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 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

Periportal and perivenous hepatocytes were isolated by the digitonin-collagenase perfusion technique. The activity of the cytosolic glutathione S-transferase was higher in perivenous cells, but the cytosolic glutathione reductase and the microsomal glutathione S-transferase activities were evenly distributed. In contrast, both the Se-dependent and the microsomal Se-independent glutathione peroxidase activity and the glucose-6-phosphate dehydrogenase activity was much lower in perivenous hepatocytes, suggesting that these cells have a lowered detoxification capacity, which may contribute to their greater susceptibility to damage by xenobiotics. The mechanism of the ethanol-induced GSH depletion in vivo was studied by incubating conventionally isolated hepatocytes. In the absence of glutathione precursors, ethanol (80 mM) did not influence the GSH content, despite accumulation of acetaldehyde (10-100 MicroM). L-Methionine or L-cysteine stimulated GSH replenishment to in vivo rates. Ethanol oxidation resulted in acetaldehyde accumulation, but did not inhibit GSH replenishment from L-methionine and even stimulated that from L-cysteine. This seems to exclude conjugation of GSH with acetaldehyde as a mechanism by which ethanol suppresses GSH levels in vivo.
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PMID:Glutathione metabolism in isolated rat hepatocytes: acinar heterogeneity of detoxifying enzymes and effects of ethanol. 342 86

The acinar distribution of glutathione S-transferase (GST), glutathione peroxidase (GPx), glutathione reductase (GR), and glucose-6-phosphate dehydrogenase (G-6-PDH) was examined by analyzing periportal (p.p.) and perivenous (p.v.) rat hepatocytes selectively isolated by the digitonin-collagenase perfusion. The cytosolic GST activity was higher in p.v. cells, but the microsomal GST and cytosolic GR were found to be evenly distributed in the acinus. In contrast, the activity of both the Se-dependent GPx and the microsomal (Se-independent) GPx, as well as G-6-PDH, was much lower in the p.v. than in the p.p. cells. The heterogeneous distribution of GST, GPx and G-6-PDH was confirmed by analyzing liver perfusion effluents collected after ante- or retrograde digitonin infusion. The relatively low activities of GPx and G-6-PDH in the p.v. cells could partly explain the susceptibility of this region to chemical injury.
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PMID:Acinar distribution of glutathione-dependent detoxifying enzymes. Low glutathione peroxidase activity in perivenous hepatocytes. 359 5

1. A method is described for extracting separately mitochondrial and extramitochondrial enzymes from fat-cells prepared by collagenase digestion from rat epididymal fat-pads. The following distribution of enzymes has been observed (with the total activities of the enzymes as units/mg of fat-cell DNA at 25 degrees C given in parenthesis). Exclusively mitochondrial enzymes: glutamate dehydrogenase (1.8), NAD-isocitrate dehydrogenase (0.5), citrate synthase (5.2), pyruvate carboxylase (3.0); exclusively extramitochondrial enzymes: glucose 6-phosphate dehydrogenase (5.8), 6-phosphogluconate dehydrogenase (5.2), NADP-malate dehydrogenase (11.0), ATP-citrate lyase (5.1); enzymes present in both mitochondrial and extramitochondrial compartments: NADP-isocitrate dehydrogenase (3.7), NAD-malate dehydrogenase (330), aconitate hydratase (1.1), carnitine acetyltransferase (0.4), acetyl-CoA synthetase (1.0), aspartate aminotransferase (1.7), alanine aminotransferase (6.1). The mean DNA content of eight preparations of fat-cells was 109mug/g dry weight of cells. 2. Mitochondria showing respiratory control ratios of 3-6 with pyruvate, about 3 with succinate and P/O ratios of approaching 3 and 2 respectively have been isolated from fat-cells. From studies of rates of oxygen uptake and of swelling in iso-osmotic solutions of ammonium salts, it is concluded that fat-cell mitochondria are permeable to the monocarboxylic acids, pyruvate and acetate; that in the presence of phosphate they are permeable to malate and succinate and to a lesser extent oxaloacetate but not fumarate; and that in the presence of both malate and phosphate they are permeable to citrate, isocitrate and 2-oxoglutarate. In addition, isolated fat-cell mitochondria have been found to oxidize acetyl l-carnitine and, slowly, l-glycerol 3-phosphate. 3. It is concluded that the major means of transport of acetyl units into the cytoplasm for fatty acid synthesis is as citrate. Extensive transport as glutamate, 2-oxoglutarate and isocitrate, as acetate and as acetyl l-carnitine appears to be ruled out by the low activities of mitochondrial aconitate hydratase, mitochondrial acetyl-CoA hydrolyase and carnitine acetyltransferase respectively. Pathways whereby oxaloacetate generated in the cytoplasm during fatty acid synthesis by ATP-citrate lyase may be returned to mitochondria for further citrate synthesis are discussed. 4. It is also concluded that fat-cells contain pathways that will allow the excess of reducing power formed in the cytoplasm when adipose tissue is incubated in glucose and insulin to be transferred to mitochondria as l-glycerol 3-phosphate or malate. When adipose tissue is incubated in pyruvate alone, reducing power for fatty acid, l-glycerol 3-phosphate and lactate formation may be transferred to the cytoplasm as citrate and malate.
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PMID:The intracellular localization of enzymes in white-adipose-tissue fat-cells and permeability properties of fat-cell mitochondria. Transfer of acetyl units and reducing power between mitochondria and cytoplasm. 439 82

