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)

By using two specific receptor assays, we identified and partially characterized receptors for transforming growth factor-beta 1 (TGF-beta 1) on porcine trabecular cells in vitro. Cultured trabecular cells were incubated with labeled TGF-beta 1 and analyzed by flow cytometry. Pretreatment with trypsin or preincubation with cold TGF-beta 1 or a neutralizing antibody to TGF-beta 1 inhibited the binding of labeled TGF-beta 1. 125I-TGF-beta 1 was cross-linked covalently to cell surface receptors on the trabecular cells. By SDS-PAGE and autoradiography, we identified three labeled macromolecular species of receptors, two of which had apparent molecular weights greater than 212 kDa and one of which had an apparent molecular weight of approximately 80-90 kDa under reducing conditions. The low and high molecular weight species probably represent type II and type III TGF-beta 1 receptors, respectively. At concentrations of 0.5 and 1 ng/ml, activated TGF-beta 1 caused retraction and a marked decrease in the rate of proliferation and in the motility of trabecular cells in vitro. Our findings implicate TGF-beta 1 in the modulation of the functional homeostasis of trabecular cells and suggest that the aqueous humor contains a level of TGF-beta 1 which, once activated, is sufficient to exert a biologic effect on the trabecular meshwork.
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PMID:Identification and partial characterization of TGF-beta 1 receptors on trabecular cells. 131 71

The binding of 125I-labelled transforming growth factor-beta 1 (TGF-beta 1) to human alpha 2-macroglobulin (alpha 2M) was studied by native PAGE and autoradiography. TGF-beta 1 bound preferentially to alpha 2M-methylamine and minimally, if at all, to native alpha 2M. Preparations of alpha 2M-proteinase complex were generated by incubating a standard concentration of alpha 2M (0.4 microM) with different concentrations of trypsin, chymotrypsin or neutrophil elastase (0.04-2.0 microM). The 125I-TGF-beta 1-binding activity depended on the initial ratio of active proteinase to alpha 2M, or r value, used to form the alpha 2M-proteinase complex. With all three proteinases, r values of 2 or greater yielded preparations with unchanged or decreased TGF-beta 1-binding activity relative to native alpha 2M. By contrast, r values near 1 yielded preparations with significantly increased TGF-beta 1-binding activity. The results of [3H]thymidine-incorporation studies performed in mouse keratinocytes were consistent with the 125I-TGF-beta-binding experiments. alpha 2M-trypsin and alpha 2M-chymotrypsin prepared at an r value of 1.0 counteracted the activity of TGF-beta 1, whereas the equivalent complexes prepared at an r value of 3.0 had no effect. As determined by SDS/PAGE, 125I-TGF-beta 1 binding to alpha 2M-methylamine was at least 80% non-covalent. Reaction of alpha 2M-methylamine with iodoacetamide or 5,5'-dithiobis-(2-nitrobenzoic acid) decreased the percentage of covalent binding but had no effect on total binding. Neuraminidase treatment had no effect on the binding of 125I-TGF-beta 1 to alpha 2M-methylamine. Cleavage of the 'bait regions' in alpha 2M-methylamine by prolonged treatment with trypsin also had no effect. These studies suggest that TGF-beta 1 binding to alpha 2M is enhanced by conformational change in the proteinase inhibitor resulting from reaction with proteinase or amine. If both proteinase-binding sites in a single alpha 2M molecule are occupied, TGF-beta 1-binding activity is decreased or perhaps eliminated.
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PMID:Binding of transforming growth factor-beta 1 to methylamine-modified alpha 2-macroglobulin and to binary and ternary alpha 2-macroglobulin-proteinase complexes. 137 Oct 50

