hymecromone and 4-methylumbelliferyl-glucuronide

Globally, millions of people who rely on groundwater for potable purposes and agriculture have been inadvertently exposed to toxic arsenic (As) because of its natural occurrence in groundwater in several countries of Asia, Europe and America. While the presence of As in groundwater and its impacts on human health have been documented in many countries, there is little information on As contamination in Pakistan. This review highlights, for the first time, the extent and severity of As-induced problems in Pakistan based on relevant published papers; discusses possible sources of As contamination of aquifers; and estimates As-induced potential health hazards in the country in relation to global data. Data from 43 studies (>9882 groundwater samples) were used to describe As variability in groundwater of Pakistan and for comparison with global data. The mean groundwater As content reported in these studies was 120 μg/L (range: 0.1-2090 μg/L; SD: ±307). About 73% of the values for mean As contents in the 43 studies were higher than the World Health Organization (WHO) permissible limit (10 μg/L) for drinking water, while 41% were higher than the permissible limit of As in Pakistan (50 μg/L). It was observed that groundwater samples in some areas of Punjab and Sindh provinces contained high As concentrations which were almost equal to concentrations reported in the most contaminated areas of the world. We predicted that the mean values of ADD, HQ and CR were 4.4 μg kg

Topics: Agriculture; Arsenic; Arsenic Poisoning; Drinking Water; Drug Contamination; Environmental Exposure; Environmental Monitoring; Europe; Groundwater; Humans; Hymecromone; Pakistan; Risk Assessment; Water Pollutants, Chemical; Water Pollution; Water Pollution, Chemical; World Health Organization

A sensitive and specific double-antibody enzyme immunoassay (EIA) for detecting an elcatonin-like immunoreactive substance (ECT-IS) in human plasma has been developed. In competitive reactions, the ECT antibody was incubated with a plasma sample (or ECT standard) and beta-D-galactosidase-linked synthetic ECT. Free and antibody-bound enzymes were separated using an anti-rabbit IgG-coated immunoplate. Enzyme activity on the plate was determined by fluorescence analysis. This immunoassay allows the detection of 20 to 300 fmol/ml (67 to 1000 pg/ml) of ECT. The EIA was applied to determine the pharmacokinetic behavior of ECT after a single intramuscular administration (20 IU). The maximum level was achieved 30 min after administration, at approximately 30 pg ECT/ml of plasma.

Topics: Adult; beta-Galactosidase; Calcitonin; Chromatography, High Pressure Liquid; Escherichia coli; Fluorescent Dyes; Humans; Hymecromone; Immunoenzyme Techniques; Injections, Intramuscular; Male; Middle Aged

The use of glycopolymer-functionalized resins (Resin-Glc), as a solid support, in column mode for bacterial/protein capture and quantification is explored. The Resin-Glc is synthesized from commercially available chloromethylated polystyrene resin and glycopolymer, and is characterized by fourier transform infrared spectroscopy, thermogravimetry, and elemental analysis. The percentage of glycopolymer functionalized on Resin-Glc is accounted to be 5 wt%. The ability of Resin-Glc to selectively capture lectin, Concanavalin A, over Peanut Agglutinin, reversibly, is demonstrated for six cycles of experiments. The bacterial sequestration study using SYBR (Synergy Brands, Inc.) Green I tagged Escherichia coli/Staphylococcus aureus reveals the ability of Resin-Glc to selectively capture E. coli over S. aureus. The quantification of captured cells in the column is carried out by enzymatic colorimetric assay using methylumbelliferyl glucuronide as the substrate. The E. coli capture studies reveal a consistent capture efficiency of 10

Topics: Biochemistry; Calibration; Carbohydrate Conformation; Escherichia coli; Glucuronidase; Hymecromone; Kinetics; Lectins; Limit of Detection; Polymers; Polysaccharides; Proteins; Resins, Synthetic; Spectroscopy, Fourier Transform Infrared; Water Microbiology

Glutamine synthetase (GS) acts as a key enzyme in plant nitrogen (N) metabolism. It is important to understand the regulation of GS expression in plant. Promoters can initiate the transcription of its downstream gene. Eichhornia crassipes is a most prominent aquatic invasive plant, which has negative effects on environment and economic development. It also can be used in the bioremediation of pollutants present in water and the production of feeding and energy fuel. So identification and characterization of GS promoter in E. crassipes can help to elucidate its regulation mechanism of GS expression and further to control its N metabolism.. A 1232 bp genomic fragment upstream of EcGS1b sequence from E. crassipes (EcGS1b-P) has been cloned, analyzed and functionally characterized. TSSP-TCM software and PlantCARE analysis showed a TATA-box core element, a CAAT-box, root specific expression element, light regulation elements including chs-CMA1a, Box I, and Sp1 and other cis-acting elements in the sequence. Three 5'-deletion fragments of EcGS1b upstream sequence with 400 bp, 600 bp and 900 bp length and the 1232 bp fragment were used to drive the expression of β-glucuronidase (GUS) in tobacco. The quantitative test revealed that GUS activity decreased with the decreasing of the promoter length, which indicated that there were no negative regulated elements in the EcGS1-P. The GUS expressions of EcGS1b-P in roots were significantly higher than those in leaves and stems, indicating EcGS1b-P to be a root-preferential promoter. Real-time Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) analysis of EcGS1b gene also showed higher expression in the roots of E.crassipes than in stems and leaves.. EcGS1b-P is a root-preferential promoter sequence. It can specifically drive the transcription of its downstream gene in root. This study will help to elucidate the regulatory mechanisms of EcGS1b tissue-specific expression and further study its other regulatory mechanisms in order to utilize E.crassipes in remediation of eutrophic water and control its overgrowth from the point of nutrient metabolism.

Topics: Base Sequence; Cloning, Molecular; DNA, Plant; Eichhornia; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Glutamate-Ammonia Ligase; Hymecromone; Nicotiana; Plant Roots; Plants, Genetically Modified; Promoter Regions, Genetic; Real-Time Polymerase Chain Reaction

4-Methylumbelliferone (4-MU) inhibits hyaluronan (HA) synthesis and is an approved drug used for managing biliary spasm. However, rapid and efficient glucuronidation is thought to limit its utility for systemically inhibiting HA synthesis. In particular, 4-MU in mice has a short half-life, causing most of the drug to be present as the metabolite 4-methylumbelliferyl glucuronide (4-MUG), which makes it remarkable that 4-MU is effective at all. We report here that 4-MUG contributes to HA synthesis inhibition. We observed that oral administration of 4-MUG to mice inhibits HA synthesis, promotes FoxP3

Topics: Animals; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Hyaluronic Acid; Hymecromone; Mice; T-Lymphocytes, Regulatory

To establish a successful infection, microorganisms have developed strategies to invade host cells. One of the most important human pathogens and the greatest cause of urinary tract infections, Escherichia coli, still do not have its invasion mechanisms fully understood. This work aims to present a new approach for detecting bacterial invasion of lineage cells, based on an enzymatic-fluorogenic method. The focus of this technique is the detection of E. coli invasion of HeLa cells, exploring β-glucuronidase, a specific constitutive enzyme of this bacterium. This enzyme hydrolyses the key substrate of this work, 4-methylumbelliferyl-β-d-glucuronide (MUG), resulting in a fluorogenic molecule, 4-methylumbelliferone. The fluorescence curve created by this method, analyzed by Tukey statistical test, demonstrated that this detection can be efficiently performed after 5 h incubation with MUG. When testing uropathogenic E. coli and E. coli isolated from human gastrointestinal microbiota, the proposed method presented similar results to those exhibited by plate counting invasion detection. Data examination by Duncan statistical test allowed the creation of an intensity range of bacterial invasion, which is part of the process of results interpretation. Detection by this enzymatic-fluorogenic method, compared to other existing bacterial invasion detection techniques, is less burdensome, more sensitive and allows fast achievement of reliable results.

Topics: Bacteriological Techniques; Cell Culture Techniques; Colony Count, Microbial; Escherichia coli; Fluorescent Dyes; Gastrointestinal Microbiome; Glucuronidase; HeLa Cells; Humans; Hymecromone; Reproducibility of Results; Substrate Specificity; Urinary Tract Infections; Uropathogenic Escherichia coli

LC-MS/MS has been proposed in various areas such as Therapeutic Drug Monitoring (TDM), Human Biomonitoring (HBM), disease diagnosis, clinical toxicology and doping control to identify and quantify chemical parents and their metabolites in biological matrices. To determine the total content of a xenobiotic (unconjugated+conjugated forms), an enzymatic hydrolysis step is required. Most studies in the literature have not controlled the effectiveness of the deconjugation process because no method has been described for that purpose. Therefore the aim of this study was to develop and validate a deconjugation probe using a LC-MS/MS method. In order to estimate deconjugation using β-glucuronidase and/or sulfatase, 4-methyl-umbelliferone (MU) and its conjugates were used as markers. Glucuronidase/sulfatase was added to plasma or urine spiked with 4-methylumbelliferyl-β-d-glucuronide (MUG) and 4-methylumbelliferyl sulfate (MUS) and umbelliferone, which was used as the internal standard. After incubation at 37°C during 90min, MU appears as a result of the deconjugation of MUG and MUS. The concentrations of the 3 markers were determined using LC-MS/MS. Trueness and precision of the LC-MS/MS method were determined by quality control analysis at three different levels of concentration covering the whole range of calibration. In both matrices, the analytical method allows quantification of the different compounds, with good linearity, trueness and precision and negligible matrix effects. The method was applied with success to deconjugation assay using active glucuronidase/sulfatase in plasma and urine. The probe developed in this study allows to ensure that enzymatic preparation is working properly in the frame of a quality system.

Topics: Chromatography, Liquid; Drug Monitoring; Glucuronides; Humans; Hymecromone; Limit of Detection; Linear Models; Reproducibility of Results; Sulfates; Tandem Mass Spectrometry

There remains a need for rapid, specific and sensitive assays for the detection of bacterial indicators for water quality monitoring. In this study, a strategy for rapid detection of Escherichia coli in drinking water has been developed. This strategy is based on the use of the substrate 4-methylumbelliferyl-β-d-glucuronide (MUG), which is hydrolyzed rapidly by the action of E. coli β-d-glucuronidase (GUD) enzyme to yield a fluorogenic 4-methylumbelliferone (4-MU) product that can be quantified and related to the number of E. coli cells present in water samples. In this study, the detection time required for the biosensor response ranged between 20 and 120 min, depending on the number of bacteria in the sample. This approach does not need extensive sample processing with a rapid detection capability. The specificity of the MUG substrate was examined in both, pure cultures of non-target bacterial genera such as Klebsiella, Salmonella, Enterobacter and Bacillus. Non-target substrates that included 4-methylumbelliferyl-β-d-galactopyranoside (MUGal) and l-leucine β-naphthylamide aminopeptidase (LLβ-N) were also investigated to identify nonspecific patterns of enzymatic activities in E. coli. GUD activity was found to be specific for E. coli and no further enzymatic activity was detected by other species. In addition, fluorescence assays were performed for the detection of E. coli to generate standard curves; and the sensitivity of the GUD enzymatic response was measured and repeatedly determined to be less than 10 E. coli cells in a reaction vial. The applicability of the method was tested by performing multiple fluorescence assays under pure and mixed bacterial flora in environmental samples. The results of this study showed that the fluorescence signals generated in samples using specific substrate molecules can be utilized to develop a bio-sensing platform for the detection of E. coli in drinking water. Furthermore, this system can be applied independently or in conjunction with other methods as a part of an array of biochemical assays in order to reliably detect E. coli in water.

Topics: Bacterial Load; Biosensing Techniques; Drinking Water; Escherichia coli; Feasibility Studies; Fluorescent Dyes; Glucuronidase; Humans; Hymecromone; Substrate Specificity; Water Microbiology; Water Quality

A method combining immunomagnetic separation and fluorescent sensing was developed to detect Escherichia coli (E. coli) O104:H4. The antibody specific to E. coli O104:H4 was immobilized on protein A-coated magnetic beads. This protein-A-anti E. coli O104:H4 complex was used to bind Fluorescein IsoThioCyanate (FITC) labeled E. coli O104:H4 antigen (whole cell) on it. The goal was to achieve a fluorescently detectable protein-A-anti E. coli O104:H4-E. coli O104:H4 complex on the magnetic beads. Fluorescent microscopy was used to image the magnetic beads. The resulting fluorescence on the beads was due to the FITC labeled antigen binding on the protein-A-anti E. coli O104:H4 immobilized magnetic beads. This visually proves the antigen-antibody binding. The fluorescent imaging results were obtained in 2 h if the minimum available bacteria in the sample were at least 10(5) CFU/ml. If no fluorescence was observed on the magnetic beads during fluorescent imaging, it indicates the bacterial concentration in the sample to be too low for it to have bound to the magnetic beads and hence no detection was possible. To detect bacterial concentration less than 10(5) CFU/ml in the sample, an additional step was required for detection. The magnetic bead complex was added to the LST-MUG (lauryl sulfate tryptose-4-methylumbelliferyl-β-D-glucuronide), a signaling reporter. The E. coli O104:H4 grows in LST-MUG and releases β-glucuronidase enzyme. This enzyme cleaves the MUG substrate that produces 4-methylumbelliferone, a highly fluorescent species. This fluorescence was detected using a spectrofluorometer. The emission peak in the fluorescent spectrum was found to be at 450 nm. The lower and upper detection range for this LST-MUG assay was found to be 2.05×10(5)-4.09×10(8) CFU/ml. The results for the LST-MUG assay for concentrations below 10(5) CFU/ml were ascertained in 8h. The advantages of this technique include the specific detection of bacteria without an enrichment step and allowing the procedure to be completed in hours rather than days.

