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At least 217 records · Page 12Linked to original sources
PubMed2006

The in vitro metabolism of irinotecan (CPT-11) by carboxylesterase and beta-glucuronidase in human colorectal tumours.

AIMS: Irinotecan (CPT-11) is a prodrug that is used to treat metastatic colorectal cancer. It is activated to the topoisomerase poison SN-38 by carboxylesterases. SN-38 is metabolized to its inactive glucuronide, SN-38 glucuronide. The aim of this study was to determine, the reactivation of SN-38 from SN-38 glucuronide by beta-glucuronidase may represent a significant pathway of SN-38 formation. METHODS: The production of SN-38 from irinotecan and SN-38 glucuronide (2.4, 9.6 and 19.2 microm) was measured in homogenates of human colorectal tumour, and matched normal colon mucosa from 21 patients). RESULTS: The rate of conversion of irinotecan (9.6 microm) was lower in tumour tissue than matched normal colon mucosa samples (0.30+/-0.14 pmol min-1 mg-1 protein and 0.77+/-0.59 pmol min-1 mg-1 protein, respectively; P<0.005). In contrast, no significant difference was observed in beta-glucuronidase activity between tumour and matched normal colon samples (4.56+/-6.9 pmol min-1 mg-1 protein and 3.62+/-2.95 pmol min-1 mg-1 protein, respectively, using 9.6 microm SN-38 glucuronide; P>0.05). beta-Glucuronidase activity in tumour correlated to that observed in matched normal tissue (r2>0.23, P<0.05), whereas this was not the case for carboxylesterase activity. At equal concentrations of irinotecan and SN-38 glucuronide, the rate of beta-glucuronidase-mediated SN-38 production was higher than that formed from irinotecan in both tumour and normal tissue (P<0.05). However, at concentrations that reflect the relative plasma concentrations observed in patients, the rate of SN-38 production via these two pathways was comparable. CONCLUSIONS: Tumour beta-glucuronidase may play a significant role in the exposure of tumours to SN-38 in vivo.

Antineoplastic Agents, Phytogenic↗
PubMed1994

Plant and algal interference in bacterial beta-D-galactosidase and beta-D-glucuronidase assays.

Several commonly occurring freshwater and marine plants and algae were screened for beta-D-galactosidase and beta-D-glucuronidase activities by using a 60-min enzyme assay based on the hydrolysis by these enzymes of 4-methylumbelliferyl-beta-D-galactoside and 4-methylumbelliferyl- beta-glucuronide, respectively. All freshwater plant extracts tested showed beta-D-galactosidase activity several at relatively high levels, and a number also showed beta-D-glucuronidase activity. A number of the macroalgae showed no activity of either enzyme, but those showing beta-D-galactosidase activity also showed beta-D-glucuronidase activity. The majority of microalgae showed some beta-D-galactosidase activity, but few showed beta-D-glucuronidase activity. Further studies, using the commercial Colilert test and the marine water formulation of Colilert, revealed that 2 of 11 of the microalgal species and several of the plant extracts tested caused positive reactions. It was concluded that several plant extracts and algae could significantly interfere with the detection of coliform bacteria and Escherichia coli with the use of rapid assays, on the basis of their production of beta-D-galactosidase and beta-D-glucuronidase, respectively. The significance of the plant and algal interferences in tests such as Colilert is dependent on the levels of enzymes released under natural conditions, the dilution which they may undergo, and the numbers of algal cells present. This also applies to interferences in rapid enzyme assays.(ABSTRACT TRUNCATED AT 250 WORDS)

Escherichia coli↗
PubMed1971

Failure of urinary beta-glucuronidase activity to localize the site of urinary tract infection.

