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)

The paper represents a summary of our studies in which in vitro perfusion of human and animal coronary vessels was carried out. Formation and uptake of lipids in perfused human coronary arteries were studied under a vairety of experimental conditions, including exposure to carbon monoxide. The effect of collagenase on lipid synthesis and transport in carotid arteries of dogs was also studied. Human plasma with hydrogen-3-labeled cholesterol and carbon-14-acetate was used to perfuse human blood vessels. Autologous plasma was employed. Inhibition of cholesterol uptake was accomplished by the addition of 7-ketocholesterol (concentrations of 0.005 to 1 mum/ml) to the perfusate. Both atherosclerotic and normal human coronary arteries incorporated 14C-acetate into lipids but failed to synthesize either cholesterol of cholesterol esters. Similar results were obtained in human saphenous veins perfused at arterial pressure. Cholesterol uptake from the perfusion fluid was demonstrated in atherosclerotic and normal human coronary arteries as well as in human saphenous veins. Carbon monoxide increased permeability of the arterial wall to cholesterol uptake. In dog arteries exposed to collagenase marked increases in cholesterol uptake were found, but total lipid synthesis was reduced; the relative synthesis individual lipids remained unchanged. The addition of 7-ketocholesterol to the perfusate reduced cholesterol uptake by the vessel by 90 percent. Inhibition of cholesterol uptake was present in all species and was not due to oxidation of cholesterol to 7-detocholesterol in the perfusate. The results illustrate that human coronary arteries as well as human saphenous veins synthesize lipids but not cholesterol. Cholesterol flux into the artery is augmented by carbon monoxide and collagenase. The data also show that active inhibition of cholesterol uptake in the arterial wall can be accomplished by competitive inhibition with 7-ketocholesterol.
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PMID:Lipid metabolism in perfused human and dog coronary arteries. 16 13

Tubular fragments from rat kidney cortex were isolated by collagenase and suspended in an incubation medium containing a combination of several renal substrates. Substrate concentrations were in the physiological range. O2 uptake, total CO2 production, and the 14CO2 production from U-14C-labeled palmitate and oleate were measured. During the first minutes of incubation the CO2 production from palmitate and oleate was 10.5% or 6.3%, respectively, of the total CO3 produced. The RQ was 0.897. A subsequent decrease of the total CO2 production at a constant uptake of oxygen indicated a rising contribution of fatty acids to the fuel of respiration. The renal preference for substrates other than longchain fatty acids is discussed.
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PMID:Contribution of long chain fatty acids to the energy supply of the rat kidney cortex. 17 58

Six normal weight subjects without any heredity of diabetes (group 1), 3 obese subjects with normal (group 2) and 9 with pathological carbohydrate tolerance (group 3) were characterized by a 2-h glucose infusion test. Adipose tissue fragments were obtained from the abdominal wall by surgical biopsy under intracutaneous anesthesia. Adipocytes were isolated by collagenase digestion and incubated in buffer containing [1-14C] glucose and different concentrations of insulin. The metabolic effect of insulin was expressed as percent increase above control 14CO2 production. Maximal CO2 raised to 207 +/- 25% and 154 +/- 9% in groups 1 and 2, respectively. These values were significantly higher than in obese subjects displaying a pathological carbohydrate tolerance (group 3; 119 +/- 6%). A negative correlation was found between blood glucose levels and biological activity of insulin on adipocytes. The results suggest that insulin sensitivity of target tissue seems to play an important role in development of carbohydrate intolerance.
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PMID:Relationship between carbohydrate tolerance, insulin secretion, and insulin sensitivity of isolated fat cells from obese protodiabetics. 36 Jul 50

