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)

A tissue culture system for rat prostatic epithelial cells was developed, and the effect of epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), and transforming growth factor beta 1 (TGF-beta 1) on these cells was evaluated. The primary culture was prepared by DNAse/collagenase dissociation of minced ventral prostates. Cells were initially plated in RPMI-1640 medium containing 10% fetal bovine serum to allow the preferential attachment of stromal cells. Twenty-four hours later, the unattached epithelial cells were replated in WAJC-404 medium supplemented with insulin (5 micrograms/ml), transferrin (5 micrograms/ml), and selenious acid (5 ng/ml). Bovine pituitary extract (BPE) (30 micrograms/ml), EGF (10 ng/ml), and TGF-beta 1 (0, 0.1, and 1.0 ng/ml) were added either alone or in combination according to experimental requirements. The rate of cell proliferation was assessed by counting the total cell number and by [3H]thymidine incorporation. Prostatic epithelial cells exhibited a bell-shaped growth curve in a span of 7-8 days, with a growth peak at day 3 or 4 of culture. Treatment of cells with EGF or TGF-alpha resulted in a concentration-dependent increase in cell growth, whereas addition of TGF-beta 1 into the culture resulted in an inhibition of cell proliferation that could be reversed with increasing concentrations of EGF. Cell death was assessed using the terminal deoxynucleotidyl transferase (TdT)-mediated immunoperoxidase-digoxigenin nick end labeling technique and the trypan blue exclusion test. Epithelial cells cultured in media containing EGF had the lowest incidence of cell death. Cells cultured in the absence of EGF demonstrated a marked increase in cells undergoing cell death. The addition of TGF-beta 1 into the EGF-depleted medium caused a further increase of cell death. These results indicated that cell proliferation and cell death in rat prostatic epithelial cells in culture could be modulated by EGF and TGF-beta 1. The former stimulated cell proliferation and prevented cell death, whereas the latter inhibited proliferation in the presence or absence of EGF and induced cell death.
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PMID:Effect of TGF-beta 1, TGF-alpha, and EGF on cell proliferation and cell death in rat ventral prostatic epithelial cells in culture. 886 96

This study was undertaken to investigate the effects of growth hormone (GH) on the in vitro maturation of the metabolism of fetal rat islets. For this purpose fetal islets were obtained from 21-day-old fetuses by mild collagenase digestion of the pancreas and cultured in RPMI 1640 supplemented with 10% fetal calf serum. After one day the medium was changed and supplemented with 1% fetal calf serum with or without GH (1 microgram/ml, human recombinant) and the islets cultured for another two days. Islets were then studied with regard to insulin secretion, (pro)insulin and total protein biosynthesis, glucose utilization and oxidation, thymidine incorporation, insulin and DNA contents and the contents of mRNAs for either insulin, adenine nucleotide translocator or cytochrome b. In addition, the activities of glucose phosphorylating enzymes and succinate-cytochrome c reductase were measured. Islets treated with GH showed increased insulin secretion in response to glucose, increased rates of glucose oxidation and utilization, increased thymidine incorporation and increased activities of succinate cytochrome c reductase and glucose phosphorylation at high glucose concentrations. There were, however, no changes in (pro)insulin and total protein biosynthesis, contents of insulin and DNA or the contents of any of the mRNAs. These combined data show that fetal beta-cells are sensitive to growth hormone with respect to glucose metabolism, insulin release and DNA replication. The increased rates of islet glucose phosphorylation may reflect glucokinase activity and explain part of the increased insulin responsiveness to glucose of the fetal rat beta-cell. These observations suggest that GH is of physiological significance for the maturation of the fetal beta-cell.
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PMID:Effects of growth hormone in vitro on the glucose metabolism of fetal rat islet beta-cells. 893 88

