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)

Previously, we used dual immunofluorescent analysis and showed that the thyroid gland from patients with Graves' disease had a reduced number of CD4+CD45RA+ cells, but an increased number of complementary CD4+CDw29+ cells. An immunohistochemical study, however, produced opposite results; interstitial lymphocytes predominantly expressed the CD45RA+ rather than the CDw29+ phenotype. Because the difference in findings may be due to differences in the techniques used, we did the following experiments: Mononuclear cells were treated with various amounts of collagenase (50-1000 mg/l) which had no effect on the cell surface antigens CD3, CD4 and CD45RA. A dual immunofluorescent study showed that the numbers of CD4+CD45RA+ and CD8+CD45RA+ cell population among CD45RA+ cell population were markedly decreased in the thyroid tissue, and that the CD45RA antigen on the intrathyroidal mononuclear cells was mainly expressed on the CD20+ cells. As the thyroid section had been fixed with acetone before immunohistochemical staining, CD45RA- cells were treated with acetone and stained with anti-CD45RA monoclonal antibody using an avidin-biotin peroxidase complex method. The results of this experiment suggest that there are cell surface molecules which react with anti-CD45RA monoclonal antibody after treatment with acetone in CD45RA- cells. The above findings confirm our previous results which showed that the thyroid glands of patients with Graves' disease have decreased numbers of suppressor-inducer T cells. Also, several problems exist in the detection of CD45RA+ cells when using an immunohistochemical method.
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PMID:CD4+CD45RA+ cells (suppressor-inducer T cells) in thyroid tissue from patients with Graves' disease. 183 59

Fibroblast-type collagenase, a neutral secretory metalloproteinase capable of cleaving interstitial collagen types I-III, is expressed by a number of different cell types including fibroblasts, macrophages, osteoblasts, and keratinoyctes. To elucidate the secretory pathway of this enzyme, we examined the ultrastructural localization of this metalloproteinase in cultured human gingival fibroblasts, particularly the routing of the enzyme from the Golgi cisternae to the cell surface utilizing rabbit polyclonal antibodies raised against human fibroblast (pro) collagenase. For this purpose, one percent glutaraldehyde followed by gentle permeabilization with saponin gave superior preservation of both cellular morphology and intracellular antigenicity. At the light microscopic level, the reacting antibodies visualized by immunofluorescence and immunoperoxidase staining were localized intracellularly in the perinuclear region reflecting the Golgi apparatus. Immunoelectronmicroscopy using the pre-embedding technique and peroxidase or immunogold staining revealed electron dense label in large vacuoles indicating extended cisternae of the Golgi field. Vesicles were noted leaving the plasma membrane in long extensions. Moreover, intact vesicle containing the antibody reaction product appeared outside the membrane. In addition, most extracellular vesicular structures appeared empty of label suggesting that the collagenase had been liberated into the extracellular space. The latter observation was supported by the fact that the label was found also on the extracellular surface of the cells indicating a (re)association of collagenase with the outer cell membrane. These data demonstrate that the pathway of interstitial collagenase in human gingival fibroblasts is similar to that of other secretory proteins.
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PMID:Intracellular secretory pathway and ultrastructural localization of interstitial procollagenase in human gingival fibroblasts. 196 15

A sensitive assay for type IV collagen degradation using an avidin-biotin sandwich technique is described. Biotinylated type IV collagen is allowed to bind to an avidin-coated microtiter plate. The solution to be assayed is incubated with the biotinylated collagen bound to the avidin plate. Collagen degraded by the solution is released into the supernatant and transferred to a second plate coated with avidin. By addition of biotinylated horseradish peroxidase to this second plate, the amount of collagen degraded is determined. Our assay requires only 0.5 microgram of type IV collagen per microtiter plate and detects nanogram quantities of bacterial collagenase activity.
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PMID:A nonradioactive assay for type IV collagen degradation. 216 Feb 4