Topics: Animals; Cattle; Culture Media; Escherichia coli; Fluorescent Dyes; Food Contamination; Hymecromone; Immunomagnetic Separation; Meat; Organic Chemicals; Spectrometry, Fluorescence; Staphylococcal Protein A

Propofol O-glucuronidation has been used as probe reaction to phenotype UGT1A9 activity in human liver, thus a sensitive and specific method for determination of propofol O-glucuronide (PG) is urgently desirable. In the current study, a new LC-ESI-MS method for determination of PG in hepatic microsomes from human (HLM), monkey (CyLM), dog (DLM), minipig (PLM), rat (RLM) and mouse (MLM) was developed and validated using 4-methylumbelliferyl-β-d-glucuronide as an internal standard (IS). PG and IS was separated by a Shim-pack XR-ODS column (100 mm × 2.0mm, 2.2 μm, Shimadzu) under gradient conditions with the mobile phase of acetonitrile and water containing 0.2% acetic acid (v/v). The mass spectrometric detection was performed under selected ion monitoring (SIM) for PG at m/z 353 and IS at m/z 351. The assay exhibited linearity over the range 0.05-30 μM for PG with the correlation coefficient of 0.9995. The intra- and inter-day precision was less than 7.2%, with accuracy in the range 93.8-107.5%. The developed method was successfully used for characterizing interspecies and human individual differences in the O-glucuronidation activity towards propofol, as well as investigating inhibitory effects of androsterone and phenylbutazone on propofol O-glucuronidation in HLM.

Topics: Acetic Acid; Animals; Calibration; Dogs; Glucuronosyltransferase; Haplorhini; Humans; Hymecromone; Kinetics; Mass Spectrometry; Mice; Microsomes, Liver; Models, Chemical; Propofol; Rats; Reproducibility of Results; Spectrometry, Mass, Electrospray Ionization

Standard incubation procedures for carrying out microsomal assays involve the use of less than 1% w/v organic solvents to minimize the potential inhibitory effects of organic solvents on metabolic activity. This presents a practical limitation for poorly soluble xenobiotics, which cannot be incubated at concentrations high enough to obtain a V(max), and therefore subsequent values for K(m) and Cl(int) cannot be calculated. Our goal was to study the application of a variety of pharmaceutical excipients to aid the solubilization of compounds in vitro in glucuronidation incubations, without affecting the reaction kinetics. In vitro glucuronidation incubations were carried out in human liver microsomes with 4-methylumbelliferone (4-MU) and the kinetics of 4-MU glucuronidation in the presence of excipients were compared to that in control incubations without any excipients. In addition, IC(75) values were calculated for each excipient. We observed that HPBCD (Hydroxypropyl-β-cyclodextrin) may be employed in in vitro glucuronidation incubations up to 0.5% w/v without affecting the Cl(int) of 4-MU. Although NMP (N-methyl-2-pyrrolidone) and DMA (N,N-dimethylacetamide); showed low IC(75) values approximately 0.1% w/v each, neither excipients altered the Cl(int) of 4-MUG (4-methylumbelliferyl-β-D-glucuronide) formation. Our studies point toward possible applications of pharmaceutical excipients to carry out in vitro glucuronidation of substrates with poor aqueous solubility, in order to estimate Cl(int) and subsequently scaled organ clearance values.

Topics: 2-Hydroxypropyl-beta-cyclodextrin; Acetamides; beta-Cyclodextrins; Excipients; Glucuronides; Glucuronosyltransferase; Humans; Hymecromone; Kinetics; Microsomes, Liver; Pyrrolidinones; Solubility

This study was designed to investigate the renal disposition of 4-methylumbelliferone (4MU) and 4-methylumbelliferyl glucuronide (4MUG) to characterise the contribution of excretion and metabolic clearance to total clearance in the kidney.. The isolated perfused kidney (IPK) from the male Sprague-Dawley rat was used in filtering and non-filtering mode to study the renal disposition of 4MU, renally generated 4MUG and preformed 4MUG. Perfusate and urine (filtering IPK only) was collected for up to 120 min and 4MU and 4MUG in perfusate and urine were determined by HPLC. Analytes were also measured in kidney tissue collected at 120 min. Non-compartmental analysis was used to derive pharmacokinetic parameters.. The concentration of 4MU in perfusate declined with a terminal half-life of approximately 120 min following administration to the filtering IPK and nonfiltering IPK. There was a corresponding increase in the concentration of 4MUG. Metabolic clearance of 4MU accounted for 92% of total renal clearance. After bolus dosing of preformed 4MUG in the perfusion reservoir of the filtering IPK, the perfusate concentration declined with the terminal half-life of approximately 260 min. The renal excretory clearance of preformed 4MUG accounted for 96% of total renal clearance. 4MU was extensively metabolized by glucuronidation in the filtering and nonfiltering IPK, and the total renal clearance of 4MU was far greater than its renal excretory clearance. This indicated that glucuronidation was the major elimination pathway for 4MU in the kidney.. The data confirmed an important role for the kidney in the metabolic clearance of xenobiotics via glucuronidation and signalled the lack of impact of impaired glomerular filtration on renal drug metabolism.

Topics: Animals; Half-Life; Hymecromone; In Vitro Techniques; Kidney; Male; Perfusion; Rats; Rats, Sprague-Dawley

The present study was undertaken to identify genetic polymorphisms of multidrug resistance-associated protein 3 (MRP3, gene name ABCC3), an ATP-binding cassette transporter that mediates the transport of substrates across the basolateral membrane into the blood, and to investigate their effects on ABCC3 expression and MRP3 function. We identified genetic polymorphisms of ABCC3 and evaluated the effects by (1) a luciferase reporter gene assay, (2) measuring mRNA levels, and (3) a human pharmacogenomics study with 4-methylumbelliferone glucuronide (4-MUG). Overall, 61 genetic variants were identified in three ethnic populations; of these variants 17 were novel (7 were non-synonymous: 61Arg>Cys, 132Gln>Stop, 221Trp>Stop, 270His>Gln, 548Leu>Gln, 600Lys>Arg, and 1324Arg>His). However, these mutations occurred at very low frequencies (max. 4.7%). The observed allele frequencies showed considerable inter-ethnic differences. The reporter gene assay indicated a significant reduction of transcriptional activity with the -1767G>A allele compared to the wild-type allele; however, a decreased expression of ABCC3 mRNA was not detected in human liver samples. A human pharmacokinetic study showed that the ABCC3 genotype in the promoter region was not associated with changes in the pharmacokinetics of 4-MUG, a substrate of MRP3. This is the first study to assess the effects of ABCC3 polymorphisms on human pharmacokinetics; however, further investigations are needed to complete the picture.

Topics: Adult; Asian; Black or African American; DNA, Complementary; Female; Gene Amplification; Genes, Reporter; Genotype; Haplotypes; Humans; Hymecromone; Luciferases; Male; Multidrug Resistance-Associated Proteins; Polymorphism, Genetic; RNA, Messenger; Transfection; White People; Young Adult

To (i) study the serogroup distribution and virulence characteristics of non-sorbitol-fermenting Escherichia coli isolates from foods of animal origin and cattle faeces and (ii) re-examine the true sorbitol and beta-D-glucuronidase (GUD) reactions of sorbitol-negative (Sor(-)) strains from MacConkey sorbitol agar (SMAC) to assess their phenotypic similarity with E. coli O157.. One hundred and thirty Sor(-)E. coli were isolated from 556 food samples and 177 cattle stool samples using cefixime tellurite-supplemented SMAC (CT-SMAC) and chromogenic HiCrome MS.O157 agar respectively. Based on typing of somatic antigen, the isolates were classified into 38 serogroups. PCR results identified about 40% strains, belonging to O5, O8, O20, O28, O48, O60, O78, O82, O84, O101, O110, O123, O132, O156, O157, O-rough and OUT as Shiga toxigenic. Majority of O5, O84, O101, O105, O123, O157, O-rough and OUT strains were enterohaemolytic. Further, 39.2% and 63.1% of Sor(-) isolates from CT-SMAC fermented sorbitol in phenol red broth and hydrolysed 4-methylumbelliferyl-beta-D-glucuronide (MUG) respectively. Members of serogroups O5, O28, O32, O81, O82, O84, O101, O-rough lacked both the sorbitol fermentation (broth test) and GUD activity and might create confusion in phenotypic identification of E. coli O157.. Sor(-)E. coli isolates from raw meat, milk, shrimp and cattle stool belonged to 38 serogroups, with E. coli O157 constituting only 14.6% of the isolates. Many of these nonclinical Sor(-) strains were potentially pathogenic. Nearly 39% of these Sor(-)E. coli from CT-SMAC fermented sorbitol in broth, indicating the need for confirmation of sorbitol reaction in broth.. Classical sorbitol utilization and GUD tests are not likely definitive tests for E. coli O157. Further improvement of differential media based on these phenotypic properties is necessary for detection of pathogenic serotypes from foods and environmental samples.

Topics: Animals; Cattle; Culture Media; Escherichia coli; Feces; Food Contamination; Food Microbiology; Hymecromone; Meat; Milk; Serotyping; Sorbitol; Virulence

The production of beta-glucuronidase (GUS) driven by the Arabidopsis small heat shock protein 18.2 promoter in liquid cultures of transgenic tobacco (Nicotiana tabacum) hairy roots is reported. Clone GD-3, showing high GUS heat induction and a moderate growth rate, was selected from 436 clones for study. Treatment of GD-3 with heat shock at 36-42 degrees C for 2 h then recovery at 27 degrees C resulted in an increase in GUS specific activity, while higher heat-shock temperatures led to a decline. These results were in accordance with the change in esterase activity, a measure of tissue viability. Using 2 h of 42 degrees C heat shock and a recovery phase at 27 degrees C, GUS specific activity increased rapidly and reached a maximum of 267.6 nmol 4-methylumbelliferyl beta-D-glucuronic acid (MU) min-1 mg-1 protein at 24 h of recovery. When tissues were continuously heated at 42 degrees C and tested without a recovery period, GUS mRNA was detectable at 2 h and peaked at 5 h, but GUS activity was not seen until 10 h and did not peak until 28 h; in addition, the maximum activity was lower than that seen after heat shock for only 30 min or 2 h, followed by recovery. This shows that recovery at normal temperature is crucial for the heat-inducible heterogeneous expression system of transgenic hairy roots. Multiple heat-shock treatments showed that this system was heat reinducible, although a gradual decline in GUS specific activity was seen in the second and third cycles.

Topics: Arabidopsis; Esterases; Gene Expression Regulation, Enzymologic; Glucuronidase; Heat-Shock Proteins; Hot Temperature; Hymecromone; Nicotiana; Plant Roots; Plants, Genetically Modified; Promoter Regions, Genetic; RNA, Messenger; RNA, Plant; Time Factors

The enzymatic defect in Pompe disease is insufficient lysosomal acid alpha-glucosidase (GAA) activity which leads to lysosomal glycogen accumulation. We recently introduced a simple and reliable method to measure GAA activity in dried blood spots using Acarbose, a highly selective alpha-glucosidase inhibitor, to eliminate isoenzyme interference. Here we demonstrate that this method efficiently detects late-onset Pompe patients who are frequently misdiagnosed by conventional methods due to residual GAA activity in other tissue types.

Topics: Acarbose; Adult; alpha-Glucosidases; Blood Specimen Collection; Cells, Cultured; Fibroblasts; Fluorometry; Glycogen Storage Disease Type II; Glycoside Hydrolase Inhibitors; Humans; Hymecromone; Isoenzymes; Substrate Specificity

Laboratory batch tests indicate that addition of sterile municipal sewage biosolids to clay soil from four depths increases the numbers of Escherichia coli isolates recoverable in EC-MUG broth (EC broth with 4-methylumbelliferyl-beta-glucuronide). This effect was most marked for the deeper soil layers, with increases of about 2.6 orders of magnitude in E. coli most probable number.

Topics: Colony Count, Microbial; Culture Media; Escherichia coli; Hymecromone; Organic Chemicals; Sewage; Soil; Soil Microbiology; Sterilization

The hepatic excretion of hydrophilic conjugates, end products of phase II metabolism, is not completely understood. In the present studies, transport mechanism(s) responsible for the biliary excretion of 4-methylumbelliferyl glucuronide (4MUG) and 4-methylumbelliferyl sulfate (4MUS) were studied. Isolated perfused livers (IPLs) from Mrp2-deficient (TR(-)) Wistar rats were used to examine the role of Mrp2 in the biliary excretion of 4MUG and 4MUS. After a 30-micromol dose of 4-methylumbelliferone, cumulative biliary excretion of 4MUG was extensive in wild-type rat IPLs (25 +/- 3 micromol) but was negligible in TR(-) livers (0.4 +/- 0.1 micromol); coadministration of the Bcrp and P-glycoprotein inhibitor GF120918 [N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide] had no effect on 4MUG biliary excretion in wild-type rat IPLs. In contrast, biliary excretion of 4MUS was partially maintained in Mrp2-deficient rat IPLs. Recovery of 4MUS in bile was approximately 50 to 60% lower in both control TR(-) (149 +/- 8 nmol) and wild-type IPLs with GF120918 coadministration (176 +/- 30 nmol) relative to wild-type control livers (378 +/- 37 nmol) and was nearly abolished in TR(-) IPLs in the presence of GF120918 (13 +/- 8 nmol). These changes were the result of decreased rate constants governing 4MUG and 4MUS biliary excretion. In vitro assays and perfused livers from Bcrp and P-glycoprotein gene-knockout mice indicated that 4MUS did not interact with P-glycoprotein but was transported by Bcrp in a GF120918-sensitive manner. In the rat liver, Mrp2 mediates the biliary excretion of 4MUG, whereas both Mrp2 and Bcrp contribute almost equally to the transport of 4MUS into bile.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Bile; Cell Line; Dogs; Hymecromone; Male; Rats; Rats, Wistar

We investigated whether the species difference in the biliary excretion activity of some Mrp2 substrates was attributable to the intrinsic transport potential or the expression level of Mrp2, especially in rat and dog. Dog Mrp2 cDNA was isolated from beagle dog liver, and a vesicle transport study was performed using recombinant rat and dog Mrp2 expressed in insect Sf9 cells. The ATP-dependent transport of 17beta-estradiol 17-(beta-D-glucuronide) ([3H]E(2)17betaG) and leukotriene C4 ([3H]LTC4), normalized by the absolute protein expression level, was similar in both Mrp2s. The Mrp2 protein expression in dog liver was only 10% of that in rat liver and was comparable with the reported difference in the biliary excretion clearance of temocaprilat as Mrp2 substrate. In contrast to LTC4, unique transport kinetics for E(2)17betaG were evident in dog Mrp2. In addition to the high-affinity site with a K(m) value of 3.25 +/- 0.10 microM, which is similar to that in rat Mrp2 (4.81 +/- 1.21 microM), dog Mrp2 has an additional low-affinity site (>>75 microM), which makes a major contribution to the transport of E(2)17betaG (65% of the total transport capacity at tracer concentration). In summary, the difference in the biliary excretion activity of Mrp2 substrates between rat and dog depends on the Mrp2 protein expression level rather than the intrinsic transport activity of the transporter molecules. The unique transport properties of glucuronide conjugates by dog Mrp2 may lead to the species difference involving the drug-drug interaction or drug-induced hyperbilirubinemia on the bile canalicular membrane.

