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)

We determined the role of cytokeratin (CK) intermediate filaments in the excretory function of hepatocytes in cultured hepatocytes containing Mallory bodies (MBs) from the livers of griseofulvin (GF)-fed mice. Hepatocytes for primary culture were obtained from GF-fed and control mice using the 0.1% collagenase perfusion method. Each component of the cytoskeleton in cultured hepatocytes and liver frozen sections was visualized by immunofluorescence. The whole mount extraction of hepatocytes was carried out using 0.5% Triton X-100. To examine the excretory function of the bile canaliculi (BC), fluorescein diacetate and horseradish peroxidase were used as visible excretory products. Thin sections of the cultured cells were made by the "pop-off" method for electron microscopic examination. Frozen sections of livers from the GF-fed mice showed that the MBs were stained with a rat monoclonal antibody to mouse CK, but the CK filaments in the cells containing MBs did not stain. The intercellular BC were reduced in number in the livers of the GF-fed mice compared with the controls. At 3 hours after seeding, hepatocytes with MBs were not stained, but by 24 hours the CK filament network stained normally in cells containing MBs. The loss of staining of the CK filaments was therefore rapidly reversible in the absence of GF in tissue culture. This reversion to normal was prevented by adding 2 x 10(-4) m GF to the culture medium. Thus, the loss of the CK filament antigenic determinants was directly maintained by GF in vitro. The extracted hepatocytes showed spherical canalicular sheaths formed by the CK filaments within the cytoplasm. This was confirmed in "pop-off" sections which revealed that the canaliculi were lined by microvilli and by the localization of actin around the canaliculi as visualized by immunofluorescence. Excretion of fluorescein diacetate into the intracytoplasmic BC was seen both in the cells from GF and control mice but uptake of horseradish peroxidase was markedly reduced by the hepatocytes from the GF-fed mice. The results show that the hepatocytes containing MBs do not form intercellular BC and excretion of fluorescein diacetate into intracytoplasmic BC is not impaired but the uptake of horseradish peroxidase is markedly reduced. The results imply that the rearrangement of the cytoskeleton induced by GF causes both structural and functional deficits in the affected hepatocytes.
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PMID:Excretory function in cultured hepatocytes from griseofulvin-treated mice. 248 Nov 50

Renewed interest in the transplantation of isolated hepatocytes into the liver as a potential therapy for liver disease has stimulated the development of methods for the identification of donor cells within the recipient organ. We describe a method for cellular tagging and in vivo identification of intraportally transplanted hepatocytes using an intracellular fluorescent dye, 5(6)-carboxyfluorescein diacetate, succinimidyl-ester (CFSE). Rat and porcine hepatocytes were isolated and labelled with CFSE. The optimal conditions for labelling consisted of a buffered saline suspension of hepatocytes (5 x 10(6) cells/mL) in 20.0 microM CFSE incubated for 15 min at 37 degrees C. In vitro, labelled hepatocytes were cultured either on fibronectin-coated chamber slides or in culture flasks. Cultures were evaluated in situ by fluorescence photomicrography or by fluorescence-activated cell sorting (FACS) after cell detachment. Cell viability was assessed serially and cultured, labelled hepatocytes retained the dye for up to 3 wk (last day of study). CFSE did not effect hepatocyte viability and there was no evidence of intercellular diffusion of the dye. In vivo, syngeneic Lewis rats underwent selective portal vein infusion of freshly isolated, labelled hepatocytes (2.0 x 10(7) cells/2.0 mL saline/animal) into the posterior liver lobes. All recipients were sacrificed 48 h and 96 h later and their livers examined. Transplanted hepatocytes were identified by fluorescence microscopy in tissue sections and by FACS following collagenase digestion of the liver tissue. CFSE persisted in a population of viable, engrafted hepatocytes. FACS analysis demonstrated that 9 +/- 3% of the hepatocytes in the posterior liver lobes were labelled 48 and 96 h after transplantation. At 96 h following transplantation, multiple engrafted hepatocytes could be observed by fluorescence microscopy around the central veins. CFSE labelling allows for both in vitro identification and in vivo localization of donor hepatocytes. Furthermore, it appears to be more stable and specific for labelling hepatocytes than other tested dyes (especially DiI).
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PMID:Carboxyfluorescein (CFSE) labelling of hepatocytes for short-term localization following intraportal transplantation. 782 77

The present study describes a method for isolating single smooth muscle cells from pig urinary bladder using a continuous resuspension device. Low concentrations of collagenase and papain were sufficient to obtain a high yield of viable smooth muscle cells, which remained viable for about 3-4 h as tested with fluorescein diacetate. Addition of fetal calf serum increased the lifespan of the isolated cells and the percentage of contractile smooth muscle cells, but caused spontaneous shortening of the cells. The length and volume of the isolated smooth muscle cells depended on the calcium concentration used in the isolation buffer solution. The isolated muscle cells were apparently relaxed if a calcium concentration less than 1.0 mmol/l was used in the isolation medium. In higher calcium concentrations the isolated cells were significantly shorter, probably as a result of a contraction caused by mechanical stimulation of the cells during the isolation procedure.
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PMID:A method for isolating smooth muscle cells from pig urinary bladder with low concentrations of collagenase and papain: the relation between calcium concentration and isolated cell length. 845 38

The study aimed to assess the effect of lipopolysaccharide (LPS) in vivo (from Escherichia coli, 2 mg/kg body weight intraperitoneally) on the production and elimination of hydrogen peroxide (H2O2) in rat hepatic endothelial and Kupffer cells. Twenty-two hours after the injection of LPS, hepatic cells were isolated by collagenase and pronase digestion followed by centrifugal elutriation, and cell-associated H2O2 was determined by flow cytometry analysis using 2',7'-dichloroflorescin diacetate (DCF-diacetate). LPS treatment did not alter the basal or phorbol myristate acetate-stimulated levels of H2O2-related fluorescence in endothelial cells; however, it doubled phorbol myristate acetate-stimulated fluorescence in Kupffer cells. Administration of varying concentrations of H202 (range, 10(-7) - 10(-4) mol/L) in vitro caused a significantly delayed increase in fluorescence in endothelial cells from endotoxemic rats as compared with cells from saline-injected animals. The 50% effective concentration of H202 was found at 1.1 x 10(-6) and 8.1 x 10(-6) mol/L on endothelial cells after saline and LPS treatment, respectively. No differences were detected in H2O2-stimulated fluorescence between resting and LPS-stimulated Kupffer cells. Administration of varying glucose concentrations in vitro significantly decreased the H2O2-stimulated fluorescence in endothelial and Kupffer cells from LPS-injected animals. Inhibition of nitric oxide synthase by in vitro administration of NG-monomethyl-L-arginine (L-NNMMA) did not alter the H2O2- or phorbol myristate acetate-stimulated responses in endothelial and Kupffer cells. As shown earlier, LPS stimulates the gene expression of GLUT1 glucose transporter, glucose-6-phosphate dehydrogenase (G6PD), superoxide dismutases, and glutathione peroxidase in hepatic endothelial cells. The present data indicate that the LPS-induced metabolic alterations are accompanied by an increased H2O2-detoxifying capacity in hepatic endothelial cells. This may represent a protective mechanism against exogenous oxidative stress caused by activated hepatic phagocytes during inflammation. Our observations are consistent with primed production of reactive oxygen species (ROS) in LPS-activated Kupffer cells.
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PMID:Endotoxin stimulates hydrogen peroxide detoxifying activity in rat hepatic endothelial cells. 878 44

New assays were compared with tritiated thymidine (3HTdR) and trypan blue dye exclusion for evaluating hepatocyte viability on cytodex 3 beads and in microcapsules, the matrices used in bioartificial liver support systems. They were the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) 5 min qualitative assay and the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4 -sulfophenyl)-2H -tetrazolium, inner salt (MTS) 1 h quantitative assay. Both tetrazolium salts are cleaved in active mitochondria, the reaction occurring, therefore, in living cells only. After bathing at 39 degrees C with MTT for 5 min, porcine or rat hepatocytes on cytodex 3 beads were detached by collagenase while those in microcapsules were released by citrate treatment or passage through a fine needle. Cell viability was determined directly by microscope. The MTT 5 min metabolic inclusion test and MTS 1 h quantitative assay results correlated closely with those obtained by the 3HTdR, trypan blue dye exclusion, and fluorescein diacetate (FDA) methods. Both the new assays are sensitive, accurate, simple, and time-saving.
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PMID:Simple and reliable methods to assess hepatocyte viability in bioartificial liver support system matrices. 912 74

Hair cells were dissociated from basila papilla of 7 to 10 week post-hatched chicks (n = 15) by collagenase digestion followed by trituration. Morphological measurements of 215 viable hair cells from 5 chicks were performed. The average dimensions of the cells were: length 20.22 +/- 3.32 microns, width 9.93 +/- 2.01 microns of tall hair cells (n = 164); length 10.97 +/- 2.29 microns, width 14.8 +/- 2.29 microns of short hair cells (n = 30); length = width = 11.93 +/- 1.74 microns of intermediate hair cells (n = 21). Using fluorescein diacetate and propidium iodide, viability of isolated hair cells from another 5 chicks was determined. With excitatory light, fluorescence of viable cells were bright green in 3 hours, while the fluorescence of live cells were attenuated to such an extent that light green shadows were showed up to 5 hours after chicks were decapitated. The results suggested that the criterion of morphological observation and dying of cells should be combined to determine viability of isolated hair cells.
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PMID:[Dissociation and morphological observation of hair cells from chick basila papilla]. 1032 34

To determine the effects of procedural modifications, 23 human islet isolations were analyzed. Isolations were divided into two groups based on the enzyme used. The influence of Liberase, with an improved method of mechanical disassociation of pancreas, was compared to an automated method using Sevac collagenase. Pancreases were processed within 10 h of cross clamping. Following ductal injection of the enzyme, tissue was placed in the digestion chamber for disassociation. Purification was accomplished using a COBE 2991 cell processor and continuous gradients of 1Hypaque EuroFicoll. Isolations in Group I (Sevac) had an average yield of 138,602 +/- 128,364 islet equivalents (IE) (2083 +/- 1679 IE/g) with a purity of 85 +/- 11%. Group II (Liberase) showed an average yield of 389,586 +/- 191,161 IE (5,958 +/- 3,083 IE/g) with a purity of 90 +/- 6.8%. Viability was confirmed by fluorescein diacetate and propidium iodide staining, static incubations, and perifusions. In conclusion, the combination of the enzyme blend, Liberase, and a more gentle system of disassociation has proven to be a more productive method of islet isolation with higher purity than the previously published methods.
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PMID:Improved method for the isolation and purification of human islets of langerhans using Liberase enzyme blend. 1062 46

Synthetic fluorogenic substrates, like the CellProbe reagents, can determine enzymes in vital human spermatozoa. These substrates will enter the cells without previous cell permeabilization and exhibit fluorescence after cleavage depending on enzyme activity. They consist of different peptide sequences, specific for the enzymes, and a fluorescein- or rhodamine 110-dye moiety. The number of positive cells and the intensity of the fluorescence can be determined by flow cytometric analysis. We investigated several enzymes (peptidases, proteinases, esterases, elastases and collagenases) in intact spermatozoa before and after cryoprotection. Semen samples with normal spermiogram parameters were cryoprotected using the freezing medium TEST yolk buffer (TYB). Fresh spermatozoa showed a marked fluorescence after incubation with the synthetic substrates for the aminopeptidase M, butyryl esterase, fluorescein diacetate (FDA)-and FDA/sodium fluoride (NAF)-esterase, ala-ala-pro-val (AAPV)-elastase, gly pro-leu-gly pro-(GPLGP)-collagenase, gly gly leu-(GGL)-subtilisin as well as lys-ala-(LA)-dipeptidyl peptidase (DPP) II. After cryopreservation the spermatozoal fluorescence increased applying substrates for butyryl esterase (P<0.05), prolyl-aminopeptidase (P<0.001) and val-lys-(VK)-cathepsin (P<0.001) most probably due to elevated enzyme activities. The activities of FDA-esterase (P<0.05) and FDA/NAF-esterase (P<0.05), AAPV-elastase (P<0.01), GPLGP-collagenase (P<0.05) and GGL-subtilisin (P<0.001) decreased after cryopreservation. The substrates for arg-gly glut-ser-(RGES)-elastase, gly phenyl-gly ala-(GFGA)-collagenase and threo-pro-(TP)-cathepsin were not cleaved before as well as after cryostorage. The substrates for subtilisin an
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PMID:Flow cytometric analysis of enzymes in live spermatozoa before and after cryostorage. 1113 45

To isolate a large number of porcine hepatocytes, we originally developed a mass preparation method that combined the usual collagenase perfusion method of a whole liver with a collagenase redigestion method of tissue fragments after liver perfusion. Using a pig of 10kg, collagenase perfusion only resulted in a yield of 63+/-78 x 10(8) total cells with a viability of 69.2+/-25.3 %, but our combined method had a yield of 167+/-31 x 10(8) total cells with a viability of 87.9+/-4.4% (mean +/- SD). Also, the combined method was applied to two pigs of 10kg body weight at the same time, and isolated 387+/-89 x 10(8) hepatocytes with a viability of 87.1+/-6.9% and a purity of 93.6+/-2.8 % in 11 experiments. We designed a large multi-capillary polyurethane foam (MC-PUF) packed-bed module containing 1 x 10(10) porcine hepatocytes on a clinical trial scale. The porcine hepatocytes in the module formed spherical multicellular aggregates (spheroids) of 200 - 500 microm diameter. Most hepatocytes forming spheroids were viable judged by fluorescein diacetate and ethidium bromide staining. The activities of ammonia removal, albumin secretion and oxygen consumption of the large MC-PUF module were the same as for a small MC-PUF module containing 2 x 10(8) porcine hepatocytes, and were maintained for at least 9 days of culture. These results show that a large MC-PUF module is successfully scaled up 50 times. In conclusion, we succeeded in developing a mass preparation method of porcine hepatocytes and a large hybrid artificial liver module on a clinical trial scale.
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PMID:Mass preparation of primary porcine hepatocytes and the design of a hybrid artificial liver module using spheroid culture for a clinical trial. 1179 50

The use of primary hepatocyte cultures as in vitro models for studying xenobiotic metabolism and toxicity is limited by the loss of liver-specific differentiated functions with time in culture and the inability of the cells to proliferate. The aim of this study was to investigate the effect of incorporating 20% chondroitin-6-sulphate (Ch6SO4), a glycosaminoglycan (GAG), into collagen gels (0.3% w/v) and crosslinking the gels with either 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) or 1,6-diaminohexane (DAH) on the expression of glutathione-S-transferases (GSTs) and the activity of cytochrome P450 in hepatocytes cultured for 48 hours and 7 days. Hepatocytes were isolated from male Sprague-Dawley rats by collagenase perfusion. Cell homogenates were immunoblotted against class alpha and pi GST subunits. To measure cytochrome P450 activity, testosterone hydroxylation was assessed. Viability of the cultured cells was assessed by confocal laser scanning microscopy using the vital stain carboxyfluorescein diacetate (CFDA). Cells cultured on gels crosslinked with EDAC were dead by 48 hours as judged by lack of CFDA-derived fluorescence and absence of GST bands on the immunoblots. The viability and morphology of the cells were unaffected by any of the other components of the substrata tested. Expression of GSTs indicated that the hepatocyte phenotype was stable for at least 48 hours. The addition of GAG did not improve the phenotype at either 48 hours or 7 days in culture, but the combination of GAG and DAH crosslinking improved GST expression in the 7-day cultures. However, the hepatocyte cytochrome P450 activity did not show any improvement on any of the gels. The combination of GAG and DAH crosslinking provided the most stable substratum environment in terms of GST expression in hepatocytes.
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PMID:The influence of glycosaminoglycans and crosslinking agents on the phenotype of hepatocytes cultured on collagen gels. 1269 29


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