes along the longitudinal axis . Section title: Rotation Mutants Educational score: 4.290440082550049 Domain: biomedical Document type: Study Language: en Rotation was defective in three mutants representing three loci in the collection. We define as rotation mutants those strains in which the pronuclei migrate as in wild-type , but in which the centrosome pair and associated pronuclei then fail to rotate. In two rotation mutants (see Table II ), the centrosome pair and associated pronuclei also fail to center, and the spindle thus assembles at 70% egg length, perpendicular to the longitudinal axis . In the third rotation mutant (see Table II ), the centrosome pair and associated pronuclei do center, and in fact move towards the anterior more so than in wild-type; as a result, the spindle assembles at 30–40% egg length, perpendicular to the longitudinal axis. In all three rotation mutants, the spindle is reoriented along the longitudinal axis by the end of anaphase , perhaps because of the physical constraints of the eggshell. As a consequence of this reorientation, roughly normal-sized daughter blastomeres are generated. However, these blastomeres often have more than one nucleus, indicative of problems in chromosome segregation. Section title: Spindle Assembly Mutants Educational score: 3.7960903644561768 Domain: biomedical Document type: Other Language: en After rotation in wild-type, the nuclear envelopes of the apposed pronuclei break down and a bipolar spindle is assembled along the longitudinal axis, in the center of the embryo . Section title: Spindle Assembly Mutants Educational score: 4.286394119262695 Domain: biomedical Document type: Study Language: en Spindle assembly was defective in five mutants representing three loci in the collection. We define as spindle assembly mutants those strains in which the pronuclear envelopes break down similarly to wild-type, but in which a bipolar spindle is not apparent thereafter . Although there is an area in the center that appears clear by DIC microscopy, this area lacks bipolarity. Consistent with the absence of a functional spindle, the cleavage furrow does not form and many small nuclei, presumably containing nonsegregated chromosomes, form as the cell returns to interphase . In a minority of spindle assembly mutant embryos, prior rotation of the centrosome pair and associated pronuclei is also defective. Section title: Anaphase Spindle Positioning Mutants Educational score: 4.292821407318115 Domain: biomedical Document type: Study Language: en After spindle assembly in wild-type, the posterior spindle pole is displaced towards the posterior during anaphase, resulting in an asymmetric spindle position along the longitudinal axis . Since the cleavage furrow in animal cells is specified by the end of anaphase to form at equal distance from the spindle poles , this posterior displacement in the one cell stage C . elegans embryo results in an asymmetric placement of the cleavage furrow . Section title: Anaphase Spindle Positioning Mutants Educational score: 4.075560569763184 Domain: biomedical Document type: Study Language: en As expected from previous work , we found that mutations at the par-3 locus abolish posterior anaphase displacement, resulting in a symmetric first cell division (Table III ). However, this lack of posterior displacement is probably a secondary consequence of the known requirement for par-3 in setting up antero-posterior embryonic polarity . To indicate this fact, par-3 is not included among the loci strictly required for cell division processes which are reported in Table II ; instead, par-3 is mentioned in Table III . Section title: Anaphase Spindle Positioning Mutants Educational score: 4.227346420288086 Domain: biomedical Document type: Study Language: en We found that anaphase spindle positioning was defective in a novel manner in nine mutants representing six loci in the collection. In these anaphase spindle positioning mutants, the spindle is usually assembled in the cell center similarly to wild-type , but is displaced to aberrant locations on the cortex during anaphase. In five anaphase spindle positioning mutants (see Table II ), the spindle exhibits an exaggerated and abrupt movement towards the posterior during anaphase . The spindle thus comes in contact with the posterior cortex and is reoriented perpendicular to the longitudinal axis by the end of anaphase, resulting in an aberrant first cleavage . In four other anaphase spindle positioning mutants (see Table II ), the spindle drifts slowly during anaphase, and ends up being positioned towards the posterior or lateral cortices. In a minority of anaphase spindle positioning mutant embryos from either set, prior rotation of the centrosome pair and associated pronuclei is defective, in which case the spindle sets up perpendicular to the longitudinal axis and tends to drift towards the anterior of the embryo. Section title: Chromosome Segregation Mutants Educational score: 4.035451412200928 Domain: biomedical Document type: Study Language: en In wild-type, the spindle segregates one chromosome complement to each daughter of the first cell division . In contrast to the other processes described here, chromosome segregation cannot be directly observed by DIC microscopy in C . elegans embryos because individual chromosomes cannot be seen. However, proper chromosome segregation can be inferred from the presence of two equally sized daughter nuclei, one in each daughter blastomere . Section title: Chromosome Segregation Mutants Educational score: 4.207822799682617 Domain: biomedical Document type: Study Language: en According to this criterion, chromosome segregation was defective in 18 mutants representing 12 loci, comprising the largest phenotypic class in the collection. We define as chromosome segregation mutants those strains in which a bipolar spindle is assembled similarly to wild-type , but generates more than one nucleus in daughter blastomeres . This indicates that the spindle is unable to properly segregate the entire complement of chromosomes to daughter cells. Perhaps some chromosomes are lagging during anaphase, and are thus not incorporated in the larger nucleus that reforms after telophase. In 15 chromosome segregation mutant strains, the first division spindle looks indistinguishable from wild-type by DIC microscopy, while it has a somewhat unusual appearance in the other three . In seven chromosome segregation mutant strains (see Table II ), some embryos have more than one female pronucleus, indicative of defects during the female meiotic divisions. Section title: Cytokinesis Mutants Educational score: 3.922809362411499 Domain: biomedical Document type: Other Language: en Cytokinesis is the last cell division process to be observed in the one cell stage embryo. In wild-type , the cleavage furrow is first specified at equal distance from the spindle poles , then ingresses and finally cleaves the cell into two unequally sized blastomeres . Section title: Cytokinesis Mutants Educational score: 4.222271919250488 Domain: biomedical Document type: Study Language: en Cytokinesis was defective in six mutants representing four loci in the collection. We define as cytokinesis mutants those strains in which the spindle is positioned by the end of anaphase similarly to wild-type , but in which the cell fails to cleave in two. In two cytokinesis mutants (see Table II ), the cleavage furrow is not visible by DIC optics and may thus not be properly specified. In the remaining four cytokinesis mutants (see Table II ), the cleavage furrow is specified in the correct location , starts to ingress , but then regresses before going to completion . In both sets of cytokinesis mutant embryos, daughter nuclei then appose in the undivided cell . Cytokinesis during the female meiotic divisions seems also affected: additional nuclei, most likely corresponding to nonextruded polar body material, join the daughter nuclei from the anterior of the embryo . Section title: Immunofluorescence Microscopy Reveals that Two Pronuclear Migration Loci Are Required for Generating Normal Microtubule Arrays and Four for Centrosome Separation Educational score: 4.105698585510254 Domain: biomedical Document type: Study Language: en Secondary screens customized for each phenotypic class will enhance understanding of the underlying cellular defect. While it is beyond the scope of this study to perform secondary screens on every phenotypic class, we wanted to examine pronuclear migration mutants further to elucidate the basis of a subdivision that was apparent from our observations with time-lapse DIC video microscopy. We had noted that a small but detectable spindle assembled around the male pronucleus and moved off the posterior cortex during mitosis in two pronuclear migration mutants. In contrast, the spindle was barely detectable or seemed altogether absent in five mutants of this class. To better understand the basis for this subdivision, we analyzed fixed wild-type and pronuclear migration mutant embryos with anti-tubulin and anti–ZYG-9 antibodies, as well as with the DNA dye Hoechst 33258. Section title: Immunofluorescence Microscopy Reveals that Two Pronuclear Migration Loci Are Required for Generating Normal Microtubule Arrays and Four for Centrosome Separation Educational score: 4.1176958084106445 Domain: biomedical Document type: Study Language: en We examined the distribution of microtubules because migration of both male and female pronuclei is abolished in the presence of the microtubule-depolymerizing agent nocodazole , as well as in zyg-9 mutant embryos, which exhibit short astral microtubules . Therefore, short or otherwise abnormal microtubules may be causing the phenotype in some pronuclear migration mutants identified here. In addition, we simultaneously examined the distribution of ZYG-9 protein, a convenient centrosomal marker which maintains this localization even after nocodazole treatment . Section title: Immunofluorescence Microscopy Reveals that Two Pronuclear Migration Loci Are Required for Generating Normal Microtubule Arrays and Four for Centrosome Separation Educational score: 4.355579853057861 Domain: biomedical Document type: Study Language: en We examined embryos both during prophase, when pronuclear migration normally takes place, and during mitosis, when a potential difference in spindle morphology may be apparent. In wild-type, the single centrosome lies initially between the male pronucleus and the posterior cortex . After centrosome duplication, the two resulting centrosomes separate and move towards the anterior, seemingly along the surface of the male pronucleus. As a result, both centrosomes lie on the anterior of the male pronucleus, facing towards the female pronucleus . The cell is in prophase at this stage, as can be seen from condensing DNA in both male and female pronuclei. During prophase, astral microtubules emanating from the centrosomes become numerous and some of them are fairly long . The dense mesh of cortical microtubules which is predominant in earlier stages is still present during prophase, but begins to disappear . During