Radioiodinated transforming growth factor-beta 1 (TGF-beta 1) bound to the plasma proteinase inhibitor, alpha 2-macroglobulin (alpha 2M), as determined by chromatography on Superose-6 and native polyacrylamide gel electrophoresis. When alpha 2M conformational change was induced with methylamine, 125I-TGF-beta 1 binding significantly increased. Intravenously injected 125I-TGF-beta 1 cleared from the circulation of mice rapidly at first; however, intravascular radioactivity stabilized near 20% of the initial level. At necropsy, radioactivity was recovered predominantly in the liver (65%); however, the density of radioactivity (disintegrations per minute/g organ wt) was highest in the lungs. Markedly different results were obtained with purified 125I-TGF-beta 1-alpha 2M-methylamine complex. Clearance of the complex occurred as a first-order process with a t1/2 of 4 min. Greater than 90% of the radioactivity was recovered in the liver. The clearance and distribution of 125I-TGF-beta 1-alpha 2M-methylamine were equivalent to those observed with 125I-alpha 2M-methylamine and 125I-alpha 2M-trypsin. The latter two radioligands clear via specific alpha 2M receptors in the liver. Large molar excesses of alpha 2M-trypsin or alpha 2M-methylamine competed with 125I-TGF-beta 1-alpha 2M-methylamine for plasma clearance. Native alpha 2M, which does not bind to the alpha 2M receptor, did not compete. The receptor binding domain of alpha 2M-methylamine was blocked by chemical modification or enzyme treatment. The resulting alpha 2M preparations still bound 125I-TGF-beta 1; however, the complexes did not clear when injected intravenously in mice. The studies presented here demonstrate that alpha 2M can mediate the plasma clearance of a growth factor via the alpha 2M receptor system. We propose that alpha 2M, the alpha 2M receptor, and proteinases may function as a concerted system to regulate TGF-beta 1 activity and the activity of related factors in vivo.
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PMID:An alpha 2-macroglobulin receptor-dependent mechanism for the plasma clearance of transforming growth factor-beta 1 in mice. 170

Using immunocytochemistry and Northern blot analysis, we investigated the role of cell morphology and reorganization of the cytoskeleton in the expression of transforming growth factor-beta 1 (TGF-beta 1) in human dermal fibroblasts. Disruption of the cytoskeleton was induced by three different agents--trypsin, ethyl-eneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), or cytochalasin--and was confirmed by staining with rhodamine-labeled phalloidin. Immunocytochemical staining with antibodies specific for TGF-beta 1 revealed a cell-shape-related induction of TGF-beta 1. Northern blot analysis of total RNA showed a significant increase in the expression of TGF-beta 1 mRNA as early as 4 h and peaking at 12 h after disruption of the cytoskeleton. Quantitative analysis of TGF-beta 1 mRNA expression at 4 h after treatment with trypsin, EGTA, or cytochalasin C showed increases of 2.6-, 3.3-, and 2.6-fold, respectively. Disruption of the cytoskeleton by trypsin, EGTA, or cytochalasin C increased mRNA for collagenase by 3.8-fold, 2.3-fold, or 2.5-fold, respectively. The expression of mRNA for tissue inhibitor of metalloproteinases I (TIMP-I) also showed a 3.2-fold increase by trypsin, a 3.6-fold increase by EGTA, and a 2.5-fold increase by cytochalasin C. Cell-shape-related induction of TGF-beta 1, collagenase, and TIMP-I genes appears to be selective, as the levels of mRNA for fibronectin and type I procollagen were not significantly altered. These data suggest that gene expression of TGF-beta 1, collagenase, and TIMP-I is governed by the status of the cytoskeleton microfilament organization, which may be a mechanism of gene regulation during cell division, migration, and differentiation, events fundamental to wound healing.
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PMID:Alteration in cell morphology triggers transforming growth factor-beta 1, collagenase, and tissue inhibitor of metalloproteinases-I expression in normal and hypertrophic scar fibroblasts. 752 43