Cryopreservation is an effective method of islet storage and may facilitate clinical trials of islet transplantation. It was the aim of the present study to evaluate the in vitro viability of cryopreserved rat islets, including the response to nonglucose secretagogues and glucose oxidation. After pancreatic digestion via intraductal injection of collagenase, 75- to 200-micron Wistar rat islets were handpicked and cultured in RPMI 1640 (glucose 11.1 mmol/L) and randomized into two groups: control (cultured 20 to 24 hours at 37 degrees C) and cryopreserved (after 20 to 24 hours of culture at 37 degrees C, islets were cryopreserved according to Rajotte's protocol: freezing velocity, -0.25 degree C/min; thawing velocity, 200 degrees C/min). In the two groups, we evaluated recovery, insulin content per islet, staining viability (ethidium bromide/orange acridine; semiquantitative scoring, measuring the viable area of the islet from 0 = less viable to 3 = more viable), insulin secretion after glucose and nonglucose secretagogues, and oxidation of D-[U-14C]glucose. The results for the control group were always higher for the following: recovery (95.4% +/- 1.2% v 83.0% +/- 2.1%, P = .00), insulin content (2,203.9 +/- 335.2 v 1,443.3 +/- 171.8 microU/islet, P = .03), insulin secretion after 5.5 mmol/L glucose (61.3 +/- 8.0 v 28.3 +/- 3.4 microU/islet/90 min, p = .00), 16.7 mmol/L glucose (151.4 +/- 16.1 v 98.7 +/- 14.1 microU/islet/90 min, p = .03), 10 mmol/L L-leucine +10 mmol/L L-glutamine (125.6 +/- 27.9 v 56.8 +/- 6.4 microU/islet/90 min, P = .05), and 10 mmol/L L-arginine (202.5 +/- 27.5 v 128.8 +/- 14.2 microU/islet/90 min, P = .01), and glucose oxidation at 5.5 mmol/L (12.5 +/- 1.1 v 7.9 +/- 0.6 pmol/islet/120 min, P = .00) and at 16.7 mmol/L (26.1 +/- 2.6 v 14.3 +/- 1.6 pmol/islet/120 min, P = .00). No significant differences in staining viability were found between groups (2.35 and 2.48, respectively, P = .55). However, cryopreserved and control islets showed a significant increase in insulin secretion and glucose oxidation after increasing the glucose concentration from 5.5 to 16.7 mmol/L. We conclude that when glucose is increased, cryopreserved islets keep the capacity to increase insulin secretion, but cryopreservation produces a significant decrease in several islet viability characteristics. This decrease may be due to a decline of beta-cell number per islet and/or a decrease in the content of insulin per beta cell.
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PMID:Cryopreservation: in vitro results in rat pancreatic islets. 918 99

Regulation of calcium balance is important in the secretory function of pancreatic islets. Ca2+-adenosine triphosphatase (ATPase) is altered in tissues of non-insulin-dependent diabetes mellitus (NIDDM) rats, and they have an impaired response to glucose, "glucose blindness." We propose that the glucose blindness of the diabetic islet is the result of defective cellular calcium metabolism. Since Ca2+-ATPase activity is important in the regulation of calcium balance, we investigated the effect of glucose and/or calcium on Ca2+-ATPase activity in pancreatic islets in vitro and compared it with the effect in freshly isolated islets from controls and from rats with NIDDM induced by streptozotocin neonatally. Islets were isolated using collagenase and were stored fresh or cultured up to 2 days in RPMI 1640 in the presence of different concentrations of glucose and calcium. Membrane Ca2+-ATPase activity, insulin secretion, and insulin content were determined. Ca2+-ATPase activity was 1.30 +/- 0.20 micromol/L Pi/microg membrane protein in normal noncultured islets and 1.02 +/- 0.15 in islets cultured in 5.6 mmol/L glucose. Ca2+-ATPase activity progressively decreased to 0.56 +/- 0.10 and 0.34 +/- 0.14 micromol/L Pi/microg membrane protein when glucose was increased in the culture media to 16.6 and 27.7 mmol/L, respectively. Decreasing glucose to 2.8 mmol/L did not alter Ca2+-ATPase activity. Increasing or decreasing the Ca2+ content of the media did not significantly change Ca2+-ATPase activity. Islets isolated from NIDDM rats had lower basal Ca2+-ATPase activity and insulin content compared with normal controls. Incubation of islets from diabetic rats in high glucose further decreased the Ca2+-ATPase content, but incubation in low glucose did not reverse it. Insulin secretion was responsive to glucose and calcium in normal islets, but was suppressed in islets from diabetic animals. From these studies, we conclude that high glucose, but not calcium, decreases Ca2+-ATPase activity in islets from normal rats. Islets from NIDDM rats with glucose blindness have decreased Ca2+-ATPase activity, likely due to the glucose status. We suggest that this decreased Ca2+-ATPase activity may contribute to the pancreatic islets' glucose blindness.
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PMID:The effect of glucose and calcium on Ca2+-adenosine triphosphatase in pancreatic islets isolated from a normal and a non-insulin-dependent diabetes mellitus rat model. 947 68

