Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A monoclonal antibody, designated HT462, is described which is specific for an antigen expressed in human T-cell leukemia/lymphoma virus (HTLV) preparations and by HTLV-infected cells. In indirect immunofluorescence assays, the antigen was detected on the surface of both HTLV-transformed producer and nonproducer cells, including cells infected in vitro with either HTLV subgroup I (HTLV-I) or HTLV-II. Normal human peripheral blood lymphocytes stimulated with phytohemagglutinin, cord blood T cells cultured with T-cell growth factor, and a variety of HTLV-negative T- and B-cell lines all lacked HT462 antigen expression. The HT462 antigen is a 52,000-molecular-weight glycoprotein, as shown by Western blotting procedures and treatment of viral preparations with neuraminidase, endoglycosidase F, and trypsin. The unglycosylated molecule is approximately 42,000 daltons. That the antigen is virus associated was demonstrated by its banding at the density of HTLV in gradients of metrizamide and by its concomitant synthesis with HTLV gag proteins after short-term culture of primary HTLV-positive leukemic cells. Differential expression of the HT462 antigen and HTLV gag-pol gene products was observed. In one case, low HT462 expression was correlated with the known inability of the particular cell line to produce syncytia in vitro. The properties of the HT462 antigen are most consistent with it being a gene product of the HTLV px region or else a cellular antigen specifically induced after viral infection. We cannot rule out, however, that the antigen is a variant cleavage product of the env gene. The monoclonal HT462 will be useful in further definition of the proteins and functions encoded by the env-px genetic sequence and in studying the biological properties of HTLV-transformed cells. Furthermore, the monoclonal, by recognizing HTLV-transformed nonproducers, will allow a greater spectrum of virus-infected cells to be detected.
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PMID:A monoclonal antibody specific for a 52,000-molecular-weight human T-cell leukemia virus-associated glycoprotein expressed by infected cells. 298 39

Fluorescently labeled (bearing N-4-nitrobenzo-2-oxa-1,3-diazole-phosphatidylethanolamine (N-NBD-PE)) reconstituted Sendai virus envelopes (RSVE) were used to study fusion between the viral envelopes and cultured living cells such as lymphoma, Friend erythroleukemia cells (FELC) and L cells. Incubation of fusogenic viruses with the above cell lines resulted in a relatively high degree (40-45%) of fluorescence dequenching. On the other hand, incubation of unfusogenic (trypsin or phenylmethylsulfonylfluoride (PMSF)-treated) RSVE with these cells led to very little (6-9%) fluorescence dequenching. The degree of fluorescence dequenching was linearly correlated to the surface density of the virus-inserted N-NBD-PE molecules. Fluorescence photobleaching recovery experiments showed that fusion of fluorescent RSVE with FELC resulted in an infinite dilution of the fluorescent molecules in the recipient cell membranes. The fluorescent probe 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (N-NBD-Cl) was covalently attached to envelopes of intact Sendai virions without significantly impairing their biological activity. Incubation of fluorescently labeled, intact Sendai virions with cultured cells resulted in about 20% fluorescence dequenching. The present data clearly indicate that fluorescently labeled Sendai virions can be used for a quantitative estimation of the degree of virus-membrane fusion.
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PMID:Fusion of fluorescently labeled Sendai virus envelopes with living cultured cells as monitored by fluorescence dequenching. 301 69

To examine whether a monoclonal antibody, TFS-4, can distinguish small-cell lung cancer from non-small-cell lung cancers, an extensive survey of fresh lung tumors, cancers from other organs, and normal tissue specimens has been carried out. The antibody has been shown to react specifically with small-cell lung cancer (15 of 15) but not with squamous cell carcinoma (0 of 20), adenocarcinoma (0 of 20) of the lung, or large-cell lung cancer (0 of 2). It reacted neither with other malignancies, including colorectal cancer, gastric cancer, and malignant lymphoma, nor with such normal tissues as trachea, lung, liver, pancreas, colon, kidney, spleen, skin, striated muscle, bone marrow, or peripheral blood cells. Interestingly, the antibody cross-reacted with central nervous tissues. The antigenic determinant on small-cell lung cancer and that on human brain were both heat labile and trypsin sensitive, but resisted treatment with neuraminidase, suggesting that they represent similar peptides. TFS-4 may be of clinical use in the diagnosis of small-cell lung cancer, while the antigen may help investigate the nature and origin of small-cell lung cancer.
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PMID:Monoclonal antibody that distinguishes small-cell lung cancer from non-small-cell lung cancer. 302 18

The 90-kDa heat-shock protein (HSP90) has been purified from mammalian tissues, mouse liver and porcine brain, with a good yield by a new method involving hydrophobic chromatography. Mouse liver HSP90 and porcine brain HSP90 were compared with mouse lymphoma HSP90 which was purified from T lymphoma cell line, L5178Y, by a modification of the previously reported method. These three HSP90s were indistinguishable from one another in amino acid composition, one-dimensional peptide mapping, elution pattern of proteolytic fragments (trypsin- or V8-protease-cleaved) in reverse-phase high-performance liquid chromatography, reactivity with the antibody against mouse T lymphoma HSP90 and the ability to bind to F-actin. The amino acid sequences of three portions (total 47 amino acid residues) of lymphoma HSP90 were determined and they were homologous to those of the corresponding portions of Drosophila HSP83A and yeast HSP90. These results suggest that HSP90 is a highly conserved protein during evolution.
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PMID:Purification and characterization of the 90-kDa heat-shock protein from mammalian tissues. 305 69