We have developed an in vitro model of human papillary collecting duct cells isolated from cadaver kidneys using methods similar to those we previously reported for the isolation of human proximal tubule cells. To date we have isolated papillary collecting duct cells from 100 normal human kidneys. Papillae were dissected and digested in Cellgro containing 400 U/ml collagenase. Cells were plated on fibronectin-coated culture flasks at a density of 10(4) live cells/ml in Cellgro supplemented with insulin and 10% fetal bovine serum. Confluent monolayers, which were able to withstand 600 mOSM for 8 h, were obtained within 10 to 15 d. Cells of primary isolates and first passages exhibited epithelial cell ultrastructure including cell junctions, microvilli, and cilia. A dark-brown reaction product was observed in these cells when stained by the immunoperoxidase method with peroxidase-labeled peanut lectin (Arachis hypogaea), which binds specifically to human distal tubule and collecting duct cells. These cells were negative for Factor-VIII (a marker for endothelial cells) and gamma-glutamyltransferase (a marker for proximal tubule cells). High activities of the glycolytic enzyme pyruvate kinase and arginine vasopressin-stimulated cAMP production in these cells are consistent with a distal nephron origin. The results indicate that human collecting duct cells can be isolated and cultured to provide an in vitro system to probe pathogenetic mechanisms of potential nephrotoxins.
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PMID:Characterization of an in vitro system of human renal papillary collecting duct cells. 216 26

In normal conditions vascular permeability is precisely regulated by mechanisms which involve among others the macromolecules of extracellular matrix of the vascular wall. Permeability for a given substance will vary according to the anatomical localisation of the vessel determining also its structure and composition. In some pathological conditions, such as inflammation or diabetes, permeability can be abnormally increased. Increased permeability can be reproduced by i.v. collagenase injection. This permeability increase can be quantified by image analysis using appropriate tracers such as FITC-dextrans or horse-radish peroxidase, on histological sections from control and collagenase treated rats, pretreated or not with procyanidolic oligomers (PCO). We studied cerebral capillaries, aorta and cardiac muscle capillaries. It could be shown that previous treatment of animals with procyanidolic oligomers prevented the permeability increase produced by collagenase injection.
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PMID:[The effect of procyanidolic oligomers on vascular permeability. A study using quantitative morphology]. 216 37

The effects of proteases on air-space clearance (AC) of small ([14C]sucrose, 342 daltons) and large (125I-neutral dextran, 70,000 daltons) solutes were studied in isolated, fluid-filled hamster lungs that were perfused in a nonrecirculating system. When instilled into the air spaces, porcine pancreatic elastase (0.1-0.4 mg/ml) and bovine pancreatic trypsin (BPT) (0.5-2.0 mg/ml), but neither Clostridium histolyticum collagenase (5.0 mg/ml) nor phenylmethylsulfonyl fluoride-inactivated BPT caused large increases in the AC of both tracer molecules. BPT-induced solute clearance was further characterized functionally and morphologically. The functional characteristics of solute AC under steady-state conditions did not indicate that transepithelial transport was diffusion-limited. Inhibition by millimolar concentrations of Zn2+ and by lung cooling, along with electron microscopic studies employing horseradish peroxidase as a macromolecule tracer, were consistent with epithelial solute transport by a vesicular mechanism (transcytosis). Solute transport from the interstitial compartment to the lung exterior was shown to occur via two pathways. By unknown mechanisms BPT caused small amounts of water to flow through an incompletely identified, extravascular pathway. In BPT-exposed lungs efflux of 125I-dextran 70 occurred almost exclusively through this pathway, whereas [14C]sucrose was transported to the lung exterior partly through this same pathway and partly through the vasculature. The large differences in the diffusion coefficients of the two tracers may have accounted for these observed patterns of solute efflux from the lung. The possible significance of our findings to the pathogenesis of experimental emphysema are discussed.
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PMID:Effect of proteolytic enzymes on transepithelial solute transport. 243 Sep 29

