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)

We have compared two components of bacterial cell walls, muramyl dipeptide (MDP) and lipopolysaccharide (LPS), for their effects on bone resorption as measured by the release of previously incorporated 45Ca. MDP is the smallest active component of peptidoglycan, whereas LPS is the active component of endotoxin. Fetal rat long bones were cultured for 5 days in a chemically defined medium supplemented with bovine serum albumin (BSA) or serum. LPS increased 45Ca release at concentrations of 0.03-1.0 microgram/ml. LPS further purified by electrolytic dialysis (ED-LPS) was active at 0.01 microgram/ml. ED-LPS was ineffective at such low concentrations in the presence of serum. The response to MDP was more variable than that to LPS, but bone resorption was stimulated at concentrations of 10(-7)-10(-5) M. MDP was less effective or inactive in medium supplemented with serum. Stereoisomers of MDP that do not have adjuvant activity caused minimal stimulation of bone resorption, whereas 6-0-steroyl MDP stimulated resorption at 10(-8) M. The stimulation of bone resorption by LPS and MDP was not inhibited by indomethacin. Both LPS and MDP increased lysosomal enzyme release in proportion to their effects on 45Ca release. LPS also markedly increased collagenase activity in the medium, but MDP did not. These results indicate that chemically different products of bacterial cell walls can stimulate bone resorption in vitro. These products may be distinguished by differences in dose response curve, serum inhibition, and collagenase release.
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PMID:Effects of two bacterial products, muramyl dipeptide and endotoxin, on bone resorption in organ culture. 629 30

Rabbit hepatocytes were isolated by a collagenase perfusion technique, and used to study the binding and endocytosis of the glycoprotein, asialo-orosomucoid, and the neoglycoprotein, Gal39-bovine serum albumin. Both of these proteins contain exposed galactosyl residues, and were avidly bound by the lectin on the hepatic parenchymal cell surface. Steady state and kinetic experiments performed at 2 degrees C and at 37 degrees C revealed the presence of two apparent classes of binding sites totalling 4.7 X 10(5) sites/cell at 2 degrees C, and 6.3 X 10(5) sites/cell at 37 degrees C. At 37 degrees C, both classes of sites participated in internalization of bound ligand. The cells were capable of internalizing about 60 000 molecules/min per cell. The process appeared to be first-order, with a rate constant k = 0.098 min-1 and t1/2 = 7.1 +/- 0.6 min. Binding could be inhibited by galactose-containing compounds, EGTA, and by anti-(hepatic lectin) immunoglobulin G. The inhibition by antibody appeared to be reversible upon removal of antibody-containing medium.
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PMID:Binding and endocytosis of glycoproteins and neoglycoproteins by isolated rabbit hepatocytes. 631 Nov 84

Recent studies of hepatitis B virus suggest that polymeric human serum albumin may facilitate the attachment of the virus via albumin receptors to hepatocytes during the infectious process. If this hypothesis is correct, hepatocytes should express binding sites for polymeric albumin. We employed the red blood cell adherence test using albumin-coated red blood cells as indicator cells on frozen sections of normal human livers to demonstrate these binding sites. Hepatocytes showed binding activity for both polymeric and monomeric albumin from different species. The receptor-ligand interaction was temperature and pH dependent, Ca++ independent and not altered by mercaptoethanol treatment. The binding activity was sensitive to neuraminidase and pronase, but resistant to trypsin, lipase and collagenase digestion. These findings suggest that human hepatocytes display species-non-specific albumin binding sites, which are glycoproteins.
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PMID:Albumin binding sites of human hepatocytes. 631 67

Triiodothyronine (T3) production from thyroxine (T4) was studied in isolated rat hepatocytes. With an initial T4 concentration of 0.56 microM, hepatocyte T3 production was 0.029 +/- 0.003 (SEM) pmoles/min/mg protein. T3 production was greater in hepatocytes than in homogenates from the same liver prepared either before or after liver perfusion with collagenase. Most T3 produced remained within the cells under the conditions employed. Hepatocyte T3 production was dependent on cell number, medium bovine serum albumin concentration and temperature. It was stimulated by dithiothreitol, and inhibited by propylthiouracil, 3,3',5'-triiodothyronine and dinitrophenol; glutathione and ouabain had no effect. Alterations in medium glucose concentration and exposure to insulin or glucagon at several glucose concentrations in vitro did not alter T3 production. These results indicate that in hepatic tissue T3 production is enhanced when intact cellular organization is present and that insulin and glucagon do not acutely influence cell production of T3 in vitro.
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PMID:Triiodothyronine production by isolated rat hepatocytes: characterization and lack of glucoregulatory hormone effects. 639 64

