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)

Isolated and cultured human hepatocytes provide a useful model for studies of the liver cell function in man. In vitro studies using human hepatocytes are scarce, due to the limited availability and the lack of suitable methods for storage. In this study, we report the effect of deep freezing storage on the viability, fine structures and albumin synthesis of human adult hepatocytes in classical culture conditions. Hepatocytes were isolated using collagenase perfusion (9 isolations). The cell yield was 4-37 X 10(8) with a viability of 60-87%. Cryopreservation was performed in medium containing 10% DMSO and 20% fetal calf serum using a Cryoson BV-4 programmable freezer (0 degree C for 5 min, followed by a freezing rate of 1.5 degrees C/min for 20 min and 7 degrees C/min for 10 min). The cells were stored for 25-275 days in the liquid nitrogen vapor phase (-150 degrees C). Within 16 h about 80% of viable cells from freshly isolated hepatocytes whereas after cryopreservation, 55% of viable cells as determined by Trypan Blue exclusion before the cryopreservation attached to plastic and survived. Electron microscopy showed well developed tight junctions, structures similar to bile canaliculi. Cell polarity was evident. However, 'bleb' formation, more lipid droplets and lysosomes were found in cryopreserved hepatocytes during a short period after thawing. At the 3rd week, cells detached and died. These changes were associated with increased secretion of lactate dehydrogenase, whereas the albumin secretion dropped (from 10 to 4 micrograms/micrograms DNA), regardless of whether hepatocytes were cultured from fresh preparations or after cryopreservation. These findings suggest the cryopreservation is a useful technique to preserve hepatocytes for in vitro studies. Nevertheless, an improved method is necessary to increase the efficiency of cell seeding after cryopreservation.
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PMID:Cryopreservation of adult human hepatocytes. The influence of deep freezing storage on the viability, cell seeding, survival, fine structures and albumin synthesis in primary cultures. 374 87

Hepatocytes from rat liver were prepared by perfusion with collagenase, and rough and smooth microsomes and mitochondria were prepared after cell disruption. By applying 1000 lb/in2 (1 lb/in2 = 6.9 kPa) in a nitrogen bomb followed by decompression, 75% of the cells were disrupted after four consecutive treatments. Intact mitochondria, and rough and smooth microsomes with little contamination were prepared from the homogenate. A more rapid disruption was attained by a short sonication with a low output, thus increasing the efficiency of homogenization. The microsomal subfractions prepared from this homogenate were comparable to those obtained after decompression. Sonication resulted in smooth microsomes, which exhibited a higher contamination with non-microsomal membranes. These, however, were partly removed by additional centrifugation with a discontinuous sucrose gradient containing divalent cations.
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PMID:Fractionation of isolated liver cells after disruption with a nitrogen bomb and sonication. 618 14

Isolated myocytes were prepared from adult canine hearts using a combined technique of myocardial perfusion followed by incubation with collagenase. More than 60% of the cells routinely excluded trypan blue dye. Disruption of the myocytes was accomplished using high pressure nitrogen cavitation. After differential and sucrose gradient centrifugation, the peak sarcolemmal fraction averaged 100-fold enrichment in ouabain-inhibited K+-stimulated p-nitrophenyl phosphatase and 82-fold in ouabain-inhibited (Na+,K+)-ATPase. These sarcolemmal membranes are enriched in phospholipid phosphorus (1.98 mumol/mg of protein) and more than 4-fold in sphingomyelin and cholesterol. Polyacrylamide gels revealed three major protein peaks at 50,000, 91,000, and 140,000 apparent molecular weights. This work demonstrates the feasibility of preparing highly pure cardiac sarcolemma from isolated adult myocytes. The problem of cellular cross-contamination due to heterogeneity of cell types in whole myocardial tissue has been circumvented. The level of enrichment exceeds all reported preparations of cardiac sarcolemma from whole myocardium and cultured myocytes. This preparation should prove to be useful as an in vitro model for studies of physiological, pharmacological, and pathological perturbations of sarcolemmal structure and function.
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PMID:Preparation and properties of highly enriched cardiac sarcolemma from isolated adult myocytes. 624 86

Vibrio alginolyticus produces an extracellular collagenase which requires specific induction by collagen or its high-molecular-weight fragments. Peptone also induces collagenase during the late exponential and early stationary growth phases. The peptone inducers have been shown to have a broad molecular weight range between 1,000 and 60,000. The peptone inducers supported slow growth of V. alginolyticus when supplied as the sole nitrogen source in minimal medium. Digestion of the peptone inducers with purified V. alginolyticus collagenase resulted in a decrease in their inducing ability, whereas digestion with trypsin or alpha-chymotrypsin did not. This indicated that induction by the inducers required the presence of collagenase-sensitive bonds. Prolonged digestion of the inducers with collagenase did not completely eliminate the inducing ability of the inducers. The peptone inducers acted as inhibitors of collagenase. A minimal medium induction system has been developed which involves resuspending cells at high density in a medium containing succinate, (NH(4))(2)SO(4), KH(2)PO(4), and the peptone inducer. Cells grown in minimal medium induce earlier than cells grown on peptone, Casamino Acids, or tryptone. Collagenase production was shown to occur for 30 to 60 min in the presence of rifampin at levels which completely inhibit the incorporation of [(3)H]uracil into trichloroacetic acid-precipitable material. Chloramphenicol completely and immediately abolished collagenase production, which together with labeling studies has confirmed that collagenase production involves de novo synthesis of the enzyme. Both glucose and Casamino Acids repressed collagenase production, although synthesis of the enzyme continued for 30 to 60 min after their addition. The repression of collagenase production by glucose and Casamino Acids was more severe than the inhibition of enzyme formation due to addition of rifampin.
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PMID:Peptone induction and rifampin-insensitive collagenase production by Vibrio alginolyticus. 624 22

