A latent form of collagenase in the involuting rat uterus and its activation by a serine proteinase.
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ABSTRACT:
SUBMITTER: Woessner JF
PROVIDER: S-EPMC1164538 | biostudies-other | 1977 Mar
REPOSITORIES: biostudies-other
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Project description:1. The earlier observation (Woessner, 1969) of oestradiol inhibition of collagen breakdown is confirmed and extended. Administration of 100mug of oestradiol-17beta/day to parturient rats strongly inhibits the loss of collagen from the involuting uterus. Three experiments show that this effect is due to an inhibition of collagen degradation rather than to a stimulation of collagen synthesis. 2. Uterine collagen was labelled with hydroxy[(14)C]-proline by the administration of [(14)C]proline near the end of pregnancy. By 3 days post partum, control uteri lost 83% of their collagen and 90% of their hydroxy[(14)C]proline. Uteri from oestradiol-treated rats lost only 50% of both total and labelled hydroxyproline, with no decrease in the specific radioactivity of the hydroxyproline. 3. Incorporation of [(14)C]proline into uterine collagen hydroxyproline in vivo was not affected by oestradiol treatment. 4. Urinary excretion of hydroxyproline was increased in post-partum control rats and decreased in oestradiol-treated rats. 5. An enzyme capable of cleaving 4-phenylazobenzyloxycarbonyl-l-prolyl-l-leucylglycyl- l-prolyl-d-arginine (a substrate for clostridial collagenase) increased in activity in the post-partum uterus and was unaffected by oestradiol treatment. 6. Uterine homogenates digested uterine collagen extensively at pH3.2. This digestion was unaffected by the oestradiol treatment. 7. Lysosomal fractions prepared by density-gradient centrifugation of uterine homogenates contained coincident peaks of cathepsin D activity and peptide-bound hydroxyproline. The cathepsin D and hydroxyproline contents of this peak were unaffected by oestradiol treatment.
| S-EPMC1178769 | biostudies-other
Project description:1. Both the post-partum involution of the rat uterus and the rapid breakdown of collagen that accompanies it are extensively inhibited by oestrogenic hormones. In the normal rat, 85% of the uterine collagen is degraded within 4 days after parturition; in rats treated with 100mug. of 17beta-oestradiol/day, only 35% of uterine collagen is broken down in the same period. 2. Similar effects are produced by diethylstilboestrol if the dose is increased tenfold. 3. Collagen breakdown is inhibited to a greater extent than is the loss of wet weight by oestradiol but not by diethylstilboestrol. 4. The oestrogens appear to act by blocking the breakdown of collagen. There is a greatly decreased concentration of free hydroxyproline in the uterus of treated animals. 5. Acid hydrolase concentrations (beta-glucuronidase, beta-galactosidase, cathepsin D and acid phosphatase) in the uterus are decreased by oestrogen treatment compared with controls, but the total amounts of these enzymes in the uterus are somewhat elevated. Oestrogens do not appear to inhibit collagen breakdown by altering the concentration and total amount of acid hydrolases.
| S-EPMC1187767 | biostudies-other
Project description:1. Total, active and latent collagenase activities were determined by direct assay of tissue homogenates. 2. The rate of collagen breakdown during post-partum involution of the rat uterus is correlated with the total activity of collagenase. Both are low at parturition, reach a maximum within 24h and fall slowly to low values of 5 days post partum. This temporal correlation strongly supports the hypothesis that collagenase participates in collagen breakdown in vivo. 3. Further support for this hypothesis is provided by the finding that oestradiol-17 beta (100 micrograms/day, intraperitoneally injected), which inhibits the breakdown of collagen by 36% during the first 4 days of involution, produces a closely corresponding decrease in total collagenase activity. 3. The effect of oestradiol in lowering collagenase activity is not due to alterations in collagen substrate, collagenase kinetic behaviour or latent-to-active enzyme conversion. 4. Of the total assayable collagenase, about 35% is fully active and 65% is in a latent form. 5. About 70% of this latent form can be activated by a serine proteinase found, together with collagenase, in the insoluble fraction of uterine homogenates.
| S-EPMC1161023 | biostudies-other
Project description:1. Homogenates of rat uteri removed 1 and 2 days post partum were centrifuged at 6000 g. Both pellets and supernatants degraded Azocoll, a general proteinase substrate, at pH 7.5. More than 80% of the total activity was in the pellet fraction. 2. Part of the pellet activity was in a latent form. Trypsin and 4-aminophenylmercuric acetate (a thiol-blocking agent) both activated this latent form, indicating that it is an enzyme--inhibitor complex. An endogenous serine proteinase activated part of the latent enzyme during the assay. 3. The enzyme activity was low before parturition and after involution; it was highest during the first 2 days post partum, when the largest losses of uterine wet weight and matrix macromolecules occur. 4. Up to 70% of the enzyme in the pellets was extracted by heating at 60 degrees C for 4 min in 0.1 M-CaCl2/0.05 M-Tris/HCl, pH 7.5. Approx. 30% of the extracted enzyme was still latent. 5. The extracted enzyme was a metalloproteinase, since it was inhibited completely by 1,10-phenanthroline, but not by inhibitors of thiol or serine proteinases. 6. The enzyme was further purified 15--30-fold by gel chromatography and precipitation with (NH4)2SO4. The apparent molecular weight, estimated by gel filtration, was 24000 for the latent form and 12000 for the active form. The pH optimum was 7--7.5. 7. The enzyme also degraded cartilage proteoglycan. This activity was studied by viscometry and the products were analysed by analytical ultracentrifugation. The major product had a mol.wt. of approx. 100000. The sites of cleavage were in the protein core, since no free oligosaccharides were detected. 8. This neutral metalloproteinase is distinct from uterine collagenase and from a uterine metal-dependent endopeptidase that hydrolyses a heptapeptide related to collagen.