Myoepithelial and secretory cells from the mammary gland of the lactating rat have been isolated, purified, and characterized. Mammary tissue was dissociated with collagenase into basket-like networks of myoepithelial cells and single secretory cells. Because of their larger size, the myoepithelial cell networks could be separated from other mammary and blood cells by differential centrifugation. Isolated secretory cells were purified by isopycnic centrifugation in 25% bovine serum albumin. The purified myoepithelial and secretory cells were viable, as shown by the incorporation of 32P into distinct macromolecules that were separable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both myoepithelial and secretory cells retained their characteristic morphology after isolation and purification, as shown by light, transmission, and scanning electron microscopies. The isolated myoepithelial cells were unique and, thus, distinguishable from other mammary cells in a number of respects; they 1) contracted in response to the addition of oxytocin, 2) bound [3H]oxytocin specifically, 3) accounted for the content of alkaline phosphatase and [Na+ + K+]ATPase in mammary tissue, and 4) reacted specifically with antiserum prepared against purified myoepithelial cells. The purified secretory cells were unique in possessing glucose-6-phosphate dehydrogenase activity. The different cell markers not only gave independent estimates of the purity of the cell fractions, but they also may be helpful in identifying mammary cells in stages of differentiation and neoplastic transformation.
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PMID:Purification and characterization of mammary myoepithelial and secretory cells from the lactating rat. 624 56

The mucosal surface of the turtle bladder consists of two major cell types, carbonic anhydrase-rich (CA) and granular (G) cells. These cells types may have specific transport functions. To separate and study these cells, they were removed from the bladder by collagenase digestion and then separated into two distant subpopulations by Ficoll discontinuous density-gradient centrifugation. By morphological criteria the lighter subpopulation (band II) consists primarily of CA cells and the denser population (band III) G cells. The distribution of two enzymes important for H+ transport, CA and glucose-6-phosphate dehydrogenase (G-6-PD), and the effect of specific transport inhibitors, acetazolamide and ouabain, on O2 consumption of these cells was examined. The CA activity of band II cells was 4.5 times greater than that of band III cells. The G-6-PD activity of band II cells was 2.2 times greater than that of band III cells. The rate of O2 consumption of band II cells, 4.73 microliters.mg protein-1.h-1, was reduced 40% by 5 X 10(-5) M acetazolamide but not by ouabain. Band III O2 consumption, 3.43 microliters.mg protein-1.h-1, was reduced 30% by 10(-4) M ouabain but not by acetazolamide. On the basis of the distribution of CA and G-6-PD and the effect of acetazolamide and ouabain on O2 consumption, we propose that CA cells are primarily responsible for H+ transport and G cells for Na+ transport.
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PMID:Specialized function of carbonic anhydrase-rich and granular cells of turtle bladder. 628

The primary objective of this study was to provide evidence for preadipocyte proliferation during culture of adipose tissue explants; a secondary objective was to compare the lipogenic activity and cellularity of adipose tissues from American Wagyu crossbred steers. Subcutaneous (s.c.) and intramuscular (i.m.) adipose tissues were obtained at slaughter from the 2nd to 6th lumbar region of the loin from Angus (n = 10) and Wagyu crossbred steers (n = 10) that had been fed for 552 d by typical Japanese production standards. Adipose tissue explants were incubated 36 h with [3H]thymidine in the absence and presence of aphidicolin (a specific inhibitor of genomic DNA replication). Adipocytes were liberated by collagenase treatment and [3H]thymidine incorporation into DNA was measured. Whereas there were no significant differences between adipose tissue depots, Wagyu s.c. and i.m. preadipocytes and stromal-vascular cells exhibited greater (P < .05) [3H]thymidine incorporation into DNA than adipocytes from Angus steers. Intramuscular adipose tissue from both breeds exhibited lower (P < .05) rates of lipogenesis from acetate both before and after long-term (36-h) incubation than s.c. adipose tissue. Furthermore, i.m. adipocytes were smaller (P < .05) than s.c. adipocytes. The activities of fatty acid synthetase and glucose-6-phosphate dehydrogenase were greater (P < .05) in Wagyu s.c. adipose tissue and less in Wagyu i.m. adipose tissue than in corresponding Angus tissues. There were no differences between breed types (P = .17) in rates of lipogenesis from acetate, either before or after explant culture.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Evidence for preadipocyte proliferation during culture of subcutaneous and intramuscular adipose tissues from Angus and Wagyu crossbred steers. 775 59

