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 sensitive lactate dehydrogenase (LDH) assay was modified to determine the cytolytic activity of Bacillus thuringiensis CryIC and CryIAc delta endotoxins to viable collagenase-dissociated midgut epithelial cells (MEC) from larvae of Spodoptera frugiperda and Spodoptera exigua. The MEC preparations from these Spodoptera sp. consisted predominantly of columnar cells (65-75%) and goblet cells (25-35%). Time course microscopy experiments indicated that only the columnar cells became swollen during CryIC toxin incubation. Also, comparative cytotoxicity studies were run with cell lines of nonmidgut origin established from S. frugiperda (SF21AE) and S. exigua (SEUCR1A). Optimum conditions for the cytotoxicity assay were similar for MEC and cell lines of both species, and were met in an assay in which 0.1-ml cell concentrations (8.5 +/- 0.5 x 10(4) cells) were incubated with toxin dilutions (0.01-20 micrograms) for 1 h at 24 degrees C at a final pH of 7.8. The Spodoptera sp. MEC were twofold more sensitive to CryIC (68% lysis) than CryIAc (32% lysis) at optimum toxin levels (2.5-5 micrograms). Also, the SEUCR1A cells were more sensitive (2.3-fold) to CryIC (70% lysis) than CryIAc (30% lysis) at optimum toxin levels of 5-10 micrograms. The SF21AE cells, however, were twofold less sensitive to CryIC (30% lysis) than SEUCR1A cells and response to CryIAc and CryIC was similar. Immunoblot analysis of either Spodoptera sp. MEC or brush border membrane vesicles (BBMV) identified seven CryIC binding proteins with molecular mass of 137, 120, 115, 68, 65, 63, and 45 kDa. Occasionally, a 148-kDa protein band was observed. The CryIAc toxin bound to two proteins on MEC and BBMV with molecular mass of 137 and 120 kDa.
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PMID:Cytolytic activity of Bacillus thuringiensis CryIC and CryIAc toxins to Spodoptera sp. midgut epithelial cells in vitro. 915 49

To investigate the hormonal control of the expression of flavin-containing monooxygenase (FMO; EC 1.14.13.8) under defined in vitro conditions, adult male rat hepatocytes were isolated by collagenase perfusion and co-cultured with rat liver epithelial cells of primitive biliary origin. The direct effect of 17beta-estradiol, testosterone, 5alpha-dihydrotestosterone (5alpha-DHT) and human growth hormone (hGH) on FMO activity was studied using this in vitro model. Optimal, non-cytotoxic hormonal concentrations were determined by measuring the lactate dehydrogenase (LDH) index. In addition, the microsomal protein content of the cultured hepatocytes was determined as a function of culture time. The female sex hormone 17beta-estradiol caused a significant decrease in FMO as a function of culture time. After 14 days of exposure, FMO activity decreased by 56%. Neither of the male sex hormones or human growth hormone had an effect on FMO activity. These results in co-cultured male rat hepatocytes support in vivo observation that 17beta-estradiol is a potent hormone involved in the negative regulation of the expression of FMO in male rat liver.
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PMID:Hormonal regulation of microsomal flavin-containing monooxygenase activity by sex steroids and growth hormone in co-cultured adult male rat hepatocytes. 977 17