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

This study investigated the effects of two putative bombesin antagonists, [D-Arg1,D-Pro2,D-Trp7,9,Leu11]substance P and [Leu13-psi-CH2NH-Leu14]bombesin, on bombesin-stimulated gastrin release from isolated canine G cells following short-term culture. Canine antral tissue was dispersed by sequential collagenase and EDTA treatment, and counterflow elutriation was used to enrich for G cells. Plates were seeded with 2 x 10(6) cells/mL in each well and cultured for 2 days prior to testing. Gastrin-containing and somatostatin-containing cells were identified by immunocytochemistry using the biotin-avidin-peroxidase method and accounted for 8.5 and 1%, respectively, of adhered cells. Basal gastrin secretion was 1.91 +/- 0.48% of total cell content. After a 2-h incubation period, bombesin (0.01-100 pM) stimulated gastrin release in a concentration-dependent fashion. The substance P analog, at a concentration of 1 microM, modestly inhibited bombesin-stimulated gastrin release from canine G cells. This analog also produced weak stimulation of basal gastrin release. In contrast, the bombesin analog, at a concentration of 1 microM, did not affect basal gastrin secretion. The bombesin analog completely blocked bombesin-stimulated gastrin release from 0.01 to 1 pM and produced greater than 50% inhibition at higher doses. The ability of the bombesin analog to directly inhibit bombesin-stimulated gastrin release from cultured canine G cells underscores its usefulness in studies involving the role of bombesin and its mammalian counterpart, gastrin-releasing peptide, in the control of gastrin cell function.
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PMID:Inhibition of bombesin-stimulated gastrin release from isolated canine G cells by bombesin antagonists. 248 58

A quantitative collagenase assay detecting soluble collagen fragments is described in this paper. Using the reagent N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) type I collagen was conjugated with horseradish peroxidase (POD) which was employed as a reporter enzyme. POD was preferentially linked to the TC B fragment in a ratio of 1.4 mol POD/mol collagen. The conjugation product was immobilized on AH-Sepharose via carbodiimide coupling to form the final collagenase substrate used in the assay. POD activity in the supernatants caused by liberated TC B fragments exhibited a linear relationship for collagenase concentrations up to 100 micrograms/ml bacterial collagenase. Over an incubation period of 4 h the lowest detection limits found were 20 ng/100 microliters for bacterial collagenase and 60 ng/100 microliters for human leukocyte collagenase. Incubation of the assay mixture with 5 micrograms trypsin resulted in 3.8% of the activity released by the equivalent amount of leukocyte collagenase. The assay developed here has been shown to be sensitive and specific for collagenase, with the additional advantage that this method is suited for simple and economic handling.
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PMID:A spectroscopic collagenase assay using peroxidase-labeled collagen. 254 Jun 72

In an attempt to clarify the Kupffer cell function in alcoholism, chronic ethanol-fed rats were investigated. The clearance of latex particles in the rat was analysed to estimate the function of the reticuloendothelial system in the liver, and the phagocytic function of Kupffer cells was measured by counting particles in the cell after isolation of non-parenchymal cells by collagenase digestion of the liver following an injection of latex particles and subsequently by staining of endogenous peroxidase activities. In addition, the number of Kupffer cells and their phagocytic function were examined histologically in fresh frozen sections of liver after an injection of particles. Serum ethanol concentration in the ethanol-fed rats was 10-60 mumol/l. The clearance of latex particles was markedly reduced in the ethanol-fed rats as compared with the paired controls (P less than 0.01). Markedly decreased-phagocytic function was found in 20% of Kupffer cells in the chronic ethanol-fed rats. The number of Kupffer cells in the ethanol-fed rats was increased as compared with the paired control rats. Chemotaxis analysis revealed that hepatocytes when incubated with ethanol, produced chemotactic factor for Kupffer cells and polymorphonuclear cells. These abnormal Kupffer cell functions may contribute to the pathogenesis of alcoholic liver disease.
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PMID:Kupffer cell function in chronic ethanol-fed rats. 269 94


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