The differentiation of renal from bladder bacteriuria is difficult on clinical grounds alone. To evaluate the correlation between site of infection and urinary beta-glucuronidase activity, 46 patients with well documented recurrent bacteriuria were studied by bilateral ureteral catheterization. Urinary beta-glucuronidase activity was also determined in 46 control subjects. In general, asymptomatic patients with renal bacteriuria, either unilateral or bilateral, had levels of enzyme activity in their urine comparable to patients with infection confined to the bladder and to normals. Only 4 of 25 patients with renal bacteriuria had significant elevations of urinary beta-glucuronidase. After localization of infection, 9 of 10 patients treated with kanamycin, a potentially nephrotoxic drug, developed significant elevations of urinary beta-glucuronidase. The results of these studies indicate that determination of beta-glucuronidase activity in urine is not useful in predicting the site of infection in patients with bacteriuria but may find a role in screening for early nephrotoxicity.

Bacteriuria↗
PubMedDate not supplied

A novel efficient method to identify beta-glucuronidase activity in rat small intestine.

BACKGROUND: Recent epidemiologic studies promote the notion that high intake of food rich in phytochemicals protects against degenerative diseases such as coronary heart diseases and cancer. Phytochemicals are detoxified in mammalian tissues by conjugation with glucuronic acid yielding less active glucuronide conjugates. However, in several tissues glucuronide conjugates are reactivated by the cleaving enzyme beta-glucuronidase. The aim of the present study was to develop a routinely manageable, rapid technique to localize the beta-glucuronidase activity in the small intestinal tissue. METHODS: Histologic slices of rat duodenum, jejunum, and ileum were incubated with a specific chromogenic beta-glucuronidase substrate, 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide (X-GlcU). After enzymatic cleavage, X-GlcU yields 5-bromo-4-chloro-3-indol, a dark blue crystalline precipitate easily monitored by light microscopic technique. RESULTS: The number and intensity of the crystals were highest in the jejunum and lowest in the ileum. In all three sections of the small intestine, the highest activity was observed at the villar tip and in the tela submucosa and only moderate activity in other layers of the intestinal tissue. CONCLUSIONS: By using the X-GlcU-technique, it could be demonstrated convincingly that beta-glucuronidase exists in all three segments of the rat small intestine. The proposed method is an efficient, simple, and convenient method to visualize beta-glucuronidase activity.

Animals↗
PubMed1994

Selective inhibition of gastrointestinal beta-glucuronidase by poly(vinylbenzyl D-glucaro(1,4)lactonate). Part 2. Poly(vinylbenzyl D-glucaro(1,4) lactonate) in vitro inhibition studies.

In vitro inhibition studies with beta-glucuronidase from purified E. coli and mouse intestinal contents indicated that the polymer, poly(vinylbenzyl D-glucaro(1,4)lactonate, is an effective beta-glucuronidase inhibitor. Purified E. coli beta-glucuronidase was inhibited by 99.6% with 177 mM D-glucaro(1,4)lactone using the polymer-inhibitor. Similarly, 95% inhibition of beta-glucuronidase activity of mouse intestinal contents was obtained with 177 mM and 50% inhibition was obtained with 31.5 mM D-glucaro(1,4)lactone based on the modified polymer. The structural requirements of an effective beta-glucuronidase inhibitor based on the structure of the polymer-inhibitor are also discussed.

Animals↗
PubMed1978

The cytochemical demonstration of beta-glucuronidase in colon neoplasms of rats exposed to azoxymethane.

Colon tumors induced with azoxymethane in male Fischer rats were cytochemically analyzed for beta-glucuronidase using naphthol AS-B1 glucuronide (6-bromo-2-hydroxy-3-naphthoyl-O-anisidine) as a substrate and hexazonium pararosanin as a diazo reagent. This method effectively localizes the bulk of beta-glucuronidase in the surface epithelium, the lamina propria and in the endothelial cells of the lymphoid sinuses and postcapillary venules. Polypoid lesions, adenocarcinomas and mucinous adenocarcinomas show no difference in the amount or in the localization of beta-glucuronidase; however, mucinous adenocarcinomas show a slight increase in the amount of beta-glucuronidase. The few tumors that did metastasize to lymph nodes did not show any difference in their enzyme patterns. Intestinal crypts that show a change in size and shape have a definite increase in beta-glucuronidase activity. An increase in the activity of this enzyme can also be seen in well defined neoplasms as opposed to normal areas of the colon.

