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)

The molecular weight fractions of 10,000-50,000 daltons prepared from "used" medium obtained during cultivation of human colon carcinoma cells (SW-48) in vitro inhibited the proliferation and DNA synthesis of these cells. Fractions exceeding 50,000 daltons were not inhibitory; those less than 10,000 daltons were cytotoxic. The inhibitory fraction did not affect either proliferation of human fibroblasts or transformation of human lymphocytes in vitro. Similar fractions from the colon mucosa of other species inhibited the proliferation of SW-48 cells, whereas extracts of dog jejunum or lung did not. This mitotic inhibition was completely reversible and could be destroyed by preincubation with trypsin. Therefore, colon cells appear to contain a cell- (but not species) specific, endogenous mitotic inhibitor or chalone.
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PMID:Evidence for a colon chalone. 13 20

B cells derived from peripheral-blood lymphocytes (PBL) and tumor-infiltrating lymphocytes (TIL) from a patient with a high serum antibody titer to autologous melanoma were transformed with Epstein-Barr virus (EBV) and evaluated for reactivity against autologous tumor. B cells producing antibody reactive with autologous tumor and unreactive with normal fibroblasts were detected both in TIL and in PBL. One cell line derived from PBL and another derived from TIL sustained production of tumor-reactive antibody for 10 weeks and over 15 months respectively. The cell line derived from PBL, 2D11, produced an antibody reactive with a trypsin-resistant antigen expressed on the cell membrane of autologous and allogeneic melanoma cell lines. The cell line derived from TIL, 1F6, produced an antibody reactive with a cell-surface glycoprotein expressed by 5 autologous melanoma cell lines derived from 5 different metastases and 16/19 allogeneic melanoma cell lines. 1F6 also showed reactivity with cell lines derived from a blue nevus, a congenital nevus, an astrocytoma, and 1/4 renal-cell carcinomas; but it was not reactive with 5 foreskin melanocyte cell lines, 2 normal fibroblast lines, 5 leukemia/lymphoma lines, 8 lung-cancer lines, 8 glioblastoma lines, or lines derived from 1 ovarian carcinoma, 1 colon carcinoma, 1 vulvar carcinoma, 1 fibrosarcoma, 1 murine melanoma, or 4 murine leukemia/lymphomas. We describe here an antibody that detects a new melanoma specificity obtained by EBV transformation of tumor-infiltrating B cells.
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PMID:Analysis of two human monoclonal antibodies against melanoma. 145 38

An enterotoxin produced by Bacteroides fragilis was purified to homogeneity and characterized as to its biological activity and basic molecular properties. Toxin preparations were prepared by growing B. fragilis VPI 13784 in brain heart infusion broth to early stationary phase, immediately precipitating the culture supernatant fluid with 70% ammonium sulfate, and stabilizing the precipitate with the protease inhibitor TPCK (tolylsulfonyl phenylalanyl chloromethyl ketone). The toxin was sequentially purified by anion-exchange chromatography on Q-Sepharose, hydrophobic interaction chromatography on phenyl-agarose, and high-resolution ion-exchange chromatography on Mono Q. The toxin appeared homogeneous as judged by polyacrylamide gel electrophoresis. The estimated molecular weight of the highly purified toxin as determined by gel filtration chromatography on Superose-12 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 19,000. It has an isoelectric point of approximately 4.5 and is stable at pHs 5 to 10. The purified toxin is stable at -20 and 4 degrees C and upon freeze-drying, but it is unstable at temperatures above 55 degrees C. It is sensitive to proteinase K and Streptomyces protease but is resistant to trypsin and chymotrypsin. The activity of the purified toxin is neutralized by antiserum to a toxigenic strain of B. fragilis but not by antiserum to nontoxigenic strains. N-terminal amino acid analysis reveal an unambiguous sequence of Ala-Val-Pro-Ser-Glu-Pro-Lys-Thr-Val-Tyr-Val-Ile-Xxx-Leu-Arg-Glu-Asn-Gly- Ser-Thr . The highly purified toxin induced a strong fluid accumulation response in the lamb ileal-loop assay as well as a cytotoxic response (cell rounding) on HT-29 colon carcinoma cells. Thus, the purified toxin can cause both enterotoxic and cytotoxic activities.
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PMID:Purification and characterization of an enterotoxin from Bacteroides fragilis. 154 60