The pathogenesis of tobacco-related periodontal diseases is not well understood. The purpose of this study was therefore to investigate smokeless tobacco extract (ST) and nicotine effects on prostaglandin E2 (PGE2) and interleukin-1beta (IL-1beta) secretion by peripheral blood mononuclear cells (PBMC, consisting of monocytes and lymphocytes) and gingival mononuclear cells (GMC). Both peripheral blood and gingival tissue adjacent to the alveolar crest were taken from non-smoking adult periodontitis patients. Gingival tissue was treated with collagenase and deoxyribonuclease and GMC and PBMC were isolated by Ficoll-Hypaque centrifugation. GMC and PBMC (100,000 cells/200 microl) were cultured for 24 hours in supplemented RPMI 1640 alone (control), or in supplemented RPMI 1640 containing 1% ST, 100 microg/ml nicotine, 1 microg/ml Porphyromonas gingivalis LPS, or 1 microg/ml P. gingivalis LPS and either 100 microg/ml nicotine or 1% ST. Enzyme immunoassays were used to quantify PGE2 and IL-1beta. Treatments were compared by repeated measures ANOVA. 100 microg/ml nicotine (7-fold, p<0.02) and 1% ST (3.5-fold, p<0.004) significantly increased secretion of PGE2 by PBMC relative to control cultures. 100 microg/ml nicotine and 1% ST, however, had no effect on IL-1beta secretion by PBMC. Enhanced PGE2 secretion also was seen when PBMC were treated with P. gingivalis LPS+ 100 microg/ml nicotine relative to P. gingivalis LPS alone (p<0.007). In contrast, 100 microg/ml nicotine significantly downregulated IL-1beta secretion by GMC relative to medium alone (p<0.008) and had no effect on PGE2 secretion by GMC. These data indicate that while nicotine and ST can stimulate PBMC to secrete PGE2, they cannot activate further mononuclear cells extracted from gingiva, possibly due to maximal previous stimulation in the periodontitis lesion.
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PMID:Nicotine and smokeless tobacco effects on gingival and peripheral blood mononuclear cells. 954 95

In this study, cultured glomerular epithelial cells (GEC) were exposed to a diabetic milieu containing glycated proteins to determine whether such proteins cause metabolic alterations that may lead to defects seen in the extracellular matrix in diabetic nephropathy. Cultured glomerular epithelial cells were cloned and maintained in RPMI media containing 10% fetal bovine serum (FBS). The medium was changed to RPMI-1% glycated FBS (experimental) or RPMI-1% control FBS, and cells were incubated for 1 or 4 d. Mitogenicity was tested by 3H-thymidine uptake. The media were collected and analyzed for collagenase activity by a quantitative fluorescence assay and by zymography. The cell layers were processed for matrix antigen (collagen I, glomerular basement membrane antigens, laminin, and fibronectin) and for the proteins of the tight junction (cadherin, desmosomal protein) by quantitative immunoperoxidase and immunofluorescence. Cell lysates were tested for cadherin and desmosomal protein by immunoblotting. Cells were also grown on 0.2-microM filter membranes to test for permeability to 3H-inulin and 125I-albumin. Glycated FBS resulted in a 1.8-fold increase in 3H-thymidine uptake in subconfluent layers accompanied by an increase in cell number. The treatment caused accumulation of laminin (18% above control, P < 0.05) and basement membrane antigens (33% above control, P < 0.05). Collagen I and fibronectin were unchanged. Exposing cells to glycated FBS changed the distribution of cadherin from a linear to a diffuse pattern associated with a decrease in cadherin observed on immunoblots. The media of glycated FBS-treated cells contained 45% lower collagenase activity (72-, 92-, and 150-kD species). Permeability to inulin increased by 550% and to albumin by 320% in glycated FBS-treated monolayers compared with controls. It is concluded that glycated proteins increased the accumulation of matrix proteins in the GEC, associated with a concomitant depression in collagenase activity. There were qualitative and quantitative changes in the tight junction protein cadherin. These matrix changes resulted in a functional defect in the permselective properties of the GEC tight junctions and manifested as increased leakage of inulin and albumin. Thus, the GEC are metabolically sensitive to the presence of glycated proteins, and this could play a role in the pathogenesis of diabetic nephropathy.
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PMID:Effect of glycated proteins on the matrix of glomerular epithelial cells. 959 77

The objective of this study was to identify the cells from rat prostate epithelium that attach and proliferate in primary culture. Minced ventral prostate was dissociated by DNAse/collagenase digestion, suspended in RPMI-1640 containing 10% fetal bovine serum, and subjected to Percoll centrifugation to separate the epithelial cells from stromal cells. With the use of lectins, it became possible to identify and monitor the fate of the dissociated epithelial cells held in suspension at 37 degrees C for several hours. In tissue sections of the rat prostate, Griffonia simplicifolia I-isolectin B4 (GSI-B4) specifically bound to basal cells, while Glycine maximus (soybean agglutinin [SBA]) was specific for secretory cells. Double staining with lectins and propidium iodide of dissociated cells revealed a preponderance of GSI-B4-positive live cells. The cells were plated in WAJC-404 medium supplemented with various factors, including insulin (5 ng/ml), transferrin (5 ng/ml), EGF (10 ng/ml), and bovine pituitary extract (30 microg/ml). Epithelial colonies that formed and proliferated from these cells also stained positively for GSI-B4 marker and for cytokeratins specific for basal cells as assessed by immunocytochemical staining. Proliferation was greater in cells grown on a collagen Type I matrix. These findings suggest that the epithelial cells that survived in suspension and proliferated in culture originated from basal cells of the rat prostate epithelium.
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PMID:The primary culture of rat prostate basal cells. 987 23