Adenocarcinoma↗
PubMed1993

Enzyme replacement with recombinant beta-glucuronidase in the newborn mucopolysaccharidosis type VII mouse.

beta-Glucuronidase injected i.v. into newborn mucopolysaccharidosis VII mice was cleared from the circulation in less than 1 h and taken up by tissues in a distribution corresponding to the location of the mannose 6-phosphate receptor. One h after a 3.5-mg/kg beta-glucuronidase injection, beta-glucuronidase levels were equal to or greater than normal in every organ examined with the exception of the brain, where 31% normal activity was present. Enzyme was detectable histochemically in the major sites of pathology for mucopolysaccharidosis VII including bone, brain, heart, and fixed tissue macrophages. The half-life of recombinant beta-glucuronidase activity in various organs of injected mucopolysaccharidosis VII mice was 1.5 to 4.5 d. These studies show that recombinant beta-glucuronidase administered to newborn mice reaches the sites of clinically important storage in murine mucopolysaccharidosis VII.

Animals↗
PubMed1986

Genetic regulation of androgen-induced accumulation of mouse renal beta-glucuronidase messenger ribonucleic acid.

Mouse kidney beta-glucuronidase is induced by androgens in a receptor-dependent fashion. Genetic regulatory mutations have been described which govern this response. In mice carrying the Gus-ra allele (A/J), the induction of enzyme activity is 3-5 times greater than in animals with the Gus-rb allele (C57BL/6J). To study this hormonal and genetic control at the molecular level, we measured changes in beta-glucuronidase mRNA concentrations in these two mouse strains using cloned cDNA. The specificity of the regulation was assessed by following changes in the concentration of kidney androgen-regulated protein (KAP) mRNA, which is also under androgen control in mouse kidney. Female mice were treated with Silastic implants that released 120 micrograms testosterone/day over a 20-day time course. Induction of specific mRNA was analyzed by either Northern blot hybridization or a more quantitative assay in which renal poly(A) RNA was covalently bound to aminophenylthioether paper discs. The induction of beta-glucuronidase mRNA was about 10-fold higher in the strain carrying the Gus-ra regulatory allele, indicating that the Gus-r locus controls the beta-glucuronidase structural gene (Gus-s) at the level of mRNA accumulation. Regulation by Gus-r was specific for beta-glucuronidase mRNA as kidney androgen-regulated protein mRNA accumulation did not differ between these two strains of mice after androgen treatment.

Animals↗
PubMed1985

Direct transfer of beta-glucuronidase from mouse macrophages to other types of cell.

Rabbit polyclonal antibodies were raised against beta-glucuronidase purified from mouse liver. This antiserum immunoprecipitated the beta-glucuronidase secreted by mouse fibroblasts but did not cross-react with the same enzyme isolated from human tissue. The beta-glucuronidase present in mouse 3T3 fibroblasts and mouse peritoneal macrophages was clearly identified by indirect immunofluorescence, using the antiserum and an FITC-conjugated second antibody, while human fibroblasts with normal levels of beta-glucuronidase activity did not fluoresce when tested with the same reagents. A range of human fibroblasts, human neuroblastoma and rat glioma cells did not fluoresce when incubated with the antibody but did fluoresce after they had been co-cultured for 24 h with mouse macrophages, showing that mouse beta-glucuronidase had been transferred from adherent macrophages into adjacent recipient cells. Transfer took place even when receptor-mediated endocytosis was blocked with a suitable competitive ligand, the transferred enzyme being visible mainly as a bright punctate fluorescence with a lysosome-like distribution. Macrophages thus have the potential to act as donors of lysosomal enzymes to a wide range of recipient cells and to transfer enzymes to them during direct cell-to-cell contact.

Animals↗
PubMed1995

Inhibitory effect of ethanol and colchicine on the intracellular processing of beta-glucuronidase which occurs in the Golgi complex.