Chloride channels that have an intermediate conductance and are outwardly rectifying were studied by the patch-clamp technique in cell-excised membrane patches from respiratory epithelial cells in primary culture (REC) of normal and cystic fibrosis tissue, HT29 and T84 human colon carcinoma cells and placenta trophoblast cells (PTC). Chloride channels were immediately activated by the exposure of the cytosolic side of the patch to a Ringer-type solution, which lacked cytosolic components normally inhibiting chloride channels in the "on" cell configuration. Tentatively, we labelled the cytosolic component (or components) responsible for this inhibition cytosolic inhibitor (CI). The presence of CI in cytosol derived from HT29 cells was shown by assaying crude cytosol extracts from these cells on Cl- channels from HT29 cells (n = 2) and REC from normal subjects and cystic fibrosis patients (n = 4). In order to examine CI further, PTC were used as a source of cytosol. The cytosol of PTC inhibited HT29 Cl- channels in a dose-dependent manner with a half-maximal inhibition observed at a 1:6 dilution (n = 11) of the native cytosol. CI from PTC was heat-stable (10 min at 100 degrees C, n = 8). When cytosol extract was partitioned into a chloroform phase, Cl- channel inhibition was shown for the lipophilic extract (n = 12) as well as for the aqueous phase (n = 10). The inhibitory potency of the lipid extract was slightly larger than that of the aqueous phase. Several separation procedures were used to determine the molecular size of CI. When CI was filtered through 30-kDa filters at 6000 rpm for 45 min, inhibitory potency was observed in the filtrate and the retained fraction (n = 3). The same was observed with 10-kDa filters (n = 6). When CI was dialysed through a 12-kDa membrane, inhibitory capacity was recovered from the dialysate. Similarly, gel filtration indicated that CI was less than 5 kDa (n = 13) and probably less than 1.5 kDa (n = 11), but greater than 700 kDa (n = 9). CI was exposed to bead-coupled hydrolysing enzymes (trypsin, non-specific protease, lipase, alpha-amylase, nucleotidase), but none of the enzymes used destroyed the inhibitory potency of CI. These data indicate that CI is present in HT29 as well as in PTC. It inhibits reversibly intermediate-conductance outwardly rectifying Cl- channels in REC, HT29, and PTC.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Inhibition of epithelial chloride channels by cytosol. 165 43

The MET proto-oncogene encodes a 190-kDa disulfide-linked heterodimeric receptor (p190 alpha beta) whose tyrosine kinase activity is triggered by the hepatocyte growth factor. The mature receptor is made of two subunits: an alpha chain of 50 kDa and a beta chain of 145 kDa, arising from proteolytic cleavage of a single-chain precursor of 170 kDa (pr170). In a colon carcinoma cell line (LoVo), the precursor is not cleaved and the Met protein is exposed at the cell surface as a single-chain polypeptide of 190 kDa (p190NC). The expression of the uncleaved Met protein is due to defective posttranslational processing, since in this cell line (i) the proteolytic cleavage site Lys-303-Arg-Lys-Lys-Arg-Ser-308 is present in the precursor, (ii) p190NC is sensitive to mild trypsin digestion of the cell surface, generating alpha and beta chains of the correct size, and (iii) the 205-kDa insulin receptor precursor is not cleaved as well. p190NC is a functional tyrosine kinase in vitro and is activated in vivo, as shown by constitutive autophosphorylation on tyrosine. The MET gene is neither amplified nor rearranged in LoVo cells. Overlapping cDNA clones selected from a library derived from LoVo mRNA were sequenced. No mutations were present in the MET-coding region. These data indicate that the tyrosine kinase encoded by the MET proto-oncogene can be activated as a consequence of a posttranslational defect.
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PMID:Defective posttranslational processing activates the tyrosine kinase encoded by the MET proto-oncogene (hepatocyte growth factor receptor). 165 24

Monoclonal antibody (mAb) 83D4 was generated using formol-fixed paraffin-embedded human breast carcinoma tissue as the immunogen. Previous studies demonstrated that it was reactive with breast carcinoma tissues, but not with normal breast. The antigen identified by mAb 83D4 was detected, using ELISA, in MCF7 breast carcinoma cell line membrane extracts, in primary breast and colon carcinoma tissue extracts and in pleural effusion fluid from patients with metastatic breast cancer. No reactivity with 83D4 was found in either human milk fat globule membranes or skimmed milk. 83D4 reactive antigen was found to be a heterogeneous high molecular weight (MW) protein (apparent Mr:300-400 to over 1000 kDa) by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The antigen was purified from MCF7 cells, breast and colon carcinomas and effusion fluid, by perchloric acid solubilisation followed by immunoaffinity chromatography with 83D4. The immunopurified antigen from MCF7 cells and pleural effusion fluid was further analysed by gel filtration and ion-exchange chromatography, which confirmed the high MW and indicated the charge heterogeneity of the reactive molecules. The 83D4 reactive antigen strongly bound to wheat-germ agglutinin and weakly to peanut lectin. No binding was found with lentil lectin or concanavalin A. Antigenic activity was strongly reduced by trypsin and subtilysin digestion and by treatment with sodium periodate, but it was not affected by neuraminidase. These results imply the glycoprotein nature of the 83D4-defined antigen and the involvement of carbohydrate, but probably not sialic acid, in the epitope. Purified 83D4 antigen did not display reactivity for mAb HMFG-1, directed against a polymorphic epithelial mucin, PEM, using ELISA, but bound mAb CC49 and weakly mAb B72.3, antibodies which define a tumour associated glycoprotein, TAG-72. Moreover CC49 and 83D4 showed similar reactivity pattern in immunoblotting assays. A double determinant radioimmunoassay confirmed that 83D4 antigen carries epitopes for mAb B72.3 and CC49. Competition radioimmunoassays clearly distinguished the 83D4 defined epitope from those recognised by B72.3 and CC49, demonstrating that antibody 83D4 identifies a unique epitope. It is suggested that the antigens identified by mAb 83D4 and by mAb B72.3 and CC49 may form part of the same family of carcinoma associated glycoproteins.
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PMID:Purification and characterisation of a breast-cancer-associated glycoprotein not expressed in normal breast and identified by monoclonal antibody 83D4. 170 94

