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)

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

Growth hormone (GH) binding and the effect of GH and insulin on glucose metabolism in rat adipocytes were studied at various time periods following hypophysectomy. Male rats were hypophysectomized at 33-34 days of age. After 6 h, 20 h or 3, 7 and 14 days adipocytes were prepared from epididymal fat pads by mild collagenase digestion (0.5 mg X ml-1, 60 min, 37 degrees C). Glucose metabolism was studied by determining the production of CO2 from [14C]glucose and the incorporation of [14C]glucose into lipids. GH binding was measured in cell aliquots using [125I]hGH. No difference in GH binding to adipocytes was observed between control rats and rats hypophysectomized or sham-operated 6 h earlier. GH binding was significantly decreased 20 h after hypophysectomy and declined further with time after hypophysectomy. Adipose tissue from normal rats is usually refractory to the insulin-like effect of GH. Adipocytes isolated from normal rats were, however, usually responsive to GH immediately after cell isolation, suggesting that refractoriness to the insulin-like effect of GH was lost during the time required for the preparation of adipocytes. The magnitude of the response to GH in adipocytes progressively declined with time after hypophysectomy. The decreased responsiveness to GH with time after hypophysectomy parallelled the decrease in GH binding. The results suggest that the pituitary, directly or indirectly, is necessary for the maintenance of GH binding sites in adipose tissue and that these binding sites are related to the insulin-like effect of GH.
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PMID:Changes in growth hormone binding and metabolic effects of growth hormone in rat adipocytes following hypophysectomy. 299 Jan 66

The TSH-responsive adenylate cyclase system was studied using porcine thyroid cells in a primary monolayer culture. Isolated porcine thyroid cells treated with collagenase were inoculated into 96 wells at the density of 5 X 10(4) viable cells/0.25 ml Ham F-12 containing 10% fetal bovine serum and cultured for 4 days in a humidified atmosphere with 5% CO2. Adenylate cyclase activities in the cells treated or non-treated with protein synthesis inhibitor were assayed in Hanks/20 mM Hepes buffer (pH 7.4) containing 1% BSA, 1 mM IBMX and various stimulants at 37 degrees C for 30 or 60 min. The reaction was stopped by adding ice-cold TCA, and cAMP content in the extract was measured by radioimmunoassay after treatment with water-saturated ether. The cultured thyroid cells had an adenylate cyclase system responsive to TSH, cholera toxin and forskolin. TSH (50 mU/ml) stimulated the activity about eight fold over the basal activity. Cholera toxin (1 microgram/ml) and forskolin (100 microM), however, were much stronger activators of the adenylate cyclase system. In the cells pretreated with cyclo-heximide (5 micrograms/ml) up to 24 hours, cAMP formation by TSH was potentiated 200 approximately 170% compared to that in non-treated cells, suggesting a suppression of an inhibitory mechanism dependent upon new protein synthesis. In contrast, forskolin (100 microM)-stimulation was greatly reduced to 30% of the control after 24-hour treatment. Cholera toxin (1 microgram/ml)-stimulation was significantly lessened or slightly reduced by the treatment. Although the ability of forskolin to act synergistically with TSH or cholera toxin was observed in non-treated cells, it was clearly unaffected and demonstrated in the cells treated with protein synthesis inhibitor. The mechanism(s) and site(s) of forskolin action still remain unclear. However, these observations are compatible with a two-site model of forskolin action. The direct activating site of forskolin appears to reside in a protein which is closely associated with the catalytic unit of adenylate cyclase system and has a relatively shorter half-life than other components of the system. The potential action of forskolin may reside in a more stable complex of an activated stimulatory guanine nucleotide binding component and catalytic unit of the adenylate cyclase system. Based on these results, it is likely that the primary monolayer culture of porcine thyroid cells is a good model to investigate the adenylate cyclase system in the thyroid, and that forskolin may potentiate the TSH-mediated stimulation of adenylate cyclase.
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PMID:[Adenylate cyclase system responsive to thyroid stimulating hormone (TSH) of porcine thyroid cells in primary monolayer cultures. Potential effect of forskolin on TSH-mediated adenylate cyclase stimulation]. 303 Aug 31

The newest knowledge on the osteoclast allows us to consider bone resorption in a global perspective, as the resultant of three successive steps that may each be individually regulated by physiopathologic or pharmacologic agents. The first involves the formation of osteoclast progenitors in hematopoietic tissues followed by their vascular dissemination and the generation of resting preosteoclasts and osteoclasts in bone. The second consists in the activation of osteoclasts at the contact of mineralized bone. Osteoblasts appear to control this step by exposing the mineral to osteoclasts and preosteoclasts and/or by releasing a soluble factor that activates these cells. In a third step, activated osteoclasts resorb both the mineral and the organic of mineralized bone through the action of agents that they secrete in the segregated zone underlying their ruffled border. The mineral appears to be solubilized by hydrogen ions secreted by an ATP-driven proton pump located at that border and fed by protons generated from CO2 by carbonic anhydrase. The removal of organic matrix, which could be prepared by osteoblast collagenase at the level of nonmineralized bone surfaces, appears dependent on acid proteinases, particularly cysteine-proteinases, secreted, together with other lysosomal enzymes, in the acid microenvironment of the resorption zone.
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PMID:Cellular biology and biochemical mechanism of bone resorption. A review of recent developments on the formation, activation, and mode of action of osteoclasts. 328 76

