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)

Cells growing on plastic or glass surfaces in vitro may be brought into suspension by proteases (e.g. trypsin) or chelating agents (e.g. EGTA). Trypsin and EGTA remove different quantities and types of molecules from cell surfaces. Previous studies have revealed that when confluent cultures of either BHK or PyBHK cells are brought into suspension by exposure to trypsin, foetal calf serum (or fibronectin) is required for cell attachment to films of denature type I collagen, but not to 3-dimensional gels of native collagen fibres. In this communication the serum requirements for the attachment of BHK and PyBHK cells to collagen substrata have been examined as a function of (a) the method used to prepare the cell suspension (EGTA or trypsin), and (b) cell density. Data are presented consistent with the view that cell surface-associated fibronectin is able to mediate cell attachment directly to films of denatured collagen.
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PMID:The effects of EGTA and trypsin on the serum requirements for cell attachment to collagens. 23 8

Elastolytic enzyme was purified and crystallized from culture fluid of Flavobacterium immotum No. 9-35. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis. The molecular weight was determined by Sephadex G-100 gel filtration to be 13,000. The isoelectric point was between pH 8.3 and 8.9. The optimum pH of the enzyme was 7.2 for elastolytic activity. The purified enzyme showed not only elastolytic activity, but also non-specific proteolytic activity against various other proteins. Milk-clotting activity was also observed. The enzyme did not act on keratin, collagen, or fourteen amino acid esters, including N-benzoyl-L-alanine methyl ester, N-benzoyl-L-arginine ethyl ester, and N-acetyl-L-tyrosine ethyl ester, which were typical substrates of pancreatic elastase [EC 3.4.21.11], trypsin [EC 3.4.21.4], and chymotrypsin [EC 3.4.21.1], respectively. However, the enzyme selectively hydrolyzed elastin when both elastin and albumin were present in the reaction mixture. The enzyme was inhibited by o-phenanthroline and various heavy metals such as cadmium, lead, zinc, and mercury. Various inhibitors, such as diisopropyl phosphofluoridate, tosyl-L-lysine chloromethyl ketone, tosyl-L-phenylalanine chloromethyl ketone, trypsin inhibitor, iodoacetamide, etc., had no effect on the elastolytic activity.
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PMID:Purification and properties of elastolytic enzyme from Flavobacterium immotum. 23 95

When cultured in-vitro, originating from different breast cancer patients, tumor cells, identified histologically as carcinoma cells, varied in their proliferation patterns and cell morphology. If exposed for brief periods to vibrio cholera neuraminidaes (VCN), the amount of sialic acid released from the cells varied from one culture to another and increased with higher enzyme concentrations. If exposed to trypsin, the amount of released proteins varied also from one culture to another. Significant difference was observed between the effect of VCN or collagenase on normal and neoplastic cell cultures. Whether human or murine cell cultures, the cell-free media harvested from cultures of neoplastic cells containing high concentrations of collagenolytic-caseinolytic-fibrinolytic and esterolytic activities. Two effects of concanavalin A (Con A) have been distinguished on thymidine incorporation, the first is a decrease in the maximal thymidine uptake, whereas the second is a shift to the maximum thymidine uptake to higher Con A concentrations. At low concentrations, alpha-1-antitrypsin (AAT) had no effect, but at high concentrations it inhibited 3H-thymidine uptake. At low concentrations human profibrinolysin inhibited and at higher concentration sit enhanced uptake of the labeled precursor. Therefore, the collagen olytic caseinolytic-fibrinolytic enzyme is a pacemaker for proliferation of human mammary carcinoma cells.
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PMID:Human mammary carcinoma cells. The enzyme pacemaker profibrinolysin. 31 26

In vitro differentiation of chick embryo brain cells was compared under several culture conditions. Morphological observations and acetylcholinesterase histochemical staining revealed that the development was similar in all conditions tested if cells have been derived from 7 days embryos. Considering the cultures from 11 days embryos, the cell dissociation by trypsin and the plastic surface proved to be the most favourable conditions in contrast to mechanical dissection and collagen surface.
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PMID:A comparative study of the differentiation of dissociated nerve cells under different culture conditions. 32 35

