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)

HeLa chromatin core particles were digested with trypsin to excise the NH2-terminal histone regions. The resulting nucleoprotein complexes were dissociated in 2.5 M NaCl; the DNA and polypeptides were then allowed to reassemble by lowering the NaCl concentration. Eighty per cent of the DNA reassociated with the polypeptides. The reassembled nucleoprotein complexes sediment at 9.7 S, have a molecular elipticity at 280 nm of 3000 degrees cm2/dmol of PO4, and contain DNase I-susceptible sites at 10 nucleotide intervals. The pattern of products generated by cross-linking the polypeptides with dimethylsuberimidate is very similar to the pattern generated by cross-linking native core particles. The results indicate that histones which lack their HN2-terminal regions retain both the features necessary for correct protein-protein interactions and the ability to fold DNA into a nucleoprotein complex resembling the chromatin core particle.
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PMID:Folding of DNA by histones which lack their NH2-terminal regions. 64 10

Mutagen damage to a cell located at some distance from the site of application of the mutagen will depend, in part, on how effectively it can penetrate through cells composing different tissues. Chinese hamster V79 spheroids were used to model mutagen "penetrability" by providing several layers of cells growing with tissue-like packing. By treating cells of intact and trypsin-dissociated spheroids at the same drug-to-cell ratio, an estimate relative sensitivity to nine mutagens was determined. The effective toxicity index (ETI) was defined as the ratio of the concentrations of mutagen required to kill 50% of cells (measured after a 1-hr treatment at 37 degrees C) in dissociated versus intact spheroids and similarly for mutation at the HGPRT locus (EMI). Values for ETI were consistent with values for EMI and varied from 0.01 for 4NQO to 2 for AF-2. Direct information on mutagen penetration was obtained for fluorescent mutagens by flow cytometry. Values for EFI (effective fluorescence index) varied from 0.07 for Hoechst 33342 to 1 for AF-2. These results can be interpreted as reflecting differences in ability of mutagens to penetrate spheroids; some direct-acting mutagens are likely to be effective only near their site of application (ie, UV, Hoechst 33342, 4NQO) while others are able to penetrate through successive cell layers with little difficulty (ie, monobromobimane, AF-2).
Environ Mutagen 1986
PMID:Patterns of mutagen binding and penetration in multicell spheroids. 309 8

We have examined the mechanism by which stromal cells from the microenvironment of the bone marrow restricted the in vitro growth of certain hemopoietic tumors. A series of leukemia cell lines was used to monitor biological activities of stromal cell lines representing five distinct subtypes. Only an endothelial-like clone derived from mouse stroma (MBA-2.1) was consistently found to produce a cell-surface-associated glycoprotein that selectively inhibited the growth of plasmacytomas. The factor, designated leukemia cell inhibitory activity (LCIA), was not detected in anchorage-dependent cells of nonhemopoietic origin. Tumors of the lymphoid lineage and plasmacytomas in particular were the most sensitive to LCIA. Myeloid, macrophage, and erythroleukemia tumors were resistant to the factor, as were normal hemopoietic target cells including pluripotent stem cells, myeloid progenitor cells, and mitogen-stimulated spleen cells. Fractionation of trypsin-released proteins from MBA-2.1 cells by gel filtration and affinity binding to concanavalin A-Sepharose revealed two types of inhibitors; one was the specific leukemia cell inhibitor (i.e., LCIA); the other, present at a lower titer, was non-target-cell specific. The high sensitivity of plasmacytomas to LCIA versus the resistance of normal stem cells may be utilized for selective elimination of plasma cell tumors from bone marrow inocula.
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PMID:Differentiation stage and lineage-specific inhibitor from the stroma of mouse bone marrow that restricts lymphoma cell growth. 345 88

