Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.31 (beta-glucuronidase)
 7,680 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We investigated the protein composition of J774-E clone macrophage phagosomes isolated at different stages of phagolysosome biogenesis. Phagosomes formed by internalizing antibody-coated Staphylococcus aureus for 3 min followed by chase for 0, 4, 9, or 15 min were isolated by density gradient centrifugation. Enrichment and purity of the phagosome preparations were quantitated by radiolabeled ligand recovery, enzyme markers, and electron microscopy. One-dimensional SDS-PAGE analyses of the isolated phagosomes revealed virtually identical protein compositions. However, Western blot analyses with antibodies directed against selected proteins of known itineraries along the endocytic pathway demonstrated distinct differences in phagosome protein compositions. Accumulating within the maturing phagosome were the 31-kD subunit of the vacuolar proton pump, cathepsin D,beta-glucuronidase, the cation dependent mannose 6-phosphate receptor, and LAMP-1. Decreasing within the maturing phagosome were the FcII receptor, the mannose receptor, and alpha-adaptin. These results indicate that although the macrophage phagosome's total protein composition changes little during phagolysosome formation, the maturing phagosome both receives and eliminates, possibly by protein recycling, specific membrane and sequestered proteins.
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PMID:Alterations in the protein composition of maturing phagosomes. 143 Feb 21

Dinitrophenol (DNP)-beta-glucuronidase and mannosylated anti-DNP IgG, which are endocytosed by the mannose receptor and delivered to lysosomes, were previously developed as probes for examination of fusion between early endosomes in a cell-free system. In this study, these probes were found to be transported by intact cells to endocytic vesicles with heavy buoyant density at different rates, as determined by Percoll gradient fractionation of cell homogenates. There was a concomitant loss of in vitro fusion activity as the ligands moved to dense compartments. In monensin-treated cells, DNP-beta-glucuronidase was retained in a light compartment corresponding to intracellular vesicles capable of fusion in vitro. Pulse-chase studies using a DNP-derivatized transferrin-alkaline phosphatase conjugate showed that a recycling ligand was always found in light intracellular vesicles that were capable of fusion to early endosomes in vitro. In contrast to cell-free systems, intact cells sequentially labeled with DNP-beta-glucuronidase and then mannosylated anti-DNP IgG showed ligand mixing in both early and late endocytic compartments. Treatment with nocodazole or colchicine did not affect the rate of DNP-beta-glucuronidase transport to heavy vesicles in intact cells, however, the extent of ligand mixing in late endosomes was decreased by microtubule disruption. Using sequentially labeled cells split into two groups, we directly compared ligand mixing in vitro to mixing by intact cells. Fusion alone does not mediate increases in vesicle density, since DNP-beta-glucuronidase/anti-DNP IgG complexes formed in vitro were found in light vesicles, while intact cells showed immune complexes predominantly in heavy vesicles. These results suggest that the density shift is an initial step in targeting to lysosomes.
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PMID:Endosomal density shift is related to a decrease in fusion capacity. 180 9

Studies were conducted to assess the mitogenic effect of lysosomal hydrolases, enzymes known to have an association with allergen- or ozone-induced airway hyperreactivity, on bovine tracheal myocytes in culture. Addition of purified human placental beta-hexosaminidase and partially purified bovine liver beta-glucuronidase resulted in the doubling of cell count after 4 d of incubation in medium M199 with 0.4% FBS. Unstimulated cells remained quiescent without a significant increase of cell count. Lysosomal hydrolases also selectively enhanced 3H-thymidine incorporation four to seven times more than that in vehicle-treated cells or cells treated with endotoxin, a common contaminant of purified enzymes. Ovalbumin (glycoprotein control), pronase, and lysozyme caused a modest but statistically insignificant increase (up to twofold) in 3H-thymidine incorporation. Elastase, collagenase and dialyzed E. coli beta-glucuronidase had no effect. The mitogenic effect of hydrolases was equally seen in quiescent, serum-depleted cells as well as in those maintained in medium with 10% FBS, suggesting that it was independent of serum factors. The effect of lysosomal hydrolases was inhibited by exposure to yeast mannan, and mannosylated human serum albumin had a mitogenic effect, suggesting the involvement of a mannose receptor. We conclude that lysosomal hydrolases may play a role in the development of the hyperplasia/hypertrophy of respiratory smooth muscle.
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PMID:Mitogenic effect of lysosomal hydrolases on bovine tracheal myocytes in culture. 183 69