Topics: Amino Acid Sequence; Animals; ATP-Binding Cassette Transporters; Biological Transport; Cell Line; Dogs; Dose-Response Relationship, Drug; Humans; Hymecromone; Insecta; Membrane Transport Proteins; Molecular Sequence Data; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Rats; Species Specificity

The relationships between the beta-D-glucuronidase (GLUase) activity, the abundance of culturable Escherichia coli and the number of viable E. coli were investigated in river and wastewater samples.. GLUase activity was measured as the rate of hydrolysis of 4-methylumbelliferyl-beta-D-glucuronide. Culturable E. coli were enumerated by the most probale number (MPN) microplate method. Viable E. coli were estimated by fluorescent in situ hybridization (FISH) coupled with a procedure of viability testing (DVC-FISH procedure). Significant correlations were found between the log of GLUase activity and both, the log culturable E. coli and the log of viable E. coli.. GLUase activity per viable E. coli gave a broadly constant value from low to highly contaminated waters while GLUase activity per culturable E. coli strongly increased at low contaminated waters because of an underestimation of the number of active E. coli by the culture-based method.. GLUase activity is a reliable parameter for the rapid quantification of viable E. coli in waters.

Topics: Bacteriological Techniques; Colony Count, Microbial; Escherichia coli; France; Fresh Water; Glucuronidase; Hymecromone; In Situ Hybridization, Fluorescence; Rivers; Water Microbiology

Tay-Sachs and Sandhoff diseases are lysosomal storage disorders that result from an inherited deficiency of beta-hexosaminidase A (alphabeta). Whereas the acute forms are associated with a total absence of hexosaminidase A and early death, the chronic adult forms exist with activity and protein levels of approximately 5%, and unaffected individuals have been found with only 10% of normal levels. Surprisingly, almost all disease-associated missense mutations do not affect the active site of the enzyme but, rather, inhibit its ability to obtain and/or retain its native fold in the endoplasmic reticulum, resulting in its retention and accelerated degradation. By growing adult Tay-Sachs fibroblasts in culture medium containing known inhibitors of hexosaminidase we have raised the residual protein and activity levels of intralysosomal hexosaminidase A well above the critical 10% of normal levels. A similar effect was observed in fibroblasts from an adult Sandhoff patient. We propose that these hexosaminidase inhibitors function as pharmacological chaperones, enhancing the stability of the native conformation of the enzyme, increasing the amount of hexosaminidase A capable of exiting the endoplasmic reticulum for transport to the lysosome. Therefore, pharmacological chaperones could provide a novel approach to the treatment of adult Tay-Sachs and possibly Sandhoff diseases.

Topics: Adult; beta-N-Acetylhexosaminidases; Cell Line; Enzyme Activation; Enzyme Inhibitors; Female; Fibroblasts; Hexosaminidase A; Hot Temperature; Humans; Hymecromone; In Vitro Techniques; Lysosomes; Molecular Chaperones; Mutation; Protein Folding; Sandhoff Disease; Tay-Sachs Disease

A prototypical characteristic of the Brassicaceae is the presence of the myrosinase-glucosinolate system. Myrosinase, the only known S-glycosidase in plants, degrades glucosinolates, thereby initiating the formation of isothiocyanates, nitriles and other reactive products with biological activities. We have used myrosinase gene promoters from Brassica napus and Arabidopsis thaliana fused to the beta -glucuronidase (GUS) reporter gene and introduced into Arabidopsis thaliana, Brassica napus and/or Nicotiana tabacum plants to compare and determine the cell types expressing the myrosinase genes and the GUS expression regulated by these promoters. The A. thaliana TGG1 promoter directs expression to guard cells and phloem myrosin cell idioblasts of transgenic A. thaliana plants. Expression from the same promoter construct in transgenic tobacco plants lacking the myrosinase enzyme system also directs expression to guard cells. The B. napus Myr1.Bn1 promoter directs a cell specific expression to idioblast myrosin cells of immature and mature seeds and myrosin cells of phloem of B. napus. In A. thaliana the B. napus promoter directs expression to guard cells similar to the expression pattern of TGG1. The Myr1.Bn1 signal peptide targets the gene product to the reticular myrosin grains of myrosin cells. Our results indicate that myrosinase gene promoters from Brassicaceae direct cell, organ and developmental specific expression in B. napus, A. thaliana and N. tabacum.

Topics: Arabidopsis; Blotting, Northern; Brassica napus; DNA, Plant; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Glucuronidase; Glycoside Hydrolases; Hymecromone; Immunohistochemistry; In Situ Hybridization; Molecular Sequence Data; Nicotiana; Plant Cells; Plants; Plants, Genetically Modified; Promoter Regions, Genetic; Recombinant Fusion Proteins; RNA, Messenger; Seeds; Sequence Analysis, DNA; Substrate Specificity

1. To elucidate the mechanisms involved in the sinusoidal efflux of sulfate and glucuronide metabolites of 4-methylumbelliferone (4MU), isolated rat liver perfusion studies were performed under several conditions. 2. The effect of sodium azide on the hepatic handling of both conjugates was examined. The net sinusoidal efflux clearance (CL(eff)) based on the unbound concentration in the liver did not change for 4MU glucuronide (4MUG) or significantly increase for 4MU sulfate (4MUS), suggesting that the sinusoidal efflux of both conjugates is not mediated by the transport systems dependent on adenosine triphosphate. 3. Under Cl(-)-depleted conditions, the CL(eff) of 4MUG significantly decreased, but the saturation of its sinusoidal efflux rather than the transport system dependent on Cl(-) might be involved because the hepatic concentration of 4MUG was extensively higher than that of the control study due to the extremely attenuated biliary excretion. The CL(eff) of 4MUS also significantly decreased, but its hepatic concentration was not different from that in the control study, suggesting that the transport system using Cl(-) is involved in the sinusoidal efflux of 4MUS. 4. The effect of glutathione was examined. CL(eff) of 4MUG was not affected by the additional glutathione, but CL(eff) of 4MUS decreased significantly, suggesting that some transport system sensitive to glutathione is involved in the sinusoidal efflux of 4MUS, but not of 4MUG. 5. Transporters such as Oatp1, Oatp2 and/or Npt1 might be involved in the sinusoidal efflux of 4MUS, but 4MUG is secreted from the sinusoidal membrane via the systems that are totally different from those for 4MUS.

Topics: Animals; Biological Transport, Active; Chlorides; Glucuronides; Glutathione; Hymecromone; In Vitro Techniques; Liver; Male; Models, Biological; Rats; Rats, Wistar; Sodium Azide; Sulfates; Xenobiotics

Escherichia coli is a routinely used microbiological indicator of water quality. To determine whether holding time and storage conditions had an effect on E. coli densities in surface water, studies were conducted in three phases, encompassing 24 sites across the United States and four commonly used monitoring methods. During all three phases of the study, E. coli samples were analyzed at time 0 and at 8, 24, 30, and 48 h after sample collection. During phase 1, when 4 degrees C samples were evaluated by Colilert or by placing a membrane onto mFC medium followed by transfer to nutrient agar containing 4-methylumbelliferyl-beta-D-glucuronide (mFC/NA-MUG), three of four sites showed no significant differences throughout the 48-h study. During phase 2, five of seven sites showed no significant difference between time 0 and 24 h by membrane filtration (mFC/NA-MUG). When evaluated by the Colilert method, five of seven sites showed no significant difference in E. coli density between time 0 and 48 h. During phase 3, 8 of 13 sites showed no significant differences in E. coli densities between time 0 and the 48-h holding time, regardless of method. Based on the results of these studies, it appears that if samples are held below 10 degrees C and are not allowed to freeze, most surface water E. coli samples analyzed by commonly used methods beyond 8 h after sample collection can generate E. coli data comparable to those generated within 8 h of sample collection. Notwithstanding this conclusion, E. coli samples collected from surface waters should always be analyzed as soon as possible.

Topics: Bacteriological Techniques; Colony Count, Microbial; Culture Media; Escherichia coli; Filtration; Fluorescent Dyes; Fresh Water; Hymecromone; Membranes, Artificial; Temperature; Time Factors; Water Purification; Water Supply

A promising development in tumor therapy is the application of non-toxic prodrugs from which the active cytostatic is released by endogenous enzymes such as beta-glucuronidase (beta-gluc). Regulation of beta-gluc expression is one crucial factor modulating bioactivation of prodrugs. Recent experiments in rats indicate regulation of beta-gluc activity by the calcium channel blocker verapamil. To further explore this phenomenon, we investigated the effect of verapamil on beta-gluc enzyme activity, protein (western blot) and mRNA expression (RT-PCR) as well as the underlying mechanisms (effects of verapamil metabolites; promoter activity) in the human hepatoma cell line HepG2. Treatment of HepG2 cells with verapamil revealed down-regulation of beta-gluc activity, protein, and mRNA level down to 50% of the control with EC(50) values of 25 microM. Effects were similar for both enantiomers. Moreover, it was demonstrated that reduced promoter activity contributes to the observed effects. In summary, our data demonstrate regulation of human beta-glucuronidase expression by verapamil. Based on our findings we hypothesize that coadministration of verapamil may effect cleavage of glucuronides by beta-glucuronidase.

Topics: Blotting, Western; Calcium Channel Blockers; Carcinoma, Hepatocellular; Cell Line, Tumor; Down-Regulation; Genes, Reporter; Glucuronidase; Glyceraldehyde-3-Phosphate Dehydrogenases; Humans; Hymecromone; Liver Neoplasms; Promoter Regions, Genetic; RNA, Messenger; Stereoisomerism; Time Factors; Transfection; Verapamil

Antibody directed enzyme prodrug therapy (ADEPT) using glucuronide prodrugs is an experimental approach to reduce systemic toxicity of anti-cancer agents. Bioactivation of such prodrugs is achieved by fusion proteins consisting of targeting moieties (e.g. ligands of tumor specific antigens) and human beta-glucuronidase. In order to test a large panel of possible beta-glucuronidase fusion proteins for their applicability in ADEPT, an easy, rapid and high-yield expression system like the baculovirus/insect cell expression system would be needed. A prerequisite for using such fusion proteins is functional and biochemical characterization of human beta-glucuronidase expressed in baculovirus-infected insect cells. Therefore, recombinant human beta-glucuronidase was expressed in Sf9 insect cells and characterized at the protein and functional level. As shown by Western blot analysis the recombinant enzyme consists of dimers with their monomers being linked via disulfide bonds. Posttranslational modifications of the monomers seem to be different as compared with mammalian cells or tissues. The enzyme is functionally active in cleaving the substrates 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid, 4-methylumbelliferyl-beta-D-glucuronide and the glucuronide prodrug HMR 1826, respectively, with similar enzyme kinetic parameters as those found in human tissues. Our data demonstrate that beta-glucuronidase expressed in Sf9 cells displays the same enzymatic features as the protein expressed in mammalian cells. Therefore, we suggest that beta-glucuronidase fusion proteins produced in this cell line will be valuable tools for testing a large panel of various targeting moieties in human tumor xenograft models or may be used for ADEPT in man.

Topics: Animals; Baculoviridae; Blotting, Western; Cell Line; Culture Media; Doxorubicin; Gene Expression Regulation, Enzymologic; Glucuronates; Glucuronidase; Histocytochemistry; Humans; Hymecromone; Indoles; Prodrugs; Protein Engineering; Recombinant Fusion Proteins; Recombination, Genetic; Spodoptera

An analytical protocol has been developed and applied for the detection of glucuronidase activity in marine waters as a rapid alternative approach to assess the microbiological quality of seawaters.. The fluorogenic substrate 4-methylumbelliferyl-beta-D-glucuronide is cleaved to a fluorescent product, methylumbelliferone, by the enzyme beta-glucuronidase, specific to Escherichia coli and closely related enterobacterial species (Shigella). The results suggest that this test is related to E. coli numbers, as estimated by immunofluorescence, more significantly than to faecal coliform numbers, obtained from culture media.. The determination of the potential rate of glucuronidase activity may be used as a diagnostic tool for the indirect estimation of the presence of E. coli in seawaters.. The method may be particularly useful in the early warning of seawater pollution, allowing the screening of coastal areas with different contamination levels in reduced time.

