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)

In order to determine the least injurious method of cell isolation, effluent perfusates from isolated rat hearts were examined for lactate dehydrogenase activity during cellular isolation procedures. Correlation of these results with the perfusates, with modifications in the perfusates, and with the yield of intact cells isolated, indicated that perfusion of isolated hearts with solutions containing bovine serum albumin and/or collagenase can result in severe cellular injury. These effects were significantly modified by the presence of calcium ion. These results indicated that the concentration of calcium ion during isolation is critical to successful isolation of calcium tolerant, functional adult heart cells.
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PMID:Release of lactate dehydrogenase during isolation of adult rat heart cells. 625 64

The response of the rat lung to a range of doses of quartz at 50 and 100 days after its administration by intratracheal instillation has been assessed by bronchopulmonary lavage. The effects on the number of polymorphonuclear leukocytes (PMN), lymphocytes and macrophages are described. In addition the concentrations of soluble protein and hydroxyproline and the activities of lactate dehydrogenase, PZ peptidase and collagenase in lavage fluid supernatants were measured and an assessment of the hydroxyproline content of recovered cells was made. Finally PZ peptidase and collagenase were assayed in PMN-enriched cell fractions and in samples obtained from short-term culture of recovered macrophages. There was a dose-dependent increase in the recovery of all three cell types, and in the amounts of lactate dehydrogenase, protein and hydroxyproline in lavage fluids, which showed no signs of resolution over the 100-day period studied. Measurements of PZ peptidase and collagenase suggested that the PMN, not the macrophages, are the major source of these degradative enzymes. The relevance of these findings with regard to the importance of PMN in quartz-induced fibrosis is discussed.
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PMID:Evidence for a dose-dependent inflammatory response to quartz in the rat lung and its significance in early changes in collagen metabolism. 631 54

Hepatocytes were prepared from rainbow trout by perfusion in situ with collagenase and hyaluronidase. Preparations normally showed high initial viability (95 +/- 5% dye exclusion, 92 +/- 5% lactate dehydrogenase retention) and gradually decreased in viability and glutathione concentration over 5 hours. Cellular metabolism of aflatoxin B1 (AFB1), a potent hepatocarcinogen, was characterized by an investigation of the following parameters: kinetics of AFB1 metabolism and DNA adduct formation, dose response, viabilities of detoxication and activation pathways with time, influence of organic solvents, and effect of variation in cell concentration. The AFB1 metabolites and DNA adducts were resolved and quantitated by high-performance liquid chromatography. From these results a standardized assay procedure was derived which we used to examine AFB1 metabolism and DNA adduct formation in hepatocytes from fish fed dietary substances known to alter the carcinogenic response to this mycotoxin. Dietary beta-naphthoflavone, which strongly inhibits AFB1 carcinogenesis in rainbow trout, dramatically and reproducibly altered AFB1 binding and metabolism in isolated hepatocytes. Overall rate of AFB1 metabolism and rates of detoxication reactions increased, whereas DNA binding decreased. Dietary cyclopropenoid fatty acids, powerful synergists and promoters of AFB1 carcinogenesis in trout, also repressed AFB1-DNA binding. Both dietary factors appeared to depress initial DNA damage by AFB1 but operated through different metabolic pathways to do so.
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PMID:Dietary modification of aflatoxin B1 carcinogenesis: mechanism studies with isolated hepatocytes from rainbow trout. 643 Dec 90

High yields of Ca2+ - stable myocytes were obtained by perfusion of adult rat heart with a buffered collagenase medium followed by mincing and three additional digestion periods. Release of lactate dehydrogenase, respiratory control, content of ATP and creatine phosphate, electrical stimulation and attachment to extracellular matrix components indicated that the sarcolemma of the isolated myocytes remained intact and that the cells maintained some of the most basic physiological functions. The myocytes maintained their rod-shape in a medium containing 2.5 mM of Ca2+ and their release of LDH was slow. Some of the myocytes were contracting spontaneously, at a low rate, in an abrupt end-to-end contraction. Other cells appeared quiescent but they were all able to respond to external electrical stimulus. The oxygen consumption was measured by a perifusion method. In different preparations the basal consumption was 14-26 nmol O2/min X 10(5) rod-shaped myocytes. Freshly isolated rod-shaped heart cells attached in 30 minutes to dishes coated with collagen type IV, laminin or fibronectin but did not attach to dishes coated with collagen type I or III or to collagen gels. Attachment occurred at the ends of the cells.
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PMID:Isolation, characterization and adhesion of calcium-tolerant myocytes from the adult rat heart. 672 24

