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)

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

Effects on Ca2+ transport of parathyroid hormone (PTH) and N6,O2'-dibutyryl adenosine 3',5'-cyclic monophosphate (DB-cAMP) were examined in the rabbit distal nephron segments including the cortical thick ascending limb of Henle's loop (CAL), the connecting tubule (CNT) and the cortical collecting tubule (CCT) by the in vitro perfusion technique. When PTH (10(-8) mol . l-1) was added to the bath, efflux of Ca2+ (pmol . mm-1 . min-1) was increased from 6.29 +/- 1.46 to 7.96 +/- 1.66 (P less than 0.02) in the CAL, and from 8.55 +/- 1.30 to 13.73 +/- 1.24 (P less than 0.001) in the CNT, respectively, without changes in influx of Ca2+. The effect of PTH on Ca2+ transport in the CAL, however, was abolished when phosphate concentration in the medium was reduced from 3.0 to 1.0 mmol . l-1. When DB-cAMP (10(-3) mol . l-1) was added to the bath, efflux of Ca2+ was also increased from 7.01 +/- 0.83 to 9.40 +/- 0.82 (P less than 0.05) in the CAL, and from 13.11 +/- 0.89 to 19.74 +/- 0.52 (P less than 0.005) in the CNT, respectively. By contrast, neither PTH nor DB-cAMP affected efflux of Ca2+ in the CCT. PTH did not affected the transepithelial voltage either in the CAL or in the CNT. But in the CNT, DB-cAMP decreased the voltage from -14.1 to -9.4 mV. The response of adenylate cyclase activity to PTH in the collagenase treated isolated nephron segments was also examined. Significant increases in adenylase cyclase activity were observed in the CAL as well as in the CNT with 10(-6) mol . l-1 PTH. These data indicate that PTH stimulates Ca2+ transport across the CNT probably via activation of the adenylate cyclase-cyclic AMP system. The hormone may also stimulate Ca2+ transport across the CAL in a special condition where plasma phosphate concentration is elevated.
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PMID:Effects of parathyroid hormone and N6,O2'-dibutyryl cyclic AMP on Ca2+ transport across the rabbit distal nephron segments perfused in vitro. 626 87

The soluble material produced from a 96-hour digest of sonicated human glomerular basement membranes with purified collagenase was shown to contain at least 12 components by sodium dodecyl sulphate-polyacrylamide gel electrophoresis with molecular weights ranging from 20 x 10(3) to greater than 200 x 10(3) daltons. Five of these components produced antigenic peaks after two-dimensional immunoelectrophoresis into agar plates containing rabbit antiserum to the solubilised glomerular basement membrane. Two groups of antigenic components were demonstrated in these gels by two-dimensional immunoelectrophoresis autoradiographic techniques utilising 125I-labelled collagenase-digested human glomerular basement membranes reacted with antibodies eluted at acid pH from the kidney of the Goodpasture's patient. This acid eluate was shown to contain contaminants of alpha 1-antitrypsin and albumin co-eluting with the antibodies bound to the glomerular basement membrane. After removal of these contaminants, the antibodies were linked to cyanogen bromide-activated Sepharose and used to affinity purify four antigenic fractions from the collagenase-digested glomerular basement membrane. These fractions were eluted by 0.2 M glycine pH 2.8 and with 0.5 M ammonium thiocyanate and had molecular weights of 22-25, 43-45, 65-70 and 205 x 10(3) daltons. The smaller molecular weight components were shown to be common to both included and excluded fractions obtained by Ultragel AcA 44 chromatography of the digested material in 10 mM phosphate pH 8. This suggests that the larger molecular weight component would be an aggregate of a smaller component. Preliminary carbohydrate and amino acid analyses indicated that the different elution procedures elicited antigens with different chemical characteristics.
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PMID:Isolation of human glomerular basement membrane antigens by affinity chromatography utilising Goodpasture's kidney antibody eluates. 627 70

Mice infected with Schistosoma mansoni develop hepatic fibrosis associated with enhanced collagen synthesis that out-paces induced collagenase activity. Administration of one dose of concanavalin A [Con A (200 micrograms)] by i.p. injection to mice at 5 or 6 weeks after infection with 50 S. mansoni cercariae decreased liver collagen content by 50% compared to levels in control-infected mice injected with either homologous immunoglobulin (200 micrograms) or phosphate-buffered saline; additional doses of Con A had no further effect. The decrease in collagen content could not be attributed to either decreased egg deposition in the liver or inhibition of liver collagen synthesis, but was coincident with a greater solubility of granulomas. Collagen contents of skin and tail were unaffected. The relative solubilities of liver collagen in 8 M urea: 10 mM dithiothreitol were greater in treated animals as compared to controls. However, the amounts of collagen solubilized were similar in both sets of animals, since the total collagen content of treated mice was 50% of the controls. A possible explanation for these results is that much of the synthesized collagen does not accumulate in treated animals, whereas it does accumulate in controls. Peak collagenase and neutral protease activities occurred at 7 weeks postinfection in treated animals, and were 2-fold greater than in controls. Similar effects were observed when succinylated Con A was administered. The results suggest that Con A may modulate host-immune responses influencing fibrogenesis in hepatic murine schistosomiasis.
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PMID:Concanavalin A reduces liver collagen accumulation in murine schistosomiasis. 627 83

