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)

Human polymorphonuclear neutrophils, when exposed to soluble or particulate stimuli, can destroy various types of cells. The aim of this study was to investigate their toxicity against hepatocytes. Human polymorphonuclear neutrophils were incubated in basal conditions and after stimulation with 5 mg per ml opsonized zymosan in the presence of rat hepatocytes isolated by collagenase digestion. Cytotoxicity was quantified by the percentage of ALT activity released by hepatocytes in culture medium. Whereas unstimulated neutrophils exhibited only minor effects, opsonized zymosan-stimulated neutrophils induced, after 16 hr incubation, a 24.0 +/- 4.1% (mean +/- 1 S.E.) ALT activity release at a neutrophil/hepatocyte ratio of 5, and a 51.7 +/- 6.8% ALT activity release at a ratio of 20. At this ratio of 20, the ALT activity release was 9.0% at 1 hr and 24.0% at 4 hr. Three proteinase inhibitors (i.e., soybean trypsin inhibitor, alpha 1-proteinase inhibitor and fetal calf serum) decrease cytotoxicity by 78, 76 and 78%, respectively. The protective effect of proteinase inhibitors was not due to a nonspecific effect of proteins, since bovine serum albumin did not decrease the toxicity of stimulated polymorphonuclear cells. The supernatant of stimulated neutrophils was also found to be toxic against hepatocytes, and again, this effect was inhibited by soybean trypsin inhibitor, alpha 1-proteinase inhibitor and fetal calf serum. Finally, the role of proteinases was supported by the demonstration of a cytotoxic effect of two purified proteinases: porcine pancreatic elastase and human neutrophil cathepsin G. The toxicity of these proteinases was also markedly reduced by the specific inhibitors used in the study.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:In vitro toxicity of polymorphonuclear neutrophils to rat hepatocytes: evidence for a proteinase-mediated mechanism. 328 86

Several studies suggest that a portion of hepatocellular nonesterified fatty acid uptake may be carrier mediated. To further investigate this process, initial rates (Vo) of [14C]oleate uptake into rat hepatocytes, isolated by collagenase perfusion and incubated at 37 degrees C with oleate in the presence of bovine serum albumin, were studied as a function of the concentration of unbound [14C]oleate in the medium. Vo was saturable with increasing unbound oleate concentration (Km = 8.3 X 10(-8) M; Vmax = 197 pmol per min per 5 X 10(4) hepatocytes) and was not inhibited by up to 40 microM sulfobromophthalein, taurocholate, or cholic acid. Oleate uptake was sodium dependent. Vo was significantly diminished when Li+, K+, choline, or sucrose were substituted for Na+ in the incubation medium and was reduced 46% by 1 mM ouabain. Uptake was also markedly reduced after exposure of cells to metabolic inhibitors (e.g., 2,4-dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone, antimycin, KCN). To evaluate the physiologic significance of the previously isolated rat liver plasma membrane fatty acid-binding protein, the effect of an antibody directed against this protein on hepatocellular [14C]oleate uptake was examined. Preincubation of hepatocytes with the IgG fraction of this antiserum inhibited Vo of [14C]oleate by up to 65% in dose-related fashion, without altering Vo for [35S]sulfobromophthalein, [14C]taurocholate, or [3H]cholate. These data indicate that at least a portion of hepatocellular oleate uptake is energy dependent, sodium linked, and mediated by a specific liver plasma membrane-fatty acid-binding protein.
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PMID:Hepatocellular uptake of oleate is energy dependent, sodium linked, and inhibited by an antibody to a hepatocyte plasma membrane fatty acid binding protein. 345 44

