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 developed a preparation of monolayer cultures of bovine parathyroid cells in order to elucidate the control mechanism of the biosynthesis and secretion of parathyroid hormone (PTH) at cellular level. Dispersion of parathyroid cells was performed by stirring minced bovine parathyroid tissues in Hanks' BSS containing 0.3 yields to 0.5 percent collagenase at 37 degrees C for 60 min. Dispersed cells were cultured at 37 degrees C in MEM-Hanks' BSS containing 10 percent fetal calf serum and 15 mM HEPES. On the 5th day of the culture, the medium was replaced with 1 percent BSA-MEM-Hanks-HEPES buffer, and the cells were incubated with 3H-leucine or in the media containing various concentrations of calcium, magnesium, PGE1, PGE2 or DBcAMP. At the end of incubation, the cells were detouched and homogenized in 8M urea, 0.2 N HCL and 0.01 M cysteine solution. The isolation of proparathyroid hormone (ProPTH) and PTH was performed through the preparation of TCA-powder followed by CMC column chromatography. PTH in the incubation medium was determined by radioimmunoassay. It was demonstrated that the monolayer cultures of bovine parathyroid cells were synthesizing ProPTH and converting it to PTH. The cultures exhibited linear secretion rates of PTH into the medium. The secretion of PTH was markedly increased by PGE1, PGE2 or DBcAMP in the range of 10(-7) yields to 10(-5)M in the former and 10(-5) yields to 10(-3)M in the latter, while calcium or magnesium changed secretion rate in the range of 0.3 yields to 4.4 mM.
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PMID:[Studies on the biosynthesis and secretion of parathyroid hormone in monolayer cultures of bovine parathyroid cells (I) (author's transl)]. 20 10

Insulin (IRI) secretion pattern of collagenase isolated islets from Wistar rats were investigated in a batch type incubation (60 min) and under organ culture conditions (up to 7 days) with different concentrations of glucose as stimulus. The B-cell response within 60 min of incubation was determined in Krebs-Ringer bicarbonate buffer with 1 mg/ml bovine serum albumin and 16 mM HEPES (KRB-HEPES) and compared with the hormone release in a culture medium (TC 199) containing 10% calf serum. Additionally the insulin content of islets before and after 7 days of culture was assayed radioimmunologically. In the presence of culture medium the insulin secretion was enhanced by 5.8 mM glucose whereas this glucose concentration does not stimulate the insulin secretion in KRB-HEPES. During organ culture the insulin secretion was identical in media with 1.0 and 5.8 mM glucose, respectively, but 15 mM glucose raised the hormon output more than 10-fold. The insulin content of islets, cultured for 7 days was decreased in the presence of 15 mM glucose up to 30% and in the presence of 5.8 mM glucose up to 13% of that of islets after isolation. The recovery rate of insulin calculated as the sum of secretion and content after cultivation at 15 mM glucose was higher than 100%, whereas the experiments in the presence of 1.0-5.8 mM glucose are characterized by a recovery rate of 25%. The results are discussed in connection with a altered intracellular breakdown of insulin in the B-cells.
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PMID:[In vitro studies on the islands of Langerhans. XI. Insulin secretion and content of isolated islands of Langerhans in the Wistar rat under short term incubation and under organ culture conditions]. 77

Endotoxin induces insulin hypersecretion in vivo, which results in hyperinsulinemia and glucose dyshomeostasis. Polymyxin-B (PMX-B), an inhibitor of protein kinase C (PKC), has been shown to ameliorate the consequences of endotoxin-induced hyperinsulinemia in vivo. To explore the mechanism for this effect in vitro, this study determined whether PMX-B could alter endotoxin-induced insulin hypersecretion in isolated pancreatic islets of Langerhans. Pancreases were obtained from fasted, male, Sprague-Dawley rats treated with either saline (control) or endotoxin (S. enteritidis B, 16.7 mg/kg, i.v.). Three hours after the experimental treatment, islets were isolated by collagenase digestion and then incubated for 1 hr in Krebs Ringer bicarbonate buffer containing 0.5% bovine albumin, 10 mM HEPES, 300 mg/dl D-glucose, phorbol 12-myristate 13-acetate (PMA, 1 microM when present), and PMX-B (1 or 10 mM when present). In the absence of PMA and PMX-B, "endotoxic" islets hypersecreted immunoreactive insulin (IRI) relative to control islets. PMA, the prototypical PKC activator, significantly increased IRI secretion from both control and "endotoxic" islets. The additional inclusion of PMX-B (1 mM or 10 mM) in the incubation media significantly reduced insulin secretion from both control and "endotoxic" islets and suppressed the insulin hypersecretion observed in "endotoxic" islets. Since insulin secretion occurs at least partially through mechanisms dependent on PKC activation, the ability of PMX-B to suppress insulin hypersecretion in "endotoxic" islets suggests that activation of PKC within pancreatic beta-cells may play a role in the excess insulin secretion and hyperinsulinemia associated with endotoxicosis.
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PMID:Polymyxin-B suppresses endotoxin-induced insulin hypersecretion in pancreatic islets. 142 25