To investigate the effect of ethanol or colchicine on the intracellular proteolytic processing of lysosomal beta-glucuronidase, which is considered to occur in the Golgi complex in the intracellular sorting pathway, three rat liver Golgi subfractions, GF-1, GF-2, and GF-3, were isolated from ethanol- or colchicine-treated rats, and the electrophoretic patterns of the extracted Golgi beta-glucuronidase on polyacrylamide gel were examined. The isolated Golgi subfractions from the drug-treated rats gave a better yield of fraction than that from the control rats. The enzymatic characterization of these three subfractions showed no significant contamination by other subcellular structures such as plasma membranes, microsomes, or lysosomes, and no inhibitory effect of the drugs was observed. On the other hand, suppressed galactosyltransferase activity, a marker enzyme of the Golgi complex, was detected in the colchicine-treated rats. The electrophoretic pattern of Golgi beta-glucuronidase on polyacrylamide gel revealed one major band which moved to the same position as the lysosomal enzyme type in the control rats. In contrast, in the ethanol- and colchicine-treated rats, Golgi beta-glucuronidase was found to have two major bands stained for enzyme activity resulting from a mixture of microsomal- and lysosomal-type enzymes. These results suggested that the post-translational modification step, during conversion from a microsomal-type enzyme to a lysosomal-type enzyme, was apparently inhibited. Taken together, these findings indicated that ethanol or colchicine administration to rats caused an inhibitory effect on the intracellular post-translational modification of Golgi beta-glucuronidase destined for targeting to the lysosomes.

Animals↗
PubMed1991

Release of liver microsomal beta-glucuronidase from hepatocytes in vitro and in vivo by organophosphates and hepatotoxic agents.

Liver microsomal beta-glucuronidase is stabilized within microsomal vesicles by complexation with the accessory protein, named egasyn. In this study, we showed that egasyn is identical to one of the carboxylesterase isozymes and organophosphorus and carbamate insecticides, acetanilide which is a specific substrate of egasyn and halothane caused a rapid dissociation of the egasyn-microsomal beta-glucuronidase complex when administered in vivo or when added in vitro to isolated hepatocytes. The dissociation was relatively specific to organophosphates, carbamates, but not pyrethroids. Dissociation of the egasyn-beta-glucuronidase complex in vivo by organophosphates was followed by massive and rapid secretion of microsomal beta-glucuronidase into plasma. From these results, we concluded that release of liver microsomal beta-glucuronidase is the most rapid and sensitive marker to organophosphorus or carbamate insecticide-induced intoxication.

Acetanilides↗
PubMed2000

Secretory phospholipases A2 induce beta-glucuronidase release and IL-6 production from human lung macrophages.

Secretory phospholipases A2 (sPLA2s) are a group of extracellular enzymes that release fatty acids at the sn-2 position of phospholipids. Group IIA sPLA2 has been detected in inflammatory fluids, and its plasma level is increased in inflammatory diseases. To investigate a potential mechanism of sPLA2-induced inflammation we studied the effect of group IA (from cobra venom) and group IIA (human synovial) sPLA2s on human macrophages. Both sPLA2s induced a concentration- and Ca2+-dependent, noncytotoxic release of beta-glucuronidase (16.2 +/- 2.4% and 13.1 +/- 1.5% of the total content with groups IA and IIA, respectively). Both sPLA2s also increased the rate of secretion of IL-6 and enhanced the expression of IL-6 mRNA. Preincubation of macrophages with inhibitors of the hydrolytic activity of sPLA2 or cytosolic PLA2 did not influence the release of beta-glucuronidase. Incubation of macrophages with p-aminophenyl-mannopyranoside-BSA (mp-BSA), a ligand of the mannose receptor, also resulted in beta-glucuronidase release. However, while preincubation of macrophages with mp-BSA had no effect on beta-glucuronidase release induced by group IIA sPLA2, it enhanced that induced by group IA sPLA2. A blocking Ab anti-mannose receptor inhibited both mp-BSA- and group IIA-induced beta-glucuronidase release. Taken together, these data indicate that group IA and IIA sPLA2s activate macrophages with a mechanism independent from their enzymatic activities and probably related to the activation of the mannose receptor or sPLA2-specific receptors. The secretion of enzymes and cytokines induced by sPLA2s from human macrophages may play an important role in inflammation and tissue damage associated with the release of sPLA2s.

