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)

This work presents a filter elution method for measuring DNA single- and double-strand breaks in primary rat hepatocytes without radioactive labeling of DNA. We have studied the effects of a series of nitrosamines and of gamma-irradiation on DNA single- and double-strand break induction. The repair of DNA single-strand breaks in the hepatocytes was measured after treatment with 60Co, 1-methyl-1-nitrosourea, and N-nitrosodimethylamine. The hepatocytes were isolated by ethylene glycol-bis(beta-aminoethyl ether)-N,N'-tetra acetic acid-collagenase perfusion and had a mean viability of 91 +/- 4% (S.D.). The isolated cells were treated for varying lengths of time with nitrosamines in suspension culture in L-15 medium containing 10% fetal bovine serum. After treatment, the cells were chilled, loaded onto 2 micrometers polycarbonate filters, and lysed in a 2% sodium dodecyl sulfate-proteinase K solution, pH 9.6. The DNA was eluted from the filter at either native or denaturing pH with fractions collected every 3 hr. The quantity of DNA in each fraction was determined by measuring the fluorescent product formed between it and diaminobenzoic acid after ethanol-sodium acetate precipitation and trapping of the DNA on 0.2-micrometer polycarbonate filters. The results show that the carcinogens, N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosodipropylamine, N-nitrosodibutylamine, and 1-nitrosopiperidine all made dose- and time-related increases in the number of single-strand breaks in rat hepatocytes. N-Nitrosodiphenylamine produced small numbers of single-strand breaks. No double-strand breaks were formed by any of the nitrosamines. Single-strand breaks induced by N-nitrosodimethylamine were repaired very slowly relative to repair of either gamma-ray of 1-methyl-1-nitrosourea-induced single-strand breaks. This system has many advantages for studying carcinogen metabolism and DNA damage in hepatocytes, one of the major target cells for many carcinogens.-
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PMID:Measurements by filter elution of DNA single- and double-strand breaks in rat hepatocytes: effects of nitrosamines and gamma-irradiation. 708 52

In an effort to evaluate further the concept of ethanol-induced lipid peroxidation, isolated rat hepatocytes obtained via collagenase perfusion were utilized. Hepatocytes were judged to be functionally intact based on measurements of adenosine-5-triphosphate, gluconeogenesis, bromosulphthalein uptake, and trypan blue exclusion. When hepatocytes were incubated with acetaldehyde, a metabolite of ethanol, at 100 mg% and 10 mg%, significant increases in lipid peroxidation resulted as measured by levels of malonaldehyde. Acetaldehyde-induced increases in malonaldehyde were reduced by pre-incubation with antioxidants such as Vitamin E (200 mg%) or glutathione (100 mg%).
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PMID:Acetaldehyde-induced lipid peroxidation in isolated hepatocytes. 710 Jun 31

The effect of ethanol on the secretion of proteins was studied in hepatocytes isolated from 24-h fasted rats and from fed rats. Hepatocytes were isolated after collagenase disruption of the liver and incubated in a standard medium containing amino acids, bovine albumin, glucose, penicillin and streptomycin in HEPES buffer. Cell viability was determined by urea production and trypan blue exclusion. When studying protein export, a model had to be chosen in which the labeling is accomplished before the addition of the test agents. Cells were incubated with [3H]valine for 2.5 and 7.5 min followed by a 15-mM valine chase and the incubates were adjusted to final concentrations of ethanol of 50 mM, 100 mM, colchicine 5-50 microM or cycloheximide 18 microM. Cells and media were harvested at various times, and counts incorporated into medium and cell protein were determined. Cycloheximide inhibited protein synthesis by 99%, decreased protein secretion by 10-20%, but did not further inihibit protein labeling when given after the chase confirming the chase's effectiveness. Colchicine inhibited protein release by 27-54% depending on the dose. With control cells labeled protein and specifically albumin appeared in the medium 20 min from the start of the pulse and this release of protein was not inhibited by 50 mM or 100 mM ethanol incubated with cells from the same animal whether the donor has been fed or fasted. The values for the ethanol-treated cells ranged from 94.0 to 113% of the control values from 30 to 120 min after the addition of the pulse. Lactate levels were markedly elevated, and urea synthesis decreased in the presence of either 50 mM EtOH or 100 mM EtOH. Thus using a method that can distinguish the effect of ethanol on synthesis from secretion, it is concluded that acute exposure to EtOH does not interfere with protein secretion.
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PMID:Protein secretion in suspensions of isolated rat hepatocytes: no influence of acute ethanol administration. 745 Apr 1