A procedure to enhance the concurrent yield of large numbers of spermatogonial, meiotic I, and meiotic II metaphases from Chinese hamster testes has been developed. Animals were injected intraperitoneally with 5, 10, 20, or 40 mg of colchicine/kg and killed 3 hr after the treatment. Both testes from each animal were excised; one testis was minced, and germ cells were isolated by using 0.1% trypsin solution; the other testis was minced, and germ cells were isolated by using a conventional procedure. Chromosomal preparations were made with a standard air-drying technique. Examination of slides revealed that with the trypsin-isolation procedure the concurrent yields of spermatogonial, meiotic I, and meiotic II metaphases were significantly higher (P less than .05) than the yields obtained with the conventional procedure. Furthermore, 40 mg of colchicine/kg produced maximum numbers of all three types of germ cell metaphases. At this maximum concentration, metaphases were stable with no evidence of structural aberrations, and the frequency of hyperploidy was not significantly different (P greater than .05) from hypoploidy.
Environ Mol Mutagen 1987
PMID:Enhancement of concurrent yield of spermatogonial, meiotic I, and meiotic II metaphase chromosomes from Chinese hamster testes. 369 87

The kidney is a key target tissue in animal and human carcinogenesis, yet there are no established short-term tests for studying the genotoxicity of chemicals in the kidney. We have developed an assay for the measurement of chemically induced DNA repair as unscheduled DNA synthesis (UDS) in isolated rat kidney cells following in vivo treatment. Male Fischer-344 rats were injected intraperitoneally with chemicals dissolved in saline or corn oil. After various treatment times, the kidneys were perfused with a collagenase/trypsin solution (CTS), minced into small pieces, and stirred in CTS at 37 degrees C for 1 hr to dissociate cells. Cultures contain a high proportion of epithelial cells from the proximal and distal tubules. Cultures were incubated for 16-18 hr with 3H-thymidine in Williams' Medium E supplemented with 20% fetal bovine serum. UDS was measured by quantitative autoradiography as net grains/nucleus (NG). The percentage of cells in repair (% IR) was defined as the percentage of cells with greater than or equal to 3 NG. Saline- or corn oil-injected controls consistently produced -3 to -5 NG with less than 1% IR. The time course of DNA repair following treatment with the direct-acting mutagen methylmethane sulfonate (MMS) or the renal carcinogen azaserine showed a peak response at 2 hr after treatment. Azaserine showed a rapid decline in UDS at 12 and 24 hr, whereas MMS exhibited a relatively high UDS level at 24 hr. The renal carcinogens methylazoxymethanol acetate, N-methyl-N-nitrosourea, and streptozotocin all yielded strong positive UDS responses. The liver and intestinal carcinogen 1, 1-dimethylhydrazine at doses up to 50 mg/kg was cytotoxic to kidney cells, but induced less than 0 NG. Treatment with 1,2-dimethylhydrazine, which induces kidney tumors in mice but not rats, also induced less than 0 NG. Treatment with o-anisidine, a weak renal carcinogen, did not induce UDS in the kidney, suggesting that it may be acting as a tumor promoter. These results demonstrate the usefulness of this assay for the detection and study of a variety of genotoxic kidney carcinogens.
Environ Mutagen 1985
PMID:Measurement of unscheduled DNA synthesis in rat kidney cells following in vivo treatment with genotoxic agents. 406 62

Mouse embryos were labeled in vivo at 10 1/2-12 1/2 days of gestation with [3H]-thymidine and subjected to DNA damage using x-ray, methylmethanesulfonate, or methylnitrosourea. DNA damage and its repair were assessed in specific cell preparations from embryos isolated at intervals thereafter using the highly sensitive method of nucleoid sedimentation, which evaluates the supercoiled state of the DNA. Repair of x-ray damage was demonstrated using trypsin-dispersed cells from whole embryos and from homogenized embryonic liver to show the validity of the analytical approach. The effects of the highly teratogenic methylnitrosourea and the much less teratogenic methylmethanesulfonate were compared in the targeted limb buds using equitoxic doses of the two alkylating agents. DNA supercoiling was fully restored after 24 hr in limb bud cells damaged with methylmethanesulfonate, while as much as 48 hr were required for full repair of methylnitrosourea damage. These results demonstrated the feasibility of studying DNA repair in embryonic tissues after damage in vivo and suggest that the potency of methylnitrosourea as a teratogen may be correlated with a prolonged period required for complete repair of DNA.
Teratog Carcinog Mutagen 1984
PMID:DNA damage and repair in mouse embryos following treatment transplacentally with methylnitrosourea and methylmethanesulfonate. 615 Dec 63