In continuation of earlier observations on the involvement of interleukin-1 (IL-1) in ovarian function, we examined the ability of IL-1 to modulate plasminogen activator (PA) activity and prostaglandin (PG) synthesis in human granulosa lutein cells (GLCs). Toward this goal, GLCs were obtained from women undergoing in vitro fertilization, preincubated with 10% fetal calf serum for 48 h, and subsequently cultured for 48 h in serum-free media in the absence or presence of IL-1 beta (10 ng/mL). Cellular PA activity was measured by plasminogen-dependent cleavage of the chromogenic substrate H-D-valyl-L-leucyl-L-lysine-p-nitroanilide (S-2251). Prostaglandin E (PGE) levels were assayed by conventional RIA. Exposure of GLCs to IL-1 resulted in a 50% increase in PGE production, a 33% suppression of PA activity, and a 75% increase in the ability of the corresponding conditioned media to inhibit exogenous urokinase activity. The inhibitory capacity was attributable to an IL-1-mediated increase in PA inhibitor type-1 (PAI-1) production, inasmuch as urokinase inhibition could be abolished by the administration of a polyclonal antihuman PAI-1 immunoglobulin G. IL-1 treatment had no effect on plasmin or trypsin inhibition. Exposure of GLCs to IL-1 receptor antagonist abolished the ability of IL-1 to enhance PA inhibitory activity and PGE production, thereby establishing specific IL-1 receptor-mediated effects. The ability of IL-1 to suppress PA activity and to produce PAI-1 persisted in the presence of indomethacin, a potent inhibitor of PG synthesis. Likewise, transforming growth factor-beta 1 suppressed the ability of IL-1 to stimulate PGE production without affecting the IL-1-induced effects on the PA system. The present findings suggest a pluripotent response of GLCs to IL-1, characterized by the induction of PAI-1 and the suppression of PA occurring concurrent with, but independent of, PG production. These observations support the potential involvement of IL-1 in the regulation of human ovulatory processes.
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PMID:Interleukin-1-mediated stimulation of prostaglandin E production is without effect on plasminogen activator activity in human granulosa lutein cell cultures. 755 90

Prostaglandins (PGs) play a major role during implantation and labor, and their level is regulated by various cytokines. Interleukin-1 (IL-1) is a known mediator of prostaglandin E (PGE) production in various cell types, including endothelial, amniotic, and endometrial cells; however, its role in the regulation of PGE production in the trophoblast cells is yet unknown. As IL-1 and PGE are both known to be synthesized in the human trophoblast cells, we examined the possibility that IL-1 regulates PG production in human trophoblast cells. To this end, use was made of first and third trimester trophoblast cells, obtained from first trimester terminations of pregnancies and elective cesarean sections. The trophoblast cells were separated by trypsin degradation and fractionation on Percoll gradients, and cultured for 18 h under serum-free conditions in the absence or presence of IL-1 (10 ng/mL). IL-1 induced a 5-fold increase in PGE production, a response that was cell density, time, and dose dependent. IL-1-induced PGE biosynthesis was prevented in the presence of either IL-1 receptor antagonist or the soluble IL-1 receptor, suggesting a receptor-mediated response. Significantly, de novo production of PGE by trophoblast cells in the absence of IL-1 was also markedly (50%) reduced by either the IL-1 receptor antagonist or the soluble IL-1 receptor, further supporting the notion that IL-1 is involved in PGE synthesis even under basal conditions. Transforming growth factor-beta 1, a putative modulator of the effects of IL-1, significantly attenuated IL-1-stimulated PGE production, supporting the possibility that transforming growth factor-beta 1 may serve as a regulator of the effects of IL-1 in trophoblast cells. These observations suggest a pivotal role of IL-1 in the regulation of PGE economy by trophoblast cells. As trophoblast cells are in intimate contact with maternal cells, understanding the regulation of PGE levels may explain crucial processes at the feto-maternal interface, including implantation of the developing blastocyst, immunosurveilance, and the initiation of labor.
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PMID:Interleukin-1 stimulates prostaglandin E production by human trophoblast cells from first and third trimesters. 774 12