Tumor necrosis factor-alpha (TNF-alpha) has been shown to contribute to the alcohol [ethanol (ETOH)]-induced alteration of hepatic function. Therefore we tested the hypothesis that the hepatic action of TNF-alpha could be due, at least in part, to alterations in TNF-alpha cell-surface receptors of hepatic parenchymal (hepatocytes) and nonparenchymal (Kupffer and sinusoidal endothelial) cells. Rats were either acutely treated with ETOH by a primed, continuous 7-hr intravenous infusion of 20% (w/v) ETOH (30 mg/100 g body weight/h) or chronically fed an ETOH-containing liquid diet (5.2% ETOH, w/v, with ETOH as 36% of total calories) for 14 weeks. Control rats in the acute group were infused with sterile saline, whereas control rats in the chronic group were fed liquid diet containing dextrin to replace ETOH in isocaloric amounts. Three hr before killing, the rats were injected intravenously with Gram-negative bacterial lipopolysaccharide [(LPS) 100 micrograms/100 g body weight] or saline. Hepatocytes, Kupffer cells, and sinusoidal endothelial cells were isolated after liver perfusion with collagenase (without pronase), separated by centrifugal elutriation, and used to determine the affinity (Kd) and capacity (Bmax) of binding sites, using recombinant human-[125I]TNF-alpha as the ligand. Two binding sites were detected on Kupffer cells and sinusoidal endothelial cells isolated from control animals: a high-affinity (Kd1, in the range of 150-200 pM), low-capacity (Bmax, in the range of 2-3 fmol/10(6) cells) binding site and a low-affinity (Kd2, in the range of 2-9 nM), high-capacity (Bmax2, in the range of 3-15 fmol/10(6) cells) binding site.(ABSTRACT TRUNCATED AT 250 WORDS)
Alcohol Clin Exp Res 1995 Apr
PMID:Tumor necrosis factor-alpha cell-surface receptors of liver parenchymal and nonparenchymal cells during acute and chronic alcohol administration to rats. 762 65

Carnitine-mediated prevention of ethanol-induced hepatic steatosis is related to the attenuation of ethanol metabolism by carnitine in the intact rat. Although carnitine retards ethanol oxidation in the intact animal, the in vitro activities of ethanol-metabolizing enzymes remain unaltered. Therefore, hepatocytes were targeted to understand the mechanism of carnitine effect on ethanol metabolism. Rat hepatocytes were isolated by a collagenase-perfusion technique and incubated in albumin-containing medium with ethanol in the presence or absence of added carnitine or related compounds. Ethanol oxidation was determined by the loss of ethanol as well as by the products formed. The rate of ethanol oxidation in the presence of carnitine was one-half the rate in the absence of carnitine (14 vs. 25 nmol.min-1.million-1 cells). It took 100 times the concentration of carnitine to equal the maximal inhibition produced by acetylcarnitine and the effect of acetylcarnitine was without a lag time. It is concluded that acetylcarnitine is the mediator of carnitine inhibition of ethanol oxidation.
Alcohol
PMID:Acetylcarnitine-mediated inhibition of ethanol oxidation in hepatocytes. 763 64

