Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.69 (APC)
 16,337 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The proteins which have been left tightly bound to the tissue culture substrate after ethylenebis (oxyethyl-enenitrilo) tetraacetic acid (EGTA)-mediated removal of normal, virus-transformed, and revertant mouse cells and which have been implicated in the substrate adhesion process have been analyzed by slab sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three size classes of hyaluronate proteoglycans were resolved in the 5% well gel; approximately half of the protein in the substrate-attached material coelectrophoresed with these polysaccharides-so-called glycosaminoglycan-associated protein(GAP). A portion of the GAP was shown to be highly heterogeneous and displaced from the polysaccharide by preincubation with calf histone before electrophoresis. The relative proportions of the proteoglycans varied in material deposited during a variety of cellular attachment and growth conditions. The remainder of the cellular protein in substrate-attached material was resolved as several major and distinct protein bands in 8 or 20% separating gels (a limited number of distinct serum proteins have also been identified as substrate bound). Protein C0 (molecular weight 220 000) was a prominent component in the material from a variety of normal and virus-transformed cells and resembled the so-called LETS or CSP glycoprotein in several respects; protein Ca was myosin-like in several respects; protein C2 was shown to be actin; and protein C1 (molecular weight 56 000) does not appear to be tubulin. Histones were also present in most preparations of substrate-attached material, particularly at high levels in transformed cell meterial, and may result from EGTA-mediated leakiness of the cell and subsequent binding to the negatively charged polysaccharide. These substrate-attached proteins were (a) prominent in substrate-attached material from many cell types in characteristic relative proportions, (b) deposited by EGTA-subcultured cells during the first hour of attachment to fresh substrate, (c) deposited by cells growing on plastic or glass substrates (three additional) components were also prominent in glass-attached material), and (d) deposited during long-term growth on or initial attachment to substrates coated wit 3T3 substrate-attached material. Pulse-chase analyses with radioactive leucine indicated that these proteins exhibit different turn-over behaviors. These results are discussed with regard to the possible involvement of these substrate-attached proteins in the substrate adhesion process, with particular interest in the interaction of cytoskeletal microfilaments with other surface membrane components and with regard to alteration of substrate adhesion by virus transformation.
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PMID:Electrophoretic analysis of substrate-attached proteins from normal and virus-transformed cells. 18 10

The nonhistone chromatin protein, C-14, was extracted from chromatin of Novikoff hepatoma ascites cells and isolated in high purity as shown by its migration as a single dense spot on two-dimensional polyacrylamide gels. Its mobility on sodium dodecyl sulfate gels is consistent with a molecular weight of approximately 70 000. The amino acid composition shows that protein C-14 has an acidic:basic amino acid ratio of 1.8. Its amino terminal amino acid is lysine. Protein C-14 stimulated the incorporation of [3H]UMP into RNA by approximately 30% when added to naked DNA and homologous RNA polymerase I. A 30% stimulation of [3H]UMP incorporation into RNA was also found when protein C-14 was added to an E. coli RNA polymerase system containing either E. coli or Novikoff hepatoma DNA.
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PMID:Isolation and characterization of nonhistone chromosomal protein C-14 which stimulates RNA synthesis. 19 58