We have recently demonstrated that many cancer cell lines produce a novel trypsinogen isoenzyme called tumor-associated trypsinogen 2 (TAT-2). It was found during a search of the target protease for tumor-associated trypsin inhibitor (TATI). We now show that degradation of subendothelial cell extracellular matrix (ECM) by four different cell lines (COLO 205 colon carcinoma, K-562 erythroleukemia, CAPAN-1 pancreatic carcinoma, and HT 1080 fibrosarcoma) can be partially inhibited by TATI or neutralizing trypsin antibodies. When cells were cultured in serum-free medium on ECM, TATI and trypsin antibodies inhibited the release of immunoreactive fibronectin fragments from ECM by 47-54 and 40%, respectively. Degradation of isotopically labeled ([3H]serine, [3H]proline, and [35S]sulfate) ECM was also significantly prevented by TATI. At its maximum, it exerted a 57% inhibition on the degradation of [3H]serine-labeled ECM. Plasminogen added exogenously to the culture medium further potentiated the proteolysis of ECM. Interestingly, addition of enteropeptidase, an activator of TAT-2, also enhanced cell-mediated proteolysis as assessed by degradation of purified fibronectin coated onto the surface of wells. Immunoblot analysis showed that enteropeptidase-mediated proteolysis generated a pattern of fibronectin fragments similar to that obtained by digestion of purified fibronectin by TAT-2. These results demonstrate the existence of a proteolytic system in tumor cells which is dependent on the activation of TAT-2. We suggest that TAT-2 is involved in a protease cascade-stimulating tumor cell invasion and degradation of extracellular matrix.
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PMID:Tumor-associated trypsin participates in cancer cell-mediated degradation of extracellular matrix. 200 30

The possible participation of tight junction formation in intercellular adhesion of tumor cells was studied in single cell suspensions of HT29 adenocarcinoma cells. Very low dose trypsin treatment (0.15 micrograms/ml, 30 min, 37 degrees C) induced rapid intercellular adhesion of suspended HT29 colon carcinoma cells. Intercellular adhesion was independent of the presence of divalent cations. Electron microscopy of freeze-fractured membrane fragments of trypsin-treated HT29 cells revealed a dramatic increase of typical tight junction structures during aggregation.
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PMID:Rapid aggregation and tight junction formation in single cell suspensions of tumor cells after very low dose trypsin treatment. 201 7

The liver is frequently colonized by metastatic tumor cells despite its dense population of macrophages (Kupffer cells). We have studied the interactions between metastatic colon carcinoma cells (DHD) and syngeneic Kupffer cells under different experimental conditions in vitro. In an adhesion assay the binding of DHD cells to Kupffer cell monolayers was shown to be time and temperature dependent, reaching a maximum level after about 90 min of incubation at 37 degrees C. In contrast, only a low level of binding could be observed at 4 degrees C. The level of binding could be increased by pretreatment of the Kupffer cells with phorbol 12-myristate 13-acetate. A firm interaction between the two cell types was shown to be dependent on the presence of calcium- and trypsin-sensitive structures on the surface of the Kupffer cells. Pretreatment of the macrophages with the cytoskeletal inhibitors colchicine and cytochalasin B was also found to reduce significantly the binding of tumor cells. This binding was also inhibited to a large extent by D-mannose and N-acetyl-D-galactosamine. The Kupffer cells were not cytotoxic against the colon carcinoma cells.
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PMID:Binding of metastatic colon carcinoma cells to liver macrophages. 249 36

Different molecular masses have been assigned to the human interferon-gamma receptor (HuIFN-gamma-R) by several authors. After extensive purification from Raji cells, this receptor was shown in a previous work to consist of two major protein species with molecular masses of 92 kD and 50 kD, as revealed by SDS-PAGE. We show here that the 50-kD band is most probably a degradation product of the 92-kD band due to a trypsin-like protease active during the purification process. The native protein of Raji cells seems, therefore, to have a molecular mass of 92 kD. The same molecular mass was found with Colo 205 cells (derived from a colon carcinoma). However, in conditions where degradation does not occur, the HuIFN-gamma-R shows a certain polymorphism: in IM-9 cells, another B-cell line, two bands exist with molecular masses of 95 kD and 85 kD, and in Wish cells, an amnion-derived cell line, one (or two) band(s) can be detected around 87 kD. This polymorphism is due at least in part to a variable extent of N-glycosylation from line to line and also within the same line, since after tunicamycin treatment of the Raji, IM-9, and Wish cells, very similar bands are obtained with a molecular mass of 72 kD.
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PMID:Molecular characterization of the human interferon-gamma receptor: analysis of polymorphism and glycosylation. 253 62


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