Synovial tissues from rheumatoid-arthritis patients were dissociated by enzymes and the resulting cells incubated overnight in tissue-culture flasks. The adherent cell population was resuspended with EDTA-trypsin, and morphological examination showed 68--80% non-lymphoid cells, most of which had the appearance of synovial lining cells. The proportion of these cells increased during subsequent culture. Between 40 and 60% of the cells exhibited marked phagocytosis, and less than 14% of the non-lymphoid cells could form EA rosettes. Further culture diminished this Fc-receptor-bearing cell population. Indirect immunofluorescence studies with rabbit anti-human collagen sera revealed membrane staining for 30--60% of the cells; this proportion usually increased to greater than 90% after 6--14 days in culture. Omitting any changes of culture medium resulted in a marked decrease in the proportion of cells staining with anti-collagen sera, whereas the viability and phagocytic ability of the cells did not significantly alter. Subsequent cell passage was followed by an increase in the proportion of cells demonstrating membrane-associated collagen, and this effect was more pronounced when a high concentration (50%) of serum supplement was used. No clear definition could be made as to whether the membrane-associated collagen represents synthesis or phagocytosis of collagen by the cells. Faint membrane staining was also observed with non-immune rabbit serum for 4--20% of the cells after the initial overnight incubation, and this usually dropped to less than 5% during prolonged culture. Rabbit antisera to human albumin, F(ab')2 fragment of IgG or alpha2-macroglobulin also gave similar results, whereas the F(ab')2 fragment of rabbit IgG antibody to human alpha2-macroglobulin was completely negative. More than 99% of the cells commonly exhibited membrane-associated beta2-microglobulin.
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PMID:Membrane characteristics of adherent cells dissociated from rheumatoid synovial tissue. 33 58

Fibronectin, the major cell surface glycoprotein of fibroblasts, is absent from differentiated cartilage matrix and chondrocytes in situ. However, dissociation of embryonic chick sternal cartilage with collagenase and trypsin, followed by inoculation in vitro reinitiates fibronectin synthesis by chondrocytes. Immunofluorescence microscopy with antibodies prepared against plasma fibronectin (cold insoluble globulin [CIG]) reveals fibronectin associated with the chondrocyte surface. Synthesis and secretion of fibronectin into the medium are shown by anabolic labeling with [35S]methionine or [3H]glycine, and identification of the secreted proteins by immunoprecipitation and sodium dodecyl sulfate (SDS)-disc gel electrophoresis. When chondrocytes are plated onto tissue culture dishes, the pattern of surface-associated fibronectin changes from a patchy into a strandlike appearance. Where epithelioid clones of polygonal chondrocytes develop, only short strands of fibronectin appear preferentially at cellular interfaces. This pattern is observed as long as cells continue to produce type II collagen that fails to precipitate as extracellular collagen fibers for some time in culture. Using the immunofluorescence double-labeling technique, we demonstrate that fibroblasts as well as chondrocytes which synthesize type I collagen and deposit this collagen as extracellular fibers show a different pattern of extracellular fibronectin that codistributes in large parts with collagen fibers. Where chondrocytes begin to accumulate extracellular cartilage matrix, fibronectin strands disappear. From these observations, we conclude (a) that chondrocytes synthesize fibronectin only in the absence of extracellular cartilage matrix, and (b) that fibronectin forms only short intercellular "stitches" in the absence of extracellular collagen fibers in vitro.
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PMID:Synthesis and extracellular deposition of fibronectin in chondrocyte cultures. Response to the removal of extracellular cartilage matrix. 36 26

Sheets of allogeneic dermal collagen measuring 20 X 15 mm and prepared by trypsin treatment of full-thickness skin were grafted under skin flaps in rats. After 2 to 5 weeks the protective recipient skin was excised and replaced by split-thickness skin isografts which remained viable on their supportive collagen beds. On average such composite grafts maintained 84 per cent of their original size over 3 to 28 weeks and in contrast with split-thickness skin grafts achieved full-thickness reconstruction of the excised skin.
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PMID:Reconstruction of full-thickness loss skin wounds using skin collagen allografts. 37 21