Topics: Bacteriological Techniques; Colony Count, Microbial; Escherichia coli; Fluorescent Dyes; Glucuronidase; Hymecromone; Seawater; Time Factors; Water Pollution

This study was undertaken to investigate interference by acids commonly found in fruit juice in Escherichia coli assays involving the use of 4-methylumbelliferyl-beta-D-glucuronide (MUG) as a fluorogenic substrate for enzyme reaction. Fluorescence intensity was negatively correlated (P < 0.001) with the volume of fresh citrus juice tested by the lauryl tryptose broth (LST)-MUG assay, and the permissible sample sizes were limited to 0.3 and 0.5 ml for fresh citrus juices with pHs of 3.3 and 3.9, respectively. In addition, false-negative results were visually observed under UV light when the E*Colite assay was used to test large volumes (5 to 10 ml per test) of fresh citrus juice or when the test broth used for the LST-MUG assay was supplemented with citric, malic, or tartaric acid at 2 to 4 g/liter. These results suggest that the size and pH of acidic samples should be controlled in MUG-based fluorogenic assays. The inhibitory effect on fluorescence was due to high acidity, which reduces fluorescence from 4-methylumbelliferone. Buffering improved the assays. When sodium bicarbonate was incorporated in the enrichment broth at 10 g/liter, the permissible sample sizes for fresh grapefruit juice (pH 3.1) increased from 0.3 to 1 ml for the LST-MUG (with 9.9 ml of broth) assay and from 3 to 10 ml for the E*Colite (with 99 ml of broth) assay.

Topics: Bacteriological Techniques; Beverages; Escherichia coli; False Negative Reactions; Fluorescent Dyes; Food Microbiology; Hydrogen-Ion Concentration; Hymecromone; Sample Size

The hydrolysis rate of 4-methylumbelliferyl-beta-D-glucuronide (MUG-HR) was determined in unamended samples, filtered samples, and in corresponding buffer resuspended filter residues of various river waters of slight to excessive fecal pollution covering a four orders of magnitude range. Regression analysis of the log MUG-HR of the unamended water samples versus the log MUG-HR of the filter residues revealed a highly significant linear relationship (R2 = 0.94; p<0.001). The median of the MUG-HR of the filtrated water samples was about 10% the MUG-HR of the corresponding unamended water samples. If MUG-HR determinations were used as a surrogate for estimating fecal coliform contamination, both the MUG-HR of the unamended water samples and the MUG-HR of the filter residues would have been equally adequate techniques at river sites of higher fecal pollution levels. However, at river locations of decreased fecal pollution, MUG-HR determination of filter residues appeared to be the more sensitive technique in order to estimate fecal coliform concentrations.

Topics: Enterobacteriaceae; Environmental Monitoring; Feces; Fluorescent Dyes; Hydrolysis; Hymecromone; Reproducibility of Results; Sensitivity and Specificity; Specimen Handling; Water Pollutants

UDP-glucuronosyltransferase (UGT) enzymes belonging to the UGT2B subfamily catalyze the transfer of glucuronic acid to a large number of endogenous compounds, particularly steroids, to facilitate their elimination from target cells. A novel human UGT2B cDNA of 1666 bp was isolated and encodes a 529-amino acid protein named UGT2B28 type I. Glucuronidation assays demonstrated that UGT2B28 type I catalyzes the conjugation of endogenous and exogenous compounds. The tissue distribution of UGT2B28 revealed the expression of the type I transcript in the liver, breast, and LNCaP cells. Two other UGT2B cDNAs were isolated, and sequence analysis led to the identification of two truncated UGT2B28 species. UGT2B28 type II differs from type I by a deletion of 308 bp in the cofactor binding domain, whereas UGT2B28 type III lacks 351 bp in the putative substrate binding domain. All UGT2B28 isoforms are encoded by a single UGT2B28 gene which has a genomic organization similar to that of the other UGT2B genes characterized thus far. Although no substrates could be identified for the shorter isoforms, the three subtypes were shown to be located in the endoplasmic reticulum and the perinuclear membrane, demonstrating that the missing domains are not required for the subcellular localization of these UGT2B proteins. However, all the domains remain necessary for observing glucuronidation activity. The expression of UGT2B28 type I in the breast and liver suggests a role of this enzyme in the androgen and estrogen metabolism in these tissues.

Topics: Alternative Splicing; Amino Acid Sequence; Androstane-3,17-diol; Androsterone; Base Sequence; Bile Acids and Salts; Cell Line; Cyst Fluid; DNA, Complementary; Enzyme Activation; Estradiol; Female; Fibrocystic Breast Disease; Glucuronides; Glucuronosyltransferase; HeLa Cells; Humans; Hymecromone; Isoenzymes; Molecular Sequence Data; Organ Specificity; RNA, Messenger; Subcellular Fractions; Testosterone

An assay to screen Escherichia coli in foods using MUG supplemented lauryl sulfate tryptose (LST) broth instead of tryptone water (TW) medium was evaluated. The results presented in this paper suggest that this method is more sensitive for lower levels of E. coli, faster (16-18 h vs. 6-10 days) and less expensive (2.454 vs. 2.887 EURO/sample) than the standard ISO procedure. Thus, this method may be beneficial for use when both fecal coliforms and E. coli analyses are required in food systems.

Topics: Colony Count, Microbial; Costs and Cost Analysis; Culture Media; Enterobacteriaceae; Escherichia coli; Feces; Fluorescent Dyes; Food Microbiology; Hymecromone; Sensitivity and Specificity; Time Factors

The relationship between the rate of beta-D-glucuronidase hydrolysis (GLUase-HR) and the E. coli concentration in rivers differing in the extent of faecal pollution was investigated. It was hypothesized that the rate of GLUase-HR is a better surrogate parameter for E. coli concentrations than estimated numbers of faecal coliforms (FC).. The GLUase-HR of the water sample filter residues was determined as the rate of cleavage of 4-methylumbelliferyl-beta-D-glucuronide. FC and E. coli concentrations were enumerated using mFC and Chromocult Coliform agar, respectively. Regression analysis revealed that a 90% variation of the variable log GLUase-HR was directly related to the variable log E. coli concentrations. The observed relationship between the log of the FC count and the log of the GLUase activity could be explained by the hydrolysis activity of the E. coli population, as E. coli is a part of the FC group.. The data suggest that the log of the GLUase-HR can be used as a surrogate parameter for the log of the E. coli concentrations.. GLUase-HR determination may provide a rapid alternative technique to estimate E. coli concentrations in freshwaters.

Topics: Colony Count, Microbial; Escherichia coli; Fresh Water; Glucuronidase; Hydrolysis; Hymecromone; Regression Analysis; Water Pollution

The detection of fluorescent colonies of Escherichia coli/total coliforms (TC) on a membrane filter is currently carried out using 4-methylumbelliferyl-beta-D-glycosides as enzyme substrates and a UV-lamp for visualization. The most rapid procedures based on this approach for the demonstration of these indicator bacteria in water take 6-7.5 h to complete. As part of efforts to further reduce the detection time, an improved two-step procedure for the fluorescence or chemiluminescence labelling of microcolonies of E. coli/TC on a membrane filter has been developed. Essential features of this approach include a separation of the bacterial propagation and target enzyme induction from the actual enzymatic labelling, the use of improved fluorogenic, i.e., 4-trifluoromethylumbelliferyl-beta-D-glycosides and fluorescein-di-beta-D-glycosides, or chemiluminogenic (i.e., phenylglucuronic- or galactose-substituted adamantyl 1,2-dioxetanes) substrates for beta-glucuronidase/beta-galactosidase, of enzyme inducers, of special membrane filters and of polymyxin B to promote the cellular uptake of the substrate. This labelling procedure has been applied in conjunction with different detection devices including a UV-lamp, CCD-cameras, X-ray film and the ChemScan((R)) RDI. Using the former three, microcolonies of pure cultures could be detected within 5.5-6.5 h, but waterborne E. coli/TC may fail to form microcolonies in this short time period, thus yielding poor sensitivity and a high false-negative rate. In contrast, a quantitative enumeration was feasible in less than 4 h with the ChemScan((R)) RDI, owing to its ability to detect both microcolonies and non-dividing single cells.

Topics: beta-Galactosidase; Colony Count, Microbial; Culture Media; Enterobacteriaceae; Escherichia coli; False Negative Reactions; Filtration; Fluorescence; Fluorescent Dyes; Galactosides; Glucuronidase; Glucuronides; Hymecromone; Luminescent Measurements; Sensitivity and Specificity; Time Factors; Water Microbiology

Beta-glucuronidase cleavage of 4-methylumbelliferyl beta-D-glucuronide generates the highly fluorescent compound, 4-methylumbelliferone. We show that other beta-D-glucuronide compounds act as competitors in this assay. The 4-methylumbelliferyl beta-D-glucuronide cleavage assay can easily be adapted to high throughput formats to detect the presence of beta-D glucuronides generated using recombinant glycosyl transferase preparations.

Topics: Binding, Competitive; Fluorescence; Glucuronates; Glucuronidase; Glucuronosyltransferase; Hymecromone; Kinetics; Recombinant Proteins

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.

Topics: alpha-Amylases; Cell Division; Chloramphenicol; Enzyme Induction; Escherichia coli; Fluorescent Dyes; Glucose; Glucuronidase; Hymecromone; Protein Synthesis Inhibitors; Starch

Capillary electrophoresis (CE) with fluorescence detection was used to determine the concentration of 4-methylumbelliferone liberated from 4-methylumbelliferyl-beta-D-glucuronide by beta-glucuronidase. Enzyme substrate saturation kinetics were studied in buffer and the pH range for the enzyme reaction was optimized. A linear relationship of initial enzyme reaction velocity as a function of peak area of enzyme product was obtained for enzyme activity ranging from 1 to 100 units. The beta-glucuronidase activity in urine was next determined. Freshly collected urine samples were dialyzed, the retentate was incubated with 4-methylumbelliferyl-beta-D-glucuronide, boiled and centrifuged. The supernatant was separated by CE in an uncoated capillary with 0.1 M sodium acetate buffer by applying a voltage of 12 kV. The product of the enzymatic reaction, 4-methylumbelliferone, was detected by fluorescence, facilitating the determination of as little as one unit of beta-glucuronidase activity in a 0.5-h incubation time, with an error of less than +/-5%.

Topics: Adult; Animals; Buffers; Cattle; Electrophoresis, Capillary; Fluorescent Dyes; Glucuronidase; Humans; Hydrogen-Ion Concentration; Hymecromone; Kinetics; Male; Sensitivity and Specificity

Fluorescence from 4-methylumbelliferyl-beta-D-glucuronide (MUG) hydrolysis is a common, rapid method for determining Escherichia coli in water and food. False-positive fluorescence occurred when either pink salmon fillets were tested or beta-glucuronidase-positive Staphylococcus species were present in other fish products. Salmon fillet, E. coli, S. xylosus, and S. warneri produced 2, 17, 39, and 43 nmol of 4-methylumbelliferone per ml, respectively, in a one-step detection broth (lauryl salts tryptose broth with MUG) for E. coli after 48 h at 35 degrees C. These false-positive reactions need to be considered when testing fish products, especially those contaminated through human handling.

Topics: Animals; Bacteriological Techniques; Escherichia coli; False Positive Reactions; Fluorescence; Glucuronidase; Hymecromone; Salmon; Seafood; Spectrometry, Fluorescence; Staphylococcus

The vascularly perfused rat intestine and liver preparations were used to examine the effect of flow (8 and 10 ml/min) on the sequential metabolism of 4-methylumbelliferone (4MU), which forms primarily the glucuronide conjugate (4MUG) in intestine and the sulfate conjugate (4MUS) in liver at low input concentrations of 4MU. In this system, a constant tracer concentration of [3H]4MU was delivered systemically at 8 or 10 ml/min to the perfused rat small intestine preparation; the portal venous outflow perfusate at 8 and 10 ml/min was collected at steady state, reoxygenated and in tum delivered to the perfused rat liver preparation from a second rat donor. The intestinal extraction ratio and formation of 4MUG were decreased from 0.57 +/- 0.07 to 0.49 +/- 0.06 and 42 +/- 5 to 36 +/- 4% input rate, respectively, upon increasing the flow rate from 8 to 10 ml/min (P < .05). These decreases were the result of the reduction in transit time with increasing flows. In contrast, hepatic 4MU conjugation was increased (from 40 +/- 7% to 48 +/- 6% input rate to intestine) upon increasing the flow rate from 8 and 10 ml/min (P < .05), attributed primarily to increased formation of the major metabolite, 4 MUS, in liver (from 35 +/- 9% to 39 +/- 9% input rate to intestine). The unusual observation on increased hepatic metabolite formation with increasing flow could be rationalized. With increased flow to the serially perfused organs, there was an increased supply of substrate to the liver, the posterior organ, because of a faster intestinal transit time. Decreased intestinal metabolism (formation of 4MUG) at increased flow was compensated by increased hepatic metabolism (formation of 4MUS), albeit attenuated because of a faster hepatic transit time. The proportions of total 4MU conjugates formed (4MUG + 4MUS) across the intestine and liver remained constant at both flow rates. Hence, a rather constant overall extraction ratio (0.98 +/- 0.004 and 0.97 +/- 0.005, P > .05) existed across the two organs. The results demonstrate that the intestine, the anterior organ, plays a regulatory role on substrate supply to the posterior organ, the liver. With an increase in flow, the contribution of the intestine will decrease, whereas the contribution of the liver will increase in the overall first-pass metabolism.

