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)

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

Nephrotoxicity is the major adverse effect produced by chronic exposure to cadmium (Cd). This injury is thought to be caused by the Cd-metallothionein complex (CdMT). In intact animals, CdMT is more efficiently taken up by the proximal tubules than CdCl2 and results in more renal damage. However, the mechanism(s) by which CdMT produces renal injury is not yet understood completely. Therefore, we used cultured renal proximal tubular cells to study the nephrotoxicity of CdMT and CdCl2. Rat kidney proximal tubules were isolated by collagenase perfusion, followed by percoll isopycnic centrifugation. 14C-alpha-methylglucose uptake and lactate dehydrogenase leakage were used as indices of nephrotoxicity. Surprisingly, CdMT was less toxic than CdCl2 to the cultured rat proximal tubule cells, as well as to cultured LLC-PK1 cells (a pig kidney proximal tubular cell line). Consistent with these observations on nephrotoxicity, 109CdMT uptake into these cultured renal cells was much less than that of 109CdCl2. Transwell cultures of LLC-PK1 cells were also used to examine the toxicity and uptake of CdCl2 and CdMT following basolateral and apical exposure. Uptake of both CdCl2 and CdMT from basolateral exposure was higher than that from apical exposure. Again, more 109CdCl2 was taken up and more cytotoxicity was observed in the CdCl2- than CdMT-exposed cells. In summary, CdCl2 is more toxic than CdMT to cultured rat kidney proximal tubules as well as LLC-PK1 cells. This is in contradiction to the greater in vivo nephrotoxic effects of CdMT than CdCl2. Therefore, cultured renal cells do not appear to be an appropriate model to study the nephrotoxicity of CdMT; transport of CdMT into proximal tubular cells in vivo does not appear to be maintained in vitro.
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PMID:Nephrotoxicity of CdCl2 and Cd-metallothionein in cultured rat kidney proximal tubules and LLC-PK1 cells. 794 May 41

The aim of the study was to investigate the effect of aging on cytoprotective properties of prostaglandins. Hepatocytes were obtained by collagenase perfusion of livers of young (4-6 mo) and old (24-28 mo) male Wistar rats. Cells were incubated for 1.5 h in Krebs-Ringer-bicarbonate buffer containing glucose and 3H-leucine in the presence of galactosamine (2.5-100 mM), PGE1, or two prostacyclin analogues: 9 beta-methylcarbacyclin and TRK-100. Cell damage was assessed by decrease in the rate of protein synthesis measured as 3H-leucine incorporation into acid precipitable material, and by increase in lactate dehydrogenase release into the medium. Hepatocytes from old rats were more susceptible to suppression of protein synthesis by GalN than cells of young ones. Preincubation of cells for 15 min with 9MC (41-560 nM) or PGE1 (10-100 nM), but not with TRK-100, before adding 10 mM GalN, led to a partial recovery of protein synthesis in both age groups. GalN increased LDH release and decreased ATP/ADP ratio to a similar extent in hepatocytes of young and old rats; both parameters were not altered by preincubation of cells with PGs. PGE1 and 9MC, but not TRK-100, elevated cyclic AMP content in hepatocytes of young but not old rats. Glucagon and forskolin similarly increased cyclic AMP content in cells of both young and old animals. These in vitro results suggest that PGE1 and some prostacyclin analogues might protect hepatocytes of both young and old rats from chemical damage, and stress the necessity for further research on cyto- and hepato-protection in the elderly.
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PMID:Prostaglandin cytoprotection of galactosamine-incubated hepatocytes isolated from young and old rats. 803 Aug 38

In this study, we evaluated cardiac myocyte viability and function under hypothermic conditions with four types of storage solutions, saline solution, Euro-Collins solution, University of Wisconsin solution, and MCDB 107 medium. Cardiac myocytes were isolated from neonatal rat ventricles by collagenase dispersion and cultured for 4 days with MCDB 107 medium. A total of 12.5 x 10(5) myocytes per culture dish was used and the myocytes were incubated at 4 degrees C for 6, 12, 18, and 24 hours in the various storage solutions. After each incubation time, creatine kinase and lactate dehydrogenase were measured in the storage solutions. The myocytes were then incubated for 24 hours at 37 degrees C to evaluate the recovery of the myocyte beating rate. In the MCDB 107 group (n = 7), the recovery ratio of myocyte beating rate was complete by 12 hours, then decreased to 44.8% of control (beating rate before hypothermic incubation) at 24 hours. The saline, Euro-Collins, and University of Wisconsin groups (n = 7 each) had significantly lower recovery ratios than the MCDB 107 group (at 12 hours: 61.0%, 32.2%, and 48.9%; at 18 hours: 0.0%, 5.5%, and 15.1% of control, respectively). Release of creatine kinase and lactate dehydrogenase in the MCDB 107 group gradually increased and at 24 hours was 143.2 mIU/flask and 486.2 mIU/flask, respectively. However, the saline and University of Wisconsin groups had significantly increased creatine kinase and lactate dehydrogenase values at 24 hours (creatine kinase: 334.6 and 319.6 mIU/flask; lactate dehydrogenase: 821.6 and 654.4 mIU/flask, respectively). The Euro-Collins group showed the greatest increase in both markers (creatine kinase: 1587.5, lactate dehydrogenase: 2106.9 mIU/flask). In summary, saline and University of Wisconsin solutions showed a beneficial effect on recovery of myocyte viability at 12 hours compared with Euro-Collins solution, however MCDB 107 medium had the best overall protective effect on cultured myocytes. Accordingly, alternate hypothermic storage solutions, such as cell-culture medium, may have protective characteristics that are suitable for cardiac preservation.
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PMID:Cardiac myocyte functional and biochemical changes after hypothermic preservation in vitro. Protective effects of storage solutions. 828 90