| S-EPMC1162032 | biostudies-other
Project description:The aim of this study was to investigate the biological characteristics and functions of a Trichinella spiralis serine proteinase (TsSerp) during larval invasion and development in the host. The full-length TsSerp cDNA sequence was cloned and expressed in Escherichia coli BL21. The results of RT-PCR, IFA and western blotting analyses showed that TsSerp was a secretory protein that was highly expressed at the T. spiralis intestinal infective larva and muscle larva stages and primarily located at the cuticle, stichosome and intrauterine embryos of the parasite. rTsSerp promoted the larval invasion of intestinal epithelial cells (IECs) and the enteric mucosa, whereas an anti-rTsSerp antibody impeded larval invasion; the promotion and obstruction roles were dose-dependently related to rTsSerp and the anti-rTsSerp antibodies, respectively. Vaccination of mice with rTsSerp elicited a remarkable humoral immune response (high levels of serum IgG, IgG1/IgG2a, IgE and IgM), and it also triggered both systemic (spleen) and local intestinal mucosal mesenteric lymph node (MLN) cellular immune responses, as demonstrated by a significant elevation in Th1 cytokines (IFN-?) and Th2 cytokines (IL-4) after the spleen and MLN cells from vaccinated mice were stimulated with rTsSerp. Anti-TsSerp antibodies participated in the killing and destruction of newborn larvae via ADCC. The mice vaccinated with rTsSerp exhibited a 48.7% reduction in intestinal adult worms and a 52.5% reduction in muscle larvae. These results indicated that TsSerp participates in T. spiralis invasion and development in the host and might be considered a potential candidate target antigen to develop oral polyvalent preventive vaccines against Trichinella infection.
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Project description:Implantation S1 family serine proteinases (ISPs) are tryptases involved in embryo hatching and uterine implantation in the mouse. The two different ISP proteins (ISP1 and ISP2) have been detected in both pre- and post-implantation embryo tissue. To date, native ISP obtained from uterus and blastocyst tissues has been isolated only as an active hetero-dimer that exhibits trypsin-like substrate specificity. We hypothesised that in isolation, ISP1 might have a unique substrate specificity that could relate to its role when expressed alone in individual tissues. Thus, we isolated recombinant ISP1 expressed in Pichia pastoris and evaluated its substrate specificity. Using several chromogenic substrates and serine proteinase inhibitors, we demonstrate that ISP1 exhibits trypsin-like substrate specificity, having a preference for lysine over arginine at the P1 position. Phage display peptide mimetics revealed an expanded but mixed substrate specificity of ISP1, including chymotryptic and elastase activity. Based upon targets observed using phage display, we hypothesised that ISP1 might signal to cells by cleaving and activating proteinase-activated receptors (PARs) and therefore assessed PARs 1, 2 and 4 as potential ISP1 targets. We observed that ISP1 silenced enzyme-triggered PAR signaling by receptor-disarming. This PAR-disarming action of ISP1 may be important for embryo development and implantation.
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Project description:Members of the genus Acanthamoeba, amphizoic protozoan parasites, are causative agents of granulomatous amoebic encephalitis and amoebic keratitis. Proteinases play a role in various biologic actions in Acanthamoeba, including host tissue destruction, pathogenesis, and digestion of phagocytosed food. Interestingly, we found that encystation of Acanthamoeba was inhibited by the serine proteinase inhibitor phenylmethanesulfonyl fluoride. In this study, we characterize a serine proteinase that is involved in mediating the encystation of Acanthamoeba. This encystation-mediating serine proteinase (EMSP) is shown to be highly expressed during encystation by real-time PCR and Western blot analysis. Chemically synthesized small interfering RNA against EMSP inhibited the expression of EMSP mRNA and significantly reduced the encystation efficiency of Acanthamoeba. An EMSP-enhanced green fluorescent protein fusion protein localized to vesicle-like structures within the amoeba. Using LysoTracker analysis, these vesicular structures were confirmed to be lysosomes. After incubation of the transfected amoeba in encystment media, small fluorescent vesicle-like structures gathered and formed ball-like structures, which were identified as colocalizing with the autophagosome. Taken together, these results indicate that EMSP plays an important role in the differentiation of Acanthamoeba by promoting autolysis.
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| S-EPMC1135800 | biostudies-other
Project description:Although mast cells play a critical role in allergic reactions, the cells are also involved in the protective immunity in the body. This study aims to investigate the role of mast cells in immune regulation during aberrant T helper (Th)2 responses. In this study, an adoptive antigen-specific Th2 response model was established with mast cell-deficient mice to test the role of mast cell in the immune regulation. Cell culture was employed to test the role of mast cells in the modulation of the expression of B cell lymphoma 6 protein (Bcl-6) in Th2 cells. The results showed that after adoptive transfer with immune cells, the mast cell-deficient mice showed stronger Th2 pattern responses in the intestine than that in the mast cell-sufficient mice. Mast cell-derived mouse mast cell protease-6 increased the expression of Bcl-6 in Th2 cells. Bcl-6 inhibited the expression of GATA-3 in Th2 cells, subsequently, forkhead box P3 was increased and the Th2 cytokines were reduced in the cells; the cells thus showed the immune regulatory properties similar to regulatory T cells. We conclude that bedsides initiating immune inflammation, mast cells also contribute to the immune regulation on Th2 polarization.
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