From PMSG-pretreated immature rats, dispersed ovarian cells were prepared with collagenase and DNase and incubated at 37 degrees C in McCoy's 5a medium under 95% air-5% CO2 atmosphere for 4 h. The activities of C17-C20 lyase measured in the 10,000 x g supernatant fluid of the cell homogenates decreased spontaneously with the lapse of time of the incubation. N,N'-Diphenyl-p-phenylenediamine (DPPD, an antioxidant) and actinomycin D inhibited the decrease most effectively. Cycloheximide was also an effective protector. Accordingly, the spontaneous decrease of the lyase activity was caused partly by an oxygen radical-mediated process and partly by a mechanism involving de novo synthesis of RNA and protein. Addition of hCG to the cells further decreased the lyase activity to about half of the control group at 4 h. DPPD itself did not affect the hCG-induced decrease of the lyase activity. However, actinomycin D and cycloheximide prevented the effect of hCG. These results indicate that de novo synthesis of RNA and protein is involved in the latter mechanism, while oxygen radical is not concerned in this process. The decrease of the enzyme activity by hCG during incubation is in agreement with the in vivo effect of hCG upon the lyase activity. On the contrary, at the end of incubation the activity of delta 5-3 beta-hydroxysteroid dehydrogenase (coupled with delta 5-delta 4 isomerase) was more than 89% of that before incubation, and the change of the enzyme activity according to the various treatments was less than 16%.
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PMID:In vitro decrease of lyase activity in rat ovarian cells during incubation: effect of hCG. 345 47

The in vitro effects of ovine PRL (oPRL) on testicular testosterone synthesis were determined using isolated, collagenase-dispersed, adult rat Leydig cells in culture. oPRL (50-1000 ng/ml) had no effect either on basal or on LH (50, 100 or 2000 pg/ml)-stimulated testosterone secretion by Leydig cells in short-term culture (4 h). 125I-oPRL binding studies revealed a single class of high affinity sites (Ka 8.7 nM) with a low capacity (Bmax 6.7 fmol/mg protein identical to approximately 980 sites/Leydig cell). Isolated Leydig cells were further purified on a continuous Percoll gradient and cultured in serum-free medium, at 34 degrees C, in 5% CO2 and 95% air. After 3 days of culture, the media were collected, the cells washed and then stimulated with hCG (3 ng/ml) for 3 h. oPRL (1-1000 ng/ml) added at plating, caused a log dose-dependent inhibition of testosterone accumulation during the 3-day culture period; the highest and most consistent inhibition (31%) was with 500 ng/ml oPRL. hCG increased the sensitivity to the inhibitory effect of PRL, 10 ng/ml oPRL causing 40% inhibition and 100 ng/ml causing a maximal inhibition of 50%. PRL in fact caused a reduction in the maximal effect (efficacy) of hCG on steroidogenesis, without significantly affecting the ED50 (sensitivity). The effects of an antiPRL receptor antibody raised by the antiidiotypic route and previously shown to bind to rat testis PRL receptors were tested. The antiPRL receptor IgG (13 micrograms/ml) mimicked the PRL inhibitory effect and acted synergistically with PRL (100 ng/ml) in inhibiting both testosterone accumulation in 3-day cultured Leydig cells and their subsequent response to hCG. In summary, a clear inhibitory effect of PRL and a synergistic effect of antiPRL receptor antibody were demonstrated on testosterone synthesis by rat Leydig cells in 3-day culture.
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PMID:Prolactin and antiprolactin receptor antibody inhibit steroidogenesis by purified rat Leydig cells in culture. 362 21

Fetal human Langerhans islets were isolated from pancreatic tissue of 2 embryos aged 20 and 22 weeks by partial collagenase digestion. The islets were kept in Eagle's medium at 37 degrees C and 5% CO2 for 10 weeks. The insulin production was continuous during this period. The two cultured fetal islet masses were transplanted to a 31-year-old diabetic man and a 58-year-old diabetic woman. They have required insulin treatment for 25 and 30 years, respectively, and have suffered from retinopathy. The transplantations were performed to the liver after tissue typing. In the case of the man the daily insulin requirement was slightly reduced after 4 months of transplantation but later (8th month) the insulin dose was half the original one. Just now (24 months) the insulin dose is on the same level. In the case of the woman the daily insulin requirement was reduced only after 8 months of transplantation. Just now (13 months) her insulin dose is 12 U less than the original one. In both of them the function of the transplanted islets was proved by measurement of serum C-peptide level and by the fundus examination (no progression of retinopathy).
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PMID:Clinical transplantation of fetal human pancreatic islets. 392 61

