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)

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

A method for measurement of glutamate dehydrogenase (GDH) activity in single renal tubules was employed to determine the distribution and regulation of GDH in tubule segments. Fresh microdissected tubules from collagenase-treated kidneys were permeabilized by hyposmotic shock and freezing. The rate of conversion of alpha-ketoglutarate, NH4+, and NADH to glutamate and NAD was measured at 37 degrees C fluorometrically. Very high activities were found in proximal tubule segments (150-210 pmol.min-1.mm tubule length-1), intermediate values (40-90 pmol.min-1.mm-1) in distal convoluted tubules, cortical thick ascending limbs, connecting tubules, medullary thick ascending limbs, and lower values (5-30 pmol.min-1.mm-1) in cortical collecting ducts, inner medullary collecting ducts, outer medullary collecting ducts, outer medullary thin limbs, and inner medullary thin limbs. To determine the effects of acid-base loading on GDH activity, 0.28 M NH4Cl (acid) or 0.28 M NaHCO3 (alkali) was added to the animals' drinking water for 7 days. Acid intake by the rats increased GDH activity in S1 and S2 proximal tubules by threefold, with no effect in other segments, including S3 proximal tubules. Alkali intake decreased GDH activity in the S3 proximal tubule by 40%, with no effect in other segments. We conclude that GDH activities are highest in proximal tubule segments and are regulated only in proximal tubule segments. Thus the results are consistent with the view that the proximal tubule is the chief site of the regulated production of ammonium in the kidney.
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PMID:Glutamate dehydrogenase activities in microdissected rat nephron segments: effects of acid-base loading. 237 92

A method is described whereby short fragments of rat kidney tubule were obtained when kidney slices were gently dispersed by exposure to collagenase and hyaluronidase. When suspended in buffered saline the fragmented tubules respired actively over a period of several hours, the rate of oxygen consumption being proportional to the amount of cell protein. Oxygen uptake was stimulated by the addition of glucose, lactate, butyrate, alpha-oxoglutarate and other substrates and was decreased by the omission of Ca(2+) from the suspending medium. With alpha-oxoglutarate as the added substrate, dinitrophenol strongly stimulated oxygen uptake. Dinitrophenol had a less-marked stimulatory effect when glucose was the added substrate, and inhibited respiration in the absence of added substrate. Oligomycin inhibited respiration and this inhibition was partially reversed by dinitrophenol. Fragmented tubules synthesized glucose from lactate at a high rate but this capacity for gluconeogenesis was abolished by dinitrophenol and by physically damaging the cells.
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PMID:Preparation and some properties of a suspension of fragmented tubules from rat kidney. 435 29

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

1. Tubule fragments were isolated by collagenase treatment of guinea pig kidney cortex. 2. 3':5'-Cyclic AMP increased gluconeogenesis from lactate, pyruvate, malate and fructose. 3. Noradrenaline decreased gluconeogenesis from lactate, pyruvate, 2-oxoglutarate and fructose. 4. Angiotensin II slightly, but significantly, increased gluconeogenesis from lactate. 5. Gluconeogenesis from glycerol as sole substrate was negligible. Gluconeogenesis from combinations of glycerol together with either lactate, pyruvate, 2-oxoglutarate or malate was appreciably greater than the sum of the glucose output observed when these substrates were added singly.
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PMID:Gluconeogenesis in guinea pig renal tubule fragments--effects of noradrenaline, 3':5' cyclic AMP and angiotensin II. 613 91

Primary cultures of liver cells isolated from adult rats by trypsin and collagenase perfusion techniques were carried out to compare cytologic and biochemical properties between the differently prepared cells. Trypsin-dispersed cells consisted of comparatively smaller cells, whereas collagenase-dispersed cells consisted of larger cells. The cell attachment efficiency on culture day 1 was about twice as high in the liver cells prepared with collagenase than those prepared with trypsin. Mature hepatocytes isolated by collagenase perfusion could be maintained in the primary culture for a longer period than those isolated by trypsin perfusion. Epithelial-like clear cells started to grow much earlier in the primary culture of the trypsin-dispersed liver cells than in that of the collagenase-dispersed liver cells. Earlier proliferation of epithelial-like clear cells could not be induced by in vitro trypsinization of the collagenase-dispersed liver cells. Both kinds of enzymatically prepared liver cells showed albumin production and exhibited glucose 6-phosphatase (D-glucose-6-phosphate phosphohydrolase, EC 3.1.3.9, G6Pase) and tyrosine aminotransferase (L-tyrosine: 2-oxoglutarate amino-transferase, EC 2.6.1.5, TAT) activities for 1 week in the primary culture. Albumin production was higher in the liver cells prepared with collagenase than those prepared with trypsin, whereas G6Pase activity was almost the same between them. TAT activity up to culture day 2 was about 3-fold higher in the liver cells prepared with collagenase than in those prepared with trypsin. Combined supplementation of dexamethasone (1 X 10(-5)M) and insulin (10 micrograms/ml) consistently improved the cell attachment efficiency and was very effective in the maintenance of mature hepatocytes in both types. Furthermore, these hormones enhanced the albumin production and TAT activity in both types.
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PMID:Comparison of cytologic and biochemical properties between liver cells isolated from adult rats by trypsin perfusion and those isolated by collagenase perfusion. 614 85

