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)

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

Active digestive enzymes are involved in the pathophysiology of acute pancreatitis. Previous studies have mainly focused on the role of trypsin in the autodigestive process. The present study compares the noxious potential of different pancreatic enzymes to damage acinar cells. Acinar cells were isolated from rat pancreas by collagenase digestion. Cell viability was studied by (1) exclusion of trypan blue, (2) release of lactate dehydrogenase, and (3) release of newly synthesized proteins identified with methionine labeled with sulfur 35. Cells were then incubated in oxygenated N-2-hydroxyethylpiperazine-N-'-2-ethanesulfonic acid-Ringer solution containing different concentrations of various active digestive enzymes. Uptake of trypan blue was the most sensitive and reliable test of cell damage when compared with release of lactate dehydrogenase or radiolabeled newly synthesized proteins. All active digestive enzymes studied caused dose-dependent cell damage. The noxious potential, however, was strikingly different for the various enzymes. Pancreatic elastase in nanomolar concentrations caused marked cell damage after 45 to 90 minutes of incubation. Lipase and chymotrypsin caused a similar damage only at micromolar concentrations, whereas even millimolar concentrations of trypsin failed to cause significant damage. The present results confirmed recent work showing that lipase and phospholipase A2 probably cause cell damage through release of free fatty acids and lysolecithin. Although activation of trypsin might be the trigger to start the activation cascade in acute pancreatitis, trypsin itself is markedly less noxious to acinar cells when compared with other digestive enzymes. Elastase by far had the greatest noxious potential of all enzymes evaluated. Studies analyzing therapeutic effects of protease inhibitors should evaluate not only the inhibitory potential against trypsin but also that against other digestive enzymes, particularly elastase.
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PMID:Active pancreatic digestive enzymes show striking differences in their potential to damage isolated rat pancreatic acinar cells. 784 75

Host responses to periodontal infections include the production of several families of enzymes that are released by stromal, epithelial or inflammatory cells. Study of these enzymes in gingival crevicular fluid may lead to insights into pathogenesis and may provide a rational basis for the development of novel diagnostic tests. However, analogous to other diagnostic interventions in dentistry and medicine, validation of host enzymes as diagnostic indicators is dependent on clear-cut demonstrations of the identity of the enzyme, reproducibility, diagnostic accuracy and clinical utility. The enzyme of interest should be readily measured over a broad range of disease severity and in varied clinical settings. Ideally, the enzyme should also be an essential component of proposed pathogenic mechanisms. In this context, the connective tissue matrix degrading enzymes elastase, collagenase and gelatinase are promising because of their apparently central role in periodontal attachment loss and disease progression. Sensitive and specific assays are also available to quantify these enzymes. Other work on enzymes associated with cell death (aspartate aminotransferase, lactate dehydrogenase) and several neutrophil lysosomal enzymes (beta glucuronidase, arylsulphatase, cathepsins) has demonstrated positive associations between enzyme levels and attachment loss and inflammation. While numerous cross-sectional studies have indicated that the levels of hydrolytic enzymes in gingival crevicular fluid parallel the severity of periodontal lesions, there are much less data on reproducibility, diagnostic accuracy and clinical utility in longitudinal studies. As appropriate study design is an essential prerequisite for establishing the efficacy of host enzymes as diagnostic tests, future clinical investigations should include: (1) individuals who would most likely benefit by early diagnosis, i.e., rapidly progressive and recurrent periodontitis cases; (2) longitudinal, cohort study designs to show that attachment loss is temporally linked with large increases in enzyme activity; (3) the use of a battery of tests to overcome intrinsic problems of low predictive values when prevalence of active disease is low. In the final analysis, the utility of host enzymes as diagnostic indicators will need to be examined in randomized controlled trials in which the question is asked: are patients better off as a result of testing?
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PMID:Host enzymes in gingival crevicular fluid as diagnostic indicators of periodontitis. 792 63


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