The glucose transporter has been identified in a variety of mammalian cell membranes using a photoactivatable carrier-free radioiodinated derivative of forskolin, 3-[125I]iodo-4-azidophenethylamido-7-O-succinyldeacetylforskoli n ([125I]IAPS-forskolin) at 1-3 nM. The membranes that were photolabelled with [125I]IAPS-forskolin were human placental membranes, rat cortical and cerebellar synaptic membranes, rat cardiac sarcolemmal membranes, rat adipocyte plasma membranes, smooth-muscle membranes, and S49 wild-type (WT) lymphoma-cell membranes. The glucose transporter in plasma membranes prepared from the insulin-responsive rat cardiac sarcolemmal cells, rat adipocytes and smooth-muscle cells were determined to be approx. 45 kDa by SDS/polyacrylamide-gel electrophoresis (PAGE). Photolysis of human placental membranes, rat cortical and cerebellar synaptic membranes, and WT lymphoma membranes with [125I]-IAPS-forskolin, followed by SDS/PAGE, indicated specific derivatization of a broad band (43-55 kDa) in placental membranes and a narrower band (approx. 45 kDa) in synaptic membranes and WT lymphoma membranes. Digestion of the [125I]IAPS-forskolin-labelled placental and WT lymphoma membranes with endo-beta-galactosidase showed a reduction in the apparent molecular mass of the radiolabelled band to approx. 40 kDa. The membranes that were photolabelled with [125I]IAPS-forskolin and trypsin-treated produced a radiolabelled proteolytic fragment with an apparent molecular mass of 18 kDa. [125I]IAPS-forskolin is a highly effective probe for identifying low levels of glucose transporters in mammalian tissues.
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PMID:Identification of the glucose transporter in mammalian cell membranes with a 125I-forskolin photoaffinity label. 306 59

Human T-T hybridomas were developed as a strategy for obtaining lymphokines that alter T-lymphocyte motility. Mitogen-stimulated human T lymphocytes were fused with cells of the human CEM lymphoma line and the supernatants derived from these fusion products were assessed for chemoattractant activity in a modified Boyden chamber assay. Supernatants from hybridoma 41B2 enhanced lymphocyte migration to 198 +/- 13% (mean +/- SEM) of control. Characterization by Sephadex G-100 molecular sieve chromatography revealed a single peak of chemoattractant activity corresponding to a molecular weight (MW) of 56,000. This activity eluted from a Sephadex QAE anion-exchange column at 4-6 mS. Subsequent isoelectric focusing in sucrose revealed an isoelectric point of 9.0-9.2. Fractions with activity after sequential molecular sieve and anion-exchange chromatography were concentrated, radiolabeled with 125I, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autoradiography revealed a band which corresponded to a MW of 14,000 (representing four similar monomeric chains) and to the region from which chemoattractant activity could be detected in eluates from slices of unstained gels run in parallel. The biological activity of this hybridoma-derived lymphocyte chemoattractant was abolished by treatment with trypsin and neuraminidase but was unaffected by heating to 56 degrees C. We conclude that certain human T-T-cell hybridomas constitutively elaborate a lymphocyte chemoattractant that appears to be physicochemically identical to a previously described human lymphokine, lymphocyte chemoattractant factor.
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PMID:A human T-T-cell hybridoma-derived lymphocyte chemoattractant factor. 309 96

Sonication of a crude rat liver membrane preparation and centrifugation at 100,000 X g yielded a supernatant which activated basal and hormone-sensitive adenylate cyclases [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. The membrane origin of the stimulatory activity was confirmed by the use of lactate dehydrogenase as a marker for contamination by cytosol. The solubility of the activating factors was verified by their passage through 0.05 micron diameter pores of Millipore filters. The membrane-derived activators were nondialyzable and destroyed by heat and trypsin in the same manner as adenylate cyclase activators detectable in cytosol. Stimulation by factors from membranes and cytosol was not additive. The amount of the activators which could be freed from membranes by sonication was 12-15% of that contained in cytosol previously separated from the membranes. Soluble activators from the two sources had limited ability to restore adenylate cyclase activity to membranes from the cyclone of S49 mouse lymphoma cells which are deficient in the enzyme's guanine nucleotide-binding stimulatory protein, Ns. Cytosol did not contain a substrate for ADP-ribosylation by cholera toxin that corresponded electrophoretically to Ns. Furthermore, purified Ns did not affect adenylate cyclase activity in preparations stimulated by the soluble activators. These findings suggest that the activating factors found in cytosol may be released from membranes during tissue homogenization. Because these protein activators can be obtained from membranes without use of detergents and can neither substitute for nor be substituted for by Ns in functional assays, they are distinct from Ns.
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PMID:Membrane association of soluble protein activators of rat liver adenylate cyclase. Evidence for distinctness from the guanine nucleotide-binding stimulating protein (Ns). 309 5