Substrate oxidation was assessed by measuring 14CO2 production from 14C-labeled substrates in proximal convoluted tubules (PCT), medullary (MTAL), and cortical (CTAL) thick ascending limb of Henle, nephron segments rich in mitochondria and characterized by active solute transport. PCT, MTAL, and CTAL were dissected from the outer cortex, outer medulla, and the medullary rays of the cortex, respectively, of collagenase-treated rat kidney slices. Tubules were incubated at 37 degrees C in 150 microliters of Krebs-Ringer-bicarbonate buffer (pH, 7.4) with 14C-labeled substrate. 14CO2 production was linear up to 4 and 2 hours in PCT and MTAL, respectively. Freeze-thawing of the tubules markedly decreased 14CO2 production, and the addition of cyanide completely abolished it. The PCT demonstrated marked 14CO2 production from labeled succinate, 2-oxoglutarate, glutamate, glutamine, and malate (approximately 10 to 45 pmoles/mm/hr) and moderate 14CO/ production from citrate (approximately 3 pmoles/ml/hr). Little 14CO2 was released from labeled glucose and lactate in PCT. These results are consistent with the existence of gluconeogenesis in this nephron segment. By contrast, MTAL and CTAL oxidized glucose, 2-oxoglutarate, lactate, glutamate, and glutamine, but not malate, succinate, and citrate. The pentose shunt pathway accounted for approximately half of the 14CO2 produced from 1-14C glucose in MTAL and CTAL. Palmitate oxidation occurred in MTAL and CTAL but minimally in PCT. The results demonstrate a distinct pattern of substrate oxidation in PCT, MTAL, and CTAL where oxidative metabolism is critical to support active solute transport.
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PMID:Substrate oxidation by isolated single nephron segments of the rat. 730 Jan 10

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

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

Background/aims: The liver apoptotic response to chronic alcohol consumption remains poorly characterized. The purpose of this study was to determine in rats the effects of chronic alcohol consumption on the relative magnitude of apoptosis in two major targets of alcohol-induced liver injury: the hepatocyte (Hep) and sinusoidal endothelial cell (SEC). Methods: Rats were fed a liquid diet containing either alcohol or isocaloric amounts of maltose-dextrin for 14 weeks. Hep and SEC were isolated by liver perfusion with collagenase followed by centrifugal elutriation. The state of the liver was assessed on the basis of light microscopic appearance, plasma liver enzymes (alanine and aspartate:2-oxoglutarate amino transferases), and the content of malondialdehyde in Hep. Apoptosis was assessed on the basis of DNA fragmentation in the whole organ (TUNEL), and caspase-3 and -8 activity in isolated cells. A mechanistic approach was also undertaken by measuring mRNA expression and the amount of protein for Fas/CD95, Fas ligand, caspase-3, Bax, Bcl-X(L), and Bcl-2 in the isolated Hep and SEC. Results: The livers of alcohol-fed rats displayed prominent steatosis. Oxidative stress was also present as reflected by an increase in the malondialdehyde content of Hep. Alcohol consumption increased apoptosis in the whole liver assessed on the basis of TUNEL procedure and in Hep and SEC as reflected by significant increase in caspase-3 activity. Of the multiple pro- and anti-apoptotic factors determined in this study, significant changes as assessed by both mRNA expression and the amount of proteins, were observed only in the SEC compartment. Conclusions: The data presented in this study indicate that: (1) chronic alcohol consumption in rats leads to a moderate augmentation of apoptosis in the whole liver and in two liver cell types which are targets for injury in alcoholic liver disease: Hep and SEC; (2) the mechanisms recruited/activated by these two types of liver cells to initiate and execute apoptosis in response to alcohol vary with the cell type.
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PMID:Chronic alcohol exposure of rats exacerbates apoptosis in hepatocytes and sinusoidal endothelial cells. 1125 13


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