anaphase , an extensive array of astral microtubules extends towards the anterior and posterior cortices , while spindle microtubules extend centrally towards the chromosomes . Section title: Immunofluorescence Microscopy Reveals that Two Pronuclear Migration Loci Are Required for Generating Normal Microtubule Arrays and Four for Centrosome Separation Educational score: 4.186373710632324 Domain: biomedical Document type: Study Language: en We found that astral microtubules were short in the first set of two pronuclear migration mutants, representing two loci (see Table II ). During prophase, astral microtubules emanating from the centrosomes were consistently shorter than in wild-type . This was still the case during anaphase . At this stage, a spindle which contained less microtubules than in wild-type was apparent . This attenuated spindle was perpendicular to the longitudinal axis and located slightly off the posterior cortex, as had been suggested by the DIC observations. Chromosomes coming from the male pronucleus were found to lie on this attenuated spindle in 19 out of 20 embryos examined for the two mutants combined . In contrast, chromosomes coming from the female pronucleus were left towards the anterior of the embryo . Section title: Immunofluorescence Microscopy Reveals that Two Pronuclear Migration Loci Are Required for Generating Normal Microtubule Arrays and Four for Centrosome Separation Educational score: 4.227974891662598 Domain: biomedical Document type: Study Language: en Interestingly, ZYG-9 protein distribution was affected in these two mutants, in a similar manner. First, ZYG-9 was localized to the anterior cortex , where it is not detected in wild-type . In some embryos, ZYG-9 was present at the anterior cortex in patches . Second, compared with wild-type, ZYG-9 signal appeared stronger at centrosomes and spindle poles . Taken together, these observations suggest that these two pronuclear migration loci are required for generating normal microtubule arrays and for proper subcellular localization of ZYG-9 protein. Section title: Immunofluorescence Microscopy Reveals that Two Pronuclear Migration Loci Are Required for Generating Normal Microtubule Arrays and Four for Centrosome Separation Educational score: 4.244287490844727 Domain: biomedical Document type: Study Language: en Somewhat unexpectedly, we found that centrosomes failed to separate in the second set of five pronuclear migration mutants, representing four loci (see Table II ). In prophase, the centrosome had duplicated, but the resulting daughter centrosomes failed to separate and remained positioned posterior of the male pronucleus . During mitosis , the two centrosomes were still in close proximity to one another and located at the very posterior of the embryo . Probably as a consequence of this failure in centrosome separation, no bipolar spindle was assembled, as had been suggested by the DIC observations. Chromosomes were not located between the spindle poles in 42 out of 43 embryos examined during mitosis for the five mutants combined . Microtubules seemed to grow preferentially or to be stabilized in the vicinity of chromosomes , and were occasionally fairly long . In some mutant embryos, centrosomes were separated to a greater extent towards the end of mitosis (data not shown). Taken together, these observations suggest that these four pronuclear migration loci are required for centrosome separation. Section title: Large Scale Mutational Analysis Reveals Spectrum of Phenotypic Classes Affecting Cell Division Processes in the One Cell Stage Embryo Educational score: 4.075434684753418 Domain: biomedical Document type: Study Language: en Mutational analysis has been successfully used to dissect a number of biological processes. Often, success has relied on having a mutant collection sufficiently extensive to recognize the full spectrum of phenotypic classes. Thus, analysis of a large number of cell division cycle mutants in S. cerevisiae was instrumental for unraveling the dependence between successive events in the cell cycle . In Drosophila , a large scale screen revealed that a limited number of gene classes pattern the early embryo . Section title: Large Scale Mutational Analysis Reveals Spectrum of Phenotypic Classes Affecting Cell Division Processes in the One Cell Stage Embryo Educational score: 4.248320579528809 Domain: biomedical Document type: Study Language: en The availability of an extensive mutant collection was also of utmost importance in this work. This allowed us to recognize that mutations affecting cell division processes in the one cell stage C . elegans embryo fall into a limited number of characteristic phenotypic classes when analyzed by DIC microscopy. The spectrum of phenotypic classes was not apparent from earlier studies relying on smaller mutant collections. Maternal-effect embryonic lethal mutations affecting aspects of C . elegans development have been identified previously, notably among three sets of temperature-sensitive mutant collections , and one set of nonconditional mutants on chromosome II . However, these studies did not focus solely on the one cell stage embryo nor on cell division processes, in contrast to what was done here. More recently, a screen specifically designed to identify mutants in cell division processes has generated 19 temperature-sensitive mutants, 10 of which display defects in early embryos . However, it is clear that this screen was far from saturation, as all 19 mutations were single alleles of different loci. In contrast, 39 loci of the chromosome III collection analyzed here are represented by multiple alleles, arguing that this collection is significantly closer to saturation. Thus, analyzing the most extensive mutant collection to date has allowed us to build on earlier studies and recognize a well-defined spectrum of phenotypic classes affecting cell division processes in the one cell stage embryo. Section title: 100–400 True Maternal-Effect Genes May Be Required Genome-wide for Cell Division Processes in the One Cell Stage Embryo Educational score: 4.299748420715332 Domain: biomedical Document type: Study Language: en Screening an extensive mutant collection has also allowed us to estimate the number of true maternal-effect genes that are required genome-wide for cell division processes in the one cell stage embryo. We found that mutations in 34 loci on chromosome III affect one of these processes. These mutations probably alter two different sets of genes; the first of these corresponds to true maternal-effect genes, which encode components required solely in the early embryo. Such genes should mutate to a maternal-effect lethal phenotype with reasonable frequency. It follows that many of the 39 multiple-allele loci analyzed in the chromosome III collection may correspond to true maternal-effect genes. Mutations in 15 (38%) of these multiple-allele loci affect a cell division process in the one cell stage embryo. The number of genes in C . elegans which mutate with reasonable frequency to maternal-effect embryonic lethality is postulated to be 300–1,000 . Assuming an even distribution of true maternal-effect genes in the genome, one can extrapolate that some 100–400 of them may be required for cell division processes genome-wide. Section title: 100–400 True Maternal-Effect Genes May Be Required Genome-wide for Cell Division Processes in the One Cell Stage Embryo Educational score: 4.1861138343811035 Domain: biomedical Document type: Study Language: en The second set comprises genes which encode components required not only in the early embryo but also later during development. The null phenotype in such genes is usually zygotic lethal. However, rare mutations in these genes may still be isolated as maternal-effect alleles if, for instance, they generate sufficient gene product to sustain a given process in the small larval and adult cells, but not in the larger embryonic blastomeres. Thus, several single- allele loci in maternal-effect mutant collections turned out to be hypomorphic alleles of essential genes . Such rare mutations are drawn from a very large pool of genes, whose size cannot be estimated with accuracy. Section title: Time-Lapse DIC Video Microscopy Permits Identification of Mutations Affecting Specific Cell Division Processes Educational score: 4.1950907707214355 Domain: biomedical Document type: Study Language: en Detailed time-lapse analysis was crucial during our screen for determining the first deviation from the wild-type sequence of events, thus identifying the earliest cell division process for which a given component may be required. It is worth noting that an early phenotypic manifestation may mask a potential requirement in a later process. For example, if a mutation results in a failure in pronuclear migration, a requirement for the corresponding locus in subsequent rotation may not be assessed. Importantly, how early the first deviation from wild-type occurs can yield clues about the type of component encoded by the corresponding locus. Indeed, one might expect that mutations affecting fundamental aspects of cytoskeletal function, such as microtubule integrity, would interfere with many cell division processes, and, thus, exhibit a very early defect. Conversely, mutations affecting components more specifically required for a later cell division process should not exhibit a very early defect. Section title: Time-Lapse DIC Video Microscopy Permits Identification of Mutations Affecting Specific Cell Division Processes Educational score: 4.172176361083984 Domain: biomedical Document type: Study Language: en In fact, our analysis demonstrates that the vast majority of mutations do not exhibit a very early defect, and may thus identify loci required for specific cell division processes. Importantly, we note that for 29 of 39 (74%) multiple allele loci, all alleles examined fall in the same phenotypic class, indicating that the encoded genes may indeed be primarily required for the corresponding cell division process. However, whether this will truly be the case remains to be determined for each locus. Indeed, some of the mutations are likely not null, and additional alleles may reveal that some loci are required for other processes as well. Moreover, in some cases, the function of a molecule in a given process may be difficult to unravel by mutational analysis. For instance, a small protein domain required for a given process may be rarely hit by mutagen. Section title: Time-Lapse DIC Video Microscopy Permits Identification of Mutations Affecting Specific Cell Division Processes Educational score: 4.075030326843262 Domain: biomedical Document type: Study Language: en Even in the event that a mutation is not null, the availability of mutations that specifically disrupt a given process can provide important insights. For example, in Drosophila , although the null phenotype of mutations in the actin binding protein profilin is embryonic lethality, female-sterile alleles at this locus revealed the role of the actin cytoskeleton in regulating transport of cytoplasmic material during oogenesis . Section title: Pronuclear Migration: An Early Process Which Relies on Fundamental Aspects of Cytoskeletal Function Educational score: 4.234155654907227 Domain: biomedical Document type: Study Language: en The earliest defect in a cell division process that we uncovered is a failure in both male and female pronuclear migration. Analysis of this phenotypic class demonstrates that the corresponding genes are required, as expected, for fundamental aspects of cytoskeletal function. In addition, analysis of the timing of nuclear envelope breakdown in this mutant class reveals the existence of a wave in cell cycle progression in the one cell stage embryo. Section title: Pronuclear Migration: An Early Process Which Relies on Fundamental Aspects of Cytoskeletal Function Educational score: 4.433598041534424 Domain: biomedical Document type: Study Language: en When newly fertilized embryos are treated with nocodazole, migration of both male and female pronuclei is abolished . Therefore, this phenotype is the first deviation from wild-type expected for mutations in loci generally required for microtubule structure or function. Accordingly, our immunofluorescence data show that astral microtubules are shorter than wild-type in mutations at two of the pronuclear migration loci identified here. Short astral microtubules likely prevent migration of the male and female pronuclei in different ways, as distinct mechanisms are thought to normally govern these processes. In wild-type, migration of the male pronucleus away from the posterior cortex may result from the movement of the associated centrosomes . Polymerization forces from astral microtubules would act against the posterior cortex to push centrosomes and associated male pronucleus away from it. In contrast, migration of the female pronucleus is postulated to result from its interaction with astral microtubules emanating from the centrosomes. Minus-end–directed motors anchored on the female pronuclear membrane would power movement along microtubules towards the centrosomes, and, thus, towards the male pronucleus . Based on these models, the failure in male pronuclear migration in the two mutants with short astral microtubules likely results from insufficient polymerization forces acting against the cortex to push centrosomes and associated male pronucleus away from it. Meanwhile, the lack of female pronuclear migration may be due to astral microtubules not reaching all the way to the female pronuclear membrane. Section title: Pronuclear Migration: An Early Process Which Relies on Fundamental Aspects of Cytoskeletal Function Educational score: 4.492254734039307 Domain: biomedical Document type: Study Language: en Intriguingly, our immunofluorescence data raise the possibility that ZYG-9 protein mislocalization is involved in generating the short astral microtubule phenotype in these two pronuclear migration mutants. Short astral microtubules are also observed in zyg-9 mutant embryos, and ZYG-9 is a member of an evolutionarily conserved family of microtubule-binding proteins which promote microtubule growth in vitro . Here, we found that ZYG-9 protein localization is abnormal in these two mutant strains. Most notably, ZYG-9 protein is present at the anterior cortex, where it is not detected in wild-type. Possibly ZYG-9 protein trapped at this location promotes abnormal microtubule growth. Consistent with this view, cortical microtubules appear denser than in wild-type at the anterior cortex in these mutant embryos . This may in turn result in less efficient microtubule growth in the cytoplasm, perhaps because a rate-limiting component is trapped at the anterior cortex along with ZYG-9. Further analysis is needed to determine whether ZYG-9 mislocalization indeed causes the short astral microtubule phenotype. In the meantime, however, the present observations demonstrate that these two pronuclear migration loci are required for proper ZYG-9 subcellular localization. Section title: Pronuclear Migration: An Early Process Which Relies on Fundamental Aspects of Cytoskeletal Function Educational score: 4.255213737487793 Domain: biomedical Document type: Study Language: en Our immunofluorescence analysis also led to the unexpected discovery that centrosome separation is a prerequisite for the migration of both male and female pronuclei, as separation is defective in mutations at the remaining four pronuclear migration loci. The unseparated centrosomes do not leave the vicinity of the posterior cortex, despite fairly long astral microtubules. Perhaps the overlap of astral microtubules emanating from two centrosomes in close proximity to one another causes steric hindrance which prevents polymerization forces from efficiently acting against the posterior cortex. Alternatively, the lack of centrosome separation may signal to prevent the subsequent migration of centrosomes away from the posterior cortex. Section title: Asynchronous Breakdown of Pronuclei in Pronuclear Migration Mutant Embryos Reveals Possible Regional Differences in Cell Cycle Progression Educational score: 4.161420822143555 Domain: biomedical Document type: Study Language: en Time-lapse observations revealed a remarkable property of pronuclear migration mutants. In all mutant embryos of this class, the nuclear envelope of the male pronucleus breaks down ∼1 min before that of the female pronucleus. A similar asynchrony had been noted in zyg-9 mutant embryos , and is observed in embryos treated with nocodazole (Gönczy, P., unpublished observations). The generality of this phenomenon suggests that it reflects a fundamental feature of the one cell stage C . elegans embryo, rather than a peculiar behavior of particular mutant strains. Section title: Asynchronous Breakdown of Pronuclei in Pronuclear Migration Mutant Embryos Reveals Possible Regional Differences in Cell Cycle Progression Educational score: 4.328214168548584 Domain: biomedical Document type: Study Language: en In sea urchin zygotes in which pronuclear fusion has been prevented, the female pronucleus always breaks down earlier than the male pronucleus . In this case, nuclear envelope breakdown is controlled at the level of each individual pronucleus, rather than by regional differences of cell cycle progression, as the female pronucleus breaks down earlier even if it is found by chance in the immediate vicinity of the male pronucleus . In contrast, in C . elegans , both pronuclei appear to undergo nuclear envelope breakdown synchronously when they happen to be juxtaposed in rare pronuclear migration mutant embryos (Gönczy, P., unpublished observations). Therefore, the asynchrony observed under conditions where the two pronuclei are distant from one another most likely results from regional differences in cell cycle progression. Section title: Asynchronous Breakdown of Pronuclei in Pronuclear Migration Mutant Embryos Reveals Possible Regional Differences in Cell Cycle Progression Educational score: 4.336215019226074 Domain: biomedical Document type: Study Language: en One possibility to explain such regional differences is that par genes, which set up polarity along the antero-posterior axis shortly after fertilization , simultaneously set up a wave of cell cycle progression emanating from the posterior pole. Another possibility is that centrosomes, which remain at the posterior of pronuclear migration mutant embryos, are preferred sites of cdc2 activation. In this context, it might be interesting to note that cyclin B1, a component of cdc2 kinase, is associated with centrosomes in HeLa cells . Perhaps a component or an activator of cdc2 kinase similarly localizes to centrosomes in the one cell stage C . elegans embryo to start a wave of cdc2 kinase activity. Section title: Centration and Rotation of Centrosomes Can Be Separated by Mutational Analysis Educational score: 3.68558669090271 Domain: biomedical Document type: Study Language: en It has been proposed that centration and rotation of centrosomes result from capture and pulling of astral microtubules by the anterior cortex . One centrosome would be pulled preferentially, thus generating a torque which induces rotation of the entire centrosome–pronuclear complex. However, to what extent centration and rotation are separable processes was previously unknown. Section title: Centration and Rotation of Centrosomes Can Be Separated by Mutational Analysis Educational score: 4.263523101806641 Domain: biomedical Document type: Study Language: en During this screen, we have identified two kinds of rotation mutants. In the first kind, represented by two mutant strains, both centration and rotation are defective. Possibly, these mutations correspond to weak alleles of loci more generally required for microtubule structure and function, since astral microtubules are required for centration and rotation . Compatible with this view, pronuclear migration is sometimes defective in one of these two strains (see Table II ). Alternatively, however, these mutations may identify components required more specifically for generating anteriorly directed pulling forces. A similar centration and rotation phenotype has been described for let-99 mutant embryos , as well as for embryos in which the C . elegans homologues of the dynactin components p50/dynamitin or p150 Glued have been inactivated by RNAi . This latter finding has reinforced the view that the minus-end–directed motor cytoplasmic dynein may power rotation by pulling astral microtubules towards the anterior cortex. Section title: Centration and Rotation of Centrosomes Can Be Separated by Mutational Analysis Educational score: 4.19123649597168 Domain: biomedical Document type: Study Language: en In the second kind of rotation mutant identified here, represented by one mutant strain, rotation alone is defective. Centrosomes centrate and move all the way towards the anterior without ever undergoing rotation, suggesting that anteriorly pulling forces are still active. Perhaps the lack of rotation in this mutant is due to the fact that forces are equally pulling on both centrosomes, and that no torque is generated under these conditions. Thus, centration and rotation can be separated by mutational analysis and, thus, likely correspond to distinct, though coupled, processes. Section title: Components Governing Spindle Assembly and Function Educational score: 4.068263530731201 Domain: biomedical Document type: Study Language: en Upon entry into mitosis, the interphase microtubule network reorganizes into a bipolar array to form the mitotic spindle. Studies using primarily Xenopus egg extracts have shown that this reorganization involves participation of microtubule motors and appropriate modulation of microtubule dynamics . However, it remains to be determined to what extent such factors govern spindle assembly in vivo. Section title: Components Governing Spindle Assembly and Function Educational score: 4.215928077697754 Domain: biomedical Document type: Study Language: en We have identified mutations at four loci in which microtubules do not assemble a bipolar spindle following pronuclear envelope breakdown. These spindle assembly mutants undergo earlier interphasic