Macrophages express a mannose-specific endocytosis receptor that binds and internalizes mannose-terminated glycoproteins. Infection of mouse peritoneal macrophages with Leishmania donovani resulted in a decrease in mannose-receptor activity. With 125I-labelled beta-glucuronidase as ligand, a 2-fold decrease in uptake rate was observed in infected cells, with no change in Kuptake. Cell-surface binding of 125I-mannose-BSA was diminished 2.5-fold after infection. The decrease in ligand binding appeared to be due to a decrease in the number of sites, with no change in affinity. Elimination of parasites from infected cells by treatment with neoglycoprotein-conjugated methotrexate resulted in an increase in receptor number. Cycloheximide suppressed the drug-treatment-mediated rise in receptor number in infected macrophages. A decrease in receptor activity was also observed in liver Kupffer cells isolated from parasite-infected mice. Binding of ligand by another carbohydrate receptor, the mannose 6-phosphate receptor, was not altered by infection. Phagocytosis of yeast cells was also not altered. These results suggest that mannose receptor synthesis in macrophages is specifically suppressed after infection with Leishmania parasites.
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PMID:Down-regulation of mannose receptors on macrophages after infection with Leishmania donovani. 185 73

Adherent cultures of rat peritoneal macrophages secrete lysozyme and the lysosomal marker enzymes beta-glucuronidase, beta-N-acetylglucosaminidase and acid phosphatase; the levels of secreted lysosomal cathepsin D, however, were found to be insignificant. Incubation of the cells at 4 degrees C for 15 min with yeast mannan or with 50 mM mannose, methyl alpha-glucopyranoside, or N-acetylglucosamine caused the concentration of cathepsin D in the culture medium to increase 30-40-fold; mannose-6-phosphate had no effect. 125I-labeled cathepsin D was prepared and the binding constant to the macrophage cell surface was determined to be KD = 27 nM. The data suggest that cathepsin D binds to the mannose receptor of macrophages and that binding to this receptor is not in equilibrium with the bulk medium.
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PMID:Binding of cathepsin D to the mannose receptor on rat peritoneal macrophages. 193 26

Ricin A chain has previously been shown to intoxicate macrophages in vitro following binding and endocytosis by the macrophage mannose receptor. In this report it is demonstrated that the intravenous injection of ricin A chain in nephrectomized rats leads to a prolonged plasma half-life for [125I]beta-glucuronidase, a ligand for the mannose receptor. Clearance of [125I]asialofetuin, a ligand for the galactose receptor of hepatocytes, was unaffected by injection of A chain. Microscopic examination of the livers of A chain-treated animals revealed a loss of phagocytic cells from the liver sinusoids. These results suggest that ricin A chain may be useful as a toxin specific for mannose receptor bearing cells of the reticuloendothelial system.
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PMID:In vivo depletion of mannose receptor bearing cells from rat liver by ricin A chain: effects on clearance of beta-glucuronidase. 244 98

Mannose receptors are expressed only in primary macrophages. Established macrophage-derived cell lines, although apparently possessing the potential to synthesize mannose receptors, do not express them on their plasma membranes. Using the drug 5-Azacytidine, mannose receptor expression was induced in the macrophage-derived mouse cell line J774. Receptor positive cells were sorted through a fluorescent activated cell sorter (FACS) prior to cloning. Clones were isolated which continuously express mannose receptors in culture. These macrophages were able to endocytose beta-glucuronidase and phagocytose yeast particles via mannose receptors. Secretion of the lysosomal enzyme beta-hexosaminidase was also reduced in proportion to the degree of mannose receptor expression.
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PMID:Generation of macrophage variants with 5-azacytidine: selection for mannose receptor expression. 244 84