Spontaneously contracting myocytes were isolated from ventricles of the adult rat heart. Hearts were perfused retrogradally via the aorta for 30 minutes at 37 degrees C with Ca2+-free phosphate-buffered saline containing collagenase and hyaluronidase. The venticles were divided into pieces and incubated 15 minutes with the enzymes. Dislodged cells were decanted, diluted with cold buffer and allowed to settle. The washed cells were then sedimented through 3% Ficoll. This procedure yielded approximately 50 mg of protein from 1 gm of heart. Viability measured by trypan-glue exclusion is 90-95%. Approximately 80% of the cells were beating. Scanning electron microscopic studies suggest that the isolated myocytes are morphologically intact. The cells oxidize glucose, pyruvate, citrate and palmitate to CO2 and synthesize protein and RNA. Uptake of glucose, 2-deoxyglucose, leucine and taurine was saturable. Glucose uptake was stimulated by insulin. The cells retained LDH and CPK as well as their capacity to oxidize substrates after 24 hours at 4 degrees C or 4 hours at 37 degrees C. After 24 hours at 4 degrees C the cells resume contracting when returned to room temperature. The procedure reported here for the isolation of spontaneously contracting, adult, rat heart myocytes provides cells with a high index of viability and greater yield than previously reported methods. The cells retain metabolic activity and withstand storage for longer periods than other described preparations.
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PMID:Isolation and characterization of myocytes from the adult rat heart. 91 20

Polyhydroxyethylmethacrylate (PHEMA) based microcarriers with different bulk structures were prepared by a phase inversion polymerization technique. PHEMA surfaces were further modified chemically by glow-discharge treatment, and biologically by covalent attachment of fibrinogen and collagen. Hepatocytes were isolated from young male Wistar rats using an in situ portal vein collagenase perfusion technique. Freshly isolated hepatocytes were seeded at 6 x 10(5) cells/mL and microcarrier concentration was 10 g/L. Stationary microcarrier cultures were carried out in standard (nontissue culture) polystyrene petri dishes in a humidified 5% CO2 incubator at 37 +/- 0.5 degrees C. Cell attachment was followed by light microscopy by taking samples from the culture medium every 30 min. Urea and protein syntheses by microcarrier-attached hepatocytes were determined by standard techniques. Nonswellable (highly cross-linked) hydrophilic PHEMA microcarriers did not support cell attachment and viability. However, swellable (low cross-linked) PHEMA microcarriers (pretreated in FBS) allowed high attachment and cell spreading. PHEMA microcarriers treated in dimethylaminoethylmethacrylate (DMAEMA) glow-discharge plasma also improved the cell attachment characteristics of the PHEMA microcarriers. The highest attachment efficiencies (immobilization yields) were observed with the biologically modified PHEMA microcarriers, especially modified with fibronectin. Metabolic activity, as estimated by urea and protein syntheses, was also higher in these microcarriers.
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PMID:Hepatocyte immobilization on PHEMA microcarriers and its biologically modified forms. 134 12

Cytochrome P-450IIE1 is induced by a variety of agents, including acetone, ethanol and pyrazole. Recent studies employing immunohistochemical methods have shown that P-450IIE1 was expressed primarily in the pericentral zone of the liver. In order to evaluate whether catalytic activity of P-450IIE1 is preferentially localized in the pericentral zone of the liver acinus, the oxidation of aniline and p-nitrophenol, two effective substrates for P-450IIE1, by periportal and pericentral hepatocytes isolated from pyrazole-treated rats was determined. Periportal and pericentral hepatocytes were prepared by a digitonin-collagenase procedure; the marker enzymes glutamine synthetase and gamma-glutamyl transpeptidase indicated reasonable separation of the two cell populations. Viability, yield and total cytochrome P-450 content were similar for the periportal and pericentral hepatocytes. Pericentral hepatocytes oxidized aniline and p-nitrophenol at rates that were 2-4-fold greater than periportal hepatocytes under a variety of conditions. Carbon monoxide inhibited the oxidation of the substrates with both preparations and abolished the increased oxidation found with the pericentral hepatocytes. Pyrazole or 4-methylpyrazole, added in vitro, effectively inhibited the oxidation of aniline and p-nitrophenol and prevented the augmented rate of oxidation by the pericentral hepatocytes. Western blots carried out using isolated microsomes revealed a more than 2-fold increase in immunochemical staining with microsomes isolated from the pericentral hepatocytes, which correlated to the 2-4-fold increase in the rate of oxidation of aniline or p-nitrophenol by the pericentral hepatocytes. These results suggest that functional catalytic activity of cytochrome P-450IIE1 is preferentially localized in the pericentral zone of the liver acinus, and that most of the induction by pyrazole of P-450IIE1 appears to occur within the pericentral zone.
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PMID:Increased catalytic activity of cytochrome P-450IIE1 in pericentral hepatocytes compared to periportal hepatocytes isolated from pyrazole-treated rats. 167 9

