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:3.4.24.3 (collagenase)
18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have recently provided evidence from studies conducted in vivo that the ovary, particularly by means of estrogen, regulates placental androstenedione (delta 4A) production during the second half of rat pregnancy. In the present study, an incubation system of dispersed rat placental cells was established to determine if estrogen acts directly on the placenta to regulate delta 4A production. Placentas were obtained on Days 14-15 of rat gestation and dispersed in Hanks' Balanced Salt Solution containing 0.1% collagenase, 0.1% hyaluronidase, 0.01% DNase, and 1% fetal calf serum. Placental cells were incubated in Medium 199 for 16 h at 37 degrees C. A time-dependent increase (r = 0.96, p less than 0.05) in the release of delta 4A occurred over the 16-h incubation period. Mean +/- SE formation of the steroid intermediate progesterone (P4) and product delta 4A was 1.17 +/- 0.78 and 1.18 +/- 0.22 ng per 10(7) cells respectively. The addition of 1-10 microM diethylstilbestrol (DES) decreased (p less than 0.05-0.01) delta 4A production, and had no significant effect on P4 or pregnenolone (P5) formation. The percent decrease in delta 4A production was 14.2 +/- 12.9, 30.9 +/- 2.3, and 55.0 +/- 4.4 with 1, 5, and 10 microM DES, respectively. Treatment of placental cells with estradiol (E2) also resulted in a decrease (p less than 0.01) in delta 4A production with no effect on P4 formation. The percent inhibition of delta 4A production was 34.2 +/- 11.1 and 77.3 +/- 5.2 with the addition of 1 microM and 10 microM E2, respectively. E2 (10 microM) produced a concomitant threefold increase (p less than 0.01) in P5 formation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The effect of estrogen on androstenedione production by rat placental cells in vitro. 292 52

In the present study, a culture system of human placental cells was established to examine the role of estrogen and androgen in progesterone (P4) formation. Normal human placentae were obtained at term, and cells were dispersed in Hank's Balanced Salt Solution (5 ml/g tissue) containing 0.1% collagenase, 0.1% hyaluronidase, 0.01% deoxyribonuclease, and 1% fetal bovine serum for 2 h at 37 C. Dispersed placental cells (10(6) cells/ml) were placed in medium 199 with modified Earle's salts (pH 7.4) containing 10% fetal bovine serum, 12.5 mM HEPES buffer, 26 mM NaHCO3, and 40 micrograms/ml Gentamycin-SO4 and incubated for 72 h at 37 C and 5% CO2 in air to allow cell attachment. Medium was then changed (time zero), and P4 formation was studied thereafter. Culture of placental cells for 96 h resulted in linear increases in P4 and estradiol (E2) formation, indicating the maintenance of cell viability and steroidogenic function. Mean +/- SE P4 formation at 48 h was 246 +/- 16 pg/micrograms DNA. To assess the role of estrogen on P4 formation, placental cells were incubated for a period of 48 h with various amounts (10(-7)-10(-4)M) of the antiestrogen ethamoxytriphetol (MER-25), the aromatase inhibitor 4-hydroxyandrostenedione (4-OHA), and/or E2. Both MER-25 and 4-OHA resulted in a dose-dependent decline (P less than 0.01) in P4 formation (greater than 80% decline at 10(-4)M MER-25 or 4-OHA). The marked reduction in P4 formation caused by 4-OHA alone was reversed by concomitant addition of E2; however, E2 alone had no effect. To assess the role of androgens on P4 formation, cells were incubated for 48 h with increasing amounts (10(-7)-10(-4)M) of androstenedione, dehydroepiandrosterone (DHA), or dihydrotestosterone. Although the formation of E2 was enhanced by DHA, formation of P4 was not affected by the aromatizable androgens DHA or androstenedione or the nonaromatizable dihydrotestosterone. The decline in P4 formation by human placental cells in culture elicited by MER-25 or 4-OHA supports the hypothesis of a regulatory role for estrogen in placental P4 formation during human pregnancy. The lack of effect of exogenous estrogen suggests that the action of estrogen on P4 formation may be permissive.
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PMID:Regulation of progesterone formation by human placental cells in culture. 294 94

We compared in vitro heparin binding activity and in vivo intravascular clearance and aortic uptake in rabbits of native, reductively methylated and heparin-complexed low density lipoprotein (LDL) in order to explore the extracellular matrix binding vs cellular metabolism of LDL. Reductively methylated LDL formed soluble and insoluble complexes with heparin which was comparable to native LDL. Reductive methylation of LDL produced only 30% reduction in aortic uptake vs 60% reduction in plasma clearance, reflecting the relatively smaller contribution of receptor-mediated pathway in aortic tissue vs whole animal. The intravascular clearance of native and heparin-complexed LDL remained essentially the same, indicating similarities in cellular metabolism of LDL in both cases. But the aortic uptake of the heparin bound LDL was 30% less than the native LDL, suggesting an inhibition in binding of heparin-complexed LDL to tissue proteoglycans. Saline extraction accounted for only part (53-66%) of the LDL preparations that were retained by the tissue while subsequent collagenase and elastase treatments extracted 3-5% and 17-22% of the materials respectively. These results favor the contribution of arterial extracellular matrix components to the retention of LDL.
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PMID:Low density lipoprotein retention by aortic tissue. Contribution of extracellular matrix. 380 Oct 86