Cryopreservation is the only available technique for long-term storage of pancreatic islets. The freezing/thawing protocol may cause considerable loss of viable islet tissue and impair its function in vivo. The aim of this study was to investigate glucose and insulin levels after transplantation of fresh and cryo/thawed rat islets. Rat pancreatic islets were isolated following intraductal collagenase injection and Ficoll gradient purification. After isolation, islets were cultured for 24 h and then either transplanted or frozen after stepwise addition of DMSO according to Rajotte et al. and stored in liquid nitrogen. After rapid thawing islets were stepwise transferred into RPMI medium and cultured for another 24 h. The recipients were athymic mice with streptozotocine-induced diabetes. Two hundred fresh (n=13) or cryo/thawed (n=15) islets were transplanted beneath the renal capsule. Glucose levels were measured for 14 days and blood samples for insulin determination were obtained 15 min after i.p. glucagon (10 mg/kg) administration on day 14. Glucose levels were normalized (<9 mmol/l) in all recipients within 3 days since transplantation. On day 14, mean fasting values+/-SE in fresh and cryo/thawed islet groups were 4.0+/-0.6 and 4.4+/-0.4 mmol/l, respectively (P>0.05). Fasting insulin levels were higher in the cryo/thaw than in the fresh islet group (1.67+/-0.33 vs 0.57+/-0.13 ng/ml; P<0.01). Post-glucagon levels did not differ significantly (1.45+/-0.24 vs 0.86+/-0.24 ng/ml; P=0.06). While glucagon significantly increased insulin levels (P<0.01) in the fresh islet group, no change in insulin levels was observed (P>0.05) in the cryo/thaw group. Immunohistochemical staining demonstrated fragmentation of viable islet tissue which was more apparent in the cryo/thaw group. We conclude that in a short-term study cryo/thawed rat islets produce higher insulin levels than fresh islets transplanted into nude mice. This may be due to better islet survival or loss of feed-back regulation.
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PMID:Increased glucagon-stimulated insulin secretion of cryopreserved rat islets transplanted into nude mice. 993 Sep 40

Culturing of islets is essential for various purposes before transplantation. It is necessary to establish optimal culture conditions for each animal species for their preservation in culture. In this study, attempts were made to preserve the Indian bonnet monkey islets in culture. The islets were isolated from monkey pancreas by the collagenase digestion method. They were separated from acinar cells by dextran density-gradient centrifugation. They were preserved in a humidified atmosphere of 5% carbon dioxide and 95% air for 7 days. The culture medium used was RPMI-1640. Various optimal conditions such as volume of the culture medium used, number of islets in one culture dish, concentration of glucose in culture medium, and fetal calf serum percentage were tested for their better preservation in culture. After the culture period, they were tested for their insulin secretory capacity by exposing them to various secretagogues. Histologic appearance of the cultured islets also was examined. Both insulin secretory characteristics and histologic structure were found to be normal.
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PMID:Optimization of culture conditions for the preservation of monkey pancreatic islets in culture. 1041 97

It is well known that the epididymis is an excellent environment to maintain sperm viability. Therefore, we used different sections of bovine epididymis (caput, corpus, and cauda) to develop epithelial cell culture monolayers to identify factors that will increase sperm survival in the freezing-thawing process. Each epididymal section was dissected and treated with collagenase to obtain epithelial cell clusters. The cells were cultured in RPMI-1640 medium with 10% serum at 38.5 degrees C. A confluent monolayer was obtained after 5-7 days in culture and preliminary characterization using cytokeratin antibody indicated that the cell culture contained 85%-95% of epithelial cells. These cellular cultures were tested for their ability to maintain motility of epididymal and frozen-thawed spermatozoa. Washed spermatozoa were added to obtain a final dilution of 1 x 10(6) spermatozoa/mL. The motility of frozen-thawed spermatozoa was also recorded after incubation in conditioned media. Our results show that cocultures of spermatozoa and epididymal cell monolayers for 24 and 48 hours were beneficial for maintaining epididymal and frozen-thawed sperm motility (36.0% and 20.4%) compared with spermatozoa cultured with fibroblast cells or in the absence of a cell monolayer (0%; P < .01). The conditioned medium provides favorable conditions for sperm motility. Results with conditioned medium on maintenance of frozen-thawed sperm motility suggest that epididymal cells in vitro secrete beneficial factors that prolong the sperm survival.
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PMID:Epididymal epithelial cells cultured in vitro prolong the motility of bovine sperm. 1110 10


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