Protein C is a vitamin K-dependent protein, which exists in bovine plasma as a precursor of a serine protease. In this study, protein C was isolated to homogeneity from human plasma by barium citrate adsorption and elution, ammonium sulfate fractionation, DEAE-Sephadex chromatography, dextran sulfate agarose chromatography, and preparative polyacrylamide gel electrophoresis. Human protein C (M(r) = 62,000) contains 23% carbohydrate and is composed of a light chain (M(r) = 21,000) and a heavy chain (M(r) = 41,000) held together by a disulfide bond(s). The light chain has an amino-terminal sequence of Ala-Asn-Ser-Phe-Leu- and the heavy chain has an aminoterminal sequence of Asp-Pro-Glu-Asp-Gln. The residues that are identical to bovine protein C are underlined. Incubation of human protein C with human alpha-thrombin at an enzyme to substrate weight ratio of 1:50 resulted in the formation of activated protein C, an enzyme with serine amidase activity. In the activation reaction, the apparent molecular weight of the heavy chain decreased from 41,000 to 40,000 as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. No apparent change in the molecular weight of the light chain was observed in the activation process. The heavy chain of human activated protein C also contains the active-site serine residue as evidenced by its ability to react with radiolabeled diisopropyl fluorophosphate. Human activated protein C markedly prolongs the kaolin-cephalin clotting time of human plasma, but not that of bovine plasma. The amidolytic and anticoagulant activities of human activated protein C were completely obviated by prior incubation of the enzyme with diisopropyl fluorophosphate. These results indicate that human protein C, like its bovine counterpart, exists in plasma as a zymogen and is converted to a serine protease by limited proteolysis with attendant anticoagulant activity.
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PMID:Human plasma protein C: isolation, characterization, and mechanism of activation by alpha-thrombin. 46 91

The protease from Russell's viper venom that activates factor X (Stuart factor), factor IX (Christmas factor), and protein C was purified by gel filtration on Sephadex G-150 and QAE-Sephadex A-50 column chromatography. The purified enzyme migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 79 000. A minimal molecular weight of 78 500 +/- 800 was determined by sedimentation equilibrium in the presence of 6 M guanidine hydrochloride. Upon reduction with 2-mercaptoethanol, a heavy chain (mol wt 59 000) and a light chain were observed. The light chain migrated as a single band (mol wt 19 000) in 7.5% polyacrylamide-sodium dodecyl sulfate gels but appeared as a doublet (mol wt 18 000 and 20 000) in 10% polyacrylamide-sodium dodecyl sulfate gels. The amino-terminal end of the heavy chain was heterogeneous and contained isoleucine, valine and serine. The amino-terminal sequence of the light chain was Val-Leu-Asp. The factor X activator contained 13% carbohydrate including 6.0% hexose, 1.7% N-acetyleneuraminic acid, and 5.3% galactosamine. Most of the carbohydrate was found to be present in the heavy chain, although some was also observed in both forms of the light chain. The factor X activator had no esterase activity toward benzoyl-Phe-Val-Arg-p-nitroanilide or benzoylarginine ethyl ester and was not inhibited by 0.05 M diisopropyl phosphorofluoridate. These data indicate that factor X activator from Russell's viper venom is a highly specific protease composed of one heavy chain and one light chain, and these chains are held together by a disulfide bond(s).
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PMID:Factor X activating enzyme from Russell's viper venom: isolation and characterization. 99 Feb 51

Thrombomodulin (TM) is a surface glycoprotein that forms a 1:1 complex with thrombin, thereby interacting to form the basis of a major physiologically relevant natural anticoagulant mechanism. Although initially described as a vascular endothelial cell receptor, TM has been reported to be present in several other cells, including megakaryocytes, platelets, monocytes, and several cultured cells. Other investigators have reported that neutrophils (PMN) may play a role in the hemostatic mechanism by supporting transformation of prothrombin to thrombin. To determine whether PMN might contribute further to the regulation of the coagulation system, we have evaluated these cells for the expression of TM. Large numbers of human leukocytes were isolated by standard techniques, and the PMN fraction was extracted and shown to be free of platelets and monocytes. Membrane preparations were affinity purified on an anti-TM-Affigel-10 matrix and the eluted material was examined by Western blotting, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and silver staining. The purified material was identical in apparent molecular weight to TM from human placenta and human umbilical vein endothelial cells (HUVEC). Using a sensitive and specific immunoassay, we estimated that there are a minimum of 5,220 +/- 1,658 molecules of TM per PMN, as compared with more than 50,000 in HUVEC. Northern analysis of RNA from PMN indicates that specific messenger RNA for TM, as identified by a single 3.8-kb band, is identical to that from HUVEC, and thereby confirms that PMN can also synthesize the receptor. Localization of TM in PMN was attempted by immunofluorescence, and the receptor was visualized only in permeabilized PMN, but was not seen on the surface of nonpermeabilized cells. Flow cytometry was also used, and could detect TM in 10% to 15% of nonpermeabilized PMN, whereas the antigen was present in greater than 80% of permeabilized cells. Biologic function of the PMN-derived TM, as tested by thrombin-dependent activation of protein C, was absent. Our results suggest that TM is synthesized by PMN, but under nonstimulated conditions, the protein is largely excluded from the membrane surface, and lacks the ability to promote activation of protein C by thrombin. TM from PMN may provide a further link between inflammation and thrombosis and may also be a significant source of plasma TM.
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PMID:Human neutrophils synthesize thrombomodulin that does not promote thrombin-dependent protein C activation. 132 11