It was reported that free fatty acids degraded from triglycerides by lipase may play a major role in acute necrotizing or hyperlipidemia-induced pancreatitis. We hypothesized that this injury may be related to the peroxidation of cell membrane phospholipids and tested this hypothesis using isolated pancreatic acini. Pancreatic acini were prepared from male Sprague-Dawley rats by collagenase digestion. Linoleic acid was added (0.1-1.0 mM) to the acinar cell suspension to induce cell injury. Acinar cell damage was measured by lactate dehydrogenase release and by trypan blue exclusion. Phosphatidylcholine hydroperoxide and alpha-tocopherol in the acinar cells were measured. Protective effects of alpha-tocopherol (0.5, 5.0 mM) against this type of cell injury were also evaluated. When isolated acinar cells were treated with linoleic acid, a significant decrease in viability was observed in a time- and dose-dependent manner. In addition, the levels of phosphatidylcholine hydroperoxide after treatment of 0.5 mM of linoleic acid were increased and levels of alpha-tocopherol were decreased significantly. alpha-Tocopherol significantly ameliorated both cellular injury (p < 0.01) and increases in phosphatidylcholine hydroperoxide (p < 0.01). These data suggest that lipid peroxidation of the cellular membrane is an important component of the pancreatic cell injury mediated by free fatty acids.
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PMID:Involvement of lipid peroxidation in free fatty acid-induced isolated rat pancreatic acinar cell injury. 982 Nov 80

The study investigates the influence of different culture conditions on attachment, viability and functional status of rainbow trout (Oncorhynchus mykiss) liver cells in primary culture. Cells were isolated by a two-step collagenase perfusion and incubated in serum-free, chemically defined minimal essential medium (MEM), (a) as a monolayer on uncoated PRIMARIA dishes, (b) as a monolayer on culture dishes coated with calf collagen type 1, and (c) in coculture with the established fish cell lines RTH-149 or RTG-2. Cell attachment was assessed from DNA and protein concentrations per dish, viability was estimated from cellular lactate dehydrogenase release, and the metabolic status was investigated by measuring activities of the phosphoenolpyruvate carboxykinase and biotransformation enzymes as well as the total cytochrome P450 contents. Seeding of hepatocytes on collagen-coated dishes did not alter cell attachment or detachment from the (culture substrate, but had a small, but not significant effect on cell viability and metabolic parameters. Coculture of liver cells and RTG-2 cells reduced hepatocyte detachment from the culture substrate, and it was associated with a significant elevation of 7-ethoxyresorufin-O-deethylase activities in the hepatic cells. Cytochrome P450 contents, however, were not altered. The coculture effect on liver cell physiology clearly depended on the type of cell line, because coculture with RTH-149 cells led to similar, but much weaker effects than obtained in cocultures with RTG-2 cells. Electron microscopical observations revealed the existence of gap junctions and possible exocytosis-like transport between cell lines and hepatocytes. The results point to the potential of coculture systems to improve physiological parameters of trout liver cells in primary culture.
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PMID:Viability and differential function of rainbow trout liver cells in primary culture: coculture with two permanent fish cells. 987 May 25

The purpose of this study was to determine if exacerbation of apoptosis precedes liver injury during chronic exposure of rats to alcohol. After 7 weeks of feeding an alcohol- or dextrin-containing liquid diet, the animals were treated with gram-negative bacterial lipopolysaccharide (1 mg x kg(-1) body weight, intravenously) or sterile saline and sacrificed 3 hr after the treatment. Alanine:2-oxoglutarate aminotransferase (ALT) and lactate:NAD oxidoreductase [lactate dehydrogenase (LDH)] were measured in plasma. The caudate lobe of the liver was resected for histology, while the rest of the organ was perfused with collagenase to isolate hepatocytes, Kupffer cells (KCs), and sinusoidal endothelial cells (SECs) by centrifugal elutriation. Hepatocyte mitochondria were isolated by differential centrifugation of the cell homogenate. Reduced and oxidized glutathione (GSH and GSSG) in isolated hepatocytes and hepatocyte mitochondria, and malondialdehyde in hepatocytes were assayed. Caspase-3 activity and Fas ligand mRNA expression were determined in hepatocytes, KCs, and SECs. Plasma ALT and LDH activity, liver histology, GSH, GSSG and their ratio, and malondialdehyde content were not affected by alcohol treatment Caspase-3 activity was significantly increased in alcohol-treated rats in all three cell types, with the lowest response observed in hepatocytes and the highest in KCs. Fas ligand mRNA expression, which had the highest level in SECs, followed by KCs and hepatocytes, was not affected by alcohol administration. Lipopolysaccharide had the following effects: an increase in ALT in both pair- and alcohol-fed rats, and LDH only in alcohol-fed rats, a decrease in GSH + GSSG levels in both mitochondria and hepatocytes, an elevation of malondialdehyde content in hepatocytes, a raise in caspase-3 activity in all groups and cell types, and an augmentation of Fas ligand expression in hepatocytes and KCs, but not in SECs. These data suggest that, during chronic alcohol consumption, an exacerbated apoptosis precedes alcohol-induced liver injury.
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PMID:Modulation of caspase-3 activity and Fas ligand mRNA expression in rat liver cells in vivo by alcohol and lipopolysaccharide. 1006 67