Rats were treated with alcohol either acutely (continuous, 7-hr intravenous infusion; blood alcohol levels approximately 35 mM) or chronically (liquid diet, 12-14 weeks). Three hr before killing, the animals received Gram-negative bacterial lipopolysaccharide (LPS) or saline. Hepatocytes, Kupffer cells, and liver sinusoidal endothelial cells were isolated by liver collagenase perfusion and centrifugal elutriation, and used for measurements of recombinant human [125I]interleukin-6 binding. Dissociation constant (Kd) and the amount of cell-surface receptors (Bmax) were measured on whole cells, at 4 degrees C. Two binding sites were detected on all three cell types: high-affinity (Kd1, from 20 to 125 pM) and low-affinity (Kd2, from 0.2 to 2 nM), with low Bmax (Bmax, from 0.4 to 12 fmol/10(6) cells) and high Bmax (Bmax2, from 10 to 210 fmol/10(6) cells). Hepatocytes displayed an 8-fold higher binding capacity for high-affinity sites (Bmax1) than the other two cell types. Acute ethanol treatment induced the following significant changes in the binding parameters: a decrease in Kd1 for hepatocytes and Kupffer cells, an increase in Bmax2 for hepatocytes, and a decrease in Bmax1 for Kupffer cells. Although the control (nonalcoholic) liquid diet per se completely suppressed the high-affinity binding sites, alcohol-containing diet induced only one change: a significant increase in Kd2 for hepatocytes. No changes in the binding parameters were seen after LPS administration to the chronically treated group. In the acute group, LPS mimicked alcohol action on hepatocyte binding parameters. Alcohol blunted LPS effects.(ABSTRACT TRUNCATED AT 250 WORDS)
Alcohol Clin Exp Res 1994 Oct
PMID:Effect of acute and chronic alcohol administration to rats on the expression of interleukin-6 cell-surface receptors of hepatic parenchymal and nonparenchymal cells. 784 8

In alcoholic liver disease, it is well-known that ethanol and its metabolites induce hepatic fibrosis. With progress in injury, the accumulation of extracellular matrix, which consists of type I, III, IV collagen and laminine, occurs in the area of hepatic central vein and perihepatocytes. In these fibrotic areas, the activated lipocytes (transitional cell and myofibroblast, etc), which may be transformed from Ito cell by fibrogenic cytokines, are increased and may play an important role in the progression of alcoholic hepatic fibrosis. Actually, a recent study indicates that chronic ethanol consumption sensitizes the response of lipocytes to TGF beta. It is observed that acetaldehyde and lactate stimulate collagen production and that acetaldehyde increases collagen mRNA expression and collagen gene transcription in cultured human fibroblast. The extracellular matrix is degraded by matrix metalloproteinases (MMPs). The collagenase activity is decreased in progression of liver cirrhosis and is regulated by fibrogenic cytokines. Acetaldehyde decreases by 50% of the collagenase mRNA expression in fibroblast. It is clear that hepatic fibrosis may progress under the balance of collagen production and degradation, which are associated with fibrogenic cytokines. Thus, in the search for mechanism of alcoholic hepatic fibrosis, it is important to elucidate how ethanol and its metabolites influence the activation of lipocytes through fibrogenic cytokines.
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PMID:[Alcoholic liver cirrhosis]. 811 91