Equine articular chondrocytes were isolated from explant cartilage cultures by digestion in a 0.075% collagenase solution for 15 to 19 hours. Cartilage from late-term fetal and neonatal foals resulted in mean chondrocyte yield of 51.99 x 10(6) cells/g of cartilage (wet weight), compared with a yield of 17.83 x 10(6) cells/g for foals 3 to 12 months old. Propagation of chondrocytes in monolayer and 3-dimensional culture was accomplished, using Ham's F-12 as the basal medium, with supplements of fetal bovine serum (10%), ascorbic acid, alpha-ketoglutarate, and L-glutamine. The medium was buffered with HEPES, and penicillin and streptomycin were added for microorganism control. In primary monolayer cultures of freshly isolated chondrocytes, the population doubling time was approximately 6 days. Dedifferentiation of chondrocytes toward a more fibroblastic-appearing cell was observed after the fifth passage (subculture), but was hastened by lower cell-plating density. Chondrocytes were frozen for periods of up to 9 months, using 10% dimethyl sulfoxide as the cryoprotectant. Cell viability of late-term fetal and neonatal foal chondrocytes after storage at -196 C decreased from 86% at 3 weeks to 31% at 12 weeks. Viability of cells derived from older foals and young adult horses was considerably better than that of cells from neonatal foals. Frozen chondrocytes can be stored for extended periods and thawed for immediate implantation or can be sustained in vitro in monolayer or 3-dimensional culture. Such cultures would be suitable for cartilage resurfacing experiments or in vitro assessment of various pharmaceuticals.
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PMID:Isolation, propagation, and cryopreservation of equine articular chondrocytes. 147 23

The studies included here identify factors affecting cartilage digestion by crude bacterial collagenase (cCGN) and describe a cartilage digestion medium that maximizes both tissue digestion rate and viable cell yield. The basal digestion medium contained 100 mM NaCl, 3 mM K2HPO4, 1 mM CaCl2, 1 mM MgSO4, 10 mM NaHCO3, 60 mM sorbitol, 5 mg/ml of dextrose, 1 mg/ml of albumin, and 2 mg/ml of cCGN in 25 mM HEPES at pH 7.2. Approximately 45% of articular cartilage tissue was digested in this basal medium in 6 h at 37 degrees C, yielding 6.8 x 10(6) viable cells per g tissue digested. The addition of 30 microM tosyllysylchloromethane (TLCM) increased the fraction of tissue digested in 6 h to 68% (p less than 0.05) and doubled viable cell yields to 13.6 x 10(6) per g tissue digested (p less than 0.05). Withholding Mg, decreasing NaCl to 70 mM, and adding 30 mM KCl increased fractional tissue digestion to 81% (p less than 0.01) and doubled viable cell yield yet again (to 29.9 x 10(6) viable cells per g tissue digested). Supplementation with TLCM increased the rate of cartilage digestion and the yield of viable cells regardless of cCGN source or lot. Additional trypsin (0.25%) inhibited tissue digestion and decreased cell yield; this effect was reversible with the addition of TLCM. The cartilage digestion medium developed in these studies (low Mg with added K and TLCM) was very effective in digesting articular, scapular, rib, and growth plate cartilage, as well as in yielding a large number of viable chondrocytes. These cells grew well in culture and maintained their chondrocytic characteristics, secreting predominantly type II collagen and large macromolecular forms of chondroitin sulfate-rich proteoglycans.
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PMID:Enzymatic isolation of chondrocytes from immature rabbit articular cartilage and maintenance of phenotypic expression in culture. 185 87

A new method for the primary monolayer cultures of adult rabbit gastric mucous cells has been developed. Rabbit gastric mucosal cells were isolated with etylenediaminetetraacetic acid and collagenase. Cells were cultured in Coon's modified Ham's F-12 medium supplemented with 10% fetal bovine serum, 15mM HEPES buffer, antibiotics, and antimycotic. The cells reached confluency on days 3-4. Histochemically 92% of the cells contained PAS positive gramules (mucous cells), 3% of cells showed a strong reaction for succinic dehydrogenase activity (parietal cells), 2% of the cells showed positive granules by Bowie staining (chief cells), and G6PDH staining was positive in 5% of the cells (surface mucous cells). Fibroblasts were rarely seen until day 7 (less than 1%). Thus rabbit cultured gastric cells were considered to be mainly comosed of mucous neck cells. These cells produced prostaglandin (PG) E2 and PGI2. Quantitatively cultured cells synthesized 1.475 +/- 0.039 ng/mg protein/hour of PGE2 and 0.244 +/- 0.042 pg/mg protein/hour of PGI2. This relatively simple and convenient technique provides a useful model for the study of cellular functions of gastric mucosa.
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PMID:A monolayer culture of gastric mucous cells from adult rabbits. 210 63