Thiol modifications during growth and differentiation of cultured normal human bronchial epithelial cells was studied by analysis of their content and redox state of low-molecular-weight thiols and protein thiols. Subculture of the cells with trypsin decreased the cellular content of the major low-molecular-weight thiol, i.e., reduced glutathione, although the glutathione content had returned to levels comparable to those before subculture already after 4 h in conjunction with cell attachment. During subsequent culture, increases in the cellular contents of glutathione, total cysteine equivalents, and total protein thiols occurred. These modifications in the amounts and redox balance of thiols were transient and preceded the major growth phase. Exposure of cells at clonal density to either diethylmaleate, a thiol-depleting agent, or buthionine sulfoximine, an inhibitor of glutathione synthesis, decreased the proliferative ability of the cells as demonstrated by a markedly decreased colony forming efficiency. Moreover, in mass cultures exposed to buthionine sulfoximine, a marked depletion of the glutathione content was again accompanied by inhibition of growth. Exposure of the cells to agents known to induce growth arrest and terminal squamous differentiation, i.e., fetal bovine serum, Ca2+, or transforming growth factor-beta 1, resulted in increased levels of reduced glutathione. No consistent alteration in the contents of the other thiols was noted. Overall, the results demonstrate consistent variations in the amounts and redox state of cellular thiols, particularly reduced glutathione, supporting a role of thiols in regulation of growth and squamous differentiation of human bronchial epithelial cells.
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PMID:Modifications of cellular thiols during growth and squamous differentiation of cultured human bronchial epithelial cells. 812 49

Two hamster pancreatic cancer cell lines, PC-1 and PC1.0, established from N-nitrosobis(2-oxopropyl)amine-induced pancreatic ductal/ductular adenocarcinomas exhibit different growth patterns. PC-1 cells, which produce well differentiated adenocarcinomas in vitro after allogeneic inoculation, form cell aggregates and characteristic island-like structures in vitro. PC1.0 cells, which produce poorly differentiated tumors in vivo, form dispersed colonies in vitro. Conditioned medium prepared from PC1.0 cells inhibits PC-1 cells from forming island-like colonies. The conditioned medium also prevents several human pancreatic carcinoma cell lines, HPAF, CD11 and CD18, from forming compact colonies. These properties are similar to those described previously as scatter factors. The scatter factor-like activity is heat-labile, acid-stable, non-dialyzable, trypsin sensitive and unaffected by reducing agents. The activity is not suppressed by addition of heparin, and it does not bind to heparin. In addition, the scatter phenomenon is not reproduced by acidic or basic fibroblast growth factor, epidermal growth factor or transforming growth factor-beta 1. Based on these findings, it appears that the scattering activity produced by PC1.0 cells differs from the scatter factors that have been identified in other systems.
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PMID:Production of scatter factor-like activity by a nitrosamine-induced pancreatic cancer cell line. 838 14

Partial-thickness defects evolving in mature articular cartilage do not heal spontaneously. This type of defect was created in the articular cartilage of adult rabbits and Yucatan minipigs, and the effects of chondroitinase ABC or trypsin, fibrin clots, and mitogenic growth factors on the healing process were examined histologically at intervals ranging from one to forty-eight weeks. The effect of chondroitinase ABC or trypsin was examined initially. Articular cartilage contains macromolecules, including proteoglycans, which render the surfaces of this tissue, and of partial-thickness defects within it, antiadhesive. Chondroitinase ABC digests the glycosaminoglycan chains of cartilage proteoglycans, and trypsin degrades their core proteins. To test the hypothesis that mesenchymal cells may be prevented from adhering to and migrating over the surfaces of partial-thickness defects by proteoglycans, we removed a superficial layer of these macromolecules from the surface of the defect with use of one of these enzymes. The treatment evoked an increase in the coverage of the defect surface with mesenchymal cells; when combined with the local application of a mitogenic growth factor (basic fibroblast growth factor, transforming growth factor-beta 1, epidermal growth factor, insulin-like growth factor-1, or growth hormone), the coverage was more extensive but mesenchymal cells did not extend into and completely fill the volume of the defect. When the surface of the defect was treated with chondroitinase ABC and the cavity of the defect was filled with a fibrin clot to furnish a matrix or scaffolding for the migration of cells therein, there was migration and proliferation of cells throughout the volume of the defect but at a low population density. Mesenchymal cells remodeled the deposited fibrin matrix, which was replaced by a loose fibrous connective tissue. When defects that had been treated with chondroitinase ABC were filled with a fibrin clot containing a mitogenic growth factor, mesenchymal cells filled the entire cavity of the defect, and the density of the cells was greatly increased, particularly when transforming growth factor-beta 1 was used. Histological studies revealed a continuous layer of mesenchymal cells extending from the synovial membrane across the superficial tangential zone of normal articular cartilage into the defect, indicating that the cells that were recruited for the repair process were of synovial origin. At forty-eight weeks, the entire cavity of the defect remained filled with a fibrous connective tissue.
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PMID:Repair of partial-thickness defects in articular cartilage: cell recruitment from the synovial membrane. 864 29

