Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.3 (collagenase)
 18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The early phase of wound healing after small central alkali burns of the guinea pig cornea was studied using electron microscopical, enzyme histochemical, and biochemical techniques. In the first phase, which was morphologically characterized by the destruction of the epithelium and keratocytes and by the infiltration of the cornea with polymorphonuclear leukocytes, an increase in the activity of lysosomal phosphatases and glycosidases (beta-D-glucuronidase, acid beta-D-galactosidase, beta-D-N-acetylglucosaminidase) was noticed. In the second phase, the cornea was invaded by capillaries and fibroblasts. In this phase, the activity of proteases (aminopeptidase M, dipeptidyl peptidase IV) increased intra- and extracellularly, suggesting that these enzymes may be involved in the turnover of the collagenous matrix and the ground substance. Using synthetic 4-methoxy-2-naphthylamine substrates and fluorescence-band detection techniques after isoelectric focusing, an increase in the activity of endopeptidases was demonstrated. The decreased activity of gamma-glutamyl transpeptidase may be linked with the activation of latent collagenase.
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PMID:The alkali burned cornea: electron microscopical, enzyme histochemical, and biochemical observations. 406 94

Microvessels were isolated from rat brain using a double collagenase treatment which removed the endothelial basement membranes. The isolate was characterized by intact luminal and abluminal membranes and an absence of pericytes and astrocyte membranes. Minimal contamination by 5'-nucleotidase, an enzyme believed exclusively localized within the plasma membranes of neuroglia, established the purity of the isolated microvessels. Enrichment of alkaline phosphatase and gamma-glutamyl transpeptidase activity in microvessel preparations supports the endothelial localization of these enzymes.
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PMID:Isolation and characterization of brain endothelial cells: morphology and enzyme activity. 610 94

Noninbred Sprague-Dawley rats were maintained on a choline-deficient diet containing 0.05% ethionine. After 10-13 weeks, livers were dispersed with collagenase, lysozyme, collagenase and hyaluronidase. Pronase, or a selected batch of trypsin. The highest yield of cells with histochemically demonstrable gamma-glutamyl transpeptidase (GGT) was obtained with trypsin. After velocity sedimentation in an isokinetic gradient of Ficoll in tissue culture medium, two modal populations of cells with histochemically demonstrable GGT were observed. The first mode contained cells that were morphologically different from hepatocytes and that may be oval cells. The second, more rapidly sedimenting modal population of cells with GGT was morphologically similar to hepatocytes as assessed with Wright's stain; the location of this population in the gradient was the same as the location of cells with the appearance of hepatocytes that lacked iron and that had decreased glucose 6-phosphatase. In multiple experiments, the purest fractions contained 71.7 +/- 3.5% cells (mean +/- SD) with the appearance of hepatocytes with histochemically demonstrable GGT.
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PMID:Separation of two populations of cells with gamma-glutamyl transpeptidase from carcinogen-treated rat liver. 611 83

Young adult male F344 rats were given 90 ppm diethylnitrosamine in drinking water for 5 weeks. Cells were obtained in suspension from the livers of these animals by in situ perfusion of collagenase. Cells were separated in a 2.7-16% (wt/wt) continuous gradient of Ficoll in tissue culture medium in the Sorvall TZ-28 reorienting zonal rotor for 10 minutes at 4 degrees C with a centrifugal force of 12 X g at the sample-gradient interface (26 X g at the gradient-cushion interface). Up to half a billion liver cells were separated without exceeding the band capacity. In all experiments the purest fractions contained more than 90% hepatocytes. After centrifugation, 72.4 +/- 6.6% of the purified hepatocytes had histochemically demonstrable gamma-glutamyl transpeptidase (GGT). When purified hepatocytes were injected into the mesenteric veins of rats given a diet containing 0.02% (wt/wt) N-2-fluorenylacetamide for 7 days and subjected to partial hepatectomy, all rats that received these cells developed foci that exhibited histochemically demonstrable GGT. Hepatocytes with histochemically demonstrable GGT make up all or part of what previously has been referred to as "liver colony-forming units." With the TZ-28 reorienting zonal rotor, these cells can be purified in biochemically preparative quantities.
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PMID:Purification and transplantation of hepatocytes from livers of carcinogen-treated rats. 612 27