The mechanism of thrombosis following intravariceal injection of sodium tetradecyl sulphate (S.T.D.) was investigated with respect to effects on the vascular endothelium, the coagulation cascade, and platelet function. Using an umbilical cord model designed to simulate blood flow over the endothelium, it was found that S.T.D. is a potent toxin for endothelial cells in that brief exposure to even low concentrations of the agent were effective in stripping endothelium over a considerable distance, exposing highly thrombogenic endothelium in the process. Effects on coagulation and platelet function were found to be dependent on concentration. Diluted S.T.D. induced a hypercoagulable state, possibly in consequence of a selective inhibition of the physiological anticoagulant, protein C, and promoted platelet aggregation. Higher concentrations inactivated the coagulation cascade and lysed platelets completely. These results suggest that intravariceal infusion of S.T.D. at considerable dilution may be at least as effective in inducing thrombosis as standard dosage, and possibly more so.
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PMID:Mechanism of thrombosis caused by sclerotherapy of esophageal varices using sodium tetradecyl sulphate. 134 80

Activation of the alternative (APC) and classical (CPC) pathways of complement by fungal (1----3)-beta-D-glucans having different degrees of branching (DB) and different conformations were examined by using human serum and plasma. The glucans used in this study were curdlan (no branch; 0/1), grifolan (one branch in every third main chain unit; 1/3), schizophyllan (1/3), SSG (1/2), and OL-2(2/3). Triple or single helix conformer of these glucans were prepared by heating at 150 degrees C or dissolution in sodium hydroxide. Activation of APC by these glucans were dependent on incubation time, concentration, molecular weight, and DB. Interestingly, the triple helix conformer of all glucans tested activated APC stronger than a single helix one. The activity of branched glucans in plasma was weaker than those in serum. On the other hand, in the case of CPC, a single helix conformer activated CPC stronger than a triple helix one, and the activity was dependent on DB. Activation of CPC by a single helix conformer was thought to be dependent on the binding of beta-glucan to immunoglobulin in serum, because the complex was clearly detected by gel permeation chromatography only in the case of single helix one. From these results, it appears that the different conformers were recognized by the host complement systems in different ways. (1----3)-beta-D-Glucan is one of the major constituents of fungal cell wall and is thought to be clearly recognized by the host immune systems.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Activation of the complement system by (1----3)-beta-D-glucans having different degrees of branching and different ultrastructures. 143 67