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.
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PMID:Measurement of unscheduled DNA synthesis in rat kidney cells following in vivo treatment with genotoxic agents. 406 62

Heart cells were obtained in suspension after incubation with collagenase and hyaluronidase in Saline A. Cardiac myocytes were separated by isopycnic centrifugation in 88.6 to 92.4% purity from other heart cells with different densities, and by velocity or rate-zonal sedimentation, in 92.8 to 97.4% purity from heart cells with different diameters. A previously described computer integration of the differential sedimentation equation was used to determine the centrifugal force, duration of centrifugation and gradient design, which would permit the separation of cardiac myocytes from other heart cells by velocity sedimentation. The myocytes continued to contract rhythmically after being recovered from the density gradients. Velocity sedimentation was superior to isopycnic sedimentation for the separation of cardiac myocytes from heart cell suspensions because it gave the most highly purified myocytes, resulted in recovery of the largest proportion of myocytes in purified fractions from the gradient and required lower centrifugal forces for shorter periods of time. The potential significance of the availability of pure cardiac myocytes is discsused.
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PMID:Separation of beating cardiac myocytes from suspensions of heart cells. 433 47

Basement membranes in human gingiva are found in dento-epithelial junction, epithelial-connective tissue junction and endothelium-connective-tissue junction where they have important attachment and filtering functions. The ability of plaque and gingiva-derived proteinases to degrade Type IV collagen, the major protein of basement membranes, was examined in vitro. The basement membrane collagen (Type IV) isolated from bovine lens capsules was incubated in the presence of enzyme samples. The degradation was assayed by the release of hydroxyproline from the insoluble substrate and by examining the peptide pattern of the residue by polyacrylamide gel electrophoresis. Salt extracts of inflamed gingival specimens degraded basement membrane collagen into soluble form and produced degradation products that were similar to those produced by human leukocyte extracts. Gingival crevicular fluid collected from patients with severe adult periodontitis also digested the substrate but the degradation pattern was different from the leukocyte and gingival extract samples. The pattern closely resembled the degradation produced by bacterial plaque extracts. A third type of cleavage was observed when collagenase from Clostridium histolyticum was incubated with basement membrane collagen. Crevicular fluid and the extracts from gingiva, leukocytes and plaque also contained gelatinase and elastase-like enzyme activities that have earlier been shown to be potent in degrading basement membrane. It was concluded that enzymes capable of degrading basement membrane collagen in gingivitis and periodontal disease may originate from both plaque bacteria and human leukocytes. It also appeared that the enzymes responsible are more likely to be gelatinase and elastase-like enzymes than specific collagenases.
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PMID:Degradation of basement membrane collagen by proteinases from human gingiva, leukocytes and bacterial plaque. 631 10

Dynamics of lipoprotein-glycosaminoglycan interactions in aortas were studied in vivo using the atherosclerotic rabbit model. Severe hypercholesterolemia and atherosclerosis were produced by relatively long-term feeding of a high cholesterol diet. [35S]Sulfate uptake by aorta was measured to assess the sulfated glycosaminoglycan metabolism while the plasma and aorta distribution of 125I-labeled LDL after intravascular injection was determined to monitor aortic LDL uptake and complex formation with glycosaminoglycans. The retention and distribution of LDL as lipoprotein-glycosaminoglycan complexes in different extracellular connective tissue elements were evaluated by extracting the tissues with saline, collagenase and elastase. Hypercholesterolemia with atherosclerosis resulted in a several-fold increase in the uptake of LDL by aorta despite a marked reduction of 125I-labeled LDL in the plasma compartment and in a significant increase in glycosaminoglycan content of aorta coupled with an increased 35S incorporation into glycosaminoglycans. Elastase-solubilized fractions from normal aortas and collagenase-solubilized fractions from atherosclerotic aortas contained maximum labeled and nonlabeled glycosaminoglycan, suggesting alterations in the make-up of fibrous structures of connective tissue matrix in atherosclerosis. Saline extraction and collagenase and elastase digestions solubilized varied proportions of lipoprotein-cholesterol and 125I-labeled LDL, thereby representing different pools of extracellular matrixbound lipoproteins. A tendency for 125I-labeled LDL to increase in collagenase- and elastase-solubilized fractions with time (4 h vs. 24 h) was noted. The occurrence of both lipoproteins and glycosaminoglycan (labeled and nonlabeled) in the ultracentrifugal floating fraction at solvent density 1.063 g/ml demonstrated that the lipoproteins solubilized by different extraction procedures occur in part as lipoprotein-glycosaminoglycan complexes. The specific activities of glycosaminoglycan in the complexes obtained by different extraction procedures differed markedly (elastase greater than collagenase greater than saline), emphasizing the presence of different pools of complexes. Thus, besides arterial cell-mediated processes, extracellular matrix components are important in affecting the retention and accumulation of LDL in atherosclerosis.
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PMID:Dynamics of lipoprotein-glycosaminoglycan interactions in the atherosclerotic rabbit aorta in vivo. 671 64