We studied the effect of rat tissue extracts on induction of lambda prophage in Escherichia coli (lambda) by L-azaserine. Hepatic and pancreatic extracts, primarily the cytosolic fraction, markedly increased the rate of induction. Hepatic extracts from lipotrope-deficient rats were somewhat more active than extracts from normal rats. The enhancing activity in normal rat hepatic cytosol was partially characterized. It reduced by about one-half the dose of azaserine required for a given purpose. The enhancement was increased by preincubating the bacterial cells with cytosol; cells retained the effect after cytosol was removed. Enhancing activity was inhibited strongly by the amino acids phenylalanine, tryptophan, and tyrosine; to lesser extents by leucine, methionine, and serine; and not at all by proline or glutamine. It was eliminated by dialysis of the cytosol and reduced by omission of nicotinamide adenine dinucleotide phosphate (NADP) from the reaction mixture. Heating the cytosol to 60 degrees C or 80 degrees C or varying the pH of the reaction mixture from 6 to 8 had no significant effect. Treating the cytosol with trypsin appeared to release an inhibitor of the activity. Glutathione, cysteine, and beta-mercaptoethanol also enhanced lambda induction by azaserine, but the cytosolic activity was not affected by the thiol-inactivating compound diethylmaleate (DEM). The results suggest that factors in cytosol interact with bacterial cells to facilitate transport of azaserine into the cells, primarily through the aromatic amino acid transport system. A small molecule, not a free thiol compound, appears to be involved. It may serve to establish reducing conditions protective for azaserine, the probable mechanism of action of sulfhydryl compounds.
Environ Mutagen 1984
PMID:Enhancing activity of rat tissue extracts for induction of lambda prophage by L-azaserine. 623 74

An in vivo/in vitro DNA repair assay has been developed to quantitate chemically induced unscheduled DNA synthesis (UDS) in rat spermatocytes utilizing autoradiography. Male Fischer-344 rats were treated by i.p. injection or gavage with a variety of genotoxic agents dissolved in dimethyl sulfoxide, corn oil, or water. At selected times after treatment, spermatocytes were isolated by trypsin digestion of testes and cultured for 24 hr in the presence of 3H-thymidine. The direct-acting genotoxicants methyl methanesulfonate (MMS) and ethyl methanesulfonate and the chemotherapeutic agent cyclophosphamide (CPA) produced positive UDS responses in spermatocytes isolated 1 hr after i.p. injection. The UDS response evoked by either CPA or MMS was maximal within 1 hr after injection and declined rapidly thereafter to control levels. Other known genotoxicants--including dimethylnitrosamine, aflatoxin B1, 2-acetylaminofluorene, 2,6-dinitrotoluene, and 1,6-dinitropyrene--failed to induce UDS, even with routes of administration and at times of exposure known to produce a positive response in hepatocytes. This negative response is consistent with these genotoxicants lack of mutagenic effect in rodent germ cells. These results demonstrate that the in vivo/in vitro spermatocyte DNA repair assay may be useful as a predictive screen for germ cell mutagens. Moreover, by its compatibility with similar assays which utilize other tissues from the same treated animal, this assay permits assessment of the organ specificity of the genotoxic response.
Environ Mutagen 1984
PMID:An assay to detect chemically induced DNA repair in rat spermatocytes. 673 43