An initial event in T cell activation is the specific adherence of T cells via their T cell receptor to the MHC peptide complex. We have studied this adherence by incubating T cells with preformed HLA DR4Dw4 peptide complexes attached to a solid support. Adherence of sodium 51Cr-labeled T cell clones specific for the influenza hemagglutinin peptide, HA 307-319, was maximal after 15 min and was specific for the HLA DR4Dw4-HA 307-319 complex. The binding was temperature dependent and could be blocked with azide or protein kinase C inhibitors, indicating that for adherence the T cells need to be metabolically active and have a functioning protein kinase C pathway. The adherence could be blocked with CD4- or CD3-reactive murine mAb, suggesting that the TCR and CD4 molecules work in concert to induce strong adherence to the HLA DR4Dw4-HA 307-319 complex. A subsequent event in T cell activation is proliferation, which is thought to need additional proteins such as IL-1 or other adhesion molecules. MHC peptide complexes coated on microtiter plates also induced proliferation in the human T cell clones. Removal of any monocytes by treatment of human T cell clones with anti-CD14 in conjunction with C, followed by purification over a nylon wool column, did not abrogate proliferation. After prolonged culture of the T cell clones in plates coated with peptide-pulsed HLA DR4Dw4 in the presence of IL-2, the T cell clones continued to proliferate in response to peptide. These results suggest that human T cell clones do not require a second signal from a monocyte or other APC to proliferate.
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PMID:Purified HLA class II peptide complexes can induce adherence and activation of peptide-specific human T cell clones. 153 49

About 30% of human plasma protein C is smaller than the predominant form as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It has been suggested that this species, referred to as beta protein C, is a degraded molecule. However, beta protein C is secreted in culture by the HepG2 cell line and is present in plasma collected directly into numerous proteinase inhibitors; the percentage of beta protein C does not change with time during culture or after blood collection. Neither thrombin, activated protein C, nor activated factor X converts the alpha form to beta in the presence or absence of calcium and phospholipids. The NH2-terminal sequences of the heavy chains of both forms are identical, and both release the same dodecapeptide and develop a functional active site when cleaved by thrombin. Both also react with antibodies to a synthetic COOH-terminal peptide. Timed digests with N-glycosidase are consistent with the interpretation that beta protein C has three N-linked oligosaccharide chains whereas alpha protein C has four. It is asparagine 329 that is not glycosylated in beta protein C since antibodies to a synthetic peptide based on the sequence around this amino acid react only with beta protein C. This site is unique in having cysteine instead of serine or threonine 2 residues distal. It is likely that the sulfhydryl group can substitute for the usual hydroxyl group as a hydrogen bond acceptor for the glycosylation reaction only until it forms a disulfide bond. The percentage of protein C that is glycosylated at this site may therefore depend at least in part on the rate of disulfide bond formation which may in turn be related to the rate of protein synthesis.
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PMID:Beta protein C is not glycosylated at asparagine 329. The rate of translation may influence the frequency of usage at asparagine-X-cysteine sites. 169 79

Sclerotherapy of bleeding esophageal varices in liver cirrhotics is a common procedure, but little is known about the possible entry of sclerosants into the systemic circulation. We injected a mixture of thrombin, sodium tetradecyl, and cefazolin and studied the effect of this sclerosant on selected hemostasis parameters. Twenty-four patients with liver cirrhosis (Child's Classification C) were studied 29 times. Blood samples were drawn before and immediately after the injection of the sclerosant. In seven patients we collected a sample 30 minutes and 24 hours after treatment. Before injection, almost all patients had elevated D-dimer, t-PA and PAI-1 levels. Fibrinogen, antithrombin, alpha-2 antiplasmin, and protein C were decreased. Only thrombin/antithrombin III complex (TAT) levels were within normal ranges. Immediately after the injection, TAT, D-dimer, and t-PA levels rose significantly (P less than 0.001, P less than 0.01, P less than 0.001), PAI-1 and PC levels decreased (P less than 0.01), while antithrombin, alpha-2 antiplasmin, and fibrinogen concentrations were unchanged. TAT and D-dimer levels were still elevated after 24 hours (P less than 0.05). These data indicate that thrombin entered the systemic circulation (elevated TAT) and that the hemostasis system was briefly systemically activated (elevated D-dimer). In spite of these changes in the hemostasis system, clinically there were no detectable thrombotic or hemorrhagic complications.
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PMID:Hemostasis activation during esophageal variceal sclerotherapy with thrombin in cirrhotics. 171


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