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)

Two murine peritoneal macrophage cell lines have been isolated by transforming primary cells with simian virus 40 (SV40) origin-deleted DNA. These lines have been maintained in continuous culture for over 8 months and have been shown to express macrophage-specific properties throughout this time. The cell lines are F4/80 positive; express Fc receptors; will phagocytose immunoglobulin-coated red cells and latex beads; stain with neutral red; and have non-specific esterase and plasminogen activator activities. Lysozyme, collagenase, prostaglandin E2, acid phosphatase and 5'-nucleotidase activities have also been detected and quantified.
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PMID:Establishment of immortalized cell lines from mouse peritoneal macrophages following transformation with SV40 early region DNA deleted at the origin of replication. 185 Nov 33

Isolation and culture techniques for hepatocytes from whole livers of the cynomolgus monkey, Macaca fascicularis, are described. Hepatocytes were isolated by two-step perfusion of livers, using collagenase with hyaluronidase; fructose and trypsin inhibitor were included to reduce cell loss. Yields from a single liver average 4 X 10(9) cells with viabilities of 90.8 +/- 5.7%. Cells, plated on collagen substrates, were assessed for changes in morphology and various marker enzyme activities over a period of 7 d in culture. Cells exhibited a morphology similar to that observed for this species in vivo; little change in attached and spread cells was observed over the length of time monitored. Enzyme activities for catalase, succinate dehydrogenase, and tyrosine aminotransferase were observed to decrease significantly (though considerable activity remained), whereas acid phosphatase and 5'-nucleotide phosphodiesterase remained unchanged. Activity of cytochrome P-450 reductase was observed to increase slightly for the first 2 d, then decrease to about 60% of initial levels. Activity of alpha-mannosidase was stable for 4 d but was observed to be increased at Day 7. Cells were observed to retain metabolic responsiveness, demonstrated by glucose production by both gluconeogenesis and glycogenolysis in response to glucagon stimulation. The monkey hepatocytes obtained by methods described here thus retain hepatocellular morphology and activity through at least 1 wk in culture without medium or culture modification.
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PMID:Isolation and culture of hepatocytes from the cynomolgus monkey (Macaca fascicularis). 197 77

A recent hypothesis for the cellular mechanism of fluid secretion by lacrimal acini has been based, in part, on the results of subcellular fractionation analyses of lacrimal gland fragments which had been incubated for a brief period in vitro. An important assumption in those studies was that the ion transporters and neurotransmitter receptors measured in isolated subcellular fractions were associated with membranes derived from the acinar cells, since these comprise the bulk of the lacrimal gland mass. This study was undertaken to validate this assumption. Acinar complexes were isolated from rat exorbital lacrimal glands by digestion with collagenase, hyaluronidase, and DNase. Although terminal intralobular duct segments and myoepithelial cells were occasionally noted, the preparations appeared to be free of larger ducts, blood cells, blood vessels, and interstitial cells. Acinar cells were then disrupted, and the homogenates underwent the fractionation procedure used previously for lacrimal gland fragment preparations. This procedure involved a sequence of analyses by differential sedimentation, isopycnic centrifugation on sorbitol gradients, and partitioning in dextran-polyethyleneglycol two-phase systems. Calculated initial specific activities for sodium/potassium adenosinetriphosphatase (Na+/K(+)-ATPase), alkaline phosphatase, acid phosphatase, and succinate dehydrogenase were identical to those obtained from fragment preparations. Major membrane populations resolved by the sequential analyses, including one believed to represent endoplasmic reticulum membranes, two believed to be derived from the acinar cell basal-lateral membrane, and two believed to be derived from the Golgi complex, corresponded closely to populations resolved from lacrimal fragment preparations. In addition to validating the previous use of lacrimal gland fragment preparations in studies of acinar cell function, these results suggest that preparations of isolated lacrimal acini will be useful for future work on neurotransmitter-receptor regulation and basal-lateral plasma membrane dynamics in the lacrimal gland.
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PMID:Analytic subcellular fractionation of acini from rat lacrimal gland. 217 90