The culture medium of certain strains of Clostridium botulinum type C contains two separable ADP-ribosyltransferases. Besides the ADP-ribosylation of actin due to botulinum C2 I toxin, a second microbial enzyme causes the mono-ADP-ribosylation of a eukaryotic protein with a molecular mass of about 20 kDa found in platelets, neuroblastoma X glioma hybrid cells, S49 lymphoma cells, chick embryo fibroblasts and sperm. The eukaryotic substrate is inactivated by heating and trypsin treatment. In contrast, the novel ADP-ribosyltransferase, which can be separated by DEAE-Sephadex chromatography, is largely resistant in the short term to trypsin digestion.
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PMID:Clostridium botulinum type C produces a novel ADP-ribosyltransferase distinct from botulinum C2 toxin. 310 Mar 33

Cytochalasin B and nitrobenzylthioinosine (NBMPR), which inhibit membrane transport of glucose and nucleosides, respectively, have served as photoaffinity ligands that become covalently linked at inhibitor binding sites on transporter-associated proteins. Thus, when membranes from erythrocytes of neonatal pigs with site-bound [3H]cytochalasin B or [3H]NBMPR were irradiated with uv light, two labeled membrane polypeptides (peak Mr values: 55,000 and 64,000, respectively) were identified. Treatment of the photolabeled membranes with endoglycosidase F increased the mobility of [3H]cytochalasin B- and [3H]NBMPR-labeled material (peak Mr values: 44,000 and 57,000, respectively) and limited digestion with trypsin yielded different polypeptide fragments (Mr values: 18,000-23,000 and 43,000, respectively). Identification of the photolabeled polypeptides as transporter components was established using monoclonal antibodies (MAbs) raised against partially purified preparations of band 4.5 from erythrocytes of adult pigs and humans. MAbs 65D4 and 64C7 (anti-human band 4.5), raised in this study, reacted with [3H]cytochalasin B-labeled material from membranes of human erythrocytes and bound to permeabilized erythrocytes but not to intact cells. MAb 65D4 also bound to erythrocytes of mice and neonatal pigs and to a variety of cultured cells (mouse, human, rat), including AE1 mouse lymphoma cells, which lack an NBMPR-sensitive nucleoside transporter. Also employed was MAb 11C4 (anti-pig band 4.5), which recognizes the NBMPR-binding protein of erythrocyte membranes from adult pigs. When membrane proteins from neonatal and adult pigs were subjected to electrophoretic analysis and blots were probed with different MAbs, MAb 65D4 (anti-human band 4.5) bound to material that comigrated with [3H]cytochalasin B-labeled polypeptides (band 4.5) from neonatal, but not adult, pig erythrocytes, whereas MAb 11C4 (anti-pig band 4.5) bound to material that comigrated with [3H]NBMPR-labeled band 4.5 polypeptides of erythrocytes from both neonatal and adult pigs. These results, which indicate structural differences in the cytochalasin B- and NBMPR-binding proteins of pig erythrocytes, establish the presence of both proteins in erythrocytes of neonatal pigs and suggest that only the NBMPR-binding protein is present in erythrocytes of adult pigs.
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PMID:Identification of glucose and nucleoside transport proteins in neonatal pig erythrocytes using monoclonal antibodies against band 4.5 polypeptides of adult human and pig erythrocytes. 314 74

A highly metastatic variant (ESb) of a methylcholanthrene-induced T lymphoma elaborates a heparan sulfate (HS) degrading endoglycosidase (heparanase) to a much higher extent than its non-metastatic parental subline (Eb). Whereas a serum-free medium conditioned by either subline contained a trypsin-like serine protease, heparanase activity was detected only in the ESb-conditioned medium (CM). ESb CM was incubated with a naturally produced, sulfate-labelled subendothelial extracellular matrix (ECM) or with a soluble, high-MW labelled proteoglycan first released from the ECM by incubation with Eb CM or with the partially purified ESb protease. Sulfate labelled degradation products were analyzed by gel filtration on Sephrose 6B. The optimal pH for degradation of ECM-bound HS was 6.2 as compared to pH 5.2 for degradation of the soluble proteoglycan. Heparanase-mediated degradation of both ECM-bound and soluble HS was inhibited by heparin. Addition of either trypsin, plasmin or to a lower extent, the purified ESb protease, stimulated between 5- and 20-fold the ESb CM-mediated degradation of ECM-bound HS but had no effect on heparanase-mediated degradation of the soluble proteoglycan. This stimulation was inhibited in the presence of heparin or protease inhibitors. These results indicate that both a protease and heparanase are involved in the ESb-mediated degradation of ECM-bound HS and that one enzyme produces a more accessible substrate for the next enzyme. This sequential cleavage is characteristic of degradation of a multimolecular structure such as the subendothelial ECM and hence cannot be detected in studies with its isolated constituents.
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PMID:Sequential degradation of heparan sulfate in the subendothelial extracellular matrix by highly metastatic lymphoma cells. 315 49


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