microtubule-dependent processes such as pronuclear migration. Therefore, the corresponding loci may encode motor proteins or regulators of microtubule dynamics which are specifically required during mitosis. Alternatively, components distinct from these may be identified by such an unbiased mutational analysis, and thus define novel mechanisms governing spindle assembly in vivo. Section title: Components Governing Spindle Assembly and Function Educational score: 4.3003764152526855 Domain: biomedical Document type: Study Language: en Analysis of spindle assembly mutants revealed that the spindle assembly checkpoint is not active in the one cell stage C . elegans embryo. We noted that, despite the absence of a bipolar spindle, all mutant embryos of this class exit mitosis and progress into the next cell cycle without significant delay. Therefore, the spindle assembly checkpoint, which prevents most eukaryotic cells with an unassembled spindle from progressing into anaphase , must be inactive in the early C . elegans embryo. This parallels observations in Xenopus where in vitro experiments have demonstrated that the checkpoint machinery is inactive in the early embryo due to a low nucleus/cytoplasmic volume ratio . Section title: Components Governing Spindle Assembly and Function Educational score: 4.258973121643066 Domain: biomedical Document type: Study Language: en We have also identified mutations in 12 loci which yield daughter blastomeres with more than one nucleus, despite the presence of a bipolar spindle during the first division. This phenotype most likely results from having nonclustered chromosomes at telophase, when chromatin induces nuclear envelope reformation. These chromosome segregation mutants are analogous to mutants in the fidelity of chromosome transmission in S . cerevisiae . Detailed analysis of the yeast mutants demonstrated that they affect distinct processes contributing to proper chromosome transmission . Similarly, appropriate secondary screens will undoubtedly break down C . elegans chromosome segregation mutants into subclasses, corresponding to distinct subprocesses, including aspects of kinetochore function. Direct visualization of individual chromosome behavior in these mutants should be particularly revealing in this regard. Section title: A Balance of Forces May Act to Properly Position the First Division Spindle Educational score: 4.309536933898926 Domain: biomedical Document type: Study Language: en Asymmetric cell divisions play a central role in the generation of cell diversity in a number of organisms . In the wild-type one cell stage C . elegans embryo, the mitotic spindle becomes asymmetrically positioned following a posterior displacement of the posterior spindle pole during anaphase. Since the cleavage furrow is specified to form equidistant from the spindle poles , this results in an asymmetric division which yields a larger anterior blastomere and a smaller posterior one. The mechanisms which mediate posterior anaphase displacement are not known. While displacement is abolished in par mutant embryos , this is probably a secondary consequence of earlier defects in antero-posterior polarity . Components downstream of the par genes must exist which translate embryonic polarity into posterior displacement. The identity of such components remains to be determined, and except for par-3 alleles, we did not isolate mutations which result in the absence of posterior displacement. Section title: A Balance of Forces May Act to Properly Position the First Division Spindle Educational score: 4.155117511749268 Domain: biomedical Document type: Study Language: en However, we did uncover a novel phenotypic class which affects posterior displacement. In anaphase spindle positioning mutant embryos, the entire anaphase spindle is usually displaced in an exaggerated manner towards the posterior. This raises the possibility that mechanisms exist in wild-type to restrict the extent of posterior displacement, and that these mechanisms are affected in anaphase spindle positioning mutants. Thus, asymmetric division of the one cell stage embryo may normally be achieved by a balance of forces: par -dependent forces which promote posterior displacement during anaphase, and forces dependent on anaphase spindle positioning loci which counteract this movement. Section title: Mutations Specific for Distinct Steps of Cytokinesis Educational score: 4.198797225952148 Domain: biomedical Document type: Study Language: en Cytokinesis involves at least two distinct steps: specification of the cleavage furrow at the cell cortex, and ingression of the furrow to cleave the cell in two. We have identified cytokinesis mutants affecting each of these two steps. In mutations at one locus, the cleavage furrow is not detectable by DIC microscopy, despite the spindle being present, suggesting a defect in cleavage furrow specification. The molecular basis of this process is still poorly understood , and the identification of components required for cleavage furrow specification is thus of great general interest. Section title: Mutations Specific for Distinct Steps of Cytokinesis Educational score: 4.398153781890869 Domain: biomedical Document type: Study Language: en In mutations at three other loci, the cleavage furrow is correctly specified, but regresses before going to completion. One of the loci which we have identified as mutating to this phenotype encodes cyk-1 , a member of the FH family of genes, some of which are also required for cytokinesis in other species . CYK-1 protein localizes to the leading edge of the cleavage furrow late in cytokinesis, where it may act by bridging actin from the contractile ring with midzone spindle microtubules . Similar cleavage furrow regression occurs in embryos lacking zen-4/CeMKLP1 function, which encodes a kinesin of the CHO family localizing to midzone spindle microtubules . CHO can bridge antiparallel microtubules in vitro , and zen-4/CeMKLP1 may thus act to stabilize midzone spindle microtubules in C . elegans . The molecular characterization of the two other loci identified here which are required for a late step in cytokinesis may help clarify the role of midzone spindle microtubules in cleavage furrow ingression. Section title: Prospects Educational score: 4.1920485496521 Domain: biomedical Document type: Study Language: en In conclusion, our work demonstrates that cell division processes in the one cell stage C . elegans embryo can be systematically dissected by mutational analysis. Collections similar to the one analyzed here exist for chromosomes II, IV, and V, corresponding to >50% of the genome in total (Schnabel, H., and R. Schnabel, unpublished observations). All of the loci identified here have been mapped by deficiencies, thus facilitating their future molecular characterization. This should be especially valuable in combination with the recently completed genome sequence and the use of RNAi to silence gene function . Thus, candidate genes in defined regions of chromosome III could be directly tested by RNAi to see if they correspond to the loci identified here by mutational analysis. This approach should lead to the rapid identification of numerous novel components required for cell division processes in metazoans. | Study | biomedical | en | 0.999997 |
10085293 | Section title: Microbial Techniques Educational score: 3.961561441421509 Domain: biomedical Document type: Study Language: en Media and genetic techniques were as described . The mating pheromone, α-factor, was added to log phase cultures at 6 μg/ml. Latrunculin-A (Lat-A) (from P. Crews, University of California, Santa Cruz, CA) was added to cultures at 400 mM. Benomyl sensitivity (a gift from E.I. du Pont de Nemours, Wilmington, DE) was tested in rich (YPD) medium. Geneticin ( GIBCO BRL ) was used at a concentration of 0.2 μg/ ml in YPD. Lists of the strains and plasmids used in this study are provided in Tables I and II . Except where indicated, all strains are congenic to W3031a. Section title: Strain and Plasmid Construction Educational score: 4.182135105133057 Domain: biomedical Document type: Study Language: en A complete deletion of the BNI1 coding sequence was created by one-step gene replacement with the selectable marker kan r , which confers Geneticin resistance to yeast . The bni1Δ::kan r containing DNA sequence was generated by PCR using oligonucleotide primers: 5′ TATCTATCTTCTGTATTGAGGAGAAACATTTTACTCAAGC - AGCTGAAGCTTCGTACGC and 5′ GAGTAAAGATGAATGTAA AGTGTATCATAAGTGATCTATAGCATAGGCCACTAGTGGAT - CTG . Deletion of the BNI1 locus was confirmed by Southern blot analysis of Geneticin-resistant transformants. This BNI1 deletion strain, hereafter referred to as bni1Δ , was used for all subsequent analyses unless specified otherwise. The deletion of ASE1 was also created by one-step gene replacement . Section title: Strain and Plasmid Construction Educational score: 4.2110209465026855 Domain: biomedical Document type: Study Language: en The centromere-based (CEN) plasmid carrying BNI1 (p182) has been described . This genomic sequence was also cloned into a CEN ARS TRP1 plasmid (pRS314) to create p1832. The COOH-terminal deletion mutant, bni1-CTΔ1 , lacks the coding sequence for amino acids 1,749–1,953 of Bni1p, and was created by the following strategy. p182 was digested with SstII which cuts BNI1 at bp 5,249 and in the polylinker, and ligated to an oligonucleotide (5′GTAGGCGGCCGCCTACGC). This created a stop codon followed by a NotI site adjacent to the SstII site in BNI1 . A CEN plasmid derived from pRS314 and an integrating plasmid derived from pRS305 contain the bni1-CTΔ1 sequence on a 6.3-kb BamHI/NotI restriction fragment. The strain containing PB1046 was used for genetic crosses, and the strain containing p2029 was used for morphological studies. Section title: Strain and Plasmid Construction Educational score: 4.124380111694336 Domain: biomedical Document type: Study Language: en The act1-116 and sla1ΔSH3#3 strains and their isogenic wild-type controls are in the S288c genetic background. To construct PY2596, the ACT1 locus was replaced by act1-116::HIS3 from pRB1537 by homologous recombination in a strain containing NUF2::GFP . This was done because previously constructed act1-116 strains contain a linked TUB2 mutation. Integration was confirmed by PCR analysis. Section title: Genetic Analysis Educational score: 4.210392951965332 Domain: biomedical Document type: Study Language: en The screen to identify mutations that were lethal in combination with an ASE1 -null allele was performed using an ADE3 sectoring assay as described . The starting strains MAT α and MAT a ase1::kan r ade2 ade3 leu2 lys2 trp1 ura3 {2μ ASE1 ADE3 TRP1} were mutagenized by transformation with a genomic library containing random insertions of a mini-Tn3lacZ- LEU2 transposon . Candidate mutants were backcrossed to demonstrate that the mutation represented a single genetic locus and to confirm that the mutation cosegregated with the LEU2 marker. The genomic site of transposon insertion was identified by direct sequencing of PCR products spanning the insertion site. These PCR products