The cytochrome P-450IIE1 (CYP2E1) isozyme activates several toxins and procarcinogens. Recent studies employing immunohistochemical and immuno-analysis techniques have shown that this isozyme is predominantly localized in the pericentral zone of the liver acinus. Experiments were conducted to evaluate whether microsomes isolated from the pericentral region of the liver display elevated catalytic activity towards effective substrates for CYP2E1 such as dimethylnitrosamine (DMN) as compared with periportal microsomes. Rats were treated with pyrazole to induce CYP2E1 and hepatocytes prepared from periportal or pericentral zones of the livers by the digitonin-collagenase procedure. Microsomes isolated from these hepatocytes had similar total P-450 contents; however, the microsomes from the pericentral hepatocytes displayed an increased DMSO binding spectrum suggesting an increased content of CYP2E1. Low Km DMN demethylase activity (but not high Km activity) as well as the oxidation of aniline and p-nitrophenol were 2- to 3-fold higher in pericentral compared to periportal microsomes. The oxidation of DMN by both microsomal preparations, as well as the increased rates obtained with the pericentral microsomes, was sensitive to inhibition by carbon monoxide as well as to other CYP2E1 substrates such as ethanol, pyrazole, or 4-methylpyrazole. Anti-CYP2E1 IgG inhibited the oxidation of DMN by both microsomal preparations 75% to 85% and prevented most of the increase found with the pericentral microsomes. Oxidation of aniline and p-nitrophenol was elevated in pericentral hepatocytes compared with periportal hepatocytes to the same extent as in the isolated microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Increased oxidation of dimethylnitrosamine in pericentral microsomes after pyrazole induction of cytochrome P-4502E1. 168 70

The purpose of this study was to measure the effects of a 10 week training, 3 week detraining cycle upon heart, muscle and adipose tissue of the rat. Specific pathogen-free female Wistar rats, 175 g at the onset of the experiments, were separated into three treatment groups; Sedentary Control (SC), Trained (T) and Detrained (DT). Animals from the T group were killed at 2, 4, 6, 8 and 10 weeks and animals from the DT group were killed at 7, 14 and 21 days after the last day of training. Unweighted swimming--6 h/day, 5 day/week, was the form of training employed. The animals, after being sacrificed, were anesthetized with nembutal (45 mg/kg body wt.) and muscle samples and heart removed. These tissues were frozen and analyzed at a later date for succinate dehydrogenase (SDH) activity (muscles), total protein (TP), total hydroxylprotein (TH) and wet and dry weight (heart). Adipose tissue was removed last, digested in collagenase (5 mg/ml) and the isolated cells used to measured 2-[3H]deoxyglucose uptake (DOG) and the conversion of D-[1-14C]glucose (C-1) and D-[6-14C]glucose (C-6) to CO2. The results of this study show that 10 weeks of endurance training induced myocardial hypertrophy (P less than 0.05) which involved increases in both TP and TH, the heart of the trained animals having 20.8% more protein and a 28.5% more hydroxlprotein than the sedentary controls. With detraining hypertrophy was lost within 21 days. Training maintained fat cell size at its pre-trained diameter, while inactivity allowed growth in the adipocytes of the control animals. The uptake of DOG and the conversion of glucose C-1 and glucose C-6 to CO2, were significantly (P less than 0.05) higher in the adipocytes of trained animals indicating that they were more responsive to insulin than the sedentary controls, which corresponded to increases in the respiratory enzyme levels of the muscles. During the first 7 days of detraining DOG uptake and both C-1 and C-6 glucose oxidation remained elevated. In conclusion the results of this study clearly demonstrate that there is a direct relationship between adiposity and training that can be related to the insulin responsiveness of the adipose tissue.
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PMID:The influence of training-detraining upon the heart, muscle and adipose tissue of female rats. 182 61