The effect of natural protease inhibitors and a chemoattractant on tumor cell invasion were studied with the use of a new in vitro quantitative assay of tumor cell penetration of native connective tissue. Human amnion membrane denuded of its epithelium is composed of a continuous basement membrane (BM) attached to a dense avascular collagenous stroma. M5076 reticulum sarcoma cells, known to be highly invasive in vivo, were placed on the BM side of the amnion connective tissue. Tumor cells penetrating the full thickness of the connective tissue barrier were collected on the stromal side with a Millipore filter. N-Formylmethionyl-leucyl-phenylalanine (FMLP) at an optimal concentration of 10(-7) M stimulated the penetration of up to 600% more tumor cells into the connective tissue after 20 hours in comparison to the number of tumor cells spontaneously penetrating in serum-free media. Natural protease inhibitors blocked both FMLP-stimulated and spontaneous invasion. A bovine cartilage extract containing inhibitors of both serine proteinases and metalloproteinases caused a 500% decrease in invasion. Furthermore, a 500% inhibition of invasion was produced by a purified collagenase (metalloproteinase) inhibitor. In contrast, soybean trypsin inhibitor and bovine serum albumin did not significantly alter the invasion rate. The protease inhibitors were nontoxic and did not reduce tumor cell proliferation, attachment to the amnion, and the rate of tumor cell migration through Nuclepore filters. These data support the hypothesis that collagenolytic metalloproteinases play a necessary role in tumor cell invasion of native connective tissue.
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PMID:Effect of natural protease inhibitors and a chemoattractant on tumor cell invasion in vitro. 675 21

A procedure is described for the isolation and cultivation of microvascular endothelium from human skin. Neonatal foreskins are pooled, washed, minced, and dissociated by a mixture of collagenase and dispase. Microvascular endothelium, liberated in the form of intact capillary fragments, is incompletely separated from fibroblasts and epidermal cells by sieving through nylon mesh, followed by velocity sedimentation on 5% bovine serum albumin. The endothelium-enriched fraction has been maintained in primary culture for up to 3 weeks. The resulting epithelioid colonies have been characterized morphologically by both light and transmission electron microscopy and manifest all of the structural features that distinguish other, large-vessel endothelia in culture. In addition, immunohistochemical studies using an indirect fluorescent antibody technique demonstrate that these cells contain the endothelium-specific product, Factor VIII antigen.
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PMID:Cultivation of microvascular endothelial cells from human preputial skin. 677 46

Adipose tissue derived from open biopsies was used to develop a system for studying insulin resistance in human tissue in vitro. Subcutaneous adipose tissue obtained from obese donors was incubated in Parker's medium 199 in the absence or presence of insulin for 24 h under sterile conditions. Adipocytes were then isolated by collagenase digestion, washed thoroughly, and incubated for 2 h with multiple insulin concentrations in Krebs-Ringer phosphate buffer with 4% bovine serum albumin. Lipolysis was estimated by measuring glycerol. Basal lipolysis in adipocytes cultured with insulin did not differ significantly from that of adipocytes cultured without insulin (2.49 +/- 0.18 vs. 2.67+/- 0.58 mumol glycerol/mmol triglyceride). The maximum acute response in adipocytes prepared from tissue exposed to insulin during culture was 55% inhibition of basal lipolysis, whereas the maximum response in cells prepared from tissue not exposed to insulin chronically was 80%. Statistical analysis by paired t test showed a significant difference (P < 0.01) in the reaction of the two groups of cells to acute exposure to insulin. The insulin dose required to produce the half-maximal effect was increased from 3 to 24 microU/ml. Thus, after chronic exposure to insulin, adipocytes were not as responsive to the acute antilipolytic action of the hormone. We conclude that chronic exposure to insulin induces insulin resistance in human adipocytes.
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PMID:Insulin-induced insulin resistance of lipolysis in human adipocytes in organ culture. 699 2