Plasminogen is present in the cornea andcan be activated to plasmin by plasminogen activator. Plasmin is able, in turn, to activate latent collagenase. This system could initiate and perpetuate the collagen degradation of corneal ulceration. This report details evidence for such a system in the cornea. Plasmin has been found to activate latent collagenase from organ cultures of ulcerating rabbit corneas and from fibroblast cultures derived from such corneas. As in the case of activation by trypsin, activation by plasmin results in the conversion of the 40,000 MW latent form to an active species of 23,000 MW. Explants of normal or alkali-burned, ulcerating corneas demonstrated plasminogen-dependent lysis of fibrin clots; frozen sections of such corneas demonstrated that lysis begins in the superficial stroma near the periphery of the cornea. Multiply freeze-thawed ulcerating corneas, but not normal corneas, showed initial lysis, not peripherally but at the ulcer region containing polymorphonuclear leukocytes. The fact that the peripheral lytic pattern existed in corneas that were obtained from eyes prefrozen in liquid nitrogen before excision of the corneas would suggest that plasminogen activator is normally contained in cells in vivo and is not made only in response to tissue injury. There was no correlation between the location of blood vessels or the presence of the corneal endothelium and the plasminogen-dependent lysis. Plasminogen activator from the ulcerating cornea and from fibroblasts was characterized by sodium dodecyl sulfate--gel electrophoresis of its cleavage products of plasminogen. The activator cleaves plasminogen into heavy- and light-chain fragments similar to those produced from plasminogen by urokinase. Plasminogen activator activity was quantitated by a new assay that restricts diffusion of the enzyme to one dimension into a narrow bore tube. The addition of plasminogen daily to cultures of ulcerating corneas resulted in earlier rises of plasminogen activator, collagenase, and collagen degradation fragments in the culture media. Although total plasminogen activator levels were not increased by the addition of plasminogen to culture, levels of both collagenase and solubilized collagen were approximately doubled. It is concluded that the plasminogen activator--plasmin system might play an important role in the destruction of stromal matrix in corneal ulceration.
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PMID:Evidence for a role of the plasminogen activator--plasmin system in corneal ulceration. 625 12

The cytochemical localization of glucose-6-phosphatase (G6Pase) and its biochemical quantification were studied in isolated and cultured adult rat parenchymal cells. Appropriate technical conditions were chosen to assume adequate ultrastructural preservation and retention of enzyme activity. Isolated hepatocytes separated by collagenase perfusion were shortly fixed in glutaraldehyde and entrapped in a pellet of fibrin. Frozen sections, 50 microns in thickness were incubated for cytochemical demonstration of G6Pase, in a slightly modified Wachstein-Meisel medium. Hepatocytes in culture, fixed for 1 min in glutaraldehyde, were impregnated in a 10% cryoprotective glycerol solution and quickly frozen in liquid nitrogen at -170 degrees C in order to induce penetration of the substrate. In these conditions, a homogeneous distribution of the enzyme was observed in both isolated and cultured cells. The cytochemical reaction appears continuous in the smooth and rough endoplasmic cisternae and in the nuclear envelope. Lead phosphate deposits, although evenly distributed, are reduced in intensity after 48 h culture. Biochemical determinations reveal the presence of a high specific enzymatic activity in isolated cells (108 nmolP/min/mg proteins), which decreases in culture, respectively to 70 and 50% of the original value, after 24 and 48 h culture. G6Pase induction by glucagon was obtained after 48 and 72 h in culture.
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PMID:Glucose-6-phosphatase distribution in isolated and cultured adult rat hepatocytes. 626 35