The study aimed to assess the effect of lipopolysaccharide (LPS) in vivo (from Escherichia coli, 2 mg/kg body weight intraperitoneally) on the production and elimination of hydrogen peroxide (H2O2) in rat hepatic endothelial and Kupffer cells. Twenty-two hours after the injection of LPS, hepatic cells were isolated by collagenase and pronase digestion followed by centrifugal elutriation, and cell-associated H2O2 was determined by flow cytometry analysis using 2',7'-dichloroflorescin diacetate (DCF-diacetate). LPS treatment did not alter the basal or phorbol myristate acetate-stimulated levels of H2O2-related fluorescence in endothelial cells; however, it doubled phorbol myristate acetate-stimulated fluorescence in Kupffer cells. Administration of varying concentrations of H202 (range, 10(-7) - 10(-4) mol/L) in vitro caused a significantly delayed increase in fluorescence in endothelial cells from endotoxemic rats as compared with cells from saline-injected animals. The 50% effective concentration of H202 was found at 1.1 x 10(-6) and 8.1 x 10(-6) mol/L on endothelial cells after saline and LPS treatment, respectively. No differences were detected in H2O2-stimulated fluorescence between resting and LPS-stimulated Kupffer cells. Administration of varying glucose concentrations in vitro significantly decreased the H2O2-stimulated fluorescence in endothelial and Kupffer cells from LPS-injected animals. Inhibition of nitric oxide synthase by in vitro administration of NG-monomethyl-L-arginine (L-NNMMA) did not alter the H2O2- or phorbol myristate acetate-stimulated responses in endothelial and Kupffer cells. As shown earlier, LPS stimulates the gene expression of GLUT1 glucose transporter, glucose-6-phosphate dehydrogenase (G6PD), superoxide dismutases, and glutathione peroxidase in hepatic endothelial cells. The present data indicate that the LPS-induced metabolic alterations are accompanied by an increased H2O2-detoxifying capacity in hepatic endothelial cells. This may represent a protective mechanism against exogenous oxidative stress caused by activated hepatic phagocytes during inflammation. Our observations are consistent with primed production of reactive oxygen species (ROS) in LPS-activated Kupffer cells.
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PMID:Endotoxin stimulates hydrogen peroxide detoxifying activity in rat hepatic endothelial cells. 878 44

An alternative method has been developed for isolating and culturing hepatocytes from livers of channel catfish. Hepatocytes are prepared using a collagenase-free perfusion system that relies on the chelating properties of ethylenediamine tetraacetic acid (EDTA). Hepatocyte yields of up to 3.6 x 10(8) cells per 100 g body weight have been achieved with initial viabilities routinely exceeding 95%. Cells isolated by this method and incubated in osmotically corrected culture medium at physiological pH have been maintained for several weeks in culture with minimal cell loss. During the first 24-48 h of culture, hepatocytes begin to link together and show structures that closely resemble those seen in intact liver (e.g. bile canaliculi, sinusoids). Cells cultured at 15 degrees C for 7 days maintain levels of glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), and lactate dehydrogenase (LDH), activity similar to those measured in vivo.
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PMID:Non-enzymatic isolation and culture of channel catfish hepatocytes. 1042 27

Oxidative stress has been implicated in the pathophysiology of myocardial failure. We tested the hypothesis that oxidative stress can regulate extracellular matrix in cardiac fibroblasts. Neonatal and adult rat cardiac fibroblasts in vitro were exposed to H(2)O(2) (0.05-5 microM) or the superoxide-generating system xanthine (500 microM) plus xanthine oxidase (0.001-0.1 mU/ml) (XXO) for 24 h. In-gel zymography demonstrated that H(2)O(2) and XXO each increased gelatinase activity corresponding to matrix metalloproteinases (MMP) MMP-13, MMP-2, and MMP-9. H(2)O(2) and XXO decreased collagen synthesis (collagenase-sensitive [(3)H]proline incorporation) without affecting total protein synthesis ([(3)H]leucine incorporation). H(2)O(2) and XXO decreased the expression of procollagen alpha(1)(I), alpha(2)(I), and alpha(1)(III) mRNA but increased the expression of fibronectin mRNA, suggesting a selective transcriptional effect on collagen synthesis. H(2)O(2), but not XXO, also decreased the expression of nonfibrillar procollagen alpha(1)(IV) and alpha(2)(IV) mRNA. To determine the role of endogenous antioxidant systems, cells were treated with the superoxide dismutase (SOD) inhibitor diethyldithiocarbamic acid (DDC, 100 microM) to increase intracellular superoxide or with the glucose-6-phosphate dehydrogenase inhibitor dehydroisoandrosterone 3-acetate (DHEA; 10 microM) to increase intracellular H(2)O(2). DDC and DHEA decreased collagen synthesis and increased MMP activity, and both effects were inhibited by an SOD/catalase mimetic. Thus increased oxidative stress activates MMPs and decreases fibrillar collagen synthesis in cardiac fibroblasts. Oxidative stress may play a role in the pathogenesis of myocardial remodeling by regulating the quantity and quality of extracellular matrix.
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PMID:Oxidative stress regulates collagen synthesis and matrix metalloproteinase activity in cardiac fibroblasts. 1112 76


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