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

Glibenclamide is a potent inhibitor of the ATP-dependent potassium channel. Opening of the ATP-dependent potassium channel is regarded as a mechanism of ischemic preconditioning. This in vitro study examines the influence of glibenclamide and glimepiride, a new sulfonylurea, on the negative inotropic action of the potassium channel opener rilmakalim in isolated ventricular myocytes. Cardiac myocytes were isolated from adult guinea pig hearts by collagenase perfusion and incubated with rilmakalim (concentration range 0.1-12.0 microM), glibenclamide (concentration range 0.03-3.0 microM) plus rilmakalim (3.0 or 7.5 microM), and glimepiride (0.03-9.0 microM) plus rilmakalim (3.0 or 7.5 microM) and paced by electrical field stimulation. Contractility of the myocytes was evaluated by digital image analysis, intracellular free calcium was determined by means of fura-2 fluorescence measurements, and cell viability was assessed morphologically as well as by measurement of lactate dehydrogenase activity. Rilmakalim reduced the systolic intracellular free calcium and contractility of ventricular myocytes in a concentration dependent manner. This effect was antagonized by glibenclamide at lower concentrations (0.3 microM) than glimepiride (3.0 microM). The smaller antagonistic action of glimepiride on the negative inotropic effect of rilmakalim as compared with glibenclamide most likely reflects a less potent inhibition of ATP-dependent potassium channels by glimepiride.
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PMID:Glimepiride (Hoe490) inhibits the rilmakalim induced decrease in intracellular free calcium and contraction of isolated heart muscle cells from guinea pigs to a lesser extent than glibenclamide. 1063 33

It has been suggested that calcium (Ca(2+)) overload and oxidative stress damage the myocardium during ischemia and reperfusion. We investigated the possible effect of varying extracellular Ca(2+)and total cell Ca(2+)on reactive oxygen species (ROS) levels in resting adult rat cardiomyocytes. Cardiomyocytes were isolated by trypsin/collagenase digestion and exposed to 1 h of hypoxia (H) (95% N(2)/5% CO(2), no glucose) and 2 h of reoxygenation (R) (95% air/5% CO(2), glucose 5.5 m M) in suspension. Cell Ca(2+)was measured by uptake of(45)Ca(2+). ROS was measured by flow cytometry of ethidium's red fluorescence formed by oxidation of dihydroethidium mostly by superoxide anion. Cell viability decreased during H and R, expressed as uptake of trypan blue, loss of rod shape morphology and release of lactate dehydrogenase. Rapidly exchangeable cell Ca(2+)was closely correlated with extracellular Ca(2+)concentration. Cell Ca(2+)was unchanged during H but increased three to four times after R. This increase was attenuated by adding 3,4-dichlorobenzamil, 10 microm at R, and amplified by adding ouabain 1 m M (from start), respectively. Levels of ROS in hypoxic cells were unchanged or slightly reduced at the end of H and increased significantly by 20% compared to control after R. Levels of ROS were significantly decreased by lowering total extracellular Ca(2+)from 1 m M to 0.1 m M or by decreasing free extracellular Ca(2+)with EGTA 0.9 m M at the onset of R. Keeping extracellular Ca(2+)constant, ROS levels were neither affected by attenuating the increase in cell Ca(2+)by DCB nor by amplifying the increase in cell Ca(2+)by ouabain. In conclusion, ROS (superoxide anion) levels increase rapidly after reoxygenation, are correlated with extracellular-free Ca(2+)and are reduced by lowering extracellular-free Ca(2+). Levels of ROS are apparently not consistently correlated with total cell Ca(2+).
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PMID:Effect of calcium on reactive oxygen species in isolated rat cardiomyocytes during hypoxia and reoxygenation. 1073 43