1. The biological activities of the proteinase-activated receptor number 2 (PAR-2)-derived peptides, SLIGRL (PP6) SLIGRL-NH2 (PP6-NH2) and SLIGR-NH2 (PP5-NH2) were measured in mouse and rat gastric longitudinal muscle (LM) tissue and in a rat aortic ring preparation and the actions of the PAR-2-derived peptides were compared with trypsin and with the actions of the thrombin receptor activating peptide, SFLLR-NH2 (TP5-NH2). 2. From a neonatal rat intestinal cDNA library, and from intestinal and kidney-derived cDNA, the coding region of the rat PAR-2 receptor was cloned and sequenced, thereby establishing its close sequence identity with the previously described mouse PAR-2 receptor; and this information, along with a reverse-transcriptase (RT) polymerase chain reaction (PCR) analysis of cDNA derived from gastric and aortic tissue was used to establish the concurrent presence of PAR-2 and thrombin receptor mRNA in both tissues. 3. In the mouse and rat gastric preparations, the PAR-2-derived polypeptides, PP6, PP6-HN2 and PP5-NH2 caused contractile responses that mimicked the contractile actions of low concentrations of trypsin (5 u/ml-1; 10 nM) and that were equivalent to contractions caused by TP5-NH2. 4. The cumulative exposure of the rat LM tissue to PP6-NH2 led to a desensitization of the contractile response to this polypeptide, but not to TP5-NH2 and vice versa, so as to indicate a lack of cross-desensitization between the receptors responsive to the PAR-2 and thrombin receptor-derived peptides. 5. In the rat gastric preparation, the potencies of the PAR-2-activating peptides were lower than the potency of TP5-NH2 (potency order: TP5-NH2 > > PP6-NH2 > or = PP6 > PP5-NH2); PP6 was a partial agonist in this preparation. 6. The contractile actions of PP6 and PP6-NH2 in the rat gastric preparation required the presence of extracellular calcium, were inhibited by nifedipine and were blocked by the cyclo-oxygenase inhibitor, indomethacin and by the tyrosine kinase inhibitor, genistein, but not by the kinase C inhibitor, GF109203X. The contractile responses were not blocked by atropine, chlorpheniramine, phenoxybenzamine, propranolol, ritanserin or tetrodotoxin. 7. In a precontracted rat aortic ring preparation, with an intact endothelium, all of the PAR-2-derived peptides caused a prompt relaxation response that was blocked by the nitric oxide synthase inhibitor, N omega-nitro-L-arginine-methyl ester (L-NAME) but not by D-NAME; in an endothelium-free preparation, which possessed mRNA for both the PAR-2 and thrombin receptors, the PAR-2-activating peptides caused neither a relaxation nor a contraction, in contrast with the contractile action of TP5-NH2. The relaxation response to PP6-NH2 was not blocked by atropine, chlorpheniramine, genistein, indomethacin, propranolol or ritanserin. 8. In the rat aortic preparation, the potencies of PP6, PP6-NH2 and PP5-NH2 were greater than those of the thrombin receptor activating peptide, TP5-NH2 (potency order: PP6-NH2 > or = PP6 > PP5-NH2 > TP5-NH2). 9. In the rat aortic preparation, the relaxant actions of the PAR-2-derived peptides were mimicked by trypsin, at concentrations (0.5-1 u ml-1; 1-2 nM) lower than those that can activate the thrombin receptor. 10. The bioassay data obtained with the PAR-2 peptides and with trypsin, along with the molecular cloning/RT-PCR analysis, point to the presence of functional PAR-2 receptors that can activate distinct responses in the gastric and vascular smooth muscle preparations. These responses were comparable to those resulting from thrombin receptor activation in the same tissues, so as to suggest that the receptor for the PAR-2-activating peptides may play a physiological role as far reaching as the one proposed for the thrombin receptor.
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PMID:Rat proteinase-activated receptor-2 (PAR-2): cDNA sequence and activity of receptor-derived peptides in gastric and vascular tissue. 876 73

An in vitro normal human epidermal keratinocytes (NHEK) model was used to study and to characterize the protease stimulated by the mustards 2-chloroethyl ethyl sulphide (CEES), 2-chloro-N-(2-chloroethyl)-N-methylethanamine hydrochloride (nitrogen mustard, HN2), and Bis-2-chloroethyl sulfide (sulfur mustard, HD). The results obtained by using a chromozym (TRY) peptide substrate protease assay showed the optimum mustard concentration and time for protease stimulation to be about 200 microM CEES, 100 microM HN2 or HD, and 16 hours. The mustard-stimulated protease was membrane-bound, and was inhibited by adding a Ca2+ chelator EGTA (2 mM), BAPTA AM (50 microM) or a serine protease inhibitor diisopropyl fluoro-phosphate DFP (1 mM), or a protein synthesis inhibitor cycloheximide (10 microM) in the extracellular medium. These results suggest that one of the mechanisms of mustard toxicity is via the stimulation of a trypsin/chymotrypsin like serine protease, which is dependent on Ca2+ and new protein synthesis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a mustard-stimulated approximately equal to 70-80 KDa protein band that was associated with protease activity which was inhibitable by EGTA, BAPTA, DFP or cycloheximide. This mustard-stimulated protein (protease) may serve as a diagnostic tool for mustard exposure as well as an assay for screening prospective antivesicant protease inhibitor drugs.
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PMID:Protease in normal human epidermal keratinocytes. 970 64


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