Our study describes a useful procedure for cryopreservation of pancreatic islet cells. The pancreatic islets of adult hamsters were collected by collagenase digestion succeeded by gradient centrifugation and were dispersed by EDTA-Dispase treatment. The dispersed cells were suspended in medium consisting of 90% Dulbecco's modified Eagle's medium and 10% fetal bovine serum, supplemented with 10% dimethyl sulfoxide or glycerol. One milliliter each of the cell suspensions containing 10(6) cells was distributed into 2 ml polypropylene tubes, processed for freezing under six cooling conditions, and stored in liquid nitrogen (-196 degrees C). Of the various conditions tested, the cells suspended in dimethyl sulfoxide-containing medium and cooled in a program freezer at a rate of 0.5 degrees C/min down to -40 degrees C, succeeded by a rate of 5 degrees C/min down to -70 degrees C, resulted in the highest recovery rate of cells, 61.2% +/- 3.1%. This rate was comparable to those of several tissue culture cell lines frozen under similar conditions. Recovered cells preserved their morphologic characteristics under light and phase-contrast microscopy and formed cell sheets in culture. Response of insulin secretion to 3 mg/ml glucose appeared 6 hours after thawing, and the response to both 3 mg/ml glucose and 10 mmol/L theophylline was recovery to the same level as nonfrozen islet cells after 12 hours. The applicability of cryopreserved cells for the detection of islet cell surface antibody was demonstrated by the indirect method of immunofluorescence using islet cell surface antibody-positive human sera.
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PMID:Cryopreservation of pancreatic islet cells. 637 Nov 65

Pancreatic islet cells of rats obtained by the collagenase digestion method were preserved from 7 to 10 days at -80 degrees C in a deep freezer or at -196 degrees C in liquid nitrogen with Dulbecco modified Eagle's medium, supplemented with 10% or 20% DMSO (dimethyl sulfoxide); thawing was performed in a 37 degree C water bath. Cryopreserved islets were morphologically almost intact, and possessed approximately 50% of the insulin secretion activity of the control groups. About 300 pancreatic islets preserved at -196 degrees C with 20% DMSO were transplanted into the portal veins of streptozotocin-induced diabetic rats. Rats recovered from the diabetic state, and the normalized condition was maintained up to 20 weeks, although 4 weeks were needed before blood and urine glucose reached normal levels after transplantation. Intact B cells were found in the transplanted islet cell masses in the liver of the recipients, but B cells of the recipient's pancreases (streptozotocin-treated rats) showed a marked decrease, as well as degenerative changes.
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PMID:Transplantation of cryopreserved pancreatic islets into the portal vein. 678 60

Rat-liver cells obtained through perfusion and collagenase treatment were frozen according to a fixed schedule and stored in liquid nitrogen at -196 degrees C. The metabolic capacity of the cells after thawing was tested by measuring their ability to achieve metabolic activation of cyclophosphamide, which in turn was measured as induction of sister-chromatid exchanges in human lymphocytes. The viability and metabolic efficiency of the frozen and thawed cells were comparable with those of fresh cells.
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PMID:Preservation of freshly isolated liver cells in liquid nitrogen at -196 degrees C. 733 87

Hepatic macrophages and endothelial cells play an important role in the clearance of endotoxin from the portal circulation. These cells are activated by endotoxin to release reactive mediators including superoxide anion, hydrogen peroxide, and nitric oxide, which have been implicated in hepatic inflammation and tissue injury. In the present studies we analyzed mechanisms regulating the production of nitric oxide by hepatic macrophages and endothelial cells following in vivo exposure to endotoxin. Rats were injected intravenously with Escherichia coli lipopolysaccharide (LPS, 5 mg/kg). Cells were isolated from the animals 48 h later by in situ perfusion of the liver with collagenase and pronase followed by differential centrifugation and centrifugal elutriation. We found that macrophages and endothelial cells from both untreated and endotoxin-treated rats readily synthesized nitric oxide following in vitro stimulation with interferon-gamma (IFN-gamma) and LPS alone and in combination. This response was dependent on l-arginine and was blocked by two nitric oxide synthase inhibitors, NG-monomethyl-l-arginine and l-canavanine. Macrophages produced more nitric oxide in response to LPS or LPS plus IFN-gamma than endothelial cells. In addition, nitric oxide production by both cell types in response to LPS plus IFN-gamma was increased after treatment of rats with endotoxin. Macrophages appeared to be more sensitive than endothelial cells to the in vivo effects of this inflammatory stimulus. Northern and Western blot analysis demonstrated that nitric oxide production by macrophages and endothelial cells in response to LPS plus IFN-gamma was due to increased expression of an inducible form of nitric oxide synthase (iNOS) mRNA and protein. Using fluorescence image analysis, iNOS protein was found to be localized in the cytoplasm of the cells. Treatment of rats with endotoxin was associated with increased expression of iNOS protein in the macrophages. The phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) also stimulated nitric oxide production by macrophages and endothelial cells from endotoxin-treated rats, although not as effectively as LPS and IFN-gamma. Macrophages were more responsive than endothelial cells to TPA. Furthermore, depletion of the cells of glutathione using buthionine sulfoximine had no major effect on nitric oxide production by macrophages but resulted in small but significant inhibition in endothelial cells. This suggests that this sulfhydryl-containing tripeptide does not regulate intracellular levels of reactive nitrogen intermediates in activated macrophages.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Distinct patterns of nitric oxide production in hepatic macrophages and endothelial cells following acute exposure of rats to endotoxin. 752 31


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