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 LIM 1863 colon carcinoma cell line grows in suspension as morphologically and functionally organized organoids in serum-containing medium. Addition of TGF-beta caused the organoids to adhere and inhibited DNA synthesis. A 20 min incubation with TGF-beta was sufficient to induce adherence and this could be inhibited by cycloheximide. The adhesion and DNA synthesis inhibition were demonstrated to be separate events. We were not able to detect any changes in matrix or cell membrane antigens. Similarly there were no changes in synthesized proteins (by two-dimensional gel electrophoresis), and no upregulation of proteoglycan. When adhered organoids were lysed from the tissue culture plastic surface, untreated organoids adhered to this surface. This 'conditioned' surface was destroyed by trypsin but not collagenase or medium from normal LIM 1863 cultures. However, the adherent phenotype was prevented when organoids were treated with transforming growth factor-beta (TGF-beta) in the presence of medium conditioned by normal LIM 1863 cultures rather than in fresh medium. The adhesion process was inhibited by an antibody (QE2E5) against beta 1 integrin although no quantitative changes in integrins were observed (by immunoprecipitation or RNA analysis). A second anti-beta 1 integrin antibody (61.2C4) inhibited LIM 1863 adhesion to collagen but not TGF-beta induced adhesion, implying that TGF-beta induced a specific conformational change or interaction of a beta 1 integrin. In this morphologically structured system TGF-beta induced a number of subtle effects including formation of new extracellular matrix and conformational change of a beta 1 integrin, rather than the major quantitative changes in cell/matrix molecules reported previously.
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PMID:Effect of TGF-beta on differentiated organoids of the colon carcinoma cell line LIM 1863. 759 Aug 99

We previously reported that murine tumor cells with a high spontaneous metastatic potential to the lung secrete higher amounts of M(r) 95,000 gelatinase (matrix metalloproteinase 9, MMP9) than do poorly metastatic cells. The present study, conducted to clarify the mechanisms underlying the increase in MMP9, revealed an autocrine factor that enhances the secretion of M(r) 95,000 gelatinase (MMP9). The secretion of MMP9 by highly metastatic colon carcinoma LuM1 cells, detected by zymography, was augmented 10-fold when cultured in medium supplemented with serum-free medium conditioned with LuM1 cells. Because the secretion of M(r) 60,000 gelatinase (MMP2), as well as total protein, by the same cells was not affected under these conditions, the augmentation appears specific for MMP9. The steady-state level of MMP9 mRNA was elevated in LuM1 cells cultured in the presence of the supernatant. The amount of the factor in the culture medium increased with time in culture, indicating that it was produced by the LuM1 cells. It was found to be heat stable but sensitive to trypsin digestion. Conditioned medium from poorly metastatic NM11 cells did not stimulate the secretion of gelatinases by NM11 cells, suggesting that autocrine stimulation of MMP9 secretion is a characteristic of metastatic cells. This factor could account for the augmented secretion of MMP9 by murine tumor cells with spontaneous metastatic potential to the lung.
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PMID:Autocrine factor enhancing the secretion of M(r) 95,000 gelatinase (matrix metalloproteinase 9) in serum-free medium conditioned with murine metastatic colon carcinoma cells. 801 88

We report the initial identification of a 37.5-kDa putative aldoketo reductase in human colon carcinoma cells. An aminoterminal trypsin fragment was sequenced and found to be identical to bovine prostaglandin F synthase in 19 of 21 amino acids. Levels of this cytosolic human aldo-keto reductase, assessed by immunoblots using polyclonal antibodies raised against this protein, increased 30-fold in cells resistant to the Michael reaction acceptor ethacrynic acid and increased with time and ethacrynic acid concentration after treatment of wild-type cells. Induction of the reductase appeared to be cell type and drug specific. It was induced by the Michael reaction acceptors dimethyl maleate, t-butylhydroquinone, and hydroquinone but not by the nitrogen mustard chlorambucil. Ethacrynic acid and dimethyl maleate induced the reductase in a second human colon cell line but not in human prostate cells. NADPH-dependent metabolism of aldoketo reductase substrates by cytosol from colon but not prostate cells was enhanced 2-3-fold when cells were grown in the presence of either ethacrynic acid or dimethyl maleate. The discrepancy between induced reductase activity and protein levels may be due to the multiplicity of constitutively expressed NADPH-dependent reductases that compete for substrate. Ethacrynic acid-resistant cells exhibited low levels of cross-resistance to Adriamycin, mitomycin C, and the bovine prostaglandin F synthase substrates phenylglyoxal and prostaglandin D2. Thus, significant overexpression of a human aldo-keto reductase structurally related to bovine prostaglandin F synthase may result from exposure of cells to Michael reaction acceptors and may give rise to an enhanced capacity to metabolize exogenous and endogenous substrates, thereby contributing to the drug-resistant phenotype.
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PMID:Overproduction of a 37.5-kDa cytosolic protein structurally related to prostaglandin F synthase in ethacrynic acid-resistant human colon cells. 831 17