Lactate dehydrogenase-containing vesicles have been isolated from the extracellular matrix of the calvaria of new-born mice. The calvariae, intramembranous ossification tissue, were removed from 2-day-old mice, followed by the separation of the extracellular matrix vesicle fraction after collagenase digestion. Lactate dehydrogenase-containing vesicles with a density different from that of matrix vesicles were detected in the matrix vesicle fraction. Lactate dehydrogenase in these vesicles did not result from cell lysis and vesicle capture during the preparation of the matrix vesicle fraction. The isoenzyme pattern of lactate dehydrogenase in lactate dehydrogenase-containing vesicles was nearly identical to that of cytosolic lactate dehydrogenase of the cell fraction. Other cytosolic enzymes were not detected in lactate dehydrogenase-containing vesicles, suggesting the presence of a mechanism for specific uptake of cytosolic lactate dehydrogenase during the in vivo formation of the vesicles. This is the first report on the presence of lactate dehydrogenase-containing vesicles in the intramembranous ossification site.
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PMID:Presence of lactate dehydrogenase-containing vesicles in an intramembranous ossifying tissue: new-born mouse calvaria. 834 86

Anterograde or retrograde perfusion of rat liver with digitonin selectively permeabilizes the periportal or the perivenous zone of the hepatic lobule. Digitonin perfusion is used to analyze the effluents released by permeabilized hepatocytes or, combined with collagenase perfusion, to obtain cell suspensions enriched in either periportal or perivenous hepatocytes. Despite the wide use of digitonin to study lobular heterogeneity, its affects on rat hepatocytes are not well documented. We therefore analyzed the effects of digitonin perfusion on the intracellular content of rat hepatocytes by combining electron microscopy, histoenzymology, immunohistochemistry, and in situ hybridization. At the concentration currently used for the study of lobular heterogeneity, digitonin perfusion induced a marked cytosolic clarification of permeabilized hepatocytes, while most organelles except mitochondria were well preserved. In the digitonin-altered zones, there was no histochemical detection of non-membrane-bound enzymes (lactate dehydrogenase, glutamate dehydrogenase), whereas membrane-bound enzymes (succinate dehydrogenase, beta-hydroxybutyrate dehydrogenase, NADPH dehydrogenase, glucose-6-phosphatase) were still detected. Immunohistochemistry and in situ hybridization revealed significant amounts of several plasma proteins (albumin, alpha 2-macroglobulin, alpha 1-inhibitor 3, alpha 1-acid glycoprotein) and their respective mRNAs in digitonin-permeabilized hepatocytes. The demonstration that digitonin-permeabilized hepatocytes retain many intracellular constituents shows that biochemical analysis of cellular effluents released from digitonin-permeabilized hepatocytes must be interpreted with caution and that the apparent characteristics of cell suspensions obtained by the digitonin-collagenase technique might be significantly altered by contamination with permeabilized hepatocytes from the opposite zone.
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PMID:Effects of digitonin on the intracellular content of rat hepatocytes: implications for its use in the study of intralobular heterogeneity. 815 38