Cultured rat embryonic skin fibroblasts phagocytosed rat mast cell granules added to the medium or released from co-cultured mast cells by rabbit anti-rat IgE or Compound 48/80. Electron microscopy of fibroblasts incubated with mast cell granules revealed that granules adjacent to the plasmalemma were engulfed by long, thin cytoplasmic processes. Internalization proceeded to fusion of encircling processes and formation of phagosomes. Microtubules and 60 A microfilaments became closely associated with the phagosomal membrane to which small vesicles and cisternae of endoplasmic reticulum fused. The rate of uptake of mast cell granules by fibroblasts was dependent upon temperature and granule concentration. Cytochalasin B inhibited granule uptake whereas colchicine and nocodazole had little effect. Phagocytosis was not influenced by actinomycin D and cycloheximide, was partially inhibited by fluoride, and was markedly inhibited by cyanide, azide, and 2,4-dinitrophenol. Supernatants from fibroblast cultures incubated with mast cell granules for 24 and 48 hr, during which period phagocytosis occurred, contained elevated levels of collagenase and beta-hexosaminidase, but normal levels of lactate dehydrogenase and superoxide dismutase. These results support the concept that immediate hypersensitivity reactions are in part terminated by phagocytosis of biologically active discharged mast cell granules by resident connective tissue fibroblasts. Further, it is suggested that a consequence of this process is an alteration in fibroblast behavior, providing a unique link between immediate hypersensitivity reactions and connective tissue responses to inflammation.
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PMID:Phagocytosis of mast cell granules by cultured fibroblasts. 684 86

The presence of neutrophils within the lung is a characteristic feature of a variety of lung diseases. To evaluate the potential role of alveolar macrophages in modulating the migration of neutrophils to the lung, normal human alveolar macrophages obtained from volunteers by bronchopulmonary lavage, were exposed for various periods of time in vitro to heat-killed microorganisms, and noninfectious particulates, immune complexes, and the macrophage supernates were evaluated for chemotactic activity. The microorganisms, noninfectious particulates, and immune complexes were chosen as stimuli for alveolar macrophages because these stimuli are representative of a spectrum of pathogenic agents that cause neutrophil accumulation in the lower respiratory tract. After incubation with each of these stimuli, alveolar macrophages released low molecular weight (400-600) chemotactic factor(s) (alveolar macrophage-derived chemotactic factor[s] [AMCF]) with relatively more activity for neutrophils than monocytes or eosinophils. Checker-board analysis of the AMCF revealed that the factor was primarily chemotactic and not chemokinetic for neutrophils. The selectivity for neutrophils vs. monocytes could not be explained by a selective deactivation of monocytes, because the AMCF was more potent in deactivating neutrophils than monocytes. Partial characterization of AMCF demonstrated it was heterogeneous with the following features: (a) stable to heating at 56 and 100 degrees C for 30 min; (b) stable over a pH range of 1.0 to 12.0 for 60 min; (c) stable after exposure to trypsin, papain, chymotrypsin, collagenase, and elastase; (d) partially inhibited by serum chemotactic factor inhibitor(s); (e) two major isoelectric points (pI 7.6 and 5.2); and (f) partially extractable into ethyl acetate, ether, and hexane. Although AMCF was, at least, partially lipid in nature, it did not appear to be similar to previously described lipid chemotactic factors (e.g., hydroxy-derivatives of 5,8,10,14-eicosatetraenoic acid); analysis by gas chromatography-mass spectrophotometry of AMCF extracted into ethyl acetate did not reveal the presence of 5,8,10,14-eicosatetraenoic acid. The macrophage supernates containing the AMCF also stimulated normal human neutrophils to release lysozyme and lactoferrin but not lactate dehydrogenase. These studies suggest that a wide variety of potentially pathogenic stimuli induce normal alveolar macrophages to generate a low molecular weight chemotactic factor(s) that preferentially attracts neutrophils. Because alveolar macrophages are normal residents of alveoli, it is likely that by releasing this factor(s) macrophages play a significant role in amplifying the inflammatory processes seen in many acute and chronic lung diseases.
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PMID:Human alveolar macrophage-derived chemotactic factor for neutrophils. Stimuli and partial characterization. 699 85

The preparation, viability, and prostaglandin (PG) binding characteristics of dispersed smooth muscle cells from the rabbit oviduct have been determined. Cell suspensions were prepared by enzymatic degradation of the intracellular matrix and subsequent mechanical dispersion with a wide bore pipette. The digestion media consisted of a modified Hanks' Balanced Salt Solution, pH 7.1, containing 1.6 U/mg wet wt elastase and 8 U/mg wet wt collagenase. This method provided a yield of single smooth muscle cells of approximately 3 x 10(6) cells/100 mg wet wt within 3-4 h of organ removal. Cell viability was determined by trypan blue dye exclusion, retention of lactate dehydrogenase, absence of 57Co-EDTA uptake, and ouabain sensitivity of cationic transport. Roughly 80% of the isolated cells remained viable after the digestion procedure. The dispersed cells specifically bound [3H]PGE2 and [3H]PGF2 alpha. Scatchard analysis of the binding data revealed separate homogenous populations of high affinity sites for both PGE2 and PGF2 alpha. The equilibrium dissociation constants and total sites per cell were 0.55 nM and 11,332 for PGE2 and 0.19 nM and 5,154 for PGF2 alpha, respectively. Specific labeled PG binding was inhibited in a concentration-dependent manner by increasing amounts of unlabeled PG. Inhibition of labeled PGE2 binding by unlabeled PGF2 alpha, and vice versa were negligible, except at high concentrations. The results indicate that smooth muscle cells can be enzymatically dispersed from the rabbit oviduct with minimal damage. Also, these cells possess distinct specific binding sites for PGE2 and PGF2 alpha that differ in regard to affinity and total number of sites per cell.
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PMID:Preparation of smooth muscle cell suspensions from the rabbit oviduct and prostaglandin binding analysis. 700 18