Human breast and colon carcinoma tissues contain a form of collagen, not described before, composed of alpha 1 chains of similar size (approximately 100 kDa) but different charge. The three constitutive chains, separated by two-dimensional electrophoresis, are a unique acidic component, undetectable in other collagen types, with an apparent isoelectric point of 4-5, and two more basic components displaying the same electrophoretic behavior as alpha 1(III) and alpha 1(I), respectively. The acidic chain is structurally distinct from alpha 1(I) and displays a cyanogen bromide-derived fragment of similar size to CB5(III). This collagen in its native state is resistant to trypsin and metalloproteinase 3, while it is fully degraded by metalloproteinases 1 and 9. Moreover, this collagen appears able to bind to laminin, as tested by affinity chromatography. The biological significance of our data is related to the finding of this collagen form not only in the tumor tissue tested but also in embryonic-fetal tissues (bovine skin and intestine and human umbilical cord). For its peculiar laminin-binding ability and occurrence in tumoral and embryonic-fetal tissues, we propose to temporarily term this new collagen form OF/LB collagen (onco-fetal, laminin-binding collagen). The presence of OF/LB collagen during development and cancer, and its absence in normal adult tissues, make this protein a potential stromal marker of malignancy.
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PMID:A new form of tumor and fetal collagen that binds laminin. 833 39

Rotaviruses were detected via electron microscopy in fecal specimens collected from school children during an outbreak of diarrhea and from a sporadic case in 1993 in Japan. All of the viruses were found to belong to human group C rotavirus by reverse passive hemagglutination assay (RPHA). These viruses replicated well in a human colon carcinoma (CaCo-2) cell line cultured in the presence of trypsin (4 micrograms/ml). This report demonstrates that human group C rotaviruses can be propagated efficiently in a cell line cultured in the presence of trypsin. The infected cells did not show any apparent cytopathic changes. However, virus was detected in the cell cytoplasm by immunofluorescence (IF) staining and in the culture supernatant by RPHA. On the basis of immune electron microscopy (IEM), virus particles collected from infected CaCo-2 cell cultures were confirmed to aggregate specifically with anti-human group C rotavirus antibody. The electrophoretic patterns of RNA segments extracted from viral particles found in the fecal specimens or infected cells were identical to those of human group C rotavirus. These results indicated that human group C rotaviruses were the causal agent of the diarrhea outbreak.
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PMID:Isolation and serial propagation of human group C rotaviruses in a cell line (CaCo-2). 882 10

Increased production of proteinases, such as matrix metalloproteinases (MMPs), is a characteristic feature of malignant tumors. Some human cancers and cell lines derived from them also express trypsinogen, but the function of the extrapancreatic trypsin has remained unclear. In this study we cloned and sequenced trypsinogen-2 cDNA from human COLO 205 colon carcinoma cells and characterized the ability of the enzyme to activate latent human type IV procollagenases (proMMP-2 and proMMP-9). As shown by cloning and N-terminal amino acid sequencing, the amino acid sequence of tumor-associated trypsin-2 is identical to that of pancreatic trypsin-2. We found that both pancreatic trypsin-2 and tumor cell-derived trypsin-2 are efficient activators of proMMP-9 and are capable of activating proMMP-9 at a molar ratio of 1:1000, the lowest reported so far. Human trypsin-2 was a more efficient activator than widely used bovine trypsin and converted the 92-kDa proMMP-9 to a single 77-kDa product that was not fragmented further. The single peptide bond cleaved by trypsin-2 in proMMP-9 was Arg87-Phe88. The generation of the 77-kDa species coincided with the increase in specific activity of MMP-9. In contrast, trypsin-2 only partially activated proMMP-2. Trypsin-2 cleaved the Arg99-Lys100 peptide bond of proMMP-2 generating 62-65-kDa MMP-2 species. Trypsin-2-induced proMMP-2 and -9 conversions were inhibited by tumor-associated trypsin inhibitor added either prior to or during activation indicating that proMMPs were not activated autocatalytically. Trypsin-2 also activated proMMPs associated with tissue inhibitor of matrix metalloproteinases, the complexes of which are thought to be the major MMP forms in vivo. The ability of human tumor cell-derived trypsin-2 to activate latent MMPs suggests a role for trypsin-2 in initiating the proteinase cascade that mediates tumor invasion and metastasis formation.
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PMID:Activation of type IV procollagenases by human tumor-associated trypsin-2. 926 Nov 9

In this study we assessed the usefulness of Caco-2 cells, derived from a human colon carcinoma, to isolate an influenza virus. Throat washings collected from 30 patients with influenza-like illnesses in Miyazaki Prefecture in 1997 were inoculated in MDCK and Caco-2 cells, 17 influenza virus strains were isolated in MDCK cells, and 20 in Caco-2 cells. Of all the viruses isolated, only one strain was identified as influenza virus type B; other strains were identified as type A (H3N2). Furthermore, some influenza viruses were isolated in Caco-2 cells also from the specimens collected between 1991 and 1997. With Caco-2 cells, each type of influenza virus was isolated effectively without the supplement of trypsin in the culture medium. These facts indicate the usefulness of Caco-2 cells as a host to isolate influenza virus as shown to be suitable in the detection of many types of enteric viruses. Caco-2 cells will serve as a useful cell line for the surveillance of infectious disease because Caco-2 cells are sensitive to a wide range of virus.
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PMID:[Use of Caco-2 cells for isolation of influenza virus]. 962 62