Second maxillary molars of hamsters were cultured in the presence or absence of 250 micrograms/ml vitamin C for periods up to 12 days. At various days, cultured explants were studied with histological and biochemical methods to investigate effects of vitamin C-deficiency on matrix production and mineralization in vitro. As biochemical parameters for protein synthesis and mineralization, uptake and incorporation of [3H]-proline, 45Ca and 32PO4 were used. To discriminate between synthesis of collagenous and non-collagenous proteins digestion of the [3H]-proline-labelled material by purified collagenase and its degree of hydroxylation were measured. Histologically, in control explants cultured with vitamin C, normal dentinogenesis, amelogenesis and mineralization in vitro were observed. In the vitamin C-deficient explants, odontoblasts produced an abnormal predentine matrix and de-differentiated. Eventually, this matrix mineralized aspecifically. In the presence of this abnormal matrix, ameloblasts failed to differentiate, which suggests that a cell-matrix type of interaction is involved in the differentiation of the pre-ameloblasts. Biochemically, in the vitamin C-deficient explants protein synthesis and collagen synthesis were reduced by about the same extent; the in-vitro produced collagens appeared under-hydroxylated and could, in degraded form, easily be extracted in formic acid. An increase of [3H]-proline solubility in formic acid in the control explants, however, paralleled enamel matrix production and was attributed to the solubilization of proline-rich enamel matrix proteins. The production of acid insoluble phosphate was not affected by vitamin C-deficiency. The uptakes of 45Ca and 32PO4 were retarded and the molar Ca:PO4 uptake ratio was lower, reflecting the histologically observed aspecific mineralization.
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PMID:A histological and biochemical study of the effect of vitamin C-deficiency on induction of amelogenesis in hamster molars in vitro. 631 49

Cells isolated from samples of human iliac crest and human femoral heads by collagenase digestion have been successfully cultured in Fitton-Jackson modified BGJb culture medium supplemented with penicillin (100 units/ml), streptomycin (100 micrograms/ml), and fetal calf serum (10%). Although only a low proportion of the cells survived the initial plating (less than 1%), cells established in culture were readily passaged. Examination of cells obtained at intervals during the collagenase digestion showed that the percentage of cells that attached increased with time of digestion. Rapid sample preparation of rat bone did not substantially increase the number of cells attaching. Thus, it seems unlikely that the low survival was due to loss of viability during sample transportation and preparation. Of several media tested BGJb supplemented with 10% fetal calf serum supported the best growth. Population doubling time averaged 104 hr. Cultured human bone cells were assayed for alkaline phosphatase activity using the azo dye method with naphthol ASTR phosphate as the substrate. A portion of the cells (19%) demonstrated high activity in all cultures examined regardless of the passage number of the culture. Autoradiography of cells exposed to [3H]thymidine showed incorporation of the label into both alkaline phosphate-positive and -negative cells. The stimulation of cell proliferation by growth factors was studied by determining the incorporation of [3H]thymidine into DNA. The specific skeletal growth factor from human bone stimulated cell proliferation several-fold with a half-maximal effect at 5 micrograms/ml. Insulin, epidermal growth factor, and a crude preparation of somatomedin C also stimulated cell proliferation.
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PMID:Characterization of cells isolated and cultured from human bone. 632 25

Adult rat heart was dissociated into a single-cell suspension by a retrograde perfusion technique with collagenase and hyaluronidase in Krebs-Ringer phosphate buffer. Long-term culture of these isolated single cardiac muscle cells was established for up to 45 days. Transmission electron microscopy and immunofluorescence analysis with monoclonal antibodies to cardiac myosin were used to examine sequentially the external and internal structural organization of the cardiac myocytes. Most of the cardiac myocytes exhibited prominent alterations in their external and internal structural organization during the first two weeks of culture. As they attached to the substrate and spread out, the myocytes assumed various shapes and sizes, with the exception of a few which maintained their original cylindrical shape. Electron microscopy of 2 to 4-day cultures revealed that most of the muscle cells contained disorganized myofibrils and surface blebs with enclosed mitochondria and myofilaments, which were eventually extruded from the cytoplasm. With progressive culture, the cardiac myocytes appeared to lose myofibrillar material; fewer myofilaments or sacromere fragments with interfibrillar mitochondria were observed in the sarcoplasm. Such cells resembled cultured embryonic or neonatal cardiac myocytes. However, some muscle cells retained closely packed, well organized myofibrils characteristic of freshly dissociated or in vivo cardiac myocytes. Immunofluorescence microscopy demonstrated that the cultured cardiac myocytes were strongly myosin positive throughout their morphological changes and subsequent maintenance in culture. Two patterns of fluorescence were observed in these cells in correlation with the fine structural evidence for myofibrillar distribution. One pattern exhibited bright fluorescence near the central region of the cell with a more weakly diffuse fluorescence throughout the cytoplasm; the other pattern was characterized by bright fluorescence throughout the sarcoplasm. Most of the myocytes retained their contractility throughout the culture period excepting the initial 24 to 48 h of cell attachment and flattening. These studies demonstrate the feasibility of maintaining contractile cardiac muscle cells from adult rats for at least 1 1/2 months in monolayer culture, although some variability in myofibrillar organization has been observed.
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PMID:Long-term cell culture of adult mammalian cardiac myocytes: electron microscopic and immunofluorescent analyses of myofibrillar structure. 635 Jun 10