Cytoskeleton not only controls cell morphology but also regulates cell growth, migration, differentiation, and gene expression, events which are fundamental to embryogenesis, carcinogenesis, and wound healing. We have recently reported that reorganization of cytoskeleton induces expression of mRNA for transforming growth factor-beta 1 (TGF-beta 1), collagenase, and tissue inhibitor of metalloproteinase-I (TIMP-I) in dermal fibroblasts. In this report we have examined the role of gene transcription in this induction. As judged by nuclear run-on assay, trypsin, EGTA (ethylene glycol-bis (beta-aminoethyl ether) N, N, N', N', tetra-acetic acid), or cytochalasin C (Chs) increased the rate of transcription of the TGF-beta 1 gene by 2.0, 2.7, and 1.6 fold, respectively, and of the collagenase gene by 5.3, 6.2, and 3.3 fold. The rate of transcription of the TIMP-I gene was increased by trypsin (4.3 fold) or EGTA (3.8 fold) but unaffected by Chs. Cytochalasin induced an increase in the rate of transcription of procollagen I (alpha 1), procollagen I (alpha 2), and fibronectin genes by 1.4, 1.5, and 1.9 fold respectively, while trypsinization or EGTA treatment had no or little effects on these gene. Since transcription of the TGF-beta 1 gene is believed to be largely governed by the activating protein 1 (AP1) complex, we also examined the expression of mRNA for c-fos and c-jun protoon-coproteins. Trypsinization induced rapid (within 30 min) and transient expression of c-fos mRNA. A 2.4 fold increase in c-jun mRNA was apparent after 4 hr and persisted for at least 24 hr. Actinomycin D (Act D) suppressed the induction of TGF-beta 1 mRNA by Chs but had less effect on the TGF-beta 1 mRNA in trypsinized cells which had been replated for 4 hr, suggesting that the half life of TGF-beta 1 mRNA is reduced in cells with a disassembled cytoskeleton. Simultaneous treatment with Chs and cycloheximide (Cxm) resulted in a superinduction of TGF-beta 1 mRNA by 88 +/- 23% (n = 4, P < 0.05), which was abrogated by preexposure to Act D. In contrast, the induction of collagenase mRNA by Chs was totally blocked by Cxm, indicating that the Cxm-mediated superinduction is selective and that protein synthesis is required for induction of this mRNA. Our results suggest that the activities of genes for proteins involved in the structure (Type I collagen and fibronectin), turnover (collagenase and TIMP-1) and regulation (TGF-beta 1) of extracellular matrix (ECM), are all governed at least in part by the status of the cytoskeleton. Since the cytoskeleton is reorganized during cell division, migration, and differentiation, these results may have implications for the regulation of ECM during such processes as embryogenesis, carcinogenesis, and wound healing.
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PMID:Cytoskeleton regulates expression of genes for transforming growth factor-beta 1 and extracellular matrix proteins in dermal fibroblasts. 925 40


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