Topics: Animals; Hymecromone; Intestinal Mucosa; Liver; Male; Perfusion; Rats; Rats, Sprague-Dawley

Concanavalin A (Con A) is the best known plant lectin, with important biological properties arising from its specific saccharide-binding ability. Its exact biological role still remains unknown. The complex of Con A with 4'-methylumbelliferyl-alpha-D-glucopyranoside (alpha-MUG) has been crystallized in space group P2(1) with cell dimensions a = 81.62 A, b = 128.71 A, c = 82.23 A, and beta = 118.47 degrees. X-ray diffraction intensities to 2.78 A have been collected. The structure of the complex was solved by molecular replacement and refined by simulated annealing methods to a crystallographic R-factor value of 0.182 and a free-R-factor value of 0.216. The asymmetric unit contains four subunits arranged as a tetramer, with approximate 222 symmetry. A saccharide molecule is bound in the sugar-binding site at the surface of each subunit, with the nonsugar (aglycon) part adopting a different orientation in each subunit. The aglycon orientation, although probably determined by packing of tetramers in the crystal lattice, helps to characterize the orientation of the saccharide in the sugar-binding pocket. The structure is the best determined alpha-D-glucoside:Con A complex to date and the hydrogen bonding network in the saccharide-binding site can be described with some confidence and compared with that of the alpha-D-mannosides.

Topics: Binding Sites; Concanavalin A; Crystallography, X-Ray; Hymecromone; Models, Molecular; Protein Binding; Protein Conformation

The use of 4-methylumbelliferyl-beta-D-glucuronide (MUG) in different solid media for the detection and enumeration of Escherichia coli in foods was evaluated by testing the effects of different substrate concentrations (50 or 100 micrograms ml-1), incubation temperatures (37 or 41.5 degrees C) and incubation times (8, 12, 24 and 48 h). Different kinds of foods, both naturally and artificially contaminated, were analysed. The use of selective media without differential substances and an incubation time of 24 h seem to be worthy of recommendation. In this case an incubation temperature of 37 degrees C would be preferred and the MUG concentration could be reduced to 50 micrograms ml-1. Incubation times shorter than 24 h, which may cause a loss of sensitivity, require higher incubation temperatures (41.5 degrees C) and MUG concentration (100 micrograms ml-1).

Topics: Colony Count, Microbial; Culture Media; Escherichia coli; Evaluation Studies as Topic; Feces; Food Microbiology; Humans; Hymecromone; Microbiological Techniques; Sensitivity and Specificity

Three commercially available kits that were supplemented with substrates for enzyme reactions were evaluated to determine their abilities to detect coliforms and fecal coliforms in foods. Japanese and U.S. Food and Drug Administration standard methods, as well as two agar plate methods, were compared with the three commercial kits. A total of 50 food samples from various retailers were examined. The levels of detection of coliforms were high with the commercial kits (78 to 98%) compared with the levels of detection with the standard methods (80 to 83%) and the agar plate methods (56 to 83%). Among the kits tested, the Colilert kit had highest level of recovery of coliforms (98%), and the level of recovery of Escherichia coli as determined by beta-glucuronidase activity with the Colilert kit (83%) was comparable to the level of recovery obtained by the U.S. Food and Drug Administration method (87%). Isolation of E. coli on the basis of the beta-glucuronidase enzyme reaction was found to be good. Levine's eosine methylene blue agar, which has been widely used in various laboratories to isolate E. coli was compared with 4-methylumbelliferyl-beta-D-glucuronide (MUG)-supplemented agar for isolation of E. coli. Only 47% of the E. coli was detected when eosine methylene blue agar was used; however, when violet red bile (VRB)-MUG agar was used, the E. coli detection rate was twice as high. Of the 200 E. coli strains isolated, only 2 were found to be MUG negative, and the gene responsible for beta-glucuronidase activity (uidA gene) was detected by the PCR method in these 2 strains. Of the 90 false-positive strains isolated that exhibited various E. coli characteristic features, only 2 non-E.coli strains hydrolyzed MUG and produced fluorescent substrate in VRB-MUG agar. However, the PCR did not amplify uidA gene products in these VRB-MUG fluorescence-positive strains.

Topics: Agar; Bacteriological Techniques; Enterobacteriaceae; Escherichia coli; Evaluation Studies as Topic; False Positive Reactions; Fluorescent Dyes; Food Microbiology; Hymecromone; Japan; Sensitivity and Specificity; United States; United States Food and Drug Administration

An internally standardized HPLC method to determine the concentration of 4-methylumbelliferone liberated from 4-methylumbelliferyl-beta-D-glucuronide by human beta-glucuronidase was developed. The assay allows the precise and rapid measurement of specific enzyme activity in human tissue homogenates. Without prior extraction the incubation mixture can be separated using a C8 column followed by fluorescence detection. The assay showed good accuracy and precision with a detection limit of 20 nM and a limit of quantification of 167 nM. The suitability of the method was shown in enzyme kinetic experiments with human liver homogenates.

Topics: Chromatography, High Pressure Liquid; Glucuronidase; Humans; Hymecromone; Liver

The use of Sorbitol MacConkey Agar supplemented with 4-methylumbelliferyl beta-D-glucuronide (MSMA), which is commonly used in the isolation of Escherichia coli O157:H7, has been shown to perform poorly when stressed cells of the pathogen are present. The incorporation of a resuscitation period (2 h at 25 degrees C) on Trypticase Soy Agar (TSA) before overlay with MSMA was found to significantly (P < or = 0.01) improve recovery of heat-stressed (52 degrees C/60 min) cells. Maximal recovery was, however, obtained by adding catalase (1000 U) to the TSA before overlaying with MSMA. This recovery protocol was shown not to result in the loss of the major known virulence factors of E. coli O157:H7 (genes encoding eae, VT1 and VT2).

Topics: Bacteriological Techniques; Base Sequence; Caseins; Culture Media; Escherichia coli; Hot Temperature; Hymecromone; Molecular Sequence Data; Protein Hydrolysates; Time Factors; Virulence

With a spectrofluorometer, the length of the incubation time required in the fluorogenic assay was reduced to 12 h. The threshold emissions for reading the fluorogenic reaction by the spectrofluorometer were 5 and 10 U for lauryl tryptose broth media containing 4-methylumbelliferyl-beta-D-galactoside and 4-methylumbelliferyl-beta-D-glucuronide, respectively. These two kinds of threshold units were equivalent to known concentrations of free 4-methylumbelliferone of 2.5 and 6 microM, respectively, in lauryl tryptose broth media.

Topics: Bacteriological Techniques; Culture Media; Enterobacteriaceae; Fluorescent Dyes; Fresh Water; Galactosides; Hymecromone; Spectrometry, Fluorescence; Time Factors; Water Microbiology

Recreational water surveillance is an important tool to prevent health hazards for the population. Therefore distinct guide and imperative values for fecal indicators are listed in the EC directive about water quality control. The detection methods, however, give laboratories some room to choose their own method, which has led to difficulties in the comparability of results. In 1989 an ad-hoc working group of the coastal countries of Germany established detection methods, which by now are obligatory for these countries. Fecal and total coliforms (FC and TC) are detected by a triplicate mpn-procedure using brilliant green-bile-lactose broth supplemented with tryptophane and 4-methylumbelliferyl-beta-D-glucuronide (BGB-MUG) as selective medium. Gas-, fluorescence- and indole-positive cultures are considered fecal coliform-positive. In the last years rises in TC but not in FC counts were observed in fresh waters. A study was carried out to evaluate the official method in another bathing season, to determine bacterial species leading to false-positive TC cultures and to compare BGB-MUG with laurylsulphate-tryptophane-MUG (LSTB-MUG). Water samples of different salinities and nutrient input were collected in weekly intervals from April to October. FC and TC concentrations were determined and all TC-positive cultures were differentiated further. The FC counts obtained by enrichment in BGB-MUG or LSTB-MUG were nearly identical, the rate of fluorescence-positive, indole-negative tubes being approximately 0.6%. Differentiation of FC-negative cultures showed a false-negative rate of 2.87% for BGB-MUG and of 8% for LSTB-MUG. During the summer months TC counts in BGB-MUG exceeded FC counts by far at most of the sampling sites. This effect was much less pronounced in LSTB-MUG; the difference between both enrichment media being significant. Differentiation of presumptive TC from BGB-MUG resulted in a high percentage of Aeromonas spp. in fresh waters. LSTB-MUG was clearly more selective for TC than BGB-MUG, but still with an average of 10% of the test tubes being false TC-positive (BGB-MUG 46%). The sensitivity of BGB-MUG was below 60% (LSTB-MUG 89%). LSTB-MUG should be preferred as enrichment medium in mpn-examination of recreational water, if no further differentiation is carried out. The selectivity for TC is better than in BGB-MUG and the only slight inhibitory effects can be tolerated.

Topics: Aeromonas; Bathing Beaches; Colony Count, Microbial; Culture Media; Enterobacteriaceae; False Positive Reactions; Feces; Fresh Water; Germany; Hymecromone; Quality Control; Recreation; Seasons; Sodium Dodecyl Sulfate; Tryptophan; Water Microbiology

Four media containing 4-methylumbelliferyl-beta-D-glucuronide were evaluated as a non-confirmatory procedure for E. coli detection in recreational water surveillance. The media included ECD-Agar for membrane filtration and laurylsulphate-tryptose, brilliant-green-bile and lactose as broth media in a three tube most probable number procedure. From six representative water sites, samples were collected weekly over a typical summer season (17.05-27.09.1994) and processed as parallels, using each media at two different incubation temperatures (36 degrees/44 degrees C). Results showed that incubation temperature had no impact on E. coli counts. Each media at a given temperature could be regarded as individual enrichment procedure. None of these enrichment procedures showed a constant and predictable higher sensitivity during the sampling period at all sites compared to the others tested. For parallel results, the rate of agreement, based upon EC-guideline (76/160/EWG) staging of recreational water quality, was 85% for membrane filtration and 75% for the MPN-procedure results. Marked differences could be observed in false-positive specificity showing correlation to the selective characteristics of the media. Subsequently lactose-broth at 44 degrees C performed worst with 30% non verifiable results, while ECD-agar and laurysulphate-tryptose-broth, both at 44 degrees C, had a nearly 100% confirmation rate. Thus, combining high specificity with no lack in sensitivity these two MUG-supplemented media seem to be best suited for E. coli detection in routine recreational water surveillance.

Topics: Colony Count, Microbial; Culture Media; Escherichia coli; Evaluation Studies as Topic; Fresh Water; Hymecromone; Quality Control; Recreation; Seawater; Sensitivity and Specificity; Water Microbiology

Two commercially available media recommended for the isolation and rapid identification of Escherichia coli from urinary tract infections were supplemented with L-phenylalanine and L-tryptophan. The non-selective medium proved suitable for the direct detection of lactose fermentation, beta-glucuronidase and phenylalanine deaminase activities, indole production and the oxidase test. It was highly efficient in making a presumptive identification at species level of the most common gram-negative urinary pathogens, E. coli, Proteus mirabilis and Pseudomonas aeruginosa, that account for c. 85% of all urinary isolates. Among the gram-positive isolates, most colonies were non-fluorescent and could be separated into staphylococci and enterococci on the basis of the catalase test. Fluorescent colonies were found to be Staphylococcus haemolyticus isolates, 61% of which were fluorescent. The selective medium proved suitable for the same biochemical tests, with the exception of indole, which was not visible against the red colour of the medium. Therefore, the differentiation of P. mirabilis from other Proteus-Providencia species was impossible on this medium.

Topics: Amino Acid Oxidoreductases; Culture Media; Electron Transport Complex IV; Fluorescent Dyes; Glucuronidase; Gram-Negative Bacteria; Gram-Positive Bacteria; Humans; Hymecromone; Indoles; Sensitivity and Specificity; Urinary Tract Infections

Enzyme assays for 4-methylumbelliferyl-beta-D-galactopyranosidase and 4-methylumbelliferyl-beta-D-glucuronidase activities were used for rapid detection (25 min) of fecal water pollution and to determine the impact of sewage discharge in coastal waters. Two coastal areas were investigated: (i) an estuary characterized by a high degree of contamination downstream of a discharge from a sewage treatment plant and a low degree of water renewal and (ii) a fjord with a low degree of pollution and a high degree of water renewal. Statistical analysis showed that a global correlation curve could be used to estimate concentrations of culturable fecal coliform bacteria in the two coastal areas, although environmental factors important for cell physiology (e.g., salinity) varied at different sampling locations. The sensitivity limit for detection of 4-methylumbelliferyl-beta-D-glucuronidase activity corresponded to bacterial concentrations on the order of 10 to 100 CFU/100 ml. The 4-methylumbelliferyl-beta-D-galactopyranosidase assay was less sensitive because of a higher rate of substrate autohydrolysis. The detection limit corresponded to bacterial concentrations on the order of 100 to 1,000 fecal coliforms per 100 ml.

Topics: beta-Galactosidase; Environmental Monitoring; Feces; Galactosides; Glucuronidase; Hymecromone; Seawater; Sewage; Substrate Specificity; Water Pollution

We describe a new method that uses a fluorogenic bioassay of the beta-glucuronidase conversion of 4-methylumbelliferyl beta-D-glucuronide (MUG) to 4-methylumbelliferone to evaluate the individual toxic effects on Escherichia coli of Al3+, Cr6+, Hg2+ and Li+. This work was designed to examine the effectiveness of this method to measure the effects of five ionic concentrations of either Al3+, Cr6+, Hg2+ or Li+, on the growth of E. coli in a minimal medium that had MUG as the only source of carbon. This method was simple and fast, and its toxicity detection sensitivity was equal to, or greater than, existing bacterial bioassays. The use of the MUG substrate minimized the danger of interference by bacteria other than E. coli. Evaluations of toxicity in samples of public drinking water proved equally sensitive.