were generated by an arbitrary primed PCR strategy . The bni1::Tn3::LEU2 allele has an insertion of the transposon at nucleotide 5,006 of the BNI1 -coding sequence. This allele has an identical phenotype to the bni1Δ::kan r -null allele described above. In addition to BNI1 , genes that affect many aspects of microtubule function in S . cerevisiae were identified by this screen (to be described elsewhere). Section title: Genetic Analysis Educational score: 4.121882438659668 Domain: biomedical Document type: Study Language: en For the bni1Δ × ase1Δ cross, 37 tetrads were dissected and 41 double mutants were recovered, all of which were temperature sensitive for growth. For the bni1Δ × dyn1Δ cross, 19 tetrads were dissected and 11 temperature-sensitive double mutants were recovered. For the bni1Δ × kar3Δ cross, 27 tetrads were dissected and 20 viable double mutants were recovered. bni1Δ kar3Δ double mutants formed smaller colonies than the kar3Δ single mutant at 23°C and were inviable at 30°C. For the bni1Δ × cin8Δ cross, 35 tetrads were dissected and 28 double mutants were recovered. For the crosses of bni1Δ with kip1Δ and smy1Δ , 23 and 17 tetrads were dissected respectively, and the double mutants were recovered at the expected frequency. Section title: Genetic Analysis Educational score: 4.0335187911987305 Domain: biomedical Document type: Study Language: en Quantitative mating assays were performed using a MAT a bni1::kan r bar1::LEU2 strain , carrying different alleles of BNI1 on a CEN plasmid. The mating partner used for the mating assays was a MATα far1-c strain (Y66) which was itself defective in mating and thus increased the sensitivity of the assay. The assays were performed in triplicate. Section title: Genetic Analysis Educational score: 4.0940399169921875 Domain: biomedical Document type: Study Language: en The strain background used for our initial genetic experiments and for our time-lapse analysis, W3031a, has a mixed random/bipolar budding pattern . Therefore, in experiments where the movement of the SPB after telophase toward the bud was followed by fluorescence microscopy, the distance between the SPB and the bud was determined by overlaying images from each time point with a differential interference contrast (DIC) image obtained after bud emergence. The spindle orientation defect of bni1Δ that we observed is not specific to this strain background because we observed a similar spindle orientation defect in a YEF473-derived bni1Δ strain that has normal axial and bipolar bud site selection (data not shown). Section title: Microscopic Analysis of Cells Educational score: 4.123409271240234 Domain: biomedical Document type: Study Language: en Nuclear morphology was observed by fluorescence microscopy in cells fixed and stained with 4′,6′-diamidino-2-phenylindole (DAPI) as described . To preserve green fluorescent protein (GFP) fluorescence for the analysis of spindle orientation, cells were fixed in culture medium with 3.7% formaldehyde for 30 min, washed, and then applied to slides. Indirect immunofluorescence was performed as described . Experiments to measure nuclear or spindle position were performed at least twice. Section title: Microscopic Analysis of Cells Educational score: 4.117867946624756 Domain: biomedical Document type: Study Language: en Budding pattern was assayed after staining bud scars with calcofluor . The strain used was a bni1::HIS3/bni1::HIS3 YEF473 diploid , containing alleles of BNI1 on a CEN plasmid. Results from these strains were compared with results from an isogenic BNI1/BNI1 diploid. The fact that BNI1 carried on a CEN plasmid does not fully complement bni1::HIS3/bni1::HIS3 suggests that the gene dosage of BNI1 may be important for bipolar bud site selection. Mating projection assays using a W3031a MAT a bni1::kan r bar1::LEU2 strain containing the bni1 alleles were performed in triplicate . Section title: Microscopic Analysis of Cells Educational score: 4.1675496101379395 Domain: biomedical Document type: Study Language: en For live cell fluorescence microscopy, cells were applied to a microscope slide and observed at 23°C. The doubling time of cells in liquid culture at this temperature was 150 min, compared with the average doubling time of 176 min for cells observed with our time-lapse fluorescence protocol. A microscope (model ECLIPSE E600; Nikon ) equipped with a 100×/1.4 NA Planapochromat objective, a 100-W mercury arc illuminator, a Z-axis focus motor (Biopoint Z-axis focus motor; Ludl Electronics), a fluorescence illumination shutter (Ludl Electronics), and an Endow GFP filter set (excitation 450–490 nm, dichroic 495, emission 500 nm LP; Chroma Optics) was used. Images were acquired with a cooled charge-coupled device camera , containing a 1280 × 1024 Sony Interline chip (Hamamatsu Photonics). At each time point a series of fluorescence images in six focal planes spaced 0.5 μm apart were collected along with a DIC image. To minimize photo bleaching and photo damage of the cells, the light from the mercury lamp was attenuated to one-eighth of maximal intensity with neutral density filters and exposure times were limited to 200–400 ms. Images were analyzed using Openlabs Software (Improvision), and all statistical analysis was performed using StatView software (Abacus Concepts). Section title: BNI1 Has a Role in Microtubule Function Educational score: 4.1277852058410645 Domain: biomedical Document type: Study Language: en An allele of BNI1 was unexpectedly isolated in a genetic screen to identify genes involved in microtubule function. The screen was for mutations that were lethal when combined with an ase1Δ allele (see Materials and Methods). ASE1 encodes a nonmotor microtubule-associated protein that localizes to the spindle midzone and promotes spindle elongation (anaphase B) . The genetic interaction between BNI1 and ASE1 suggested that BNI1 might have a role in maintaining the integrity of the microtubule cytoskeleton. Section title: BNI1 Has a Role in Microtubule Function Educational score: 4.1163554191589355 Domain: biomedical Document type: Study Language: en A deletion allele of BNI1 was created (see Materials and Methods). The bni1Δ ase1Δ double mutant strain displayed a marked temperature-sensitive growth defect . Because this strain grew at a near normal rate at 23°C, we were able to test its sensitivity to the microtubule depolymerizing agent, benomyl. In contrast to the control strains, the bni1Δ ase1Δ strain had a strikingly increased sensitivity to benomyl (5 μg/ml). bni1Δ and ase1Δ mutations therefore have an additive detrimental effect on microtubule function. Section title: bni1Δ Cells Have a Defect in Spindle Orientation Educational score: 4.251189708709717 Domain: biomedical Document type: Study Language: en Because Bni1p is localized at the bud cortex and septum of mitotic cells and the shmoo tip in mating cells (Evangelista, M., and C. Boone, unpublished results) we considered the possibility that BNI1 affects microtubules through a role in spindle orientation and/or nuclear positioning. Here, defects in the location of the nucleus within the mother cell or defects in nuclear segregation that result in binucleate mother cells will be referred to as “nuclear position defects.” Defects in the alignment of the spindle with the mother–bud axis will be described as “spindle orientation defects.” The role of Bni1p in nuclear positioning was initially tested by examining bni1Δ and control cells stained with the DNA-binding dye, DAPI. Whereas mitosis in wild-type cells results in the faithful distribution of nuclei to mother and daughter cells , mitosis in a significant fraction of bni1Δ cells was abnormal, resulting in two nuclei in the mother cell. The fraction of binucleate mother cells in bni1Δ strains increased at 37°C . Section title: bni1Δ Cells Have a Defect in Spindle Orientation Educational score: 4.187655448913574 Domain: biomedical Document type: Study Language: en We next tested whether the binucleate phenotype of bni1Δ cells was due to a defect in spindle orientation. bni1Δ and control strains were grown to early logarithmic stage and the angle between the spindle and the mother– bud axis was measured . A chimeric gene expressing a fusion between Nuf2p, a spindle pole body protein, and GFP was introduced into both strains, allowing visualization of the poles by fluorescence microscopy . In wild-type cells, more than 60% of cells had spindles oriented within 30 degrees of the mother–bud axis . By contrast, spindle orientation in bni1Δ cells appeared to be random . Furthermore, the distance between the neck-proximal SPB and the bud neck was significantly greater in bni1Δ than in BNI1 cells: 1.0 and 0.7 μm, respectively . Thus, bni1Δ has a defect in spindle orientation. Section title: Genetic Interactions Suggest that BNI1 and KIP3 Are in the Same Pathway Educational score: 4.423425674438477 Domain: biomedical Document type: Study Language: en The entire set of Saccharomyces cerevisiae microtubule-based motor proteins has been defined. The genome contains a single gene encoding a dynein heavy chain, DYN1 , and six genes encoding kinesin-related proteins, KIP1, KIP2, KIP3, KAR3, CIN8, and SMY1 . Strains lacking any one of these genes are viable; however, combinations of different null alleles result in lethality. Only Dyn1p, Kip2p, Kip3p, and Kar3p are implicated in cytoplasmic microtubule function . Single mutant kip3Δ and dyn1Δ strains have the most striking effect on nuclear position. kip3Δ and dyn1Δ affect distinct steps in nuclear positioning: kip3Δ cells have a spindle orientation defect, whereas dyn1Δ cells have a defect in the insertion of the spindle across the bud neck. The fact that kip3Δ dyn1Δ double mutant strains are not viable suggests that there are two partially redundant pathways for spindle orientation . Section title: Genetic Interactions Suggest that BNI1 and KIP3 Are in the Same Pathway Educational score: 4.147632598876953 Domain: biomedical Document type: Study Language: en Double mutants were constructed to test if BNI1 was in the KIP3 pathway. Consistent with the similar spindle orientation defect observed in bni1Δ and kip3Δ strains, the bni1Δ kip3Δ strain was viable and grew indistinguishably from single mutant strains (Table III ). The percentage of binucleate mother cells in the bni1Δ kip3Δ strain was also not increased relative to either single mutant strain . By contrast, bni1Δ produced a striking synthetic interaction when combined with dyn1Δ . The bni1Δ dyn1Δ strain had a growth defect at 23°C and was inviable at 14° and 37°C . Also, the bni1Δ dyn1Δ strain had a marked increase in binucleate mother cells relative to the single mutant strains . Like the bni1Δ ase1Δ strain, the bni1Δ dyn1Δ strain had increased sensitivity to benomyl . Section title: Genetic Interactions Suggest that BNI1 and KIP3 Are in the Same Pathway Educational score: 4.033170700073242 Domain: biomedical Document type: Study Language: en The genetic interactions we observed between bni1Δ and the other motor gene deletions paralleled those observed with kip3Δ (Table III ). Like kip3Δ , bni1Δ is lethal when