Aniline Compounds↗
PubMed1999

Human and Escherichia coli beta-glucuronidase hydrolysis of glucuronide conjugates of benzidine and 4-aminobiphenyl, and their hydroxy metabolites.

Individuals exposed to carcinogenic aromatic amines excrete arylamine N- and O-glucuronide metabolites. This study assessed the susceptibility of selected glucuronides to hydrolysis by human and Escherichia coli beta-glucuronidase. N- or O-glucuronides were prepared with the following aglycones: benzidine, N-acetylbenzidine, N'-hydroxy-N-acetylbenzidine, N-hydroxy-N-acetylbenzidine, N-hydroxy-N,N'-diacetylbenzidine, 3-hydroxy-N,N'-diacetylbenzidine, 3-hydroxy-benzidine, 4-aminobiphenyl, N-hydroxy-4-aminobiphenyl, and N-hydroxy-N-acetyl-4-aminobiphenyl. The (3)H- and (14)C-labeled glucuronides were prepared with human or rat liver microsomes using UDP-glucuronic acid as cosubstrate. Each of the 10 glucuronides (6-12 microM) was incubated at pH 5.5 or 7.0 with either human recombinant (pure) or E. coli (commercial preparation) beta-glucuronidase for 30 min at 37 degrees C. Hydrolysis was measured by HPLC. Reaction conditions were optimized, using the O-glucuronide of N-hydroxy-N,N'-diacetylbenzidine. Both enzymes preferentially hydrolyzed O-glucuronides over N-glucuronides and distinguished between structural isomers. With E. coli beta-glucuronidase at pH 7.0, selectivity was demonstrated by the complete hydrolysis of N-hydroxy-N-acetyl-4-aminobiphenyl O-glucuronide in the presence of N-acetylbenzidine N-glucuronide, which was not hydrolyzed. Metabolism by both enzymes was completely inhibited by the specific beta-glucuronidase inhibitor saccharic acid-1,4-lactone (0.5 mM). The concentration of human beta-glucuronidase necessary to achieve significant hydrolysis of glucuronides was substantially more than the amount of enzyme reported previously to be present in urine under either normal or pathological conditions. The bacterial enzyme may hydrolyze O-glucuronides, but not N-glucuronides, in urine at neutral pH. Thus, the nonenzymatic hydrolysis of N-glucuronides by acidic urine is likely a more important source of free amine than enzymatic hydrolysis.

Aminobiphenyl Compounds↗
PubMed1999

Implications of alpha-amylase production and beta-glucuronidase expression in Escherichia coli strains.

Two Escherichia coli strains in which alpha-amylase production differed were used to study in depth some characteristics related to beta-glucuronidase induction by starch. The beta-glucuronidase background activity in Luria broth medium was comparable for the two isolates, but only amylase positive S1 was able to grow on starch molecules supplied as the sole carbon source. In this case growth resulted at higher beta-glucuronidase levels (p < 0.01) with respect to basal activity and the induced expression was maximal (6.1-fold) when cultures reached the stationary phase. Growth in the presence of a protein synthesis inhibitor (chloramphenicol) was associated with a marked reduction of activity. The beta-glucuronidase activity of amylase negative M94 remained unchanged during starvation on starch medium, but an induced response was observed with methylumbelliferyl-glucuronide. These results further support the hypothesis that starch metabolism is involved in the complex beta-glucuronidase regulation of E. coli strains. This is relevant not only for basic research but also to investigating gut microbial enzymology.

Cell Division↗
PubMed2001

beta-D-glucuronidase is associated with goat sperm cytoskeleton.

Glycosidase activities were detected as detergent-insoluble after sequential extractions of goat sperm with Triton X-100. Seventy percent of total beta-glucuronidase activity was found in the detergent-insoluble fraction. This portion of beta-glucuronidase was resistant to extractions in the presence of 1 M KCl, chaotropic agents, colchicine, or cytochalasin B, being only partially solubilized by 3 M KCl or DNAse I treatment. The treatment with 0.1% sodium deoxycholate was effective, releasing 73% of the enzyme activity. Treating the deoxycholate extract with DNAse I resulted in a change in the elution profile of beta-glucuronidase as judged by gel filtration chromatography. A polyclonal antibody was developed against pancreatic beta-glucuronidase, and the sperm enzyme was strongly inhibited by the IgG fraction of this antibody. Western blot analysis showed that the same protein correspond to both Triton-soluble and insoluble enzyme. Results demonstrate that beta-glucuronidase is tightly bound to the Triton X-100 resistant fraction, suggesting that the enzyme is associated to sperm cytoskeleton. J. Exp. Zool. 289:146-152, 2001.