Hepatic ethanol (EtOH) metabolism has been assumed to involve hepatocytes differently, according to their location in the hepatic acinus. This study's aim was to gain information on plasma membrane (PM) order parameter in periportal (PP) and perivenular (PV) hepatocyte-enriched fractions isolated by a digitonin-collagenase perfusion technique from rats pair-fed for 6-8 wk liquid diets containing either EtOH or isocaloric carbohydrates. Fluorescence polarization (P) studies have been performed to measure PM order parameter of PP and PV hepatocytes cultured for 2-6 h on glass cover slips and labeled with 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH), a specific probe for PM of living cells. Fluorescence polarization and microscopy indicated that TMA-DPH is a suitable probe to study PM order parameter in subconfluent rat hepatocyte monolayers where it labeled, after a rapid incorporation, PM of cells. In pair-fed control rats, PM order parameter was lower in PP hepatocytes than in PV cells (P = 0.366 +/- 0.013 vs. 0.381 +/- 0.021, respectively, P < 0.02; n = 7). In EtOH-treated rats, these zonal differences tended to disappear (P = 0.419 +/- 0.012 in PP cells vs. 0.417 +/- 0.007 in PV cells; n = 7). In addition, the order parameter was significantly higher either in PP or PV hepatocytes compared with pair-fed control animals (P < 0.002 and 0.003, respectively). A 30-min culture of cells in the presence of 40-200 mM EtOH significantly decreased the PM order parameter of hepatocytes isolated from pair-fed control rats with respect to EtOH-treated animals both in PP and PV cells (P < 0.01 and 0.02, respectively; n = 4).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Plasma membrane order parameter in periportal and perivenular hepatocytes isolated from ethanol-treated rats. 814 2

Chronic alcoholism represents a high risk for fractures and osteopenia. Previous histomorphometric studies reported a decreased bone formation, but it has never been established whether ethanol has a direct toxic effect on osteoblasts. This present in vitro study was performed on human osteoblast cells derived from bone explants after collagenase digestion. The direct effect of ethanol was determined after 4 days of exposure to various doses, ranging from 0.01 to 5 g/l on the alkaline phosphatase (AP) activity, osteocalcin secretion and [3H]thymidine incorporation. The influence of the duration of exposure to 0.8 g/l ethanol was also determined. A significant and dose-dependent decrease in the cell proliferation was observed. AP activity was significantly decreased by high doses of ethanol (2-5 g/l). A biphasic effect of ethanol was noted on osteocalcin secretion according to the dose: it decreased at doses lower than 0.8 g/l and increased at the highest concentrations. At the dose of 0.8 g/l, whatever the duration of exposure, the decrease of the proliferation was of the same magnitude and no significant change in AP activity was observed. Significant ethanol-induced effects on osteocalcin secretion were observed only after 4 and 8 days of exposure. These data demonstrate that ethanol may have a direct toxic effect on osteoblast activity and proliferation. This could be one of the mechanisms of alcohol-induced osteopenia which has a multifactorial pathophysiology.
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PMID:In vitro evaluation of dose-effects of ethanol on human osteoblastic cells. 825 69

The tectorial membrane is a gel-like, acellular connective tissue overlying the microscopic organ of Corti--the auditory sensory structure. It is instrumental in the sound-synchronous deflection of the stereocilia of the hair cells, a central event in auditory transduction. It is well established that collagen, primarily type II, constitutes the major protein of the tectorial membrane, with smaller amounts of types IX and XI also present. However, conclusive information on the proteoglycans in this structure is lacking. Tectorial membranes were extracted with a 4 M guanidine--HCl solvent, and proteoglycans isolated after ethanol precipitation and collagenase treatment. A colorimetric assay based on the binding of the cationic dye safranin O to glycosaminoglycans, in combination with enzymatic techniques, detected significant amounts of chondroitin sulfate and keratan sulfate (0.29 and 0.17% on a wet weight basis, respectively). Agarose-polyacrylamide electrophoresis of chondroitinase-digested samples revealed a core protein with a similar molecular mass to that of the large cartilage proteoglycan aggrecan. This proteoglycan reacted with the antibody 3-B-3 (recognizing modified chondroitin 6-sulfate linkage region oligosaccharides). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed several low molecular mass proteins which reacted with 5-D-4, specific for keratan sulfate, one of which showed characteristics of fibromodulin. Comparison of the quantitative aspects of various connective tissue components of tectorial membrane with other type II collagen-containing structures revealed that this tissue resembles highly hydrated cartilage.
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PMID:Uronic acid-containing glycosaminoglycans and keratan sulfate are present in the tectorial membrane of the inner ear: functional implications. 827 27


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