Str. griseus protease hydrolyzes essentially insoluble collagen of bone tissue, with 34.5% of protein solubilized and 6.0% of peptide bonds splitted. 60.0 M of N-terminal amino acids is formed per 10(5) g of protein, out of them 16.8 in the fraction of free amino acids, 32.3 M in the fraction of soluble DNP-peptides and 10.9 M in that of insoluble DNP-peptides. Under the effect of trypsin the amount of collagen changing to the soluble form is thrice as low and the splitted peptide bonds are ten times as low as in case of the Str. griseus protease action. The peptide bonds incorporating the N-end of serine, threonine, glycine are more available for protease. It is supposed that under used conditions Str. griseus protease hydrolyzes not only telepeptides but also the main molecule of collagen.
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PMID:[Study of bone tissue insoluble collagen hydrolysis by Streptomyces griseus protease using the method of N-terminal analysis]. 40 89

A method to isolate and to serially cultivate rabbit skin epithelial cells from adult trunk skin has been developed. Using a collagen gel as substrate and trypsin and EDTA to dissociate cells, nonproliferative primary cultures of rabbit cells may be converted to proliferative populations, and at least 3 serial passages achieved. In the presence of large concentrations of methotrexate (up to 1000 microgram/ml), epithelial cells in primary culture show no decrease in their ability to attach, spread, or keratinize. Following conversion to proliferative populations by trypsin and EDTA low concentrations of methotrexate (1 microgram/ml) are strongly cytotoxic. When the incorporation of 32PO4(3-) and 3H-thymidine into DNA of proliferative and nonproliferative cells is compared, the incorporation of 32PO4(3-), but not that of 3H-thymidine, correlates with changes in cell number and DNA content. In both primary and serially cultivated cells, L-serine is required for optimal growth.
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PMID:Isolation and serial cultivation of rabbit skin epithelial cells. 41 41

The elaboration of leukocyte chemotactic factors by human fibroblasts was studied. 12 lines of normal fibroblasts obtained by skin biopsy and then cultured in vitro produced chemoattractants (assessed by modified Boyden-chamber techniques) for both peripheral blood polymorphonuclear leukocytes and monocytes (obtained by Hypaque-Ficoll and dextran sedimentation). Chemotactic activity was not present performed in fibroblasts, and cycloheximide blocked its elaboration. The chemotactic activity of crude-culture supernate was heat stable (56 degrees C for 30 min), trypsin- and pronase-sensitive, and neuraminidase resistant. Characterization of the chemotactic activity by gel filtration (Sephadex G-75) showed two active fractions, one with mol wt greater than 100,000 and the other less than 10,000. In studies designed to relate these chemotactic factors to collagen, we have confirmed that type I collagen and alpha 1-chain; are chemotactically active for monocytes but not polymorphonuclear leukocytes. However, the chemotactic activity in fibroblast-culture media was media was distinct from collagen in that it attracted neutrophils, it was not precipitated by 25% ammonium sulfate, and it was resistant to collagenase treatment; ascorbic acid, in concentrations known to stimulate fibroblast collagen synthesis, had no effect on the elaboration of the chemotactic factors. Furthermore, amino acid analysis of Sephadex G-75 fractions with chemotactic activity failed to reveal amino acids such as hydroxyproline characteristic of collagen. In addition to the chemotactic factors secreted by fibroblasts, a heat-resistant factor (30 min at 56 degrees C) which generated the chemotactically active fragment of C5 (C5a) from human serum was also secreted. The elaboration of mediators of the inflammatory and immune responses by fibroblasts may initiate and(or) modulate local skin inflammatory reactions and play a protective role in vivo.
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PMID:Polymorphonuclear leukocyte and monocyte chemoattractants produced by human fibroblasts. 43 25


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