Rat serum albumin has been labeled with dilactitol-125I-tyramine, (125I-DLT) a radioactive tracer which remains entrapped within lysosomes following cellular uptake and degradation of the carrier protein. Similar kinetics of clearance from the rat circulation were observed for albumin labeled conventionally with 125I or 125I-DLT-albumin, both proteins having circulating half-lives of approximately 2.2 days. In contrast, the recovery of whole body radioactivity had half-lives of approximately 2.2 and 5.1 days, respectively, for the two protein preparations, indicating substantial retention of degradation products derived from catabolism of 125I-DLT-albumin. Measurement of total and acid-soluble radioactivity in tissues 2 or 4 days after injection of 125I-DLT-albumin revealed that skin and muscle accounted for the largest fraction (50-60%) of degradation products in the body. Fibroblasts were identified by autoradiography as the major cell type containing radioactive degradation products in skin and muscle. Fibroblasts were isolated from skin by collagenase digestion, followed by density gradient centrifugation. The amount of acid-soluble radioactivity recovered in these cells was in excellent agreement with that predicted based on acid precipitation of solubilized whole skin preparations. These studies demonstrate for the first time that fibroblasts are a major cell type involved in the degradation of albumin in vivo.
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PMID:Identification of fibroblasts as a major site of albumin catabolism in peripheral tissues. 351 16

Primary breast adenocarcinomas obtained from ten patients were enzymatically digested using collagenase (1 mg/ml), hyaluronidase (1 mg/ml), elastase (0.1 mg/ml) and DNAse (0.2 mg/ml). The tumor cells were labeled with 3H-thymidine and, in some cases, with 3H-estradiol. The isolated cells were submitted successively to a Ficoll-Hypaque and a bovine serum albumin gradient, from which 12 fractions were obtained. In each fraction, several characteristics were determined: carcinoembryonic antigen (CEA), thymidine (dThd) incorporation, and estrogen receptors (ER). Three main cellular subpopulations were characterized: An intermediate density subpopulation (1.046-1.054 g/ml), in which the proliferating cells are concentrated. In this subpopulation a small number of CEA-positive cells are present, but ER containing cells are virtually absent. A high-density, small cell subpopulation that concentrates most of the ER-containing cells. This subpopulation lacks proliferating cells, but CEA-containing cells are abundant. A low-density subpopulation, lacking proliferating cells and with scarce ER-positive cells, although CEA-positive cells are frequent. These findings strongly suggest that proliferating cells lack ER.
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PMID:Determination of DNA synthesis, estrogen receptors, and carcinoembryonic antigen in isolated cellular subpopulations of human breast cancer. 352 93

The effects of a range of commercially available proteases and glycosidases on blastocyst development and hatching were examined on rabbit embryos cultured from the morula stage in a defined medium supplemented with charcoal-treated bovine serum albumin. The proteases tested were trypsin, alpha-chymotrypsin, thrombin, elastase, plasmin, papain, clostripain, collagenase, Streptomyces griseus protease and cathepsin C. The glycosidases tested were neuraminidase, alpha-mannosidase, beta-galactosidase and hyaluronidase. None of these enzymes appeared to stimulate blastocyst growth. The only enzymes which digested the embryonic investments, the zona and mucin coat, sufficiently to cause complete blastocyst hatching were trypsin and Streptomyces griseus protease at relatively low concentrations (250 ng/ml) and chymotrypsin and elastase at higher concentrations.
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PMID:A survey of the effects of proteases and glycosidases on culture of rabbit morulae to blastocysts. 353 6