Bacteroides fragilis isolates from intestinal and non-intestinal infections, normal flora and the environment were examined for properties linked with interactions among cells in vitro. Different adhesion molecules were detected in agglutination assays with human erythrocytes and tests for auto-agglutination and adherence to human colon carcinoma cells (HT29). There was no correlation between these properties, indicating that independent molecules are involved. Treatment with trypsin, heat or EDTA inhibited agglutination and adherence, suggesting that these molecules are proteins. The lack of correlation with the origin of the strains did not permit any of these activities to be recognised as virulence markers. The expression of fragilysin, a protease associated with damage to intestinal cells and bacterial translocation, was examined. Only those strains from patients with diarrhoea expressed this protease activity in assays with HT29 cells and this was confirmed by specific PCR for the bft gene. The activity of fragilysin as an enterotoxin was confirmed in the rabbit intestinal ligated loop assay. The association of this property only with strains from intestinal infections indicates that it is too early to suggest this protease as a determinant factor of B. fragilis invasiveness.
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PMID:Expression of Bacteroides fragilis virulence markers in vitro. 1053 43

Studies in different liver-derived cells in culture indicate that apolipoprotein (apo) B-100 production is regulated largely by intracellular degradation and the ubiquitin-proteasome pathway is a major mechanism for the degradation. The proteasomal degradation of apoB-100 was postulated to be an intrinsic property of the protein that occurs even in the presence of optimal amounts of lipids supplied to the cell. We examined apoB-100 and apoB-48 biogenesis in CaCo2, a human colon carcinoma cell line. To our surprise, apoB-100 and apoB-48 were quantitatively secreted by CaCo2 cells; essentially none of the newly synthesized apoB was degraded before secretion in a 2-h period whether the cells were cultured on filter or on plastic. Furthermore, although ubiquitin immunoreactivity was readily detected in the intracellular apoB isolated from HepG2 cells, little or no ubiquitin was detectable in the intracellular apoB from CaCo2 cells. The amounts of free ubiquitin and total and non-apoB ubiquitinated proteins were comparable in HepG2 and CaCo2 cells, indicating that CaCo2 cells have the necessary machinery for tagging ubiquitin chains onto cellular proteins for proteasomal degradation. Incubation in lipoprotein-deficient serum did not induce apoB degradation, but the addition of a microsomal triglyceride transfer protein inhibitor led to apoB degradation in CaCo2 cells. Finally, similar proportions of apoB polypeptide in isolated microsomes from CaCo2 and HepG2 cells were accessible to exogenously added trypsin, indicating that the mere exposure of apoB nascent chains to the cytosolic compartment is insufficient to cause the proteasomal degradation. Therefore, the intracellular degradation of apoB is not an intrinsic property of the protein, and the phenomenon is neither universal nor inevitable. The unconditional use of apoB as a paradigm for intracellular protein degradation is not warranted.
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PMID:Apolipoprotein B, a paradigm for proteins regulated by intracellular degradation, does not undergo intracellular degradation in CaCo2 cells. 1066 May 49

Lovastatin and simvastatin are HMG-CoA reductase inhibitors widely used as antihyperlipidemic drugs, which also display antiproliferative properties. In the present paper, we provide evidence that both lovastatin and simvastatin are modulators of the purified bovine pituitary 20 S proteasome, since they mildly stimulate the chymotrypsin-like activity and inhibit the peptidylglutamylpeptide hydrolyzing activity without interfering with the trypsin-like activity. However, those effects are only observed when the closed ring forms of the drugs are used, while the opened ring form of lovastatin acts as a mild inhibitor of the chymotrypsin like activity. The closed ring form of lovastatin is much more potent as a cytotoxic agent on the Colon-26 (C-26) colon carcinoma cell line than the opened ring form, which is only mildly cytostatic. Moreover, neither the cytotoxic effects nor the effects on 20 S proteasome activities are prevented by mevalonate, which by itself inhibits the trypsin-like activity of the proteasome. Neither the opened ring nor the closed ring form of lovastatin induces an accumulation of ubiquitin-protein conjugates, which is observed after treatment with lactacystin, a selective proteasome inhibitor. In contrast with the opened ring form of lovastatin, the closed ring form induces the disappearance of detectable p27(kip1) from C-26 cells. Altogether, our results indicate that the closed ring form of lovastatin induces cytotoxic effects independent of its HMG-CoA inhibiting activity, however, those effects are mediated by a complex modulation of proteasome activity rather than by inhibition of the 20 S proteasome.
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PMID:Lovastatin and simvastatin are modulators of the proteasome. 1108 75


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