combined with kar3Δ . bni1Δ did not show a significant synthetic interaction with either kip1 , cin8Δ , or smy1Δ . This pattern of genetic interactions observed with bni1Δ suggests that BNI1 and KIP3 function in the same pathway. Section title: Defective Movement of the SPB to the Incipient Bud in bni1Δ Cells Educational score: 4.105428695678711 Domain: biomedical Document type: Study Language: en Because of the spindle orientation defect in bni1Δ and kip3Δ strains, we determined whether movement of the SPB to the incipient bud site was defective in bni1Δ cells as previously observed in kip3Δ cells. Time-lapse fluorescence microscopy of live cells expressing either Nuf2p– GFP or GFP–Tub1p (the major α tubulin gene in S . cerevisiae ) was used to examine SPB movement early in the cell cycle in both bni1Δ and control cells (see Materials and Methods). Section title: Defective Movement of the SPB to the Incipient Bud in bni1Δ Cells Educational score: 4.251528263092041 Domain: biomedical Document type: Study Language: en SPB movements in wild-type haploid cells were observed from the end of mitosis (telophase) through bud emergence and SPB separation . At the end of mitosis the poles were extended 8–10 μm and located near the cell periphery. After spindle disassembly, the poles usually moved ∼1–2 μm toward the center of the cell . In the wild-type strain, this initial movement of the SPB was followed by a concerted movement of the pole toward the site of the incipient bud. This movement of the SPB toward the bud occurred on average within 100 min ( n = 6), and once the SPB had moved toward the bud its position became relatively stable. SPB separation usually occurred after the old SPB was positioned near the bud. Although the SPB underwent some oscillations as it moved toward the bud, large movements away from the bud were not observed in wild-type cells . Section title: Defective Movement of the SPB to the Incipient Bud in bni1Δ Cells Educational score: 4.225426197052002 Domain: biomedical Document type: Study Language: en In a significant fraction of posttelophase bni1Δ cells, prolonged movement of the SPB away from the bud was observed . In Fig. 3 b, the daughter cell (white arrow) shows this abnormal movement of the SPB (for example, 14.2–121.1 min). In the cells that exhibited the phenotype, the SPB spent almost as much time moving away from the bud as toward the bud. The more random direction of movement of the SPB with respect to the incipient bud in bni1Δ cells was similar to the abnormal SPB movements previously observed in kip3Δ cells . This phenotype was detected in four out of 10 cells and was observed in both mother and daughter cells. The mother cell in Fig. 3 b is an example of a bni1Δ cell with relatively normal SPB movement. As in kip3Δ cells, the SPB eventually assumed a relatively stable position near the bud, suggesting that other factors may stabilize the interaction of the SPB with the bud neck. Section title: Abnormal Spindle Rotation and Transiting of Short Mitotic Spindles in bni1Δ Cells Educational score: 4.198662281036377 Domain: biomedical Document type: Study Language: en In addition to the defect in the movement of the unduplicated SPB toward the bud, we observed strikingly abnormal movements of short (1.5 μm) spindles that were unique to bni1Δ cells. In another series of experiments, spindle movements before anaphase were observed in bni1Δ and wild-type cells. In wild-type cells, short spindles aligned along the mother–bud axis as expected from our analysis of fixed cells and previous imaging of live cells . These short spindles displayed some oscillations relative to the bud neck. However, short spindle movement in these cells was limited and only rarely were large rotations (90–180°) of the spindle observed. At anaphase, the spindles in wild-type cells were correctly inserted into the bud neck, and spindle elongation occurred through the bud neck . Section title: Abnormal Spindle Rotation and Transiting of Short Mitotic Spindles in bni1Δ Cells Educational score: 4.159719467163086 Domain: biomedical Document type: Study Language: en By contrast, the spindles of bni1Δ cells underwent much more noticeable oscillations, and large rotations of the spindle were frequently observed. In the example shown in Fig. 5 b the spindle underwent four 90–180° rotations in a 40-min time period before anaphase (crossing of lines with open and filled circles). Furthermore, in bni1Δ cells the short spindle frequently underwent what we refer to as transiting: movements of the entire short spindle into the bud or back into the mother cell. In the example shown in Fig. 5 b and Fig. 6 , transiting of short spindles back and forth between the bud and mother cell was observed four times in the interval before anaphase . Like the defect in the initial SPB movement toward the bud observed in our other series of experiments, this phenotype was not completely penetrant (four out of 10 cells). Section title: Abnormal Interactions between Microtubules and the Cortex in bni1Δ Cells Educational score: 4.279449939727783 Domain: biomedical Document type: Study Language: en Recent live-cell fluorescence microscopy experiments suggest that spindle or SPB movement is driven by cytoplasmic microtubule contacts with the cell cortex . Three types of contacts between cytoplasmic microtubules and the cell cortex have been correlated with changes in the position of the poles or spindles. Apparent pushing forces are generated when cytoplasmic microtubules remain in contact with the cortex while polymerizing. Apparent pulling forces are generated when they depolymerize while maintaining contact with the cortex. Finally, lateral movements of the SPB or spindles are seen as microtubules sweep across the cortex . Section title: Abnormal Interactions between Microtubules and the Cortex in bni1Δ Cells Educational score: 4.173457145690918 Domain: biomedical Document type: Study Language: en To determine if the spindle movements in bni1Δ cells were due to abnormal interactions of cytoplasmic microtubules with cortical sites, image sets of wild-type and bni1Δ cells expressing GFP::Tub1p were acquired over short time intervals (30–60 s) using exposures that enabled us to clearly visualize cytoplasmic microtubules. In the wild-type cells with short spindles, the pattern of microtubule interactions with the cell cortex and the associated pattern of SPB and/or spindle movement were similar to that described above. Most notably, pulling events in our wild-type strain were observed in cells with short spindles when microtubules interacted with the bud cortex (five events observed over 140 min in five cells); however, such pulling events toward the mother cell were not observed (0 events over 140 min in five cells). Furthermore, in different experiments, we did not observe transiting of preanaphase spindles in seven wild-type cells, observed for an average time of 57 min per cell, over longer time intervals (data not shown). Although we did not observe spindle transits in our wild-type strain, low frequency spindle transits have been reported in some wild-type strains . Section title: Abnormal Interactions between Microtubules and the Cortex in bni1Δ Cells Educational score: 4.328876972198486 Domain: biomedical Document type: Study Language: en We next examined microtubule interactions with the cell cortex in bni1Δ cells . Unlike the control strain, pulling events between the cortex and the SPB in both the bud and mother cell were frequently observed (14 events toward bud and 10 events toward mother observed over 147 min in four out of 12 cells). An example of such interactions is shown in Fig. 7 . In this cell, from time points 8.9– 9.8 min, a microtubule end remained at a fixed position at the mother cell cortex whereas the microtubule shrank from 5.1 to 0 μm. A concomitant 5.6-μm movement of the spindle toward the mother cell cortex was observed. Again, from 15.8–16.6 min, a microtubule with one end at the mother cell cortex shrank from 4.2 to 1.7 μm, whereas the spindle moved 2.1 μm toward the cortex. Overall, we observed a strong correlation between pulling events and the abnormal transiting of short spindles between the mother and the bud in bni1Δ cells. Therefore, although microtubules in the bni1Δ cells still interact with the cortex, the inability to restrict pulling interactions toward the bud suggests that the microtubule cytoskeleton of the bni1Δ strain fails to sense the cell's polarity. Section title: Bni1p Is Not Required for Anaphase Spindle Elongation or Cell Cycle Progression Educational score: 4.149177551269531 Domain: biomedical Document type: Study Language: en Because loss of BNI1 affects cytoplasmic microtubule function, we next tested whether nuclear microtubule function was affected by measuring the kinetics of anaphase spindle elongation. Two major stages of anaphase have been observed in S . cerevisiae . There is an initial fast stage of elongation from an ∼2-μm spindle to an ∼4-μm spindle. The rate of elongation then slows as the spindle reaches maximal extension at 8–10 μm. Anaphase kinetics was measured in bni1Δ and wild-type cells . Fast elongation was 0.55 μm/min ± 0.14 ( n = 7) in the bni1Δ strain and 0.48 μm/min ± 0.10 ( n = 5) in the wild-type strain. Slow elongation was 0.14 μm/min ± 0.04 ( n = 7) in the bni1Δ strain and 0.12 μm/min ± 0.04 ( n = 5) in the wild-type strain. Thus, loss of BNI1 does not have a significant effect on anaphase spindle elongation. Section title: Bni1p Is Not Required for Anaphase Spindle Elongation or Cell Cycle Progression Educational score: 4.183279991149902 Domain: biomedical Document type: Study Language: en Because the bni1Δ mutant could affect spindle orientation either through a direct effect on the cytoskeleton or indirectly by perturbing the cell cycle, we next tested whether bni1Δ strains were delayed in any specific stage of the cell cycle. bni1Δ strains grew slightly slower than control strains at 23°C. The distribution of cells throughout the cell cycle was first characterized by examining the distribution of cell morphologies in cultures of logarithmically growing cells (Table IV ). The numbers of unbudded G1 cells, budded cells with undivided nuclei, and postanaphase cells were nearly identical in bni1Δ and the wild-type strain. The ratio of cells in G1 and G2 cell cycle phases were also similar in both strains by FACS ® analysis (data not shown). Finally, we analyzed our time-lapse experiments to measure the intervals between spindle disassembly and the initiation and completion of budding in bni1Δ and control cells. After completion of anaphase, the bni1Δ and control strains initiated budding on average at 87 and 89 min, respectively. Budding was completed on average at 138 and 150 min, respectively, in these strains. Section title: Bni1p Is Not Required for Anaphase Spindle Elongation or Cell Cycle Progression Educational score: 4.028039932250977 Domain: biomedical Document type: Study Language: en Therefore, although we cannot rule out a subtle effect on cell cycle progression in bni1Δ cells, our analysis shows that the cells do not exhibit