We describe a method of culturing intact porcine thyroid follicles for physiological de novo thyroid hormone formation; the roles of cAMP and protein kinase-C in thyroid hormone formation were also studied. Thyroid follicles were obtained by digesting minced porcine thyroid tissue with 0.04% collagenase and cultured in Coon's Modified Ham's F-12 medium supplemented with 0.5% calf serum, 0.5 mU/ml TSH, other standard hormones, and 3 antibiotics (6H medium). On the fourth day of culture, 6000-8000 follicles/well were plated in 12-well culture dishes. On the sixth day, thyroid hormone formation was carried out by incubating thyroid follicles with 0.5 microM KI in the presence of 6H medium for 2 days in a 5% CO2-95% air incubator at 37 C. To examine the effects of cAMP and protein kinase-C on de novo thyroid hormone formation, follicles were incubated with KI in the presence of 1-2.5 mM (Bu)2cAMP, 10 microM forskolin, 2 microM prostaglandin E2 (PGE2), or 0.5-1 microM 12-O-tetradecanoylphorbol-13-acetate in TSH-free medium for 2 days. The amount of newly formed thyroid hormone was measured by RIA of T3 content in the Pronase digest of thyroid follicular cells. Thyroid follicles cultured in 6H medium had normal polarity of the membrane, determined by electron microscope, and thyroid cAMP was responsive to the alteration of TSH. In this culture system cAMP alone was sufficient to form thyroid hormone. 12-O-Tetradecanoylphorbol-13-acetate, a protein kinase-C stimulator, disrupted thyroid follicles and inhibited cAMP-mediated thyroid hormone formation. The integrity of follicular structure was also required for thyroid hormone formation in this culture system. This study introduces perhaps the most physiological culture system for de novo thyroid hormone formation. Our data provide direct evidence that thyroid hormone formation is linked to cAMP and that the protein kinase-C system acts as an inhibitor of thyroid hormone formation.
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PMID:Physiological de novo thyroid hormone formation in primary culture of porcine thyroid follicles: adenosine 3',5'-monophosphate alone is sufficient for thyroid hormone formation. 215 8

The binding and degradation of 125I-hIGF-I by isolated sheep hepatocytes have been examined. Hepatocytes were isolated by collagenase perfusion of 32-55 kg wether lambs and were incubated at 20 or 37 C at pH 7.4 in a 95% O2/5% CO2 atmosphere. Maximal binding was obtained at 60 min and declined slightly over the following 60-min period at both 20 and 37 C. Degradation of 125I-hIGF-I by the hepatocytes was minimal with 10-12% degradation over a 120-min period at 37 C. The lysosomal inhibitors chloroquine (0.2 mM), leupeptin and ammonium chloride had no significant effects on 125I-hIGF-I degradation or binding. At 20 C (60-min incubation), half maximal inhibition of 125I-hIGF-I binding was obtained with 8.4 +/- 1.1 nM hIGF-II, 16 +/- 2.4 nM hIGF-I, 36 +/- 6.2 nM oIGF-II, and 60 +/- 5.9 nM oIGF-I. Ovine insulin (0.01-10 uM) had no effect on 125I-hIGF-I binding. These observations suggest that IGF-I binds to the type II IGF receptor. The low molecular weight sheep serum IGF binding protein inhibited binding of 125I-hIGF-I to hepatocytes in a dose-dependent manner with half maximal inhibition occurring at 16.5 micrograms/ml, but did not affect IGF-I degradation. The current studies show that IGF-I interacts with ruminant hepatocytes via type II IGF receptors. The liver is not a major site of IGF-I degradation and the observed degradation is nonlysosomal and independent of receptor interaction.
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PMID:Interaction of insulin-like growth factors (IGF) with isolated sheep hepatocytes. I. Binding and degradation of IGF-I. 216 12


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