Pediatric liver transplantation is successful but donor scarcity is a major limitation. We are studying hepatocyte transplantation as an alternative to provide functional hepatic replacement. This report details the study of rat liver perfusion for optimal harvest of hepatocytes and cell implantation. We performed 128 rat liver perfusions using a technique modified from the two-step enzymatic perfusion described by Seglen. We examined variations in the perfusion, rate, time, antegrade versus retrograde, pulsatile versus continuous flow, temperature, collagenase type, and variables of buffer composition. We have found optimal cell yield and viability under the following conditions: in situ perfusion, continuous flow at 25 cc/min, retrograde perfusion via the inferior vena cava, water bath temperature 38 degrees C, Boerhinger-Mannheim collagenase using a nonoxygenated HEPES based perfusion buffer, pH 7.4, for the initial perfusion and the same buffer with 4.8 mmol/L CaCl2 for the collagenase perfusion. These conditions consistently generate cell harvests of 500 to 700 x 10(5) cells/g of liver tissue with cell viability between 85% and 95%.
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PMID:Studies in rat liver perfusion for optimal harvest of hepatocytes. 215 93

As a first step in the study of membrane transport characteristics of aortic endothelial cells the content of the two main cations, Na and K, was determined in cultured cells from bovine and porcine origins. The Na and K contents of cultured endothelial cells, dissociated by scraping or trypsin and collagenase treatment and subsequently separated through oil (25% dodecyl-, 75% dibutyl-phthalate), were more than 20-fold higher and five-fold lower, respectively, than those of undissociated cells. Based on daily determination of cell Na, K, and protein contents, the following findings were made. (1) Steady-state levels of Na and K were not reached in subconfluent, confluent or post-confluent monolayers. Instead, intracellular K content varied by up to two-fold, and intracellular Na by more than six-fold with marked 'peaks' after confluency. (2) Increasing the number of passages decreased cellular Na but not K content. (3) In cells cultured with 25 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) the protein content was decreased by five-fold. (4) The K/Na ratio was dependent on the number of passages and buffers used and varied daily. (5) Cell Na decreased and K increased exponentially with the seeding density. These data not only reveal significant changes of ion transport parameters during manipulations of endothelial cell cultures, but moreover suggest unsynchronized development of ion transport systems and/or their intermittent activation and deactivation as reflected in the variations observed in cellular cation composition.
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PMID:Changes in cation composition of aortic endothelial cells in culture. 285 54

Intrapancreatic activation of proteases is believed to play a major role in the pathogenesis of acute necrotizing pancreatitis. Several authors have questioned, however, the central role of trypsin in autodigestion of the pancreas. To clarify the direct effects of pancreatic enzymes and other related factors on acinar cells, we used the model of isolated pancreatic acini. Acini were prepared from male Wistar rats by collagenase digestion. Protein synthesis was measured by incubation of acini with [35S]methionine. Acini were resuspended thereafter in fresh buffer and further incubated for 30-90 min under various conditions [e.g., with pancreatic homogenates, ascites (from rats with pancreatitis induced by sodium taurocholate), pure pancreatic enzymes, and other factors]. The percentage of release of newly synthesized proteins into the culture medium was regarded as a biochemical parameter of cellular integrity. A morphologic score of cellular integrity was obtained via light microscopic evaluation of acini at the end of the various incubations by measuring the degree of cell lysis, loss of cell granules, ballooning, formation of vacuoles, and karyopyknosis. When normal [35S]methionine-labeled pancreatic acini were incubated with various factors, the percentage of release of labeled proteins into the medium was as follows: incubation with HEPES/Ringer's buffer, 1.8%; hemorrhagic pancreatic ascites, 3.8%; pancreatic homogenates, 2.0%; lipase, 1.8%; phospholipase A2, 3.0%; phospholipase A2 + lecithin, 3.2%; trypsin, 2.5%; 5% olive oil, 1.8%; ascites + olive oil, 78.3%; ascites + homogenized epididymal fat, 79.9%; lipase + olive oil, 32.0%; pancreatic homogenates + olive oil, 28.0%; diolein, 2.65%; and oleic acid, 62.9%. The cellular release of radiolabeled proteins showed an inverse correlation with cellular integrity as shown by light microscopy. We postulate that interstitial release of degradation products from triglycerides by lipase causes cellular disruption. Whereas phospholipase A2 and proteases do not seem to be very harmful in the early phases of cellular damage, lipase may play a major role in acute necrotizing pancreatitis.
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PMID:Role of pancreatic enzymes and their substrates in autodigestion of the pancreas. In vitro studies with isolated rat pancreatic acini. 291 45

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


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