To further understand the mechanism of PTH effects on bone and bone cells, we have analyzed the effect of PTH on specific protein phosphorylation in cells isolated from neonatal mouse calvaria. Four populations of cells (I-IV), isolated by sequential digestion with chromatographically purified bacterial collagenase isozymes and neutral proteinase, were cultured overnight. Alkaline phosphatase activity was greater than acid phosphatase activity in all four populations. PTH stimulated cyclic AMP production in all four populations, although the effect was greatest in populations II and III. Cultured cells were treated with PTH for up to 15 minutes. Cytosolic and membrane fractions were obtained and assayed for in vitro protein phosphorylation. No hormonal effects were found in membrane fractions. In cytosol fractions, treatment of the population II cells for 10-15 minutes with 0.1 microM PTH decreased the subsequent protein phosphorylation of an 85,000 Mr protein. In contrast, PTH treatment increased in vitro phosphorylation of both the 85,000 and 35,000 Mr proteins in population III cells. Phosphorylation of the 35,000 Mr protein was cyclic AMP-dependent. All of the phosphoproteins appeared to be phosphorylated solely on serine or threonine residues except the 85,000 Mr protein which may also contain significant amounts of phosphotyrosine. Therefore, some of the effects of PTH are cyclic AMP-mediated and other effects may be mediated through tyrosine phosphorylation. These data indicate that PTH has differential effects on in vitro protein phosphorylation in two separable populations of isolated neonatal mouse calvarial cells and support a hypothesis that multiple osteoblastlike cells exist in vivo.
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PMID:Differential effects of parathyroid hormone on protein phosphorylation in two osteoblastlike cell populations isolated from neonatal mouse calvaria. 249 26

T-cell subsets extracted from chronically inflamed periodontal tissues were identified using monoclonal antibodies, and their functional activity was analysed using the autologous mixed lymphocyte reaction (AMLR). Tissue was obtained from a total of 33 adult periodontitis (AP) patients and 6 normal/marginal gingivitis (N/MG) patients. All AP patients had received repeated oral hygiene instruction and root planing prior to the surgery, and the majority (30 out of 33) had at least one site with greater than 6 mm loss of attachment from the cementoenamel junction within the surgical field. The N/MG patients had no loss of attachment, and probing depths were less than 3 mm. Single cell suspensions were obtained following collagenase digestion (90 minutes at 37 degrees C) and mechanical disruption of the tissue. T-cell subsets were identified using an indirect immunofluorescence assay on cells obtained from 19 AP patients and the 6 N/MG patients. The mean (+/- standard error) helper:suppressor (T4:T8) ratio for the AP patients was found to be 0.94 +/- 0.48 compared with 1.65 +/- 0.16 for the N/MG group and 1.51 +/- 0.12 for peripheral blood controls. HLA-DR positive macrophages were identified and were found to include both acid phosphatase (AcP) positive and adenosine triphosphatase (ATPase) positive populations. Functional analysis was carried out using cells extracted from the remaining 14 AP patients. Cells from six of these 14 patients were found to be capable of spontaneous proliferation. Co-culture experiments using autologous T and non-T populations revealed that cells from only four patients were able to respond in an AMLR while those from only one of the 14 patients were able to stimulate the AMLR.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Phenotypic and functional analysis of T cells extracted from chronically inflamed human periodontal tissues. 295 90

A technique is reported here for the quantitative extraction of live cells from the lung interstitium; it involves the incubation of slices of perfused lung in a mixture containing optimal concentrations of collagenase, DNAse, and fetal calf serum, followed by the simultaneous recovery and fractionation of cells released from the tissue matrix on a six-step discontinuous percoll gradient. Yields in the order of 10(8) viable cells per gram of lung were routinely achieved with tissues from rat, mouse and guinea-pig. Preliminary characterization of these cells has been performed in the rat by histological techniques (Giemsa staining, transmission electron microscopy), cytochemistry (acid phosphatase, esterase, peroxidase), by the capacity to bind monoclonal antibodies directed at lymphocyte surface markers, and by a range of functional tests. The cells comprised, on average, 32% macrophages, 44% lymphocytes (T and B cells and large granular lymphocytes), with small numbers of eosinophils, mast cells and epithelial cells. Transmission electron microscopy revealed minimal ultrastructural damage to extracted cells, with such functions as phagocytosis, FcR activity, mitogen responsiveness, antigen presentation, and NK-cell activity, being readily demonstrable. In addition, these activities segregated into defined areas of the six-step density gradient.
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PMID:Preparation of interstitial lung cells by enzymatic digestion of tissue slices: preliminary characterization by morphology and performance in functional assays. 298 30

In osteoarthritis, despite increased matrix synthesis, there is a reduction of both major matrix components, proteoglycan and collagen. This study suggests that this is the result of enhanced degradative activity intrinsic to the cartilage. Because osteoarthritis is a focal disease, histologic controls were used to measure the severity in different areas of the cartilage and in different specimens. Neutral proteoglycanase and collagenase were both described in human cartilage, and their levels matched the severity of the disease as did acid phosphatase, a marker of lysosomal enzymes. Articular cartilage collagenase has an inhibitor in the cartilage and has negligible activity in normal cartilage. This was found not to be a lysosomal enzyme. A model of osteoarthritis was studied and found to have the same biochemical pattern as human disease. Using this method, inhibitors of degradative enzymes were used as a treatment. The chelator of metallic cations EDTA was found to have a significant effect on reduction of degradative enzyme activity and altered the arthritic process.
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PMID:Degradative enzyme systems in osteoarthritic cartilage. 298 65