A long-term cell culture system for adult cardiomyopathic hamster cardiac muscle cells has been established. The diseased and control hearts were dissociated into single cell suspension with the modifications of our previous technique using collagenase and hyaluronidase as applied to the dissociation of the adult rat heart. The postperfusion of the diseased heart with Krebs-Ringer phosphate buffer and bovine serum albumin was very helpful in obtaining greater yield of viable diseased muscle cells; the cells were cultured for 4 wk. Approximately 60% of the myocytes from the diseased heart and 85% of the myocytes from the normal heart attached to the substrates and survived throughout the culture period. Approximately 60 to 70% of the cardiac myocytes from the diseased and control hearts were bi- or multinucleated; 30% of the diseased and 80% of the normal myocytes showed rhythmic contractility. Electron microscopy revealed the presence of two kinds of cardiac muscle cells in the diseased cell culture on the basis of their myofibril content: one with scanty myofibrils and another with abundant myofibrils. Myocytes with sparse myofibrils showed certain characteristic features that included autophagic vacuoles, amorphous matrix of fine filamentous texture, scattered strips of myofibrils, and abnormal organization of the Z-line. Cardiac muscle cells with abundant myofibrillar content contained unorganized myofibrils in certain sarcomeres. These studies demonstrate the feasibility of maintaining diseased cardiac muscle cells from adult cardiomyopathic hamsters for at least 4 wk in monolayer culture.
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PMID:Isolation, long-term culture, and ultrastructural characterization of adult cardiomyopathic cardiac muscle cells. 357 Oct 99

Bilirubin (BR) and organic anionic dyes such as sulfobromophthalein (BSP), indocyanine green (ICG) and rose bengal (RB) enter the hepatocyte by a specific non-sodium-dependent membrane transport system. Two analogous but distinct Na+-dependent transport systems effect the uptake of conjugated bile acids such as taurocholate (TC) and free fatty acids such as oleate, respectively. The mechanism of uptake of the acetanilidoiminodiacetic acid (HIDA) class of biliary scintiscanning agents is unknown. Accordingly, rat hepatocytes were isolated by collagenase perfusion of the liver and differential centrifugation, and incubated with 99mTc-diisopropyl-HIDA (99mTc-DISIDA) alone, or in the presence of various concentrations of BSP, ICG, RB, oleate or TC, with and without bovine serum albumin (BSA). Initial uptake velocity (Vo) was determined from the initial slope of the cumulative radioactivity/time curve. In albumin free media, uptake of 99mTc-DISIDA was temperature- and pH-dependent, with maximal uptake at 37 degrees C. There was virtually no uptake of inorganic 99mTc. In incubations containing 15-1500 microM 99mTc-DISIDA, Vo was saturable, with estimated Vmax = 65 nmoles/min/10(6) hepatocytes, and Km = 1200 microM. In keeping with its weak albumin binding, 99mTc-DISIDA Vo was only minimally influenced by equimolar concentrations of BSA. 99mTc-DISIDA Vo was inhibited by BR, BSP, ICG and RB, as well as by a rabbit antibody to the rat liver plasma membrane BSP/BR binding protein. Surprisingly, Vo was also inhibited by TC and oleate, but not influenced either by ouabain or by substitution of Li+ for Na+ in the medium.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Studies of the hepatocellular uptake of the hepatobiliary scintiscanning agent 99mTc-DISIDA. 379 5