The preparation, viability, and prostaglandin (PG) binding characteristics of dispersed smooth muscle cells from the rabbit oviduct have been determined. Cell suspensions were prepared by enzymatic degradation of the intracellular matrix and subsequent mechanical dispersion with a wide bore pipette. The digestion media consisted of a modified Hanks' Balanced Salt Solution, pH 7.1, containing 1.6 U/mg wet wt elastase and 8 U/mg wet wt collagenase. This method provided a yield of single smooth muscle cells of approximately 3 x 10(6) cells/100 mg wet wt within 3-4 h of organ removal. Cell viability was determined by trypan blue dye exclusion, retention of lactate dehydrogenase, absence of 57Co-EDTA uptake, and ouabain sensitivity of cationic transport. Roughly 80% of the isolated cells remained viable after the digestion procedure. The dispersed cells specifically bound [3H]PGE2 and [3H]PGF2 alpha. Scatchard analysis of the binding data revealed separate homogenous populations of high affinity sites for both PGE2 and PGF2 alpha. The equilibrium dissociation constants and total sites per cell were 0.55 nM and 11,332 for PGE2 and 0.19 nM and 5,154 for PGF2 alpha, respectively. Specific labeled PG binding was inhibited in a concentration-dependent manner by increasing amounts of unlabeled PG. Inhibition of labeled PGE2 binding by unlabeled PGF2 alpha, and vice versa were negligible, except at high concentrations. The results indicate that smooth muscle cells can be enzymatically dispersed from the rabbit oviduct with minimal damage. Also, these cells possess distinct specific binding sites for PGE2 and PGF2 alpha that differ in regard to affinity and total number of sites per cell.
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PMID:Preparation of smooth muscle cell suspensions from the rabbit oviduct and prostaglandin binding analysis. 700 18

Though sex steroids are found to influence thyroid pathogenesis in human and in animals, their role in normal thyroid growth and thyrocyte proliferation is not yet understood fully. The present study is addressed to know the effect of testosterone and estradiol on the basal and TSH-induced thyrocyte proliferation in immature and adult rats in vitro. The male and female Wistar rats were gonadectomized (GDX) and one group of GDX rats were supplemented with either testosterone or estradiol. After the experimental period, the rats were sacrificed by decapitation and thyroid glands were removed, washed in Hank's Balanced Salt Solution (HBSS), pH 7.4 and digested with the enzyme mixture containing 0.08% collagenase and 0.12% dispase in HBSS. The isolated follicles were washed thrice with Dulbecco's modified Eagle's medium (DMEM) containing 0.5% fetal bovine serum (FBS), and were cultured in Falcon's tissue culture flasks containing 5 ml DMEM with FBS (5%) transferrin (5 microg/ml), hydrocortisone (10(-8) M), somatostatin (10 microg/ml), insulin (10 microg/ml) and glycyl-L-histidyl-L-lysine acetate (10 microg/ml). The cells (2.5 x 10(4)) were exposed to various exponential doses of TSH or testosterone (6.25-800 ng/ml) or estradiol (6.25-800 pg/ml). It is suggested from the present study that both TSH and sex steroids enhance thyrocyte proliferation. The mitogenic effect of TSH is greater than that of sex steroids. Sex steroids modulate TSH-induced cell proliferation in a gender-specific manner.
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PMID:Testosterone and estradiol differentially regulate TSH-induced thyrocyte proliferation in immature and adult rats. 1199 29

In this study a detailed description of the equine hepatocyte isolation procedure is presented. Livers were obtained from horses slaughtered at the local slaughterhouse. For blood removal and liver preservation the following steps are suggested: perfusion with the oxygenated HBSS (0-2 degrees C, with continuous flow of 500-800 ml/min for 3-6 min), protection from ischemia injury by flushing with ice-cold University of Wisconsin Solution (UW, flow rate of 500-800 ml/min), and finally immersion of the liver lobe in UW solution (2 degrees C) during its transport to the laboratory. For equine isolated hepatocyte preparation a "three-step" perfusion procedure was elaborated: rewarming, chelating and collagenase perfusion. We found optimal cell yield and viability under the following conditions: rewarming with UW (38 degrees C) for 8-14 min, chelating with calcium free Hanks' Balanced Salt Solution (HBSS, 38 degrees C) supplemented with 1 mM ethylene glycol-bis[beta-aminoethyl esther]-N,N,N'N'-tetracetic acid at the flow rate of 450 ml/min for 6 min and enzymatic digestion with HBSS supplemented with 0.1% collagenase at 38 degrees C and 450 ml/min flow rate for 8-27 min. These conditions consistently generated cell harvests of 21 x 10(6)+/-4.86 cells/g of perfused liver tissue with viability of 82.7%+/-10.2.
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PMID:Preparation of equine isolated hepatocytes. 1459 53


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