The present study was designed to improve the dispersed adrenal cell technique for determining adrenocorticotrophic hormone (ACTH) concentrations in small amounts of rat plasma. Priming with ACTH, incubation with methyl-isobutylxanthine, or dexamethasone pre-treatment were employed as modifications. Of these, only dexamethasone pre-treatment increased the sensitivity of the assay. The adrenal fragments obtained from 10-12 adult male rats pre-treated with dexamethasone (100 micrograms/kg B.W.) one hour before sacrifice, were digested with collagenase and deoxyribonuclease solution for 30 minutes. The dispersed cells were collected by centrifugation and resuspended in Krebs-Ringer bicarbonate buffer containing 0.2% glucose and 0.5% bovine serum albumin. Aliquots of cell suspension (3-4 X 10(4)/tube) were incubated with various doses of ACTH1-24 or the eluate of plasma samples at 37 degrees C for 2 hours in an atmosphere of 95% O2/5% CO2 in a Dubnoff shaker. The quantity of corticosterone produced was measured fluorimetrically. The assay is precise (lambda = 0.06), extremely sensitive (10 fg/tube), and convenient. One skilled technician can handle 15 to 20 plasma samples per day using 10 rats as the source of assay cells. ACTH can be measured in as little as 10-50 microliters of eluate.
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PMID:An improved dispersed adrenal cell assay for corticotropin in rat plasma. 608

A superfusion method consisting of fully recovered, dissociated pituitary cells adhering to Cytodex beads has proved useful in monitoring the dynamics of hormone secretion over time. Male rat anterior pituitaries were dissociated with collagenase and Viokase, then cultured in the presence of Cytodex beads for 3-5 days, during which time the cells attached firmly to the surface of the beads. The bead-attached cells were stable and could be transferred to any vessel without the need for centrifugation or further trypsinization. For this application, the bead-attached cells were packed in a column and superfused with a low bicarbonate buffer requiring no CO2 gassing. Viability was more than 95% after 48 h in the column. The cells responded in a normal physiological manner to hypothalamic releasing and inhibitory peptides. The ED50 was 0.3 nM for somatostatin and 1.2 nM for gonadotropin-releasing hormone. A postinhibitory rebound of GH secretion was observed after the discontinuation of large doses of somatostatin. LH secretion reached maximal levels within 6 min after 10 nM gonadotropin-releasing hormone, but started declining after 2 h of continuous stimulation and dropped close to baseline within 18 h. GH release was significantly increased by prostaglandin E2, 3-isobutyl-1-methylxanthine, and 8-bromo-cAMP. LH secretion increased 5-fold in response to 1 mM 8-bromo-cAMP, but showed little increase during prostaglandin E2 or 3-isobutyl-1-methylxanthine stimulation. The cocarcinogen phorbol myristate acetate (12-O-tetradecanoyl-phorbol-13-acetate) induced secretion of all pituitary hormones and continued to do so for hours after a short pulse. The superfusion system is simple to operate and has proven effective in studying transient phenomena, desensitization, and short term kinetics of secretagogues.
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PMID:Superfusion of rat anterior pituitary cells attached to Cytodex beads: validation of a technique. 615 98

Purified Leydig cells were obtained from testes of mature male pigs by collagenase treatment and mechanical dispersion, followed by Percoll (0-90%) density gradient centrifugation. The cells recovered at 40-45% Percoll were applied to a second gradient of 15 ml of Percoll (10-60%) to yield three bands, one major and two lesser in numbers of cells. Incubations were then made with 0.25-1.0 X 10(6) cells at 34 degrees C for 3 h in 95% O2: 5% CO2, with or without human chorionic gonadotrophin (hCG) added to the medium. Steroid concentration was determined by radioimmunoassays. The steroids measured in the media were testosterone, dehydroepiandrosterone sulfate (DHAS) and estrone sulfate (E1S). Lesser amounts of dehydroepiandrosterone (DHA) and estrone (E1) were found. Stimulation by hCG led to an increase in apparent steroid production for all steroids, including estrogens, with the greatest quantities seen with DHAS (greater than 200 ng/1 X 10(6) cells/3 h). Cells in the major band gave the best response. These results show that Leydig cells are a significant site of estrogen production in the boar testis and that this organ is a source of an abundant supply of such cells.
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PMID:Estrogen and androgen production by purified Leydig cells of mature boars. 622 67


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