Pancreatic islets from normal C57BL/KsJ-+/+-mice and diabetic C57BL/KsJ-db/db-mice were collagenase-isolated and incubated with 33P-labelled inorganic phosphate. No significant difference was observed in phosphate uptake between normal and diabetic mouse islets whether the animals were young (6-8 weeks) or old (27 +/- 9 weeks). When 33P-labelled islets were perifused with non-radioactive medium, all types of islets exhibited a brisk and transient peak of phosphate release in response to a change of glucose concentration from 2.8 to 16.7 mmol/l. Expressed in absolute terms, both the basal and peak efflux of phosphate appeared to be diminished in the diabetic mice. In relative terms (peak over basal), the glucose-stimulated phosphate efflux was not lower in diabetic than in normal mice. At both a low (3 mmol/l) and a high (20 mmol/l) glucose concentration, the production of 3H2O from D-[5-3H]glucose was reduced in old but not in young diabetic mouse islets. The percentage increase in glucose metabolism in response to a rise in glucose concentration from 3 to 20 mmol/l was about the same in all types of islets. The results add to previous observations of disturbed ionic fluxes in the pancreatic islets of diabetic KsJ-db/db-mice. These effects are probably not due to gross alterations in glycolytic metabolism but more probably reflect alterations in the function of the beta-cell plasma membrane.
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PMID:Phosphate flush and glucose metabolism in pancreatic islets of young and old diabetic mice (C57BL/KsJ-db/db). 637 50

The effects of alterations in extracellular calcium concentration on prostaglandin (PGE) and thromboxane (TXB2) syntheses were studied in isolated epithelial cells from the urinary bladder of the toad, Bufo marinus. In epithelial cells prepared using collagenase, basal iPGE synthesis was greater than iTXB2 synthesis. Increasing extracellular calcium from zero to 1 mM increased iPGE synthesis and decreased iTXB2 synthesis equivalently such that total conversion of endogenous arachidonate to these two metabolites was unaltered. Vasopressin stimulated iPGE and iTXB2 syntheses when the incubation buffer contained 1 mM calcium but had no effect in the presence of 0.4 microM calcium. In contrast, using an EDTA isolation method, basal iPGE and iTXB2 syntheses were equal in the presence of zero calcium. Increasing extracellular calcium concentration to 1 mM caused a greater enhancement in iTXB2 synthesis compared to iPGE. Increasing extracellular calcium to 2 mM was associated with a decline in iPGE and iTXB2 syntheses back to the levels observed with no calcium added to the medium. The effect of increasing the calcium concentration was greater in phosphate than in bicarbonate buffer. In a Tris buffer the effect of altered calcium was almost completely abrogated. These studies demonstrate that the choice of buffer and alterations in extracellular calcium concentration differentially alter basal arachidonic acid metabolism to prostaglandins and thromboxane in isolated toad urinary bladder cells. The results suggest that there may exist several endogenous pools of arachidonic acid which are differentially influenced by calcium. Furthermore, the pool sensitive to vasopressin has an absolute requirement for calcium.
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PMID:Alterations in extracellular calcium concentration differentially influence prostaglandin and thromboxane synthesis in epithelial cells from the toad urinary bladder. 642 55

Morphologic features of the cell surface were studied in isolated cardiac myocytes obtained from 12 and 48 week old Spontaneously Hypertensive Rats (SHR) and normotensive Wistar-Kyoto (WKY) control rats. Hearts obtained from ether-anesthetized animals were perfused with Ca++-free Hanks' solution containing EGTA and 0.1% collagenase. The hearts were minced and the suspended cells fixed in 2% phosphate buffered glutaraldehyde. Cells mounted on Nuclepore membrane filters were dehydrated in alcohol and critical point dried for SEM. Quantitative evaluation of myocyte length, width and volume was done with a sonic digitizer on H and E stained cells by light microscopy. At both ages studied there was a significant increase in heart weight to body weight ratio, systolic blood pressure and cell size in SHR compared to WKY controls. In the older animals there appeared to be increased numbers of cell junction areas and deep grooves in the cell surface which appeared more pronounced in SHR than in WKY. The cell surface of the myocytes from 48 week old animals had deep capillary grooves surrounded by protruding longitudinal bundles of myofibrils. These changes would result in increased surface area of the larger cells and diminish the effect of increased cell size on diffusion distance from capillary to tissue. These changes in cell morphology were interpreted as providing a protective effect against development of functional impairment in the hypertrophied hearts.
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PMID:Surface morphology of isolated cardiac myocytes from hypertrophied hearts of aging spontaneously hypertensive rats. 644 76


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