This paper presents a study on the structure and function of Kupffer cells (KC) and liver endothelial cells (LEC) isolated by a simple and rapid technique involving 1) perfusion of the liver with collagenase; 2) cell separation by means of density centrifugation in Percoll; and 3) cell culture, taking advantage of the fact that KC and LEC differ in their preferences for growth substrate. The KC, which attach and spread under serum-free conditions on surfaces of glass or plastic during the first 15 min in culture exhibit a typical macrophage-like morphology including membrane ruffling and a heterogenous content of vacuoles. Moreover, these cells express (a) Fc receptors (FcR) for binding and phagocytosis of erythrocytes covered with immune globulin G (E-IgG), and (b) complement receptors (CR) for binding and serum dependent phagocytosis of erythrocytes covered with either human C3b or mouse inactivated C3b (iC3b). The cells also bind fluid phase fluoresceinated C3b. Approximately 30% of the KC express immune response-associated (Ia)-antigens. The LEC attach and spread on fibronectin coated surfaces, but not on glass or plastic surfaces, during the first two hours in culture with or without serum, and are morphologically distinct from KC. Cultured LEC are well spread out with no membrane ruffling and with numerous large vesicles surrounding the regularly shaped nucleus. These cells bind, but do not ingest E-IgG via the FcR, but no binding of fluid phase C3b or particle fixed C3b or iC3b can be observed. Incubation of LEC with fluorescein amine conjugates of ovalbumin or formaldehyde treated serum albumin, but not with fluoresceinated native serum albumin, results in accumulation of fluorescence specifically localized in the large perinuclear vesicles. Neither KC nor any other cell types tested have the ability to accumulate fluorescence upon incubation with these compounds. Ia-antigens are not present on the LEC. Cytochemical demonstration of unspecific esterase, acid phosphatase, and peroxidase reveals different patterns and intensities of staining in KC as compared to LEC.
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PMID:Functional and morphological characterization of cultures of Kupffer cells and liver endothelial cells prepared by means of density separation in Percoll, and selective substrate adherence. 299 96

Cathepsins B and D, beta-galactosidase, and acid phosphatase activities were found to be decreased in the regenerating rat liver, the reduction being maximal around the peak of hepatocyte mitoses (30 h). To investigate whether these changes could be heterogeneously distributed among hepatic cells, total cell populations from control or two-thirds hepatectomized rat livers were dissociated by the collagenase perfusion technique and analysed by different procedures. Isopycnic centrifugation in a Metrizamide gradient satisfactorily resolved hepatocytes and non-parenchymal cells from control animals but was not adequate when applied to 30-h regenerating liver cells. Colchicine treatment of the hepatectomized animals, resulted in substantial accumulation of phase M-hepatocytes. Subpopulations considerably enriched in fast-sedimenting phase M-cells were obtained by sedimentation at 1 g of the total liver cell population, and subsequently analysed by isopycnic equilibration. Phase M-hepatocytes were shown to have markedly reduced levels of beta-galactosidase, acid phosphatase, and cathepsin B activities in comparison, not only with control hepatocytes, but also with those parenchymal cells which were not metaphase-arrested in the same regenerating livers. Therefore, in partially-hepatectomized rats, hepatocytes progressing up to metaphase in the first mitotic cycle exhibited a selective depletion of lysosomal enzyme activities. The mechanism(s) underlying this change remain(s) presently unknown.
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PMID:Cellular distribution of lysosomal hydrolase activities in the regenerating rat liver. 308 41

Col 1(I), a collagenase-resistant segment of the amino-terminal propeptide of pro alpha 1(I) chains, is known to inhibit collagen synthesis in cultured skin fibroblasts and also in a cell-free protein synthesizing system by reducing the translation of procollagen mRNA. These findings prompted us to explore the fate of exogenous Col 1(I) in the cellular processing of human skin fibroblasts using colloidal gold labeled protein (Col 1(I)-Au). Distribution of Col 1(I)-Au on the cell surface was studied by the platinum-carbon replication technique. Three different types of binding pattern could be observed: 1) Binding sites in the form of a fibrillar network, 2) those in the form of clusters, and 3) solitary bound gold conjugates. The latter two cases were determined to be specific. The intracellular routing of Col 1(I)-Au was studied by thin sections. Specifically bound gold conjugates were found in coated pits and after the initiation of the internalization process in coated vesicles and endosomes. Acid phosphatase cytochemistry revealed that only a small amount of Col 1(I)-Au is delivered to lysosomes. The bulk of gold conjugates is present even after prolonged incubation at 37 degrees C in acid phosphatase-negative compartments of the cell. Our data suggest a mechanism in which Col 1(I) initially is bound to the cell surface and subsequently internalized via the coated pit-coated vesicle pathway.
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PMID:Binding and uptake of Col 1(I), a peptide capable of inhibiting collagen synthesis in fibroblasts. 316 32


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