A method has been developed for the purification of Kupffer and endothelial cells from rat liver by collagenase enzyme perfusion followed by centrifugal elutriation. After intravenous injection of a soluble antigen, [3H]azoaniline bovine serum albumin ( [3H]BSA), its distribution was studied in isolated cell populations from liver and spleen tissue of two aged groups of male F-344 rats. In young adult rats (6-8 months) both sinusoidal cell types contained the same amount of [3H]BSA; however, in older rats (22-24 months) the amount of antigen in the endothelial cells was significantly decreased. In comparison to the liver, the spleen retained only a small fraction of the injected dose. In order to assess the catabolic properties of both Kupffer and endothelial cells, supernatants obtained from in vitro cell culture were evaluated for both biological and physiochemical properties. Antigen was almost completely degraded by both cell types as determined by gel filtration and did not directly stimulate BSA-primed lymphocytes in vitro; however, these supernatants were shown to enhance the lymphoproliferative response of primed lymphocytes to additional antigen exposure. Kupffer cell receptors, Fc and C3, assayed by direct rosetting, did not vary with age; endothelial cells also possessed Fc receptors that were found to be unchanged with age. These studies are an initial attempt to better define our previous finding of defective antigen handling with aging by use of isolated pure cell populations.
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PMID:Antigen handling in aging. II. The role of Kupffer and endothelial cells in antigen processing in Fischer 344 rats. 716 18

Rat hepatocytes were isolated by perfusion with a Ca2+ free solution, followed by a buffer containing Ca2+ and collagenase. They specifically and avidly bind 125I-asialo-orosomucoid. At 4 degrees C, cells in minimal essential medium, incubated in the presence of saturating concentrations of 125I-asialo-orosomucoid, bound 500,000 molecules/cell. Addition of 10% horse serum to the binding reaction reduced the number bound to 200,000. Prewashing of the cells in 1% bovine serum albumin reduced the number of bound molecules by 60%. In all cases, the Kd value was approximately 7 x 10(-9) M. At 37 degrees C, the rate of uptake of 125I-asialo-orosomucoid (approximately 0.1 pmol/min/10(6) cells) is proportionately reduced by the presence of serum or albumin preparatons. Thus, it appears that the absolute number of asialoglycoprotein receptors on the surface of rat hepatocytes is not readily determined in the presence of serum or albumin and is about 500,000/cell.
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PMID:Difficulties in the quantification of asialoglycoprotein receptors on the rat hepatocyte. 741 Apr 10

Euro-Ficoll and bovine serum albumin (BSA) are two of the most commonly used density gradient media for the purification of pancreatic islets. Euro-Ficoll is based upon Euro-Collins, a cold storage medium, and must, therefore, be used at 4 degrees C. The ionic composition of BSA, however, is likely to contribute to hypothermic cellular swelling, and this may influence the efficiency of islet purification using this medium at 4 degrees C. Experience in this laboratory also suggested that batch-to-batch variation in islet purity using BSA was related to differences in BSA osmolality. The aim of this study was to assess the effect of gradient medium temperature and osmolality on the purification of human and porcine islets using BSA. Pancreata were collagenase-digested, and islets were purified on continuous linear density gradients of BSA. The distribution of insulin and amylase in each gradient was assayed, and used to calculate the median density of islets and exocrine tissue, and the efficiency of islet purification (% amylase contamination at a fixed insulin yield), using: 1) gradient osmolalities of 300, 400, and 500 mOsm/kg H2O (seven porcine pancreata), and 2) gradients at 4 degrees C and at 22 degrees C (eight human and seven porcine pancreata). Increase in density gradient osmolality produced increases in porcine exocrine tissue density which exceeded changes in islet density, resulting in improved islet purity, maximal at a BSA osmolality of 400 mOsm/kg H2O. For human pancreata there was no significant change in pancreatic tissue densities nor islet purity with temperature.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Pancreatic islet purification using bovine serum albumin: the importance of density gradient temperature and osmolality. 751 74


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