The Sertoli cell is thought to play a significant role in the hormonal regulation of spermatogenesis within the rat testis. Little, however, is known about Sertoli cell function in man, largely because of the difficulties associated with the isolation of pure cell populations from human tissue. We have now developed a rapid and reproducible technique for establishing a human Sertoli cell monolayer culture. This has involved mechanical separation of the tissue, sequential trypsin and collagenase enzyme digestion, and final disruption of tubules by passage through a wire mesh grid. Using this technique, primary cultures can be maintained for up to 45 days. Ultrastructural studies of these cells have demonstrated the presence of the perinucleolar spheres, cell to cell junctional complexes, abundant lipid droplets, and smooth endoplasmic reticulum, all characteristic of Sertoli cells. Furthermore, biochemical markers of animal Sertoli cells, androgen-binding protein and gamma-glutamyl transpeptidase, have also been identified in these human cells. Concentrated media electrophoresed on nondenaturing gels containing 2 nM [3H]dihydrotestosterone produced a single peak of bound activity which coelectrophoresed with rat androgen-binding protein. This binding activity persisted despite media changes, thus ruling out contamination by serum binding proteins; fresh media lacked demonstrable binding activity. Using a colorimetric assay, these cells were also found to contain significant gamma-glutamyl transpeptidase activity compared to human foreskin fibroblasts and Leydig cells. Enzyme activity increased in a characteristic dose-response fashion in the presence of FSH (0.05-0.5 microgram/ml) and dibutyryl cAMP (0.1-1 microgram/ml), but not with LH or testosterone. These data offer the first demonstration of human Sertoli cells in monolayer culture and their production of a marker specifically regulated by FSH.
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PMID:Characterization of human Sertoli cells in vitro. 612 20

Male F344 rats were continuously fed 0.05% phenobarbital (PB)- or 0.02% 2-acetylaminofluorene (2-AAF)-containing diet for 3 or 8 weeks. The hepatocytes were isolated by a collagenase-perfusion technique and changes in the phalloidin-sensitivity of the cells were examined. After an 8-week treatment with PB or 2-AAF, the phalloidin-sensitivity, in terms of formation of cytoplasmic blebs over the cell surface, was reduced significantly in both groups. The degree of insensitivity to phalloidin induced by PB was found to be similar to that induced by 2-AAF. Two weeks after cessation of the PB-feeding, the sensitivity had recovered to the control range, while the decreased sensitivity induced by 2-AAF persisted for at least 2 weeks after the cessation of 2-AAF-feeding. Furthermore, cytochemical examinations of normal and 2-AAF-treated hepatocytes revealed that the degree of sensitivity decreased in the order of normal hepatocytes, 2-AAF-treated gamma-glutamyl transpeptidase-negative hepatocytes, and 2-AAF-treated gamma-glutamyl transpeptidase-positive hepatocytes. The decreased sensitivity of the latter two cell types also persisted for 2 weeks after the cessation of carcinogen treatment.
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PMID:Effects of phenobarbital-feeding on the phalloidin-sensitivity of rat hepatocytes. 613 50

The tumorigenic roles of specific types of cells that emerge during chemical hepatocarcinogenesis in rats could potentially be determined if the specific types of cells could be purified adequately. Type II cells, defined (J. M. Jacobs, T. P. Pretlow, N. Fausto, A. M. Pitts, and T. G. Pretlow, J. Natl. Cancer Inst., 66: 967-973, 1981) as small, slowly sedimenting cells with histochemically demonstrable gamma-glutamyl transpeptidase, have oval nuclei similar in size to those of lymphocytes. Adult male F344 rats were fed a choline-deficient diet containing 0.05% ethionine for 4 weeks. Liver cells were obtained in suspension by in situ perfusion of collagenase. A two-step process, i.e., sedimentation in an isokinetic gradient followed by free-flow electrophoresis, was used to purify type II cells. We obtained preparations of cells with 32.3 +/- 5.0% (S.D.) type II cells, 13.4 +/- 2.0% erythrocytes, 52.9 +/- 3.3% small nucleated cells, and only 1.4 +/- 0.3% hepatocytes. The small nucleated cells were Kupffer cells and cells smaller than Kupffer cells that included many lymphocytes and unidentified cells similar in size to lymphocytes. Because both the electrophoretic mobilities and the rates of sedimentation of type II cells and hepatocytes are different, there is some advantage to be obtained from the sequential use of these techniques that exploit independent differences in the physical properties of these cells.
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PMID:Purification of cells from livers of carcinogen-treated rats by free-flow electrophoresis. 613 4

Oval cells and biliary epithelial cells were isolated from livers of rats fed a choline-deficient diet containing 0.1% ethionine and from normal rat livers, respectively. Nonparenchymal cell suspensions prepared from these livers by collagenase perfusion followed by digestion of undissociated tissue with 0.1% collagenase, 0.1% Pronase, and 0.004% DNase I were separated into six fractions by centrifugal elutriation. Cells in each fraction were characterized histochemically for gamma-glutamyl transpeptidase, peroxidase, alkaline phosphatase, and glucose-6-phosphatase activities, and for albumin and alpha-fetoprotein by immunocytochemical methods. Cells from Fraction 5 of the elutriation procedure had various features predicted for oval cells and were selected for further studies. The cell yield in this fraction, from each preneoplastic liver, was 5.7 X 10(7) cells, 93 +/- 2% of which were gamma-glutamyl transpeptidase positive, 6 +/- 1% peroxidase positive, 61% albumin positive, and 29% alpha-fetoprotein positive. Cells in this fraction have a median diameter of 13.1 micron and are diploid and cycling. The majority of these cells has morphological features characteristic of biliary epithelial cells, although some cells display features intermediate between duct cells and hepatocytes. Nucleic acid hybridization using specific probes revealed that these cells contain albumin and alpha-fetoprotein messenger RNAs, while hepatocytes from normal and preneoplastic liver contain only albumin messenger RNA. Biliary cells obtained from normal livers do not contain albumin messenger RNA. The large-scale purification and characterization of cell populations from preneoplastic livers is an important step in elucidating the cellular derivation of liver tumors.
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PMID:Isolation of oval cells by centrifugal elutriation and comparison with other cell types purified from normal and preneoplastic livers. 669 43

Alcoholic and, to a lesser extent, nonalcoholic patients with liver disease have serum antibodies to acetaldehyde-protein adducts produced in vitro. These antibodies presumably reflect the presence of adducts in the liver, but the protein that triggers this immune response has not been identified. To study this, we measured the reactivity of cytosolic proteins to rabbit IgG developed against a P-450 2E1-acetaldehyde adduct, isolated from alcohol-fed rats, that recognizes acetaldehyde-modified epitopes in proteins. Adducts were determined on Western blots by scanning densitometry of antibody-linked alkaline phosphatase activity in 4 normal livers and in needle biopsy specimens from subjects with liver disease, 17 alcoholic and 14 nonalcoholic. In all livers, except for a normal one, we found a reactive protein of at least 200 kD, similar to the collagen-acetaldehyde adduct we reported to be markedly increased in rats with experimentally induced cirrhosis. The immunostaining intensity in the alcoholic patients with liver disease was eightfold (p < 0.01) and that in nonalcoholic patients with liver disease was fourfold, greater (p < 0.02) than the weak staining in normal livers; it correlated with the degree of inflammation and serum AST or gamma-glutamyl transpeptidase activities. The adduct was reproduced on incubation of normal cytosolic proteins with 2.5 mmol/L acetaldehyde, whereas higher concentrations yielded many additional adducts; the adduct also reacted with IgG antibody to rat collagen type I and disappeared after digestion with collagenase, suggesting that the target protein is a form of collagen.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Collagen-acetaldehyde adducts in alcoholic and nonalcoholic liver diseases. 791 86

An in vitro model to establish primary and subcultures of rat kidney proximal tubule (RPT) cells is described. After excising the kidneys and separating the cortex, the cortical tissue is digested with the enzymes DNAse-collagenase (Type I) resulting in a high yield of viable RPT Cells. The isolated RPT cells are then seeded onto rat tail collagen-coated surfaces and grown to confluency in a serum-free, hormonally defined medium. The cell yield can be increased by transferring the conditioned medium on Day 1 to more rat tail collagen-coated surfaces. RPT cell attachment and morphology was better on rat tail collagen-coated surfaces than on bovine collagen Type I coated surfaces. The culture medium was a 1:1 mixture of Ham's F-12 and Dulbecco's modified Eagle's medium supplemented with bovine serum albumin, insulin, transferrin, selenium, hydrocortisone, triiodothyronine, epidermal growth factor, and glutamine. The RPT cells became confluent in 7-10 d, at which point they could be subcultured by trypsinizing and growth in the same medium. In some studies, 10 ng/ml cholera toxin was added to the culture medium. We could passage the RPT cells up to 14 times in the presence of cholera toxin. The cells were investigated for activity of several markers. The cells were histochemically positive for alkaline phosphatase and gamma-glutamyl transpeptidase activity and synthesized the intermediate filament pankeratin. The RPT cells displayed apically directed sodium-dependent active glucose transport in culture. Hence, the RPT cells retain structural and functional characteristics of transporting renal epithelia in culture. This rat cell culture model will be a valuable tool for substrate uptake and nephrotoxicity studies.
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PMID:Normal rat kidney proximal tubule cells in primary and multiple subcultures. 879 58


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