a prominent delay at a specific stage of the cell cycle. This is consistent with the idea that loss of BNI1 function affects spindle orientation through direct effects on the cytoskeleton rather than indirect effects on the cell cycle. Section title: Bud6p and the Bud6p-binding Domain of Bni1p Are Required for Spindle Orientation and Bipolar Bud Site Selection Educational score: 4.284908294677734 Domain: biomedical Document type: Study Language: en Bni1p associates with other proteins that are required for bipolar bud site selection . As an initial test to determine if these associations are required for spindle orientation, we characterized a BNI1 allele (bni1-CTΔ1) lacking the COOH-terminal domain of Bni1p that interacts with the actin-associated protein Bud6p . The bni1-CTΔ1 strain is proficient both for shmoo formation and mating (Table V ). By contrast, bni1-CTΔ1 did not complement the bud site selection defect of a bni1Δ/bni1Δ diploid strain (Table V ). Furthermore, bni1-CTΔ1 cells had a significant defect in spindle orientation . Like bni1Δ cells, the spindles in bni1-CTΔ1 cells were also located farther from the bud neck and were highly variable in position and, like the bni1 null allele, the bni1-CTΔ1 allele produced a striking temperature-sensitive growth defect when combined with ase1Δ or dyn1Δ . bni1-CTΔ1 ase1Δ and bni1-CTΔ1 dyn1Δ strains were also more sensitive to benomyl, although the effect was less pronounced in the bni1-CTΔ1 ase1Δ strain . Section title: Bud6p and the Bud6p-binding Domain of Bni1p Are Required for Spindle Orientation and Bipolar Bud Site Selection Educational score: 4.193017959594727 Domain: biomedical Document type: Study Language: en Spindle orientation was also affected in cells lacking Bud6p. At 23°C, the mean spindle orientation angle in a bud6Δ strain was 24.0°, compared with 17.8° in an isogenic wild-type . At 37°C, the mean angle in bud6Δ was 28.3° in contrast to 20.6° in the wild-type . These results demonstrate that Bud6p and the Bud6p interaction domain of Bni1p are required for spindle orientation and bipolar bud site selection, but suggest that Bud6p is less important for spindle orientation than Bni1p. Section title: The Role of Actin in Spindle Orientation and Bipolar Bud Site Selection Educational score: 4.1361470222473145 Domain: biomedical Document type: Study Language: en Because Bni1p and Bud6p are both thought to be regulators of actin function, we next characterized the nature of actin's role in spindle orientation and nuclear positioning. Previous studies demonstrated that actin mutations interfere with normal nuclear position . To determine if actin is required for spindle orientation, we treated logarithmically growing wild-type cells with Lat-A for a brief period (5 min), fixed the cells, and then measured spindle orientation . Staining with rhodamine-phalloidin revealed a complete loss of polymerized actin after treatment with Lat-A (data not shown). By contrast with mock-treated cultures , the 5-min treatment with Lat-A–randomized spindle orientation. Thus, actin is required for spindle orientation in normal growing cells. The rapid effect on spindle orientation suggests that the role of actin in controlling spindle orientation may be direct. Section title: The Role of Actin in Spindle Orientation and Bipolar Bud Site Selection Educational score: 4.266877174377441 Domain: biomedical Document type: Study Language: en Genetic experiments have provided evidence for a specialized actin structure that mediates bipolar bud site selection . Many, but not all, mutations that affect the actin cytoskeleton affect bipolar bud site selection. A sla1ΔSH3#3 strain with abnormal cortical actin structure did not have a defect in bipolar bud site selection . Strains with this sla1 allele accumulated abnormal cortical actin aggregates and like sla1Δ strains, were temperature-sensitive for growth. Like sla1Δ , these strains were also supersensitive to osmotic stress at 23°C (data not shown). On the other hand, two “pseudo-wild-type” actin alleles, act1-116 and act1-117 , exhibited normal growth and cortical actin structures but had a defect in bipolar bud site selection . The reciprocal phenotypes of these mutants suggested that a specific actin structure might mediate bipolar bud site selection. This postulated structure (or substructure) might also contain bipolar bud site determinants since these proteins are known to interact with actin. Section title: The Role of Actin in Spindle Orientation and Bipolar Bud Site Selection Educational score: 4.114060401916504 Domain: biomedical Document type: Study Language: en The work by Yang et al. together with our analysis of bni1Δ and bni1-CTΔ1 prompted us to test the hypothesis that similar actin structure(s) required for bipolar budding are required for spindle orientation. We first examined spindle orientation in the sla1ΔSH3#3 strain. As shown in Fig. 9 , c and d, spindle orientation in sla1ΔSH3#3 cells was indistinguishable from that of the isogenic wild-type strain. This demonstrates that alterations in the actin cytoskeleton per se do not affect spindle orientation and supports the idea that a specific actin structure may be required for both spindle orientation and bipolar bud site selection. Section title: The Role of Actin in Spindle Orientation and Bipolar Bud Site Selection Educational score: 4.228609085083008 Domain: biomedical Document type: Study Language: en We next examined spindle orientation in the act1-116 strain. For our analysis we chose act1-116 of the two pseudo-wild-type actin alleles previously studied because it has the most noticeable effect on bipolar bud site selection . The mean angle for spindle orientation in the act1-116 strain was significantly greater than that for the isogenic wild-type strain . The percentage of cells with obviously misoriented spindles was also greater in the act1-116 strain (10.4% with spindle orientation between 60° and 90° compared with 3.4% for the wild-type strain). Although the spindle orientation defect in act1-116 was small, it was consistent with the magnitude of the defect in bipolar bud site selection pattern (35% of act1-116/ act1-116 diploid cells had a random budding pattern; data not shown). The fact that a mutant strain with no detectable cortical actin defect ( act1-116 ) disrupts spindle orientation and bipolar bud site selection, whereas another mutant strain ( sla1ΔSH3#3 ) with abnormal cortical actin does not, supports the idea that spindle orientation and bipolar bud site selection may be mediated by a similar actin-containing structure. Section title: The Role of Bni1p in Spindle Orientation Educational score: 4.233294486999512 Domain: biomedical Document type: Study Language: en The formins are important for cytokinesis and aspects of development in many organisms . On the molecular level, formins are thought to be regulators of the actin cytoskeleton, but their exact role in actin function is not defined. Here we report that the yeast formin, Bni1p, is required for orientation of preanaphase mitotic spindles in S . cerevisiae . Our analysis of the spindle positioning defect in bni1Δ cells suggests that Bni1p is required to assemble or transport cortical microtubule-binding sites into the bud. Section title: The Role of Bni1p in Spindle Orientation Educational score: 4.1647491455078125 Domain: biomedical Document type: Study Language: en The idea that Bni1p has a role in spindle orientation was prompted by genetic interactions we observed between bni1Δ and mutations in genes encoding components of the microtubule cytoskeleton. The localization of Bni1p to the bud cortex early in mitosis and to the septum late in mitosis (Evangelista, M., and C. Boone, unpublished observation) suggested that Bni1p would most likely regulate cytoplasmic microtubules. Indeed, we observed that in bni1Δ cells the orientation of preanaphase spindles was random with respect to the mother–bud axis. Section title: The Role of Bni1p in Spindle Orientation Educational score: 4.256943702697754 Domain: biomedical Document type: Study Language: en Our real-time analysis demonstrated two striking phenotypes in bni1Δ cells. First, Bni1p was required for the initial directed movement of the SPB toward the incipient bud site. Unlike wild-type cells, where the SPB makes a concerted (but not absolutely direct) movement toward the incipient bud site, the SPB in bni1Δ cells spent almost as much time moving away from the incipient bud site as toward it. Consistent with the idea that a partially redundant pathway is able to compensate for the loss of BNI1 (in some cells better than others), this defect was observed in four out of 10 cells. This phenotype is very similar to that previously observed in kip3Δ cells , and parallels the common spindle orientation defect observed in fixed populations of bni1Δ and kip3Δ cells. Section title: The Role of Bni1p in Spindle Orientation Educational score: 4.23130989074707 Domain: biomedical Document type: Study Language: en The polarity of the cell is established before bud emergence. Bni1p and other proteins are localized to the incipient bud before bud emergence . Initial studies of fixed populations of cells detected interactions between cytoplasmic microtubules and the incipient bud site in unbudded cells . These observations suggested that the microtubule cytoskeleton must be able to sense and respond to polarity cues even before the bud emerges. This study and previous fluorescence time-lapse microscopy studies of wild-type cells demonstrate that the SPB does move toward the bud site before bud emergence . The finding that Bni1p was required for this initial movement of the SPB suggests that Bni1p is either directly involved in the physical interactions between cytoplasmic microtubules and the cortex, or that it regulates these interactions. Section title: The Role of Bni1p in Spindle Orientation Educational score: 4.280633449554443 Domain: biomedical Document type: Study Language: en It is important to note that, although there was a profound delay in movement of the SPB in bni1Δ cells toward the incipient bud, the SPB eventually assumed a position close to the bud before SPB separation. Therefore it is possible that other proteins can also mediate the cortical interaction, but that they assemble at cortical sites later than Bni1p. Alternatively, there may be a Bni1p-independent pathway for spindle orientation. Because of the genetic evidence of functional overlap between BNI1 and DYN1 , cytoplasmic dynein is one candidate to mediate an alternate mechanism for spindle orientation. Section title: The Role of Bni1p in Spindle Orientation Educational score: 4.28713321685791 Domain: biomedical Document type: Study Language: en A second striking phenotype that was unique to bni1Δ cells was the abnormal rotations of the preanaphase spindle and transiting of these spindles between the mother and the bud. The transiting motions of the spindle were correlated with shortening of the cytoplasmic microtubules that maintain contact with the cell cortex. We interpret these movements to be the result of pulling forces between