Animals↗
PubMedDate not supplied

Breast milk beta-glucuronidase levels in hyperbilirubinemia.

Breast milk beta-glucuronidase was thought to be one of the etiological factors in the pathogenesis of late-onset breast-milk jaundice, but results of these studies are conflicting. In this study breast milk beta-glucuronidase levels were determined in groups with physiologic jaundice, early breast-feeding jaundice and late breast-milk jaundice. No difference in beta-glucuronidase levels of these three groups was found in samples taken on the 4th and 15th days of life. beta-glucuronidase activity in breast milk declined from the 4th to 15th day in all groups. These results imply that factors other than breast milk beta-glucuronidase activity should be investigated to reveal the pathogenesis of late-onset breast-milk jaundice.

Glucuronidase↗
PubMedDate not supplied

[Serum beta-glucuronidase activity in polytrauma patients and in centrifuged autopsy blood samples].

The serum activity of beta-glucuronidase was investigated in 58 patients after severe trauma as well as in 43 autopsy cases. In 10 cases the enzyme activities in postmortem blood samples from the femoral vein were compared to those present in the correspondent heart blood samples. An elevated activity of beta-glucuronidase was observed in 14% of the patients within the first 36 h after severe trauma increasing to 62% in blood samples collected later on. The activity of beta-glucuronidase in the heart blood samples was always higher than in the corresponding sample from the femoral vein. In cases of prolonged post-mortem interval an elevated activity might have been due to bacterial contamination. In postmortal blood samples from the femoral vein an elevated enzyme activity was found in 70% of the study material. The results of the preliminary study on the activity of beta-glucuronidase in blood samples frequent in forensic routine work indicated that an elevated enzyme activity might be present for the following scenery: after severe trauma, in alcohol/drug abuse, presence of putridity/autolysis, presence of inflammatory processes, in diabetes as well as in carcinoma diseases. The significance of elevated beta-glucuronidase activity concerning alterations of unconjugated drug concentration due to in vitro cleavage of O-glucuronides should be investigated.

Autopsy↗
PubMed2002

[Molecular evolution of beta-glucuronidase in vitro: obtaining thermotolerant GUS gene].

The Escherichia coli beta-glucuronidase gene (gus) has been developed as a reporter gene for plants, and has been widely used for over a decade. Both chromogenic and fluorogenic GUS substrates have been synthesized, allowing rapid nonradioactive assays. The use of the Escherichia coli enzyme beta-glucuronidase (GUS) as a reporter in gene expression studies is limited by some plants and plant-associated bacteria express endogenous glucuronidase activities. The use of the enzyme as a reporter in transgenic plants is limited by high false positive. Laboratory evolution methods were used to enhance the thermostability and activity of the beta-glucuronidase. Using plasmid pBI121 as template, a 1.8 kb specific product was amplified and cloned into the vector pBluescript SK. The result of nucleotide sequence analysis was the same as reported. In vitro recombination (DNA shuffling), which involves DNase I digestion, primerless PCR, and primer PCR was used to generate mutant libraries. The mutant GUS3-3 gene was isolated after three rounds of mutation, DNA shuffling, and screening. The GUS3-3 enzyme can resistant high temperature up to 80 degrees C for 30 min. The nucleotide sequence analysis showed 99.2% homology between the GUS-ck gene from pBI121 and GUS3-3 gene. The deduced amino acid sequence demonstrated that 11 amino acid was changed. The Tm value of GUS3-3 is 80 degrees C and increased by 25 degrees C above GUS-ck (55 degrees C). The researches indicated the feasibility of the molecular evolution of beta-glucuronidase in vitro to improve enzymatic thermostability.

Amino Acid Sequence↗