Rat cardiac myocytes were isolated by heart perfusion in the presence of collagenase and incubated in the absence of presence of oxygen. As a result of anoxia, there was a gradual increase in plasma membrane permeability, noted as a decrease in trypan blue exclusion frequency, leakage of cytosolic lactate dehydrogenase and intracellular accumulation of the isotope compound 99Tcm-gluconate. The changes in plasma membrane permeability properties were preceded by a marked decrease in cellular ATP level and an increased proportion of contracted myocytes. The ability of the myocytes to resynthesize ATP and to recover from the anoxic injury upon reoxygenation decreased gradually with the length of initial anaerobic incubation during the first 25 min and disappeared after 30 min of anoxia, indicating that the anoxic injury to the isolated rat cardiac myocytes becomes irreversible after 25--30 min of anoxia. It is suggested that a decreased energy level is of primary importance for the initiation of cell injury in anoxia and that it is followed by cell contracture and subsequently by a disturbed plasma membrane function, cell swelling and death. This experimental model system of isolated viable rat cardiac myocytes is suitable for problems dealing with reversibility of myocytic injury.
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PMID:Isolated rat cardiac myocytes as an experimental tool in the study of anoxic cell injury. Effect of reoxygenation--a preliminary report. 720 17

Biochemical and morphological properties of rat hepatic parenchymal cells isolated without calcium were compared to cells isolated by adding calcium to the isolation medium at the time of addition of collagenase. Calcium contents of the two cell preparations were 4.5 +/- 0.3 and 10.5 +/- 0.5 nmol/mg dry wt, respectively (P les than 0.001). Magnesium content of both preparations was 37 nmol/mg dry wt. Potassium contents were 92 and 154 meq/l, respectively (P less than 0.001). Potassium content of calcium-deficient cells increased to 161 meq/l following incubation for 30 min in a medium containing 1.6 mM ionized calcium. When incubated in a medium containing a subphysiologic concentration of ionized calcium, calcium-deficient cells rapidly lost the ability to exclude trypan blue and to retain lactate dehydrogenase activity. As contrasted to calcium-sufficient hepatocytes, calcium-deficient cells failed to accumulate alpha-aminoisobutyric acid by active transport and lacked microvilli and nuclear contents. This study supports simultaneous addition of calcium and collagenase to the isolation medium as a means for preserving physical, functional, and morphological integrity of isolated hepatic parenchymal cells.
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PMID:Deleterious effects of calcium deprivation on freshly isolated hepatocytes. 724 60

In order to evaluate the suitability of cytopathological criteria in isolated fish hepatocytes as endpoints in (eco)toxicological research, liver cells isolated from rainbow trout (Oncorhynchus mykiss) by collagenase perfusion were exposed in vitro for up to 5 days to sublethal dilutions of two seepage water samples collected from garbage dumps. Hepatocytes were analysed with respect to acute (lactate dehydrogenase leakage) and sublethal toxicity (electron microscopy, stereology). In addition, acute toxicity (24 h) was tested in the piscine fibrocytic cell line R1 by means of crystal violet staining and neutral red retention. Acute toxicity in R1 cells and isolated hepatocytes could only be documented for sample I at dilutions of 1:2 and 1:4. This difference in toxicity could be corroborated by cytological alterations in isolated hepatocytes, which could be documented for dilutions of 1:100 and 1:8 in samples I and II, respectively. Ultrastructural changes were time- and dose-dependent and included reduction of hepatocellular volume, disturbance of intracellular compartmentation, modified heterochromatin distribution, transformation of rough endoplasmic reticulum into concentric membrane whorls, proliferation of lysosomes and cytoplasmic vacuoles, as well as reduction of hepatocellular glycogen. Although several hepatocellular reactions were found after exposure to either sample, the syndrome of ultrastructural alterations allowed clear differentiation between the two samples. Results illustrate that cytological effects far below macroscopically detectable damage can be discovered not only in intact fish, but also in fish cell culture systems. On the basis of the data presented, a multi-tiered test procedure for aquatic toxicity assessment exclusively based on tests with fish cell culture systems is proposed: (1) rapid screening for acute toxicity with permanent cell lines; (2) short-term tests with more complex, yet more sensitive systems such as primary hepatocytes with straightforward biochemical endpoints; (3) prolonged exposure of isolated hepatocytes in combination with ultrastructural and biochemical investigations as a sensitive tool to detect adverse effects at environmentally relevant toxicant concentrations.
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PMID:Acute and sublethal toxicity of seepage waters from garbage dumps to permanent cell lines and primary cultures of hepatocytes from rainbow trout (Oncorhynchus mykiss): a novel approach to environmental risk assessment for chemicals and chemical mixtures. 772 25


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