The model of rat primary hepatocytes incubated in DMEM/F12 (Ham) medium was used for studying the influence of the cAMP-effectors epinephrine (100 microM), norepinephrine (100 microM), glucagon (1 microM) and isoproterenol (1-1000 microM) as well as the synthetic cAMP-analogon dibutyryl-cAMP on the metabolism of metallothionein. Liver parenchymal cells isolated by a two-step collagenase perfusion were incubated with DMEM/F12 containing 5% (v/v) fetal calf serum (FCS) and 20 microM zinc in Petri dishes. Experiments were initiated after a 24 h equilibration period by adding the agonists for 18 h. MT in hepatocyte homogenates was quantified by the 109Cd-hemoglobin-binding assay. Cell viability was assessed by the activity of the cytosolic enzyme lactate dehydrogenase (LDH) liberated into the culture medium and by trypan blue exclusion. Isoproterenol and glucagon produced a significant increase of cytosolic MT about 50%. In contrast, incubation with epinephrine and norepinephrine did not lead to any significant effects in the amount of hepatic metallothionein. Simulating the influence of cAMP by dibutyryl-cAMP (500 microM) did not affect the content of hepatic metallothionein. To examine transcriptional and translational regulatory effects supplementation of cycloheximide (0.1-500 microM) and actinomycin D (0.1-100 microM) showed a total inhibition of the agonist induced amounts. Particularly in combination with isoproterenol low LDH activities reflected a high viability of hepatocytes. In conclusion, in primary hepatocyte cultures cAMP-mobilizing-agonists like isoproterenol and glucagon indicate an independent effect on the MT-metabolism. This is possibly due to the de novo synthesis of the protein because suppression by actinomycin D can be observed. However, cAMP-effectors do not seem to be involved in the induction of metallothionein because theophylline and dibutyryl-cAMP did not affect MT-metabolism by suppressing the phosphodiesterase or by stimulating the cAMP-cascade.
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PMID:Influence of cAMP-effector-agonists on the synthesis of metallothionein in rat primary hepatocytes. 858 45

An hepatocyte culture system was developed for potential use in toxicological studies in vitro. Rat hepatocytes were isolated by two-step collagenase perfusion and cultured on Vitrogen-coated Permanox dishes in a modified Chee's medium containing 1 microM dexamethasone and 1% dimethylsulfoxide. The cells remained highly viable for at least 10 d as determined by lactate dehydrogenase release and total protein levels. Albumin secretion into the medium, as a measure of differentiated function, was maintained at elevated levels over the course of 10 d in culture. A number of CYP activities were determined by the analysis of testosterone metabolism in freeze-thawed cells, diazepam metabolism in live cells, and specific assays for CYP 1A1/2, 2B1/2, 2E1, and 3A. Results of these assays indicated that a wide range of CYP isozymes were maintained, some activities were enhanced under the conditions of culture and some activities were inducible. Activities of the phase II enzymes, glutathione S-transferase and UDP-glucuronosyltransferase, and glutathione levels were also maintained in the cultured hepatocytes for at least 6 d. These results strongly support the use of this hepatocyte culture system for in vitro toxicological studies.
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PMID:Characterization of a primary hepatocyte culture system for toxicological studies. 872 45

The phagocytic ingestion of reference strains and clinical isolates of Fusobacterium nucleatum, Porphyromonas gingivalis, and Treponema denticola by polymorphonuclear leukocytes (PMNs) and the concomitant release of PMN granule proteinases were studied by specific functional and immunological assays. PMNs were incubated with the microorganisms anaerobically at 37 degrees C for indicated time periods. The suspensions and pellets were used for phagocytic ingestion assay and electron microscopic study, respectively. The supernatants were used for the measurements of the amounts and activities of the released PMN enzymes including PMN gelatinase (MMP-9), collagenase (MMP-8), serine proteases (elastase and cathepsin G), and lactate dehydrogenase (LDH). Both fluorescence microscopy and transmission electron microscopy showed that F. nucleatum, P. gingivalis and T. denticola were ingested by the PMNs in comparable numbers. However, measurements of the enzymes released from the triggered PMNs revealed major differences among the three species. High amount of elastase was released from the PMNs triggered by F. nucleatum, but not by P. gingivalis or T. denticola. The treatment of PMNs with P. gingivalis whole cells resulted in the release of gelatinase partly in the 82 kD active form, suggesting proteolytic activation of the degranulated 92 kD proMMP-9. The 82 kD active form of gelatinase was not detected upon triggering the PMNs with F. nucleatum and T. denticola. The PMN-bacteria interaction did not result in release of LDH from triggered PMNs indicating the proteinase release was not due to the PMN cell death. The results show that the susceptibilities of the 3 potentially periodontopathogenic microorganisms, F. nucleatum, P. gingivalis and T. denticola to phagocytic ingestion are not directly related to the amounts and activities of PMN enzymes released during the bacteria-PMN interactions. As PMN degranulation is considered as one of the major pathogenic mechanisms in periodontitis, the observed differences among the microorganisms may be important virulence characteristics of these species.
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PMID:Release and activation of human neutrophil matrix metallo- and serine proteinases during phagocytosis of Fusobacterium nucleatum, Porphyromonas gingivalis and Treponema denticola. 914 46


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