Using 4-month-old fetal bovine tissue, the properties of the tibia epiphyseal cartilage matrix vesicles, a type of endochondral ossification tissue, were compared with those from tracheal cartilage. The matrix vesicle fractions, obtained by collagenase digestion and differential centrifugation, were subjected to sucrose-density-gradient centrifugation. Alkaline phosphatase activity, protease activity, and lacatate dehydrogenase activity were assayed for the marker enzyme of the matrix vesicles. Matrix vesicles containing alkaline phosphatase, metalloprotease, and lacatate dehydrogenase were found in the tibia epiphyseal cartilage at a density of 1.11 g/ml. In surprising contrast, we also found matrix vesicle-like vesicles with a high density of 1.24 g/ml in the tracheal cartilage. These also contained alkaline phosphatase and lactate dehydrogenase, but not metalloprotease. The electrophoretic profiles of the lactate dehydrogenase isoenzymes from the matrix vesicle and matrix vesicle-like vesicles were identical with those of chondrocyte cytosolic lactate dehydrogenase. Aldolase, aspartate: 2-oxoglutarate aminotransferase, alanine: 2-oxoglutarate aminotransferase, glucose-6-phosphatase, glutamate dehydrogenase, catalase, and cytosolic enzymes except for lactate dehydrogenase were not detected in these vesicles. These results suggest the presence of a mechanism for specific uptake of cytosolic lactate dehydrogenase in both vesicles. In this study, a new type of matrix vesicles without protease was found in the tracheal cartilage, a kind of permanent cartilage, but not in the tibia epiphyseal cartilage, which is replaced by bone tissue.
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PMID:A new type of matrix vesicles is found in fetal bovine tracheal cartilage. 1099 57

Chicken liver is lack of ascorbic acid biosynthesis system, different from mammals and highly evoluted birds. Chicken hepatocytes cultured without ascorbate was expected to have lower ascorbate amounts than physiological levels. Intracellular was decreased as compared with intact liver by cell preparation performed with in situ collagenase perfusion. We added ascorbate to a primary culture of chicken hepatocytes in order to restore the amount of ascorbate. Serum-free Leivobitz's L-15 medium which do not contain ascorbate was used for control medium. Cells were cultured with several concentrations of ascorbate for 24 or 48 h. After ascorbate supplementation for 24 to 48 h, cellular ascorbate concentration increased depending on the dose of medium ascorbate. Medium lactate dehydrogenase activity derived from hepatocytes, an index of cell injury, decreased upon 5-100 mg/l of ascorbate supplementation for 48 h. Tyrosine aminotransferase activity, an index of liver function, increased following culture with 50 and 100 mg/l ascorbate for 48 h. The activities, however, decreased by supplementation with 1000 mg/l of ascorbate. In conclusion hepatocytes lost intracellular ascorbate during preparation by in situ collagenase perfusion. Supplementation of ascorbate restored cellular ascorbate concentration, lowered cell injury and raised tyrosine aminotransferase activitv in primary cultured chicken hepatocytes. Ascorbate treatment for 48 h at 50 mg/l was the best combination in this study for primary culture of chicken hepatpcyte with non-serum L-15 medium
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PMID:Ascorbic acid supplementation to primary culture of chicken hepatocytes with non-serum medium. 1108 76


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