Antigen-retaining follicular dendritic cells (FDC) have been identified and studied in sections of lymph nodes and spleen, but studies of these cells in culture have been extremely limited. The purpose of this study was to establish techniques to release these fragile cells from mouse lymph nodes in a viable state and to identify these cells routinely in lymph node cell suspensions. FDC were obtained from passively or actively immunized popliteal lymph nodes of mice injected in the footpads with 125I-labeled human serum albumin (HSA) or horseradish peroxidase (HRP). Lymph nodes were removed 1 hr after the footpads had been injected with collagenase. After another hour of incubation in vitro with collagenase, protease, and deoxyribonuclease, FDC were released by gentle teasing and enriched by centrifugation on a low density bovine serum albumin (BSA) or Percoll gradient. Most FDC with the associated radiolabel floated at densities greater than 1.06 g/ml on BSA or Percoll gradients. Slides of the FDC-enriched fraction were prepared, using a cytobucket which allowed the cells to be affixed to glass slides by centrifugation in a less disruptive manner than by cytocentrifugation. FDC that were air-dried and fixed with 3% glutaraldehyde had a characteristic pink acidophilic cytoplasm after Wright's staining, and had a faintly basophilic euchromatic nucleus frequently with peripherally-clumped chromatin. In addition, these cells were large and irregularly shaped (up to 60 micron long). Fixation of FDC with 0.6% paraformaldehyde/ 0.9% glutaraldehyde on poly-L-lysine-coated slides resulted in a preservation of FDC which made possible visualization of long dendritic processes by Nomarski optics. Antigen presence on the cell surface was confirmed by autoradiography and, in the case of HRP, was also visualized enzymatically using diaminobenzidine. In contrast to resident peritoneal macrophages or some contaminating lymph node macrophages present on the same slides, FDC did not phagocytize opsonized sheep red blood cells (SRBC) or adhere to plastic surfaces although they did form rosettes with opsonized SRBC. Cell marker studies indicated FDC have a distinctive phenotype. They were positive for Ia, Fc receptor, and leukocyte common antigen, but negative for Thy-1, Ly-1, Ly-2, endogenous Ig, Mac-1, Mac-2, Mac-3, and F4/80, and negative to weakly positive for nonspecific esterase. Cultured FDC remained viable and retained radiolabeled antigen-antibody complexes on their surfaces and were significantly enriched for FDC.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Follicular dendritic cells in suspension: identification, enrichment, and initial characterization indicating immune complex trapping and lack of adherence and phagocytic activity. 396 23

Maintenance of functional estrogen receptors in culture has been accomplished in chick oviduct cells by manipulating the estrogen exposure before tissue dissociation. Tissue from chicks pre-treated with daily 17-beta-estradiol injections for 2 weeks or with 2 weekly diethylstilbestrol implants can be established in culture using a variety of enzymes. Tissue from animals with chronic estrogen stimulation must be withdrawn from hormone in culture at least 4 days before the digestion procedure. When tissue is digested using collagenase and pancreatin buffered by bovine serum albumin (Fraction V), large quantities of virtually fibroblast-free cultures can be established. The estrogen and progesterone receptors remain intact at normal levels using this procedure. The receptors have maintained biological function as evidenced by two hormone-dependent measurements. The first was an increase in the amount of ovalbumin mRNA transcribed in response to estrogen supplementation of the cultures compared to cultures with no estrogen. The second function was an increase in ovalbumin protein secreted into the medium upon estrogen stimulation. The protein increment demonstrated that the hormone-induced levels of mRNA were functional and capable of being translated.
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PMID:Retention of estrogen receptors in vitro requires limited estradiol exposure in vivo. 397 26

Previous studies have established that 17 beta-estradiol is the principal luteotropic hormone in the rabbit. However, a direct effect of 17 beta-estradiol on rabbit luteal cell progesterone production has been difficult to show in vitro. The goal of this study was to develop a system in which the effect of estrogen on luteal cell progesterone production could be studied in vitro. To that end, a dissociated rabbit luteal cell preparation was developed using collagenase and the resultant isolated cells were studied using a perifusion system. Optimization of the cell digest procedure revealed that: inclusion of 2% bovine serum albumin in our optimal dissociation medium increased cell yield; and animals killed by cervical dislocation maintained more stable levels of progesterone during a 7-h perifusion compared to animals killed with barbituate-induced euthanasia (euthobarb). When dissociated luteal cells were perifused with medium, stable progesterone output (greater than 80% of initial levels) was observed for 5-6 h, after which medium progesterone concentrations declined. The inclusion of 17 beta-estradiol (10(-8) M) in the perifusion medium maintained progesterone output at control levels for up to 15 h. However, the maintenance of progesterone was not noted until after 5 h of perifusion, suggesting that the effect of estradiol may be time dependent. Thus, this investigation describes a rabbit luteal cell dissociation technique and perifusion system that may be used to examine the mechanism through which estradiol acts to maintain rabbit luteal progesterone production.
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PMID:Steroidogenic effect of 17 beta-estradiol on rabbit luteal cells in vitro: estrogen-induced maintenance of progesterone production. 404 31


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