Early steps of receptor-mediated endocytosis appear to require the fusion of endosomes with each other. Recently, these fusion events have been reconstituted in vitro using vesicle preparations from J774 macrophages which have internalized ligands via the mannose receptor (Diaz, R., Mayorga, L., and Stahl, P. (1988) J. Biol. Chem. 263, 6093-6100). The present studies indicate that endosomes first form clusters when incubated under fusogenic conditions. Aggregation state was determined by electron microscopy using vesicles containing ligand-coated colloidal gold of different sizes previously internalized via the mannose receptor. Aggregation required cytosol and ATP. Afterwards, the limiting membranes of the vesicles composing these aggregates undergo multiple fusion and bring about the formation of large diameter vesicles that maintained the same density as endosomes when analyzed by Percoll gradient sedimentation. These large diameter vesicles were no longer fusogenic in the fusion assay. Multiple fusion was determined morphologically by the co-localization of three different size colloidal gold vesicles inside endocytic vesicles and biochemically by the fusion-dependent formation of triple immune complexes between three endocytic ligands internalized by receptor-mediated endocytosis: anti-dinitrophenol mouse IgG and dinitrophenol-derivatized beta-glucuronidase, ligands for the mannose receptor, and aggregated rabbit anti-mouse IgG, a ligand for the macrophage Fc receptor.
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PMID:In vitro clustering and multiple fusion among macrophage endosomes. 275 8

Receptor-mediated endocytosis and receptor recycling involve a series of intracellular membrane fusion events that appear to play an important role in the regulation of the overall rate and efficiency of the process. An endosome-endosome fusion assay is described using two ligands that (i) rapidly and efficiently enter the endosomal compartment via the macrophage mannose receptor and (ii) that mutually recognize each other. Dinitrophenol-derivatized beta-glucuronidase (DNP-beta-glucuronidase), a ligand for the mannose receptor, was endocytosed by one population of J774 E clone cells, and mannose-derivatized monoclonal anti-DNP IgG (Man-IgG) was internalized by a second set of cells. Both ligands were localized in endosomes as determined by fractionation on Percoll gradients. Incubation of vesicles prepared from the two set of cells resulted in vesicle fusion as indicated by the formation of DNP-beta-glucuronidase-Man-IgG complexes. Under standard conditions, fusion was time-, ATP-, and temperature-dependent. KCl was required for fusion. Fusion required both cytosolic- and membrane-associated proteins. N-Ethylmaleimide treatment of cytosol inhibited fusion. Proton ionophores and amines had no effect on the fusion reaction. ATP-dependent fusion was only observed between early endocytic compartments. While in the presence of a Ca2+ chelator fusion was ATP-dependent, in its absence fusion was also observed in an ATP-independent fashion.
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PMID:In vitro fusion of endosomes following receptor-mediated endocytosis. 336 Jul 75

We used the A-chain of the toxin ricin (RTA) as a toxin specific to Kupffer cells in mice. RTA is specifically taken up by the mannose receptor present exclusively in macrophages. Kupffer cells were quantitated by shifts in beta-glucuronidase clearance and microscopic counts of cells which phagocytosed India ink. When compared to saline controls, 20 mg/kg of RTA intraperitoneally (divided over 4 days) or intraportally (single doses) significantly prolonged the t 1/2 half-life of beta-glucuronidase by 270 +/- 37 and 210 +/- 8%, respectively. Kupffer cell numbers were significantly decreased by 27 +/- 8 and 33 +/- 16%. This effect persisted for at least 3 days after toxin administration. Despite effects on Kupffer cell number, minimal histological damage to liver, spleen, lung, and heart was noted. Higher doses of RTA or doses potentiated by ureteral ligation to prevent renal clearance resulted in prohibitive mortalities and histologic liver damage. Doses of Hura crepitans inhibitor, a toxin similar to RTA but not mannose-receptor specific, did not affect Kupffer cell numbers. We conclude that RTA given both intraperitoneally and intraportally at low doses is toxic specifically to Kupffer cells. Kupffer cell numbers can be indirectly measured by beta-glucuronidase clearance.
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PMID:Use of ricin A-chain to selectively deplete Kupffer cells. 339 96


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