Topics: Aluminum; Biological Assay; Chromium; Escherichia coli; Fluorescent Dyes; Glucuronidase; Hymecromone; Lithium; Mercury; Water Pollutants, Chemical

An enzyme capture assay (ECA) for rapid identification of Escherichia coli in blood cultures by using beta-D-glucuronidase as a marker was developed. Microdilution plates coated with antiglucuronidase were used to capture this enzyme from the cell lysates of blood cultures which showed growth of gram-negative bacteria. The assay, using 4-methylumbelliferyl-beta-D-glucuronide as a fluorogenic substrate, had a detection limit of 0.1 ng/ml (3 x 10(-13) M) for the enzyme; this was approximately equal to a cell concentration of 10(6) CFU of E. coli per ml. Among 212 blood cultures showing growth of gram-negative bacteria, 77 specimens were found to contain E. coli by conventional culture procedures and 73 samples were positive by ECA. Among the 135 blood cultures from which E. coli was not isolated, ECA gave one false-positive (Salmonella enteritidis) reaction. Thus, the sensitivity and specificity for the identification of E. coli in blood cultures by ECA were 94.8% (73/77) and 99.3% (134/135), respectively. From the finding of positive growth in the culture bottle, the assay can be completed within 4 h. In view of the high rate of isolation of E. coli from bacteremic patients, the test can be performed in parallel with conventional culture protocols; this may shorten the identification time for E. coli, and proper antimicrobial treatments may be started 24 h earlier than when results of conventional identification systems are used.

Topics: Antibodies, Bacterial; Bacteremia; Bacterial Proteins; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Escherichia coli Infections; Fluorescent Dyes; Glucuronidase; Hymecromone; Sensitivity and Specificity; Time Factors

Topics: Drug Stability; Hymecromone; Solubility

GM2-gangliosidosis B1 variant, considered a rare disorder with a wide geographical and ethnic distribution, appears to be exceptionally frequent in Portugal. In order to establish a carrier detection method for this disease we have determined the ratio of enzymatic activities against 4MUGS and 4MUG in urine from B1 variant obligate carriers and controls, using the total extract and the Hex A immunobound to a monoclonal antibody. The Hex A immunoassay was applied to the identification of carriers in B1 variant families and the results obtained were compared with those from DNA analysis. The reliability and feasibility of the Hex A immunoassay make it a suitable method for B1 variant carrier screening, which is particularly important for the prevention of this severe neurological disease in the population at risk.

Topics: Adolescent; Adult; Aged; beta-N-Acetylhexosaminidases; Child; Child, Preschool; Female; Genetic Carrier Screening; Genetic Variation; Hexosaminidase A; Humans; Hymecromone; Male; Middle Aged; Tay-Sachs Disease

We describe late infantile Tay-Sachs disease with high residual hexosaminidase A activity in two siblings of a Syrian Druze family. The patients' leukocytes had 26% of normal hexosaminidase A activity when tested with the conventional fluorogenic substrate 4-methyl-umbelliferyl-2-acetamido-2-deoxy-beta-D-glucopyranoside (4-MUG) and only about 10% when assayed with the sulfated substrate, 4-methyl-umbelliferal- beta-N-acetyl-glucosamine-6-sulfate (4-MUGS). According to the standard procedure of the heterozygote screening program (employing 4-MUG and heat inactivation), the parents were not diagnosed as an at-risk couple since the father was classified as a noncarrier. However, both parents' levels were clearly within the carrier range on the basis of 4-MUGS. The unique catalytic characteristics of the patients' enzyme forward the assumption that the affected sibs are B1 variants. The parents' enzymatic levels, together with their known consanguinity, might indicate that these patients are homozygotes for the rare mutation and not genetic compounds as has been documented for most of the infantile B1 variants. To the best of our knowledge this is the first reported case of B1 variant in a child of that extraction.

Topics: beta-N-Acetylhexosaminidases; Female; Genetic Carrier Screening; Hexosaminidase A; Humans; Hymecromone; Infant, Newborn; Male; Syria; Tay-Sachs Disease

4-Methylumbelliferyl-beta-D-galactoside and 4-methylumbelliferyl-beta-D-glucuronide were added to MacConkey broth and their diagnostic powers for total coliforms (TC) and Escherichia coli, respectively, were tested by membrane filtration at primary isolation. Examining water samples from different sources proved the usefulness of fluorogenic rather than reference media both as regards recovery efficiency and rapidity (possible within 12 h) of analyses. The recoveries obtained by fluorogenic and conventional tests for both TC and E. coli were correlated. Values were comparable in surface water samples, while a higher sensitivity of fluorogenic media was observed in samples of shallow contaminated ground water. Results seem to indicate that the use of fluorogenic membrane filtration analysis for colimetric indicators could be favourably considered especially for sanitary surveying of drinking water.

Topics: Colony Count, Microbial; Culture Media; Enterobacteriaceae; Escherichia coli; Filtration; Galactosides; Hymecromone; Water Microbiology; Water Supply

A two-step membrane filter procedure was evaluated to determine the ability to differentiate Escherichia coli from other coliform bacteria recovered from water. M-Endo LES agar incubated at 35 degrees C for 24 +/- 2 h was used as the initial isolation medium. Membranes containing coliform colonies were transferred to nutrient agar plus 4-methylumbelliferyl beta-D-glucuronide (MUG) and incubated for an additional 4 h at 35 degrees C. Escherichia coli colonies were distinguished by fluorescence when viewed under a long-wavelength ultraviolet light. A total of 119 MUG-positive colonies were isolated from 15 water sources, of which 115 (96.6%) were identified as E. coli. An examination of 182 pure culture environmental E. coli isolates revealed that 167 isolates (91.8%) exhibited fluorescence on the nutrient agar plus MUG medium. Survivors of E. coli cultures exposed to chlorination were also capable of producing a positive MUG reaction.

Topics: Bacterial Typing Techniques; Chlorine; Colony Count, Microbial; Culture Media; Disinfection; Enterobacteriaceae; Escherichia coli; Fluorescence; Fresh Water; Hymecromone; Micropore Filters; Water Microbiology

The uidA gene, which encodes the beta-glucuronidase enzyme, was detected in 97.7% of 435 Escherichia coli isolates from treated and raw water sources by DNA-DNA hybridization; 92.4% of the strains expressed the translational product in 4-methylumbelliferyl-beta-D-glucuronide-containing media after reinoculation. Upon initial isolation from water samples, the minimal medium o-nitrophenyl-beta-D-galactopyranoside-4-methylum-belliferyl -beta-D-glucuronide preparations failed to detect more than 50% of the E. coli isolates that possessed uidA gene. Treated water gave the lowest recovery, with Colilert producing 26% positive samples and Coliquik producing 48% positive samples. There appears to be no relationship between the intensity of the autoradiographic signals of the uidA gene and the expression of beta-glucuronidase activity. Therefore, another variable such as physiological condition of the bacteria could be responsible for the nonexpression of the enzyme activity.

Topics: Autoradiography; Culture Media; Escherichia coli; False Negative Reactions; Glucuronidase; Hymecromone; Water Microbiology

A factor in colon carcinogenesis might be the partial defeat in colon epithelial cells of the protective enzymic barrier against xenobiotics, via bile acid inhibition of enzymes that detoxify mutagens. The applicability of aspects of this concept to glucuronosyltransferase, a phenol detoxification enzyme, was tested in a colon cancer cell line. Inhibition of glucuronidation of the test substrate, 4-methylumbelliferone, occurred at bile acid concentrations found in faecal water, and depended on pH for some bile acids. Lithocholate was the most inhibitory: the concentration causing 50% inhibition of the initial rate of glucuronidation (IC50) was about 3 microM at pH 7.4 and at pH 6.2. The inhibitory potency of deoxycholate and chenodeoxycholate increased when pH decreased, but still remained less than that of lithocholate: the IC50 for deoxycholate was 88.5 microM at pH 7.4, and 14.8 microM at pH 6.2, and for chenodeoxycholate the IC50 was 67.4 microM at pH 7.4, and 21.7 microM at pH 6.2. Cholate did not cause appreciable inhibition. The inhibitory effects were additive when lithocholate was present together with either deoxycholate or chenodeoxycholate. The results provide a mechanism for the comutagenicity of bile acids, a feature of which is the inter-relation of bile acid comutagenicity specifically with mutagens that are inactivated by a bile acid-inhibitable enzyme. The results are also in accord with the view that high concentrations of bile acids in solution in faecal water, especially lithocholate, are a risk factor for colon cancer.

Topics: Bile Acids and Salts; Chenodeoxycholic Acid; Colon; Colonic Neoplasms; Deoxycholic Acid; Dose-Response Relationship, Drug; Fluoresceins; Fluorescence; Fluorescent Dyes; Glucuronates; Glucuronic Acid; Glucuronosyltransferase; Glutathione Transferase; Humans; Hymecromone; Lithocholic Acid; Permeability; Tumor Cells, Cultured; Xenobiotics

Futile cycling between 4-methylumbelliferone and its sulfate and glucuronide conjugates was examined in the single-pass perfused rat liver preparation. The steady-state hepatic extraction ratio of 4-methylumbelliferone was found to be high (0.97) at a low input concentration of 0.005 mumol/L (tracer), with a net 4-methylumbelliferyl sulfate/4-methylumbelliferyl glucuronide ratio of about 5:1; at 63 mumol/L the steady-state extraction ratio had remained constant despite a shift from net sulfation to net glucuronidation. At higher input 4-methylumbelliferone concentrations, saturation was evidenced by a decreased steady-state extraction ratio and reduced net sulfation and net glucuronidation. Because 4-methylumbelliferyl sulfate and 4-methylumbelliferyl glucuronide deconjugation would result in an intracellular accumulation of 4-methylumbelliferone, the phenomenon was monitored with a shift in tracer [3H]4-methylumbelliferone metabolism from sulfation to glucuronidation with increased intracellular 4-methylumbelliferone concentration. When 4-methylumbelliferyl sulfate (0 to 890 mumol/L) or 4-methylumbelliferyl glucuronide (0 to 460 mumol/L) was delivered simultaneously with tracer [3H]4-methylumbelliferone to the rat liver, notable desulfation of 4-methylumbelliferyl sulfate (18% to 38% rate in) but little deglucuronidation of 4-methylumbelliferyl glucuronide (1.2% to 2.1% rate in) was observed. With 4-methylumbelliferyl sulfate, 4-methylumbelliferone and 4-methylumbelliferyl glucuronide were readily found as metabolites, whereas with 4-methylumbelliferyl glucuronide, levels of the metabolites, 4-methylumbelliferone and 4-methylumbelliferyl sulfate, were much reduced. 4-Methylumbelliferyl sulfate and not 4-methylumbelliferyl glucuronide shifted tracer [3H]4-methylumbelliferone metabolism from [3H]4-methylumbelliferyl sulfate to [3H]4-methylumbelliferyl glucuronide formation in a concentration-dependent fashion. The steady-state extraction ratio for 4-methylumbelliferyl sulfate (0.1 to 0.3) was comparatively higher than that for 4-methylumbelliferyl glucuronide (0.05), and it was found to increase with concentration, an observation explained by the nonlinear protein binding of 4-methylumbelliferyl sulfate. Biliary excretion rates for 4-methylumbelliferone and 4-methylumbelliferyl sulfate were proportional to their input or net formation rates, regardless of whether 4-methylumbelliferone, 4-methylumbelliferyl glucuronide or 4-methylumbelliferyl sulfate was ad

Topics: Animals; Bile; Blood Proteins; Hymecromone; Liver; Male; Models, Biological; Perfusion; Protein Binding; Rats; Rats, Sprague-Dawley

Contrary to the general belief that cimetidine and ranitidine spare glucuronidation of drugs, some authors have indicated that both cimetidine and ranitidine could inhibit the glucuronidation of paracetamol (acetaminophen) by cultured rat hepatocytes. Thus, we tested the effect of three histamine H2-receptor blockers (cimetidine, ranitidine and famotidine) on the glucuronidation of 7-hydroxy-4-methylcoumarin (7-OH-4-MC) by human liver microsomes. None of the drugs studied produced significant inhibition of the glucuronidation of 7-OH-4-MC when used at concentrations up to 1.5 mM. Thus, even the new H2-receptor blocker, famotidine, spares glucuronidation. These findings further support the previous reports which suggest that glucuronide conjugation in humans is spared by H2-receptor blockers.

Topics: Cimetidine; Famotidine; Glucuronosyltransferase; Histamine H2 Antagonists; Humans; Hymecromone; Microsomes, Liver; Ranitidine; Uridine Diphosphate Glucuronic Acid

A total of 449 Escherichia coli isolates in treated and raw water sources were submitted to DNA-DNA hybridization using seven different DNA probes to detect homology to sequences that code for Shiga-like toxins I and II; heat-stabile and heat-labile toxins, adherence factors EAF and eae, and the fimbrial antigen of entero-hemorrhagic E. coli. Fifty-nine (13%) of the isolates demonstrated homology with one or more specific DNA probes. More than 50% of the isolates in treated water were not recovered in MMO-4-methylumbelliferyl-beta-D-glucuronide media designed for detection of this indicator.

