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 method is described for introducing and expressing cloned genes in isolated hepatocytes. Primary rat hepatocytes isolated by collagenase perfusion were transfected in suspension with plasmid pSV2CAT by electroporation. Forty-eight hours later, soluble extracts from transfected hepatocytes showed chloramphenicol acetyltransferase activity comparable to that obtained in rat hepatoma cell line H4AzC2 by calcium phosphate or DEAE-dextran transfection. The latter two methods could not be used successfully for primary hepatocytes because of cytotoxicity of these reagents. This indicates that electroporation is a useful method to obtain transient expression of foreign genes in primary epithelial cells, such as rat hepatocytes, which are difficult to maintain in cell culture.
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PMID:Use of electroporation to introduce biologically active foreign genes into primary rat hepatocytes. 346 23

The migration of ascites and cultured hepatoma cells, AH109A of rat (Donryu) origin and MH134 of mouse (C3H), was enhanced by collagen degradation products (CDP) in vitro using the modified Boyden chamber, but not by collagen. Both tumor cells demonstrated somewhat increased motility in the presence of CDP regardless of whether or not there was a gradient present, but the maximum response was seen in the presence of a gradient. MH134 cells responded more effectively to CDP than AH109A cells and showed similar migratory responses to type I and IV collagen degradation products (CDP-I and -IV). Synthetic di- or tripeptides containing hydroxyproline were less chemotactic for MH134 cells than CDP-I and -IV. Both proteases (collagenase and trypsin) and MH134 cells could degrade a collagen substrate and generate CDP. These findings suggest that CDP released during the process of invasion may play a role in the migration of tumor cells and consequent formation of metastases.
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PMID:Enhanced migration of tumor cells in response to collagen degradation products and tumor cell collagenolytic activity. 353 92

The uptake and internalization of a triglyceride emulsion by rat hepatocytes in culture less than 24 hr was either inhibited or uninfluenced by apoE. ApoE significantly increased the uptake of these emulsions in later cultures. Specific low density lipoprotein (LDL) binding was similar for hepatocyte monolayers prior to and after 24 hr. Rat hepatocytes in culture for 2 days, which were treated with collagenase, detached and then replated within 1 hr and were apoE-responsive in 2 hr. Heparin inhibited the apoE stimulation in both hepatocytes and hepatoma monolayers. Heparin wash of hepatocytes or hepatoma cells incubated with apoE-[14C]triolein emulsions at 4 degrees C resulted in a considerable loss in radiolabeled cell lipid. A similar wash after 37 degrees C incubations produced little loss suggesting internalization. Hepatocytes had lower affinity but similar apoE-emulsion binding capacity compared to hepatoma cells. Triolein emulsions with apoE were significantly more rapidly metabolized by the hepatocyte than unsupplemented emulsions. The apoE-mediated hepatocyte lipid uptake was inhibited by apoC proteins. High molar ratios of free fatty acid/albumin also suppressed hepatocyte apoE-mediated lipid uptake. Both rat high density lipoprotein (HDL) and LDL inhibited with a potency directly related to their content of apoE. Human LDL and HDL without apoE also inhibited the interaction with less potency than the rat lipoproteins. Human HDL inhibition was diminished after removal of apoC proteins.
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PMID:Effect of apoE on triglyceride emulsion interaction with hepatocyte and hepatoma G2 cells. 362 37

DNA synthesis of adult rat parenchymal hepatocytes alone in primary culture can be stimulated only by the addition of humoral growth factors to the culture medium. However, when parenchymal hepatocytes were cocultured with nonparenchymal liver cells from adult rats, their DNA synthesis was markedly stimulated in the absence of added growth factors or calf serum. DNA synthesis of parenchymal hepatocytes was not stimulated by conditioned medium from nonparenchymal liver cells and was greatest when the parenchymal cells were plated on 24-h cultures of nonparenchymal liver cells. A dead feeder layer of nonparenchymal cells was almost as effective as a feeder layer of viable nonparenchymal cells. These results suggest that the stimulation of DNA synthesis in parenchymal hepatocytes was not due to some soluble factors secreted by nonparenchymal liver cells but to an insoluble material(s) produced by the nonparenchymal liver cells. This insoluble material(s) was collagenase- and acid-sensitive, suggesting that it was a protein containing collagen. The effect of nonparenchymal liver cells was specific: coculture with hepatoma cells, liver epithelial cells, or Swiss 3T3 cells did not stimulate DNA synthesis in parenchymal hepatocytes. Added insulin and epidermal growth factor showed additive effects with nonparenchymal cells in the cocultures. These results suggest that DNA synthesis in parenchymal hepatocytes is stimulated not only by various humoral growth factors but also by cell-cell interaction between parenchymal and nonparenchymal hepatocytes, possibly endothelial cells. This cell-cell interaction may be important in repair of liver damage and liver regeneration.
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PMID:Stimulation of growth of primary cultured adult rat hepatocytes without growth factors by coculture with nonparenchymal liver cells. 365 56

N-13A malignant ascitic hepatoma, induced by 4-dimethylaminoazobenzene in Wistar rats, does not produce distant mestastases when transplanted in vivo. The mixed co-cultures of N-13A tumor cells and several tissue explants of newborn Wistar rats show selective adhesivity between tumoral and hepatic epithelial cells, but not with fibroblast-like cells of nervous tissue, kidney or diaphragm. In short-term co-cultures, N-13A cells in contact with rat hepatocytes prepared by the collagenase perfusion technique, display a selective adherence capacity with the production of abundant microvilli and fingerlike protrusions (microspikes) which are elaborated by the neoplastic cells. Groups of tumoral cells tightly envelop the free surface of the cultured hepatocytes. Tight-junction formations are observed, and immature desmosomes and polydesmosomic systems are also seen between both tumoral and normal cells.
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PMID:Cell-to-cell interaction between N-13A ascitic hepatoma cells and normal cells maintained in short-term co-cultures: an optical, transmission and scanning electron microscopical study. 388 Jan 56

The beta-adrenoceptor density and the activities of adenylate cyclase and cyclic AMP phosphodiesterase were examined to compare AH13 cells having lower beta-adrenergic responsiveness with other rat ascites hepatoma cells and normal rat liver cells. Normal rat liver cells used were cultured for 24 hr after the collagenase digestion of liver. The density of binding sites of 3H-dihydroalprenolol in AH13 cell plasma membrane was very similar to the density in AH44 and normal liver cell membrane, but that in AH130 cell plasma membrane was about 10-fold greater than those in the other three cell lines. The activity of cyclic AMP phosphodiesterase was about 2.5- to 7-fold higher in hepatoma cells than in rat liver cells, but this enzyme activity of AH13 cells was not especially high among the hepatoma cells examined. The basal adenylate cyclase activity was lower in AH44 cells, but was higher in AH13 and AH130 cells than in rat liver cells. However, adenylate cyclase of AH13 cells was hardly activated by isoproterenol, while the enzyme of the other cells was activated 3- to 5-fold. On the other hand, adenylate cyclase of each cell line including AH13 was activated 4- to 14-fold by NaF. From these results, it is suggested that AH13 cells can hardly produce cyclic AMP by the beta-adrenergic stimulation because of the disordered interaction of beta-adrenoceptors with adenylate cyclase.
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PMID:Studies on responsiveness of hepatoma cells to catecholamines. I. Lack of beta-adrenergic responsiveness in rat ascites hepatoma AH13 cells. 609 61

gamma-Glutamyl transpeptidase (gamma GT) positive hepatocytes were isolated from F344 male rats fed 2-acetylaminofluorene. The isolation procedure is rapid and highly selective for cells exhibiting gamma GT on their surface. Suspensions of liver cells obtained from perfusion in situ with a collagenase solution were incubated on Petri dishes coated with affinity purified rabbit anti-gamma GT antibody. gamma GT-positive cells bound to the dish within fifteen minutes and could be recovered as viable cells. Approximately 15% of the gamma GT-positive cells are isolated using this procedure. Novikoff hepatoma cells, a gamma GT-positive cell line, were used to define the parameters of the assay. The binding was both time and temperature dependent. Binding of cells to the anti-gamma GT antibody coated dishes was 50% inhibited by 2.8 mM sodium azide and 86% inhibited by 4.6 mM.
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PMID:Isolation of gamma-glutamyl transpeptidase positive hepatocytes during the early stages of hepatocarcinogenesis in the rat. 612 30

Two Fischer 344 rat hepatoma cell strains, JM1 and JM2, have been isolated from a primary hepatocellular carcinoma. Primary tumor formation was induced in a two-thirds partially hepatectomized rat by a single low dose (70 mg/kg of diethylnitrosamine followed by chronic phenobarbital administration (0.1 g/100 ml drinking water). The primary tumors were passed three times by subcutaneous implantation of tumor fragments into the inguinal region of syngeneic recipients. The fourth pass was by injection of tumor cells directly into the livers of recipient rats. Several weeks later, the tumor-containing rat livers were subjected to collagenase perfusion. Two cell lines emerged from tissue culture of the cells isolated by perfusion. Each cell line was cloned by serial dilution. Cells JM1 and JM2 were tumorigenic when injected into syngeneic rats. The tumors, which arose from injected cell strains, exhibited several characteristics of hepatocellular carcinoma. Morphology was examined by light and electron microscopy. Histochemical studies of JM1 and JM2 cells grown in vitro and in vivo were done. The levels of tyrosine aminotransferase and three microsomal enzymes of importance to drug and carcinogen metabolism were investigated. To our knowledge, this is the first report of cell strains derived from an initiation promotion protocol in rats.
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PMID:Establishment of two rat hepatoma cell strains produced by a carcinogen initiation, phenobarbital promotion protocol. 613 63

Bovine capillary endothelial cells have been found to respond to several stimuli by producing increased amounts of plasminogen activator and latent collagenase. These stimulators include the tumor promoter tetradecanoyl phorbol-13-acetate as well as crude preparations from a human hepatoma, bovine retinae, and mouse adipocytes, all of which are known to contain angiogenic factors. Endothelial cells and skin fibroblasts do not respond to these stimuli in the same way, indicating a specificity of the response. In addition, inhibitors of plasmin and vertebrate collagenase have been isolated from cartilage, a tissue resistant to neovascularization. We have proposed that these specific protease inhibitors confer on cartilage its antiangiogenic properties.
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PMID:The involvement of proteases and protease inhibitors in neovascularization. 617 34

Soluble proteoglycans (SPG) were extracted from bovine (BCC) and human (HCC) costal cartilages by the dissociative method using 4 M guanidinium chloride (GuHCl). Proteoglycans which are resistant to extraction (RPG) were obtained following collagenase digestion or hydroxylamine treatment of the cartilage residues. Similarly, SPG were extracted from bovine metaphyseal and cortical bone using EDTA. The RPG were extracted from the bones using hydroxylamine. Density gradient fractionation under dissociative conditions of cartilage SPG and RPG followed by chromatography on Sepharose 2B revealed that A1D1 RPG are smaller than the SPG. SPG reacted with either collagenase or hydroxylamine are also smaller than the parent SPG. A1D1 fractions obtained from BCC-SPG and RPG or from mixtures of SPG and acid-soluble collagen are free of hydroxyproline. Hydroxyproline is not completely separated from HCC-RPG. Density gradient fractionation of bone proteoglycans and Sepharose chromatography of the A1 and A1D1 fractions showed that those obtained from metaphysis are larger than those from cortical bone. This was attributed to the presence of calcified cartilage in metaphyseal bone. The A1D1 fractions of the metaphyseal proteoglycans seemed to undergo self-association since this fraction is larger than the A1 fraction from which it is derived. Cortical bone proteoglycans do not behave similarly. Density gradient purification under dissociative conditions failed to separate hydroxyproline from the proteoglycans obtained from bone. It is hypothesized that in bone proteoglycans and collagen might be linked.
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PMID:Studies on extractable and resistant proteoglycans from metaphyseal and cortical bone and cartilage. 626 Mar 14


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