the cortex and the spindle, because the shortening microtubule consistently maintained contact with the cell cortex. These spindle movements did not appear to result from pushing forces, because we did not observe consistent contact between the cortex and cytoplasmic microtubules from the distal SPB. Section title: The Role of Bni1p in Spindle Orientation Educational score: 3.9685542583465576 Domain: biomedical Document type: Study Language: en What was most remarkable about the bni1Δ cells was that these apparent pulling movements were observed with approximately equal frequency toward the mother cell body as toward the bud. Apparent pulling interactions between cytoplasmic microtubules and the bud cortex were observed in wild-type cells during spindle orientation. However, apparent pulling movements of the spindle into the mother cell were not observed in our wild-type samples. Section title: The Role of Bni1p in Spindle Orientation Educational score: 4.388830184936523 Domain: biomedical Document type: Study Language: en The simplest explanation for the high frequency of transiting of preanaphase spindles in bni1Δ cells is that Bni1p is required to concentrate microtubule-binding sites to the bud cortex while spindle orientation occurs. In the absence of Bni1p, these microtubule-binding sites would no longer be asymmetrically localized and would be distributed evenly between the mother and the bud. Bni1p could participate in the assembly or transport of microtubule-binding sites into the bud. Other models are also possible. For examples, cortical attachment sites might be evenly distributed throughout the cell, but Bni1p might specifically alter those in the bud, therefore biasing pulling movements toward the bud. Interestingly, we have found that the second formin in yeast, Bnr1p, is not required for spindle orientation (Lee, L., and D. Pellman, unpublished results). This is consistent with Bnr1p's specific localization to the septal regions and not the bud cortex . Section title: The Genetic Pathways Controlling Spindle Orientation Educational score: 4.292372226715088 Domain: biomedical Document type: Study Language: en Two major pathways for spindle positioning have been identified. As discussed above, the kinesin Kip3p is required for the initial migration of the SPB toward the incipient bud site before SPB duplication . Cytoplasmic dynein is required for insertion of the preanaphase spindle into the bud neck, but not for the initial alignment of the spindle with the axis of division . These pathways are at least partially overlapping, because cells lacking both KIP3 and DYN1 are inviable . The pattern of genetic interactions that we observed between bni1Δ and null alleles of different motor genes was remarkably similar to the pattern of genetic interactions previously observed with kip3Δ . These findings together with the fact that bni1Δ kip3Δ double mutant cells were viable and did not have an exacerbated defect in nuclear segregation relative to the single mutant cells, place KIP3 and BNI1 in the same genetic pathway. Section title: The Genetic Pathways Controlling Spindle Orientation Educational score: 4.220306396484375 Domain: biomedical Document type: Study Language: en There are several molecular models that can explain the relationship between BNI1 and KIP3 . One possibility is that Kip3p is a minus end–directed motor, and that the pulling of the spindle toward the bud is mediated by Kip3p tethered to Bni1p. Arguing against this idea is the fact that Kip3p is not detected at the cell cortex . Also, Kip3p and Bni1p do not coimmunoprecipitate under a variety of conditions, and the localization of Kip3p is not altered in a bni1Δ mutant (Ellingson, E., L. Lee, and D. Pellman, unpublished results). However, it remains possible that there is a physical interaction that is transient or of low affinity. Section title: The Genetic Pathways Controlling Spindle Orientation Educational score: 4.357856273651123 Domain: biomedical Document type: Study Language: en Another explanation for the similar phenotypes and genetic interactions observed with bni1Δ and kip3Δ strains is that Kip3p may promote microtubule dynamics that are required for cortical microtubule capture. Dynamic microtubules could be required to increase the number of encounters between the ends of the cytoplasmic microtubules and the cortex, thereby increasing the likelihood that the microtubules will find a binding site. Finally, Kip3p might mediate microtubule depolymerization after the cytoplasmic microtubule is tethered to the bud cortex through interactions with another protein. Microtubule depolymerization in this manner would generate a pulling force on the spindle. Consistent with the idea that Kip3p has an important role in controlling microtubule dynamics, kip3Δ cells have longer cytoplasmic microtubules and are resistant to microtubule-depolymerizing agents . Interestingly, we have noted that bni1Δ cells also have longer cytoplasmic microtubules than control cells, but we have not characterized microtubule dynamics in bni1Δ cells in detail (Lee, L., and D. Pellman, unpublished observation). Section title: Bipolar Bud Site Determinants, Actin, and Cortical Microtubule Capture Educational score: 4.529599189758301 Domain: biomedical Document type: Study Language: en Several lines of evidence suggest that other bipolar budding determinants in addition to Bni1p may be involved in spindle orientation. First, several genes required for bipolar budding appear to define a functional group based on similar mutant phenotypes, similar genetic interactions, and similar localization of the encoded proteins . Furthermore, there is evidence for physical interactions among at least four of these proteins. Bni1p and Spa2p interact by two-hybrid analysis and in vitro, and the localization of Bni1p is dependent upon Spa2p function . There is also evidence for physical interactions between Spa2p, Pea2p, and Bud6p . Second, we found that bud6Δ cells exhibit a defect in spindle orientation. Third, cells expressing Bni1-CTΔ1p, which lack the Bud6p-interacting domain of Bni1p, are defective in bipolar budding and spindle orientation but not Bni1p mating functions. Although Bud6p is necessary for normal spindle positioning, our results demonstrate that it is not as important as Bni1p. This suggests that the biochemical mechanism for cortical microtubule capture may be complex, perhaps involving several partially redundant factors. Section title: Bipolar Bud Site Determinants, Actin, and Cortical Microtubule Capture Educational score: 3.9772870540618896 Domain: biomedical Document type: Study Language: en The actin cytoskeleton is also required for both spindle orientation and bipolar budding . We have extended previous studies by demonstrating that spindle orientation in normal logarithmically growing cells becomes random within 5 min of depolymerizing cellular actin with Lat-A. The rapid effect of Lat-A on spindle orientation argues that the role of actin may be direct. Section title: Bipolar Bud Site Determinants, Actin, and Cortical Microtubule Capture Educational score: 4.344216346740723 Domain: biomedical Document type: Study Language: en Our findings are also consistent with the idea that actin structures postulated to be required for bipolar budding , are also required for spindle orientation. The supposition that a specific actin structure is required for both bipolar budding and spindle orientation is based on several lines of evidence. First, a mutation that results in abnormal cortical actin ( sla1ΔSH3#3 ) does not affect bipolar budding or spindle orientation, demonstrating that alterations in the actin cytoskeleton per se do not perturb these two processes. Second, an act1-116 allele that does not alter cortical actin or cell growth is defective for both bipolar budding and spindle orientation. Third, both bni1Δ and bni1-CTΔ1 cells have normal appearing actin structures (Lee, L., and D. Pellman, unpublished data) but are defective for spindle orientation and bipolar bud site selection. Together, these findings are consistent with a model where Bni1p is a component of an actin complex that mediates bipolar bud site selection and spindle orientation. Section title: Bipolar Bud Site Determinants, Actin, and Cortical Microtubule Capture Educational score: 4.390810012817383 Domain: biomedical Document type: Study Language: en Although our results suggest that Bni1p is necessary to concentrate microtubule/cortical interaction sites into the bud, the molecular nature of this postulated microtubule-binding site remains to be defined. As discussed above, one possibility is that the interaction is mediated by transient association of a microtubule motor such as Kip3p with the cortex. Another possibility is that cytoplasmic microtubules are captured by microtubule-binding proteins located at cortical sites. One candidate cortical protein that might have microtubule-binding activity is Kar9p . The kar9Δ phenotype partially overlaps with that of bni1Δ . Furthermore, Kar9p is mislocalized in bni1Δ cells, although not obviously to cortical sites in the mother cell . Another candidate for a cortical interaction site is coronin . Coronin colocalizes with actin patches in vivo and, interestingly, binds to both microtubules and actin in vitro. Since most of the proteins required for spindle orientation are not essential, it seems likely that there may be several cortical proteins that interact with microtubules. Section title: Bipolar Bud Site Determinants, Actin, and Cortical Microtubule Capture Educational score: 4.267320156097412 Domain: biomedical Document type: Study Language: en Whatever the nature of the cortical microtubule-binding sites, it is likely that their activity is regulated during the cell cycle. One plausible mechanism to achieve this would be through Rho-type GTPases. Because Bni1p links Rho-type GTPases to the actin cytoskeleton , it is interesting to note that genetic evidence suggests a role for Rho2p and the GDP/GTP exchange factor Rom2p in microtubule function . Whether the interaction between Bni1p and the activated forms of GTPases is required to maintain spindle orientation remains to be determined. Section title: Bipolar Bud Site Determinants, Actin, and Cortical Microtubule Capture Educational score: 4.270864486694336 Domain: biomedical Document type: Study Language: en The results described here define an important role for an S . cerevisiae formin, Bni1p, in regulating spindle position. Whether formins in other organisms have similar functions is unknown. However, we note that mutations in the genes encoding the Drosophila formins, cappuccino and diaphanous , result in abnormal microtubule structures . cappuccino mutant oocytes contain long and thick microtubule bundles around the cell cortex. During male meiosis in diaphanous mutants the structure of both the actin contractile ring and the central spindle is disrupted. These findings raise the possibility that formins have a conserved role in microtubule function. | Study | biomedical | en | 0.999996 |
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