Topics: Bacterial Toxins; Culture Media; DNA Probes; Enterotoxins; Escherichia coli; Fresh Water; Genes, Bacterial; Hymecromone; Oxygenases; Species Specificity; Thiogalactosides; Virulence; Water Microbiology

A rapid fluorogenic medium was evaluated for the detection of Escherichia coli in dairy products. The medium was capable of detecting Esch. coli after 7.5 h incubation at 41.5 degrees C. Samples of pasteurized milk (136), raw milk (63), soft cheese (60) and pasteurized cream (39) were examined with media based on 4-methylumbelliferyl-beta-D-glucuronide (MUG-7) and Violet red bile agar and there were no significant differences between the numbers of Esch. coli detected on the two media. MUG-7 medium had a specificity of 98.6% and the small number of organisms giving a false positive reaction were identified as Klebsiella pneumoniae. The incidence of false negative results was approximately 2%. MUG-7 medium was suitable for pour plate, spread plate and membrane filtration methods. Possible applications of the method are discussed.

Topics: Animals; Cheese; Culture Media; Dairy Products; Escherichia coli; False Negative Reactions; False Positive Reactions; Fluorescent Dyes; Food Microbiology; Hymecromone; Milk; Predictive Value of Tests

Multiplex polymerase chain reaction (PCR) and gene probe detection of target lacZ and uidA genes were used to detect total coliform bacteria and Escherichia coli, respectively, for determining water quality. In tests of environmental water samples, the lacZ PCR method gave results statistically equivalent to those of the plate count and defined substrate methods accepted by the U.S. Environmental Protection Agency for water quality monitoring and the uidA PCR method was more sensitive than 4-methylumbelliferyl-beta-D-glucuronide-based defined substrate tests for specific detection of E. coli.

Topics: Base Sequence; DNA, Bacterial; Electrophoresis, Agar Gel; Enterobacteriaceae; Escherichia coli; Genes, Bacterial; Humans; Hymecromone; Molecular Sequence Data; Polymerase Chain Reaction; Water Microbiology

For rapid identification of Escherichia coli, we evaluated Fluorocult MacConkey Agar, Fluorocult Laurylsulfate Broth and Bactident E. coli, which are incorporating fluorogenic substrate, MUG (4-methylumbeliferyl-beta-D-Glucuronide) that specifically reacts with E. coli. To assess the specificity and sensitivity of Fluorocult MacConkey Agar and Laurylsulfate Broth, beta-D-glucuronidase; beta-GUR activities of 264 strains from urine including 72 of E. coli were investigated. For both media, sensitivity was 92% and specificity was 100%. When there was 10(8) c.f.u./ml of E. coli in urine specimen, incubation times required for positive fluorescence by Fluorocult MacConkey Agar, Laurylsulfate Broth, and Bactident E. coli were 8 h, 4 h and 15 min, respectively. Influence of drugs in urine to fluorescence reaction was not observed.

Topics: Bacteriological Techniques; Colony Count, Microbial; Culture Media; Cystitis; Escherichia coli; Escherichia coli Infections; Evaluation Studies as Topic; Fluorescent Dyes; Humans; Hymecromone; Sensitivity and Specificity; Urine

The performance capabilities of two commercial 4-methylumbelliferyl-beta-D-glucuronide preparations were evaluated for the detection of Escherichia coli from water samples. Eighty-three water samples were collected from a treated water reservoir, and 32 samples were collected from untreated surface water. There was a statistically significant difference between the two commercial preparations compared with the Standard Methods membrane filtration fecal coliform (MFC) method for the detection of E. coli from treated water samples. However, there was no difference between the two methods and the MFC test for E. coli detection from the untreated surface water samples. The disagreement between the two commercial products and the MFC method was primarily due to the occurrence of false-negative results with the two commercial products. The data indicate that the occurrence of false-negative samples could be attributed to impaired substrate specificity and sensitivity of the two tests for E. coli detection. There was no apparent relationship between the occurrence of false-negative results and heterotrophic plate counts in samples.

Topics: California; Colony Count, Microbial; Escherichia coli; Feces; Filtration; Fluorescent Dyes; Humans; Hymecromone; Membranes, Artificial; Sensitivity and Specificity; Water Microbiology; Water Pollutants; Water Supply

In the monitoring of bathing waters according to the EC-guideline, total coliform and faecal coliform bacteria were obtained from 97 samples of river waters by the comparison of two fluorogenic assays using MUG-Lauryl Sulfate Broth and MUG-BRILA Broth with an usual method with MacConkey Broth, Tryptophane Broth and BRILA Broth. MUG-BRILA Broth had the lowest average counts and proved to be not an appropriate medium. Compared with the usual method, however, MUG-Lauryl Sulfate Broth yielded similar high counts, reduced the investigation period from four to two days and diminished the expenditure of work by one third. In addition, the indole test for the confirmation of presumptive faecal coliforms might be abolished. Therefore, the examination with MUG-Lauryl Sulfate Broth can be recommended.

Topics: Colony Count, Microbial; Culture Media; Enterobacteriaceae; Fluorescent Dyes; Fresh Water; Germany; Humans; Hymecromone; Sodium Dodecyl Sulfate; Swimming; Water Microbiology

A medium containing the fluorogenic substrate 4-methylumbelliferyl-beta-D-glucuronide was developed for the isolation and identification of Escherichia coli within 7.5 h and was based on the detection of beta-glucuronidase. Optimum conditions for the rapid development of fluorescent colonies were determined. The optimum temperature was 41.5 degrees C. Development of fluorescence was delayed when carbohydrates were incorporated into the medium. Water samples were used to evaluate the medium by surface plating and membrane filtration. The frequency of false-negative results was 6.1% and false-positives were 3.7% for freshwater samples. The false-positive organisms were identified as Klebsiella spp. and Shigella sonnei. The potential applications of the medium are discussed.

Topics: Carbohydrate Metabolism; Culture Media; Escherichia coli; False Negative Reactions; False Positive Reactions; Fluorescent Dyes; Fresh Water; Glucuronidase; Hymecromone; Predictive Value of Tests; Seawater; Water Microbiology

Recently, Escherichia species other than Escherichia coli have been isolated from potable water. Environmental isolates as well as clinical isolates of E. adecarboxylata, E. blattae, E. fergusonii, E. hermannii, and E. vulneris were assayed for the enzyme beta-glucuronidase by using EC MUG medium and the Colilert system. None of the isolates were positive for the enzyme by either method.

Topics: Escherichia; Fluorescent Dyes; Glucuronidase; Hymecromone; Water Microbiology; Water Supply

A direct high-performance liquid chromatographic (HPLC) assay was developed for the separation and determination of 4-methylumbelliferone (4MU) and its glucuronide (MUG) and sulfate (MUS) conjugates in the cell-free perfusate ("plasma") from in situ perfused rat intestine-liver preparation. In addition, a procedure was developed to extract and determine 4MU in the whole blood perfusate. Perfusate plasma containing an internal standard (umbelliferone) was precipitated with methanol (1:4, v/v), and injected into a reversed-phase HPLC system with gradient elution. 4MU and the same internal standard were also extracted directly from the whole blood perfusate with ethyl acetate and injected into a reversed-phase HPLC system with isocratic elution. Inter- and intra-day precision studies (n = 5 for each) for both the plasma and whole blood procedures demonstrated relative standard deviation of less than 10% at all concentrations studied. The compounds were stable in either the plasma or blood extracts at room temperature for up to 72 h. The procedures were successfully used to analyze perfusate samples obtained from the single-pass in situ perfusion of rat intestine-liver system with either trace (0.95 nM) or 32.3 microM concentrations of 4MU. The intestine was responsible for the formation of most of the MUG formed by the intestine-liver preparation during steady-state perfusion with either input concentration of 4MU.

Topics: Animals; Chromatography, High Pressure Liquid; Drug Stability; Erythrocytes; Humans; Hymecromone; Intestines; Liver; Perfusion; Rats

The anticoagulant phenprocoumon is mainly metabolized in humans to hydroxylated metabolites and their glucuronides. A method is described for the determination of phenprocoumon, 4'-hydroxyphenprocoumon, 6-hydroxyphenprocoumon, 7-hydroxyphenprocoumon, and their glucuronide and sulphate conjugates in human urine. Reversed-phase high-performance liquid chromatography is performed after selective extraction with disposable quaternary amine columns of untreated, and beta-glucuronidase- or sulphatase-treated urine samples. Urinary excretion data are presented for total, glucuronidated, sulphated and free phenprocoumon, 4'-hydroxyphenprocoumon, 6-hydroxyphenprocoumon and 7-hydroxyphenprocoumon in twelve patients after an average daily dosage of 1.3-4.2 mg phenprocoumon.

Topics: Adult; Aged; Chromatography, High Pressure Liquid; Glucuronidase; Humans; Hydrolysis; Hymecromone; Indicators and Reagents; Male; Middle Aged; Phenprocoumon; Spectrophotometry, Ultraviolet; Sulfatases

Fluorogenic procedures were used with the substrate 4-methylumbelliferyl-beta-D-glucuronide (MUG) to identify Escherichia coli. Most strains produced beta-glucuronidase and, thus, were MUG positive. A 20-min procedure was developed to detect glucuronidase activity in 1,295 bacterial cultures, representing 23 genera, of strains that were isolated from clinical specimens. Very few organisms other than E. coli were MUG positive. Of 682 E. coli strains that were isolated, 630 (92.4%) were MUG positive. When an additional 188 E. coli serotype O157 isolates were examined, 155 E. coli O157:H7, 10 E. coli O157:H-, and 1 E. coli O157:H (rough) isolate were MUG negative. All 166 cultures were verocytotoxin positive. Of the remaining 22 E. coli O157 isolates, 2 isolates were O157:H-, 1 isolate was O157:H (rough), and 19 isolates were other H types (H6, H16, H19, H25, H42, and H45); these 22 isolates were MUG positive. All 22 cultures were verocytotoxin negative. The rapid MUG procedure can be used to predict verocytotoxin-positive isolates of E. coli O157; that is, there is a very good likelihood that MUG-negative E. coli O157 isolates are verocytotoxin positive.

Topics: Bacterial Toxins; Bacteriological Techniques; Cytotoxins; Escherichia coli; Fluorescent Dyes; Gram-Negative Bacteria; Humans; Hymecromone; Serotyping; Shiga Toxin 1

The Autoanalysis Colilert (AC) test was compared with the membrane filter (MF), 10-tube multiple-tube fermentation (MTF) technique, and the presence-absence test as described in Standard Methods for the Examination of Water and Wastewater for the detection and enumeration of total coliforms in water. The methods were evaluated with 31 samples from seven different sources. Each sample was analyzed by each of the techniques, using replicate 100-ml sample volumes. A total of 582 confirmed tubes were positive by the MTF test, and 533 tubes were positive by the AC test. Statistical analysis of the most-probable-number comparability data showed a statistically significant difference in the number of positive tubes, with the MTF test resulting in more positive tubes. There were no statistically significant differences in precision between the two methods. All the methods were comparable in detection of total coliforms. Levels of heterotrophic bacteria generally encountered in drinking water did not interfere with detection or enumeration of coliforms by the AC test.

Topics: beta-Galactosidase; Colony Count, Microbial; Enterobacteriaceae; Fluorescent Dyes; Humans; Hymecromone; Nitrophenylgalactosides; Water Microbiology

A comparison was made with different chromogenic and fluorogenic substrates, 4-methylumbelliferyl-beta-D-glucuronide (MUG), 4-nitrophenyl-beta-D-glucuronide (PNPG), 4-methylumbelliferyl-beta-D-galactopyranoside (MUGA), 2-nitrophenyl-beta-D-galactopyranoside (ONPG), 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-GAL), for the rapid and simultaneous enumeration of total coliforms and E. coli in water samples, based on 2 commercially available culture-media. The combination of the chromogenic compound X-GAL (for detecting coliforms) and of the fluorogenic compound MUG (for detecting E. coli) incorporated either into ECD agar or into lauryl sulfate broth proved to be most useful. The optimum concentration of the X-GAL/MUG supplement was (50 micrograms/ml/70 micrograms/ml) for the solid medium (EMX agar) and (60 micrograms/ml/70 micrograms/ml) for the fluid medium (LMX broth). As a result of the examination of 244 Enterobacteriaceae strains isolated from water samples and clinical material, it was shown that the use of EMX agar (LMX broth) had several advantages over conventional methods. A routine method for the analysis of water samples was proposed involving the EMX agar and the LMX broth.

Topics: Chromogenic Compounds; Culture Media; Enterobacteriaceae; Escherichia coli; Fluorescent Dyes; Galactosides; Glucuronates; Hymecromone; Indoles; Nitrophenylgalactosides; Water Microbiology

Topics: Escherichia coli; Food Microbiology; Hymecromone; Umbelliferones

The glucuronidation kinetics of 4-methylumbelliferone (4MU) and 1-naphthol (1NP) have been investigated in human liver microsomes to determine the validity of using these compounds as probes for specific UDP-glucuronosyltransferase (GT) activities in human liver. 4MU glucuronidation followed Michaelis-Menten kinetics, whereas 1NP glucuronidation kinetics were biphasic. Cross inhibition studies were performed with 4MU and 1NP to determine the relationship between 4MU glucuronidation and the two phases of 1NP glucuronidation. 4MU glucuronidation was competitively inhibited by 1NP but 4MU inhibited only the high affinity component of 1NP glucuronidation. There was good agreement between the apparent Km values for 4MU and the high affinity component of 1NP glucuronidation and their respective apparent K1 values determined in the cross inhibition studies. These data suggest that the same form(s) of human liver GT is involved in 4MU glucuronidation and the high affinity component of 1NP glucuronidation. A number of compounds known to be specific substrates for purified rat liver GTs were screened for inhibitory effects on 4MU glucuronidation in human liver microsomes. 4-Nitrophenol, 2-aminophenol and androsterone inhibited 4MU glucuronidation whereas bilirubin, chloramphenicol, digitoxigenin monodigitoxoside, morphine, oestrone and testosterone had no effect. 4-Nitrophenol and 2-aminophenol were competitive inhibitors of 4MU glucuronidation but the inhibition of 4MU glucuronidation by androsterone followed atypical kinetics. Overall, the substrate specificity of the human liver 4MU/high affinity 1NP-GT activity appears to be broadly similar to that of the 3-methylcholanthrene inducible rat hepatic microsomal GT.

Topics: Glucuronates; Glucuronosyltransferase; Humans; Hymecromone; In Vitro Techniques; Kinetics; Microsomes, Liver; Naphthols; Substrate Specificity; Umbelliferones; Uridine Diphosphate Glucuronic Acid

Three fluorogenic methylumbelliferone (MU) substrates were evaluated for rapid detection of total and fecal coliform bacteria (TC and FC) in drinking water. 4-MU-beta-D-galactoside, MU-heptanoate, and MU-glucuronide were used to determine enzyme activity as a surrogate measure of coliform concentration. Coliforms occurring in river water and in potable water artificially contaminated with raw sewage were tested. The initial rate of hydrolysis (delta F) of MU-beta-D-galactoside showed promise as an indicator of TC and FC within 15 min. delta F of MU-glucuronide was insufficient in the 15-min assay, and combinations of the MU substrates did not enhance delta F. A direct membrane filter method incorporating MU-beta-D-galactoside into an agar medium allowed the detection of as few as 1 FC per 100 ml within 6 h.

Topics: Colony Count, Microbial; Enterobacteriaceae; Fluorescent Dyes; Galactosides; Glycosides; Hydrolysis; Hymecromone; Sewage; Umbelliferones; Water Microbiology

Topics: False Positive Reactions; Humans; Hymecromone; Serum Albumin; Spectrophotometry; Umbelliferones

Evidence was found for UDPglucuronyltransferase-catalysed deconjugation of p-nitrophenol-, 4-methylumbelliferone- and phenolphthalein-glucuronides. The evidence is based on the following observations: 1, deconjugation is UDP-dependent and the reactions show Michaels-Menten kinetics with respect to UDP and glucuronide saturability; 2, UDP-glucuronic acid was identified as reaction product; 3, all studies were done in the presence of a beta-glucuronidase inhibitor; 4, induction profiles, using 3-methylcholanthrene and phenobarbital as inducing agents, were identical for conjugation and deconjugation reactions. Optimal deconjugation rates for p-nitrophenol- and 4-methylumbelliferone-glucuronides were at pH 5.1 and for phenolphthalein-glucuronide at pH 6.5. Only conjugation reactions showed latency; the corresponding deconjugation reactions were not latent. UDPglucuronyltransferase is a group of oligomeric isoenzymes with different molecular masses. The molecular masses of the isoenzyme species catalysing the forward and reverse reactions were determined by radiation-inactivation analysis. The molecular masses of the isoenzyme species mediating the catalyses of deconjugation reactions were significantly smaller than those mediating catalyses of conjugation reactions: 66 +/- 4 kDa vs. 109 +/- 7 kDa for p-nitrophenol; 82 +/- 8 kDa vs. 105 +/- 6 kDa for 4-methylumbelliferone; and 74 +/- 8 kDa vs. 159 +/- 14 kDa for phenolphthalein. This suggests that for catalyses of deconjugation reactions only part of a UDPglucuronyltransferase isoenzyme is needed, whereas for forward reactions the complete isoenzymes are required.

Topics: Animals; Glucuronates; Glucuronosyltransferase; Hydrogen-Ion Concentration; Hymecromone; Isoenzymes; Kinetics; Male; Microsomes, Liver; Molecular Weight; Phenolphthaleins; Rats; Rats, Inbred Strains; Uridine Diphosphate Glucuronic Acid

The dose-dependent first-pass hepatic metabolism and pharmacokinetics of 4-methylumbelliferone (4-MU) were studied at four dose levels (17.6 micrograms-5.29 mg) in rats. 4-MU was given intravenously and intraportally to determine the availability of 4-MU. The availability increased from 0.18 to 1.31 when the dose was increased from 17.6 micrograms to 5.29 mg/rat. The total body plasma clearance of 4-MU was accounted for mostly by the hepatic conjugative metabolism. The contribution of renal clearance to total plasma clearance was 11-35%, depending on the dose. The marked dose-dependency of availability may thus be explained by the saturable conjugative metabolism of 4-MU.

Topics: Animals; Biological Availability; Chromatography, Thin Layer; Dose-Response Relationship, Drug; Femoral Vein; Hymecromone; Injections, Intravenous; Kinetics; Liver; Male; Portal Vein; Protein Binding; Rats; Rats, Inbred Strains; Time Factors; Umbelliferones

Most flavobacteria tested with the fluorogenic substrate 4-methylumbelliferyl-beta-D-glucuronide possessed beta-glucuronidase (GUD), but when some of the same strains were tested with the API ZYM gallery, all were negative for GUD. Conflicting reports also appear in the literature about starch hydrolysis among flavobacteria. We observed that the results obtained can depend on the medium used and the length of incubation. Our results indicate that GUD activity and starch hydrolysis are more widely distributed in the genus Flavobacterium than previously reported.

Topics: Culture Media; Flavobacterium; Glucuronidase; Hydrolysis; Hymecromone; Starch

Plate count-monensin-KCl (PMK) agar, for enumeration of both gram-negative bacteria and Escherichia coli, is composed of (per liter) 23.5 g of plate count agar, 35 mg of monensin, 7.5 g of KCl, and 75 mg of 4-methylumbelliferyl-beta-D-glucuronide (MUG). Monensin was added after the medium was sterilized. The diluent of choice for use with PMK agar was 0.1% peptone (pH 6.8); other diluents were unsatisfactory. Gram-negative bacteria (selected for by the ionophore monensin) can be used to judge the general quality or sanitary history of a commodity. E. coli (differentiated by its ability to hydrolyze the fluorogenic compound MUG) can be used to assess the safety of a commodity in regard to the possible presence of enteric pathogens. Pure-culture studies demonstrated that monensin completely inhibited gram-positive bacteria and had little or no effect on gram-negative bacteria. When gram-negative bacteria were injured by one of several methods, a few species (including E. coli) became sensitive to monensin; this sensitivity was completely reversed in most instances by the inclusion of KCl in the medium. When PMK agar was tested with food and environmental samples, 96% of 535 isolates were gram negative; approximately 68% of colonies from nonselective medium were gram negative. PMK agar was more selective than two other media against gram-positive bacteria and was less inhibitory for gram-negative bacteria. However, with water samples, KCl had an inhibitory effect on gram-negative bacteria, and it should therefore be deleted from monensin-containing medium for water analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Bacteriological Techniques; Culture Media; Escherichia coli; Food Microbiology; Furans; Gram-Negative Bacteria; Hot Temperature; Hymecromone; Monensin; Water Microbiology

An assay procedure to screen for Escherichia coli in foods by using 4-methylumbelliferyl-beta-D-glucuronide (MUG) incorporated into lauryl tryptose (LST) broth was evaluated. The beta-glucuronidase produced by E. coli cleaves the MUG substrate to yield a fluorescent end product. E. coli-negative samples can be identified by lack of fluorescence in LST-MUG within 24 h. MUG was not inhibitory to coliforms and E. coli. Over 1,400 food and dairy samples were tested to compare the standard three-tube most-probable-number procedure with the MUG-containing or non-MUG-containing LST procedure. LST-MUG testing detected a greater number of E. coli, with a lower false-positive rate (1.4%) and in a shorter time, than did the standard procedure. All false-positive results in the LST-MUG testing were attributable to beta-glucuronidase-producing staphylococci. No false-negative result was encountered. Use of MUG in LST broth obviates the EC broth step, allowing a 2.5-day procedure to a completed E. coli test versus the present 4- to 6-day standard most-probable-number method.

Topics: Bacteriological Techniques; Escherichia coli; Fluorescent Dyes; Food Microbiology; Glucuronidase; Hymecromone

The role of the liver in the conjugation of 4-methylumbelliferone (4MU), mainly glucuronidation, was investigated in the rat in vivo. The liver extracted 4MU almost completely (97%) during steady-state infusion, as measured by the difference between 4MU concentration in portal and hepatic venous blood. Previously, it was shown that the intestinal region extracts 40% of the 4MU of the incoming arterial blood. The liver and the gastrointestinal region are so efficient that their conjugation activity can account for total body clearance of 4MU (50-60 ml/min per kg). These results and other evidence on extrahepatic conjugation of phenolic substrates suggest that glucuronidation may be limited to the liver, (the kidney) and the gastrointestinal region, while sulfation may occur more widespread throughout the body. Protein binding studies showed the sulfate conjugate to be even more protein-bound than unconjugated 4MU, while 4MU glucuronide was much less bound to rat plasma proteins.

Topics: Animals; Blood Proteins; Hymecromone; Infusions, Parenteral; Injections, Intravenous; Intestinal Mucosa; Liver; Male; Protein Binding; Rats; Rats, Inbred Strains; Umbelliferones

Glucocerebrosidase was isolated from bovine brain by cholate extraction, ammonium sulfate fractionation, acid precipitation at pH 5.35, and hydrophobic chromatography. The purification is about 2400-fold with a specific activity of about 286,000 nmole/hr/mg protein. Molecular weight as determined by chromatography on Bio-Gel P-200 was 138,000. On SDS-polyacrylamide gel electrophoresis the enzyme protein resolved into two bands with apparent molecular weights of 63,000 and 56,000. These bands are cross-reactive to monospecific polyclonal antibody to homogeneous human placental glucocerebrosidase. The enzyme was found to be a complex glycoprotein based on its lectin binding specificity. Brain enzyme was found to be similar to placental glucocerebrosidase in its pH optima, heat stability at 52 degrees C, and substrate affinity. Enzyme kinetics were measured in the presence of conduritol-beta-epoxide, an irreversible inhibitor, and gluconolactone, a competitive inhibitor.

Topics: Animals; Brain; Cattle; Gluconates; Glucosidases; Glucosylceramidase; Glucosylceramides; Hot Temperature; Hydrogen-Ion Concentration; Hydrolysis; Hymecromone; Kinetics; Lactones; Molecular Weight

Escherichia coli is the most common gram-negative microbe isolated and identified in clinical microbiology laboratories. It can be identified within 1 h by oxidase, indole, lactose, and beta-glucuronidase tests. The oxidase and indole tests are performed as spot tests, and lactose fermentation is read directly from MacConkey agar. It was found that 4-methylumbelliferyl-beta-D-glucuronide could be incorporated directly into a modified MacConkey agar to directly detect the presence of beta-glucuronidase. Other characteristics of MacConkey agar were not affected. The incorporation of 4-methylumbelliferyl-beta-D-glucuronide into modified agar obviated the need for manufacture, quality control, and incubation of reagent-containing test tubes. The time needed to identify E. coli strains was reduced from 1 h to 5 min, and the ability to detect this species in mixed specimens was also enhanced.

Topics: Agar; Culture Media; Escherichia coli; Fermentation; Fluorescence; Glucuronates; Glucuronidase; Hymecromone; Indoles; Lactose; Oxidoreductases; Time Factors

A fluorogenic assay procedure with 4-methylumbelliferyl-beta-D-glucuronide incorporated into lauryl sulfate broth was evaluated to detect and confirm the presence of Escherichia coli in foods. Fluorescence is indicative of the presence of E. coli; extensive biochemical confirmation is unnecessary with this assay. The 4-methylumbelliferyl-beta-D-glucuronide assay was tested concurrently with our present methodology for detection of E. coli on 270 samples of raw ingredients and powdered food products. Total agreement between the two methods was 94.8%; there was a false-positive rate of 4.8% and no false-negatives. We found the 4-methylumbelliferyl-beta-D-glucuronide assay to be rapid, accurate, simple to perform, and inexpensive.

Topics: Escherichia; Escherichia coli; Fluorescent Dyes; Food Microbiology; Hymecromone; Species Specificity; Spectrometry, Fluorescence

Topics: Chromatography, High Pressure Liquid; Humans; Hymecromone; Kinetics; Umbelliferones

Rapid assays for Escherichia coli were developed by using the compound 4-methylumbelliferone glucuronide (MUG), which is hydrolyzed by glucuronidase to yield a fluorogenic product. The production of glucuronidase was limited to strains of E. coli and some Salmonella and Shigella strains in the family Enterobacteriaceae. For immediate confirmation of the presence of E. coli in most-probable-number tubes, MUG was incorporated into lauryl tryptose broth at a final concentration of 100 micrograms/ml. Results of both the presumptive test (gas production) and the confirmed test (fluorescence) for E. coli were obtained from a variety of food, water, and milk samples after incubation for only 24 h at 35 degrees C. Approximately 90% of the tubes showing both gas production and fluorescence contained fecal coliforms (they were positive in EC broth incubated at 45 degrees C). Few false-positive reactions were observed. The lauryl tryptose broth-MUG-most-probable-number assay was superior to violet red bile agar for the detection of heat- and chlorine-injured E. coli cells. Anaerogenic strains produced positive reactions, and small numbers of E. coli could be detected in the presence of large numbers of competing bacteria. The fluorogenic assay was sensitive and rapid; the presence of one viable cell was detected within 20 h. E. coli colonies could be distinguished from other coliforms on membrane filters and plates of violet red bile agar if MUG was incorporated into the culture media. A rapid confirmatory test for E. coli that is amenable to automation was developed by using microtitration plates filled with a nonselective medium containing MUG. Pure or mixed cultures containing E. coli produced fluorescence within 4 h (most strains) to 24 h (a few weakly positive strains).

Topics: Bacteriological Techniques; Culture Media; Escherichia coli; Fluorescent Dyes; Food Microbiology; Glucuronidase; Hymecromone; Water Microbiology

When isolated hamster lