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)

Hepatocytes isolated by the collagenase digestive method were transplanted into the spleens of syngeneic rats. Morphology and function of the hepatocytes in the spleen were investigated for 12 to 17 months after transplantation. The transplanted hepatocytes proliferated and reconfigured in the spleen without direct perfusion of portal venous blood and with the presence of an intact host liver. Fourteen to 17 months after transplantation, the hepatocytes which had formed a demarcated nodule occupied approximately 40% of the area of the splenic parenchyma without undifferentiation on microscopic examination. However, the weight of the hepatized spleen did not increase beyond the weight of a normal spleen and the weight of the host liver that had normal morphology also did not differ from a normal liver. Light and electron microscopic studies demonstrated differentiated cord structure and normal architecture for each heptocyte. Furthermore, the hepatized spleen synthesized albumin and glycogen as demonstrated by immunofluorescence and histochemical studies. Ammonia tolerance and indocyanine green clearance tests revealed functioning hepatocytes in the spleen proper. These results indicate that our experimental model lends itself well to investigations in cell growth mechanism and that hepatocellular transplantation has potential clinical application to compensate for impaired hepatic function.
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PMID:Morphology and function of isolated hepatocytes transplanted into rat spleen. 51 33

1. Isolated parenchymal cells were prepared by collagenase perfusion of livers from fed rats that had been previously injected with [(3)H]leucine to label liver proteins. When these cells were incubated in a salts medium containing glucose, gelatin and EDTA, cellular integrity was maintained over a period of 6h. 2. Cells incubated in the presence of 2mm-leucine to minimize radioactive isotope reincorporation released [(3)H]leucine into the medium at a rate accounting for the degradation of 4.5% of the labelled cell protein per h. 3. Degradation of [(3)H]protein in these cells was inhibited by insulin and by certain amino acids, of which tryptophan and phenylalanine were the most effective. 4. Protein degradation was decreased by several proteinase inhibitors, particularly those that are known to inhibit lysosomal cathepsin B, and by inhibitors of cell-energy production. 5. Ammonia inhibited degradation, but only at concentrations above 1.8mm. Aliphatic analogues of ammonia were effective at lower concentrations than was ammonia. 6. High concentrations of ammonia inhibited degradation by 50%. The extent of this inhibition could not be increased further by the addition of the cathepsin B inhibitor leupeptin, which by itself inhibited degradation by approx. 30%. 7. The sensitivity of proteolysis in isolated hepatocytes to these various inhibitory agents is discussed in relation to their possible modes of action.
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PMID:Inhibition of protein degradation in isolated rat hepatocytes. 88 Feb 45

Hydrolysis of arginine into urea and ornithine (Orn) was observed to take place in several segments of the rat nephron including cortical and medullary pars recta of the proximal tubule (PST) and collecting duct (CD). This work was now extended to the adult mouse and rabbit. Representative nephron segments, obtained by microdissection of collagenase-treated kidneys, were incubated with L-[guanido-14C]arginine (216 microM). Addition of urease produced 14CO2 + 2 NH3 from the newly formed urea released in the incubate. 14CO2 was trapped in KOH and counted. In both species, as well as in the rat, the PST was the site of the highest urea + Orn production, with an intensity increasing from cortex to medulla. For other nephron segments, the pattern was not similar in all species. Significant production of urea + Orn was observed in the proximal convoluted tubule and the medullary thick ascending limb in the rabbit, but not in the CD of either the rabbit or the mouse. The functional significance of this urea + Orn production remains unclear. The total amount of urea generated intrarenally by this reaction does not seem sufficient to play a significant role in the urinary concentrating mechanism. It may be assumed that Orn could be further metabolized to polyamines and play a role in maintaining cell integrity and function in the PST, especially in its medullary part, exposed to hypertonicity and poor oxygen supply.
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PMID:Localization of urea and ornithine production along mouse and rabbit nephrons: functional significance. 144 76

A study was conducted to investigate morphologic as well as metabolic characteristics of microcarrier-attached hepatocytes in culture, and also to evaluate the effect of intraperitoneal transplantation of the microcarrier-attached hepatocytes on acute hepatic failure in rats induced by D-galactosamine (GalN). Rat hepatocytes were isolated by collagenase perfusion, and cultured on collagen-coated microcarriers. Protein synthesis estimated by [14C] leucine incorporation was four-fold higher in microcarrier culture than in cell suspension. The rates of albumin, transthyretin and bile acid syntheses in hepatocytes cultured on microcarriers were similar to those in monolayer culture. When microcarrier-attached hepatocytes were intraperitoneally transplanted into rats with Galn-induced acute liver failure, a marked improvement in survival rate was observed as compared with control rats which received injections of microcarriers alone (80% vs 0% beyond 6 days of transplantation). Mean serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), methionine and glucose levels were similar in both groups, while serum bilirubin and ammonia levels were lower (P less than 0.1, P less than 0.05) in rats transplanted with the microcarrier-attached hepatocytes. Immunohistochemical examinations revealed that the transplanted hepatocytes around microcarriers had albumin synthesis activity, whereas almost no albumin synthesis was demonstrated in recipient liver. In conclusion, intraperitoneal transplantation of the microcarrier-attached hepatocytes will provide sufficient metabolic support, representing detoxication of ammonia (and presumably bilirubin) and synthesis of albumin, to allow GalN-damaged liver function to restore. Microcarrier culture of isolated hepatocytes seems to be one of the most appropriate tools for an artificial liver support.
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PMID:Effects of intraperitoneal transplantation of microcarrier-attached hepatocytes on D-galactosamine-induced acute liver failure in rats. 168 85

To determine the relationship between the expression of bone proteins and the formation of mineralized-tissue matrix, the biosynthesis of non-collagenous bone proteins was studied in cultures of fetal-rat calvarial cells, which form mineralized nodules of bone-like tissue in the presence of beta-glycerophosphate. The temporal pattern of protein synthesis in both mineralizing and non-mineralizing cultures was studied by metabolic labelling with [35S]methionine, 35SO4(2-) or 32PO4(3-) over a 5-day period. After a 24 h labelling period, the culture media were harvested and the cell layers extracted sequentially with aq. 0.5 M-NH3, followed by 4 M-guanidinium chloride (GdmCl), 0.5 M-EDTA and a second extraction with 4 M-GdmCl. Protein associated with collagenous bone matrix was analysed after digestion with bacterial collagenase. On the basis of [35S]methionine labelling, the major proteins extracted from the mineralizing matrix were secreted phosphoprotein-1 (SPP-1; osteopontin), bone sialoprotein (BSP) and a 14 kDa phosphoprotein. The presence of SPP-1 and BSP in the conditioned media of both mineralizing and non-mineralizing cultures and their incorporation into the mineralizing nodules indicated that these proteins associate with preformed mineral crystals. However, some BSP was also present in GdmCl extracts and, together with a 35 kDa sulphated protein, was released from a bacterial-collagenase digestion of the tissue residue in both non-mineralizing and mineralizing cultures. Two forms of sulphated SPP-1 were identified, a highly phosphorylated 44 kDa species being the predominant form in the mineralized matrix. The BSP was more highly sulphated but less phosphorylated than SPP-1. Bone SPARC (secreted protein, acid and rich in cysteine) protein (osteonectin) was present almost entirely in the conditioned media and did not incorporate 32PO4(3-) or 35SO4(2-). The SPP-1 and the 14 kDa protein were susceptible to thrombin digestion, the 44 kDa SPP-1 being specifically cleaved into 28 and 26 kDa fragments. The fragments were labelled uniformly with [35S]methionine, but the 28 kDa fragment incorporated more 35SO4(2-), but less 32PO4(3-), than the 26 kDa fragment. These studies demonstrate that SPP-1 and BSP are the major osteoblast-derived bone proteins to bind to the bone mineral. That BSP also binds to the collagenous bone matrix indicates a potential role for this protein in linking the hydroxyapatite with collagen.
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PMID:Biosynthesis of bone proteins [SPP-1 (secreted phosphoprotein-1, osteopontin), BSP (bone sialoprotein) and SPARC (osteonectin)] in association with mineralized-tissue formation by fetal-rat calvarial cells in culture. 200 15

Measurements of the effects of NH3/NH4+ on glucose-induced electrical activity in beta-cells from microdissected mouse islets of Langerhans and on intracellular pH in single collagenase-isolated islets pre-loaded with a fluorescent pH probe were performed and are reported here. Application of NH3/NH4+ (15 mM) in the presence of glucose (11 mM) promptly hyperpolarized the beta-cell membrane, reduced input resistance by 60% and blocked electrical activity. These changes were paralleled by an increase in islet fluorescence indicative of a cytosolic pH increase. Removal of NH4Cl initially stimulated electrical activity, which returned to resting level with a time constant of 51 s. Concomitant with the removal of NH4Cl there was a drop in pHi followed by a slow return to resting level with a time constant of 83 s. The results suggest that the [Ca2+]-dependent K+ channel in the beta-cell membrane is activated by a rise in cytosolic pH.
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PMID:Modulation of K+ conductance by intracellular pH in pancreatic beta-cells. 242 60

Periportal and perivenous hepatocytes were isolated from rat liver by digitonin/collagenase perfusion for investigating the acinar heterogeneity of amino acid transport activities related to glutamine and ammonia metabolism. Immunocytochemical staining of the respective subpopulations for glutamine synthetase demonstrated that periportal subpopulations were essentially free of glutamine synthetase-positive cells, whereas perivenous subpopulations showed a 2- to 3-fold enrichment of glutamine synthetase-positive hepatocytes. The high perivenous/periportal ratio of 59 found for glutamine synthetase activity as well as the perivenous/periportal ratios of other marker enzymes further indicated the good separation of periportal and perivenous cells. alpha-Aminoisobutyric acid, histidine and glutamate were used to determine the distribution pattern of amino acid transport systems A, N and G-, as well as of the sodium-independent uptake of these compounds 1 hr after isolation and after maximal hormonal stimulation during primary culture. The strong heterogeneity of the sodium-independent transport of histidine, characterized by higher perivenous transport rates [perivenous/periportal ratio: 1.5 (1 hr) to 3.5 (48 hr)], suggests a significant role of facilitated diffusion, presumably in glutamine export. Conversely, the strong heterogeneity of the sodium-dependent glutamate transport (System G-) characterized by higher uptake rates in nonstimulated [perivenous/periportal ratio: 6.6 (1 hr)] and in hormonally treated perivenous hepatocytes (perivenous/periportal ratio: 2.2) reflects its possible significance with respect to the substrate availability for glutamine synthesis. The observed heterogeneities provide a basis for understanding how substrate fluxes related to glutamine metabolism might be established and regulated.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Different capacities for amino acid transport in periportal and perivenous hepatocytes isolated by digitonin/collagenase perfusion. 256 97

Viable Hepatocytes were isolated from adult canine liver by in situ collagenase perfusion, and cultured on collagen coated borosilicate glass plates (100 X 200mm) at confluent cell density. The medium of hepatocytes in the primary culture was L-15 supplemented with aprotinin 5000U/L, proline 30mg/L, insulin 10(-8)M, dexamethasone 10(-8)M, glucagon 10(-8)M, and h-EGF 10ng/ml. Long-stroke type bioartificial liver module consisted of 200 glass plates with hepatocytes. It contained 6 billion primary cultured cells in total, that is almost equivalent to 30% of the normal canine liver. All hepatocytes in the module were quite viable during 2 weeks in the perfusion culture, and maintained various liver functions at a high level. Gluconeogenesis was 368.0 +/- 15.4mg/module/hr, albumin synthesis was 19.1 +/- 2.5mg/module/day, ureogenesis was 3.7 +/- 0.1mg/module/hr, and ammonia metabolism was 8.4mg/module/hr. Moreover, those functions were maintained at least 2 weeks in the canine plasma as well as in the culture medium with hormones. This hybrid bioartificial liver may exert various liver functions like a liver in situ.
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PMID:[Hybrid bioartificial liver using canine hepatocytes in primary culture]. 276 24

Bacteroides gingivalis W50 was grown in a chemostat under steady-state conditions at pH 7.5 +/- 0.2 and a constant growth rate of 6.9 h for periods of up to 6 weeks (146 bacterial generations) in a complex medium. Hemin was capable of limiting the growth of cells up to a concentration of approximately 0.5 micrograms/ml since higher concentrations of hemin did not increase cell yields; cells grew in the absence of exogenously added vitamin K1. Only a limited number of amino acids was metabolized during growth, but because none of these was totally depleted, the limiting nutrient under hemin excess conditions was probably a peptide. A range of fermentation products was produced under all conditions of growth; higher concentrations of cytotoxic metabolites such as propionate and butyrate were formed under hemin excess conditions, although more ammonia was released under hemin limitation. When viewed by electron microscopy, cells grown under hemin limitation appeared to be either coccobacillary or short rods and possessed few fimbriae per cell, but large numbers of extracellular vesicles could be seen both surrounding the cell surface and free in the environment. In contrast, cells grown under hemin excess conditions were more commonly coccus shaped and were more heavily fimbriated but had fewer extracellular vesicles. Marked differences were found in the susceptibility of mice to infection with cells grown under different concentrations of hemin. Cells transferred to media without any added hemin were avirulent, whereas those grown under conditions of hemin limitation (0.33 and 0.40 micrograms/ml) produced a 20 and 50% mortality in mice, respectively. In contrast cells grown under hemin excess always caused 100% mortality in mice, although this virulence was dose dependent. When virulent, the bacteria caused an extensive, spreading infection with necrosis of the skin and subcutaneous tissues. Collagen disintegration was seen histologically, implying a role for collagenase production in the pathogenicity of these bacteria.
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PMID:Effect of hemin on the physiology and virulence of Bacteroides gingivalis W50. 369 84

To study kinetics and principles of cellular uptake of 13N-ammonia, a marker of coronary perfusion in myocardial scintigraphy, heart muscle cells of adult rats were isolated by perfusion with collagenase and hyaluronidase. Net uptake of 13N, measured by flow dialysis, reached equilibrium within 20 sec in the presence of sodium bicarbonate and carbon dioxide (pH 7.4, 37 degrees C). Total extraction, 80 sec after the reaction start, was 786 +/- 159 mumol/ml cell volume. Cells destroyed by calcium overload were unable to extract 13N-ammonia. Omission of bicarbonate and carbon dioxide reduced total extraction to 36% of control. 13N-Ammonia uptake could also be reduced by 50 muM 4,4' diisothiocyanostilbene 2,2' disulfonic acid, by 100 micrograms/ml 1-methionine sulfoximine, and by preincubation with 5 muM free oleic acid. These results indicate that in addition to metabolic trapping by glutamine synthetase, the extraction of 13N-ammonia by myocardial cells is influenced by cell membrane integrity, intracellular-extracellular pH gradient, and possibly an anion exchange system for bicarbonate. For this reason, the uptake of 13N-ammonia may not always provide a valid measurement of myocardial perfusion.
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PMID:Kinetics of 13N-ammonia uptake in myocardial single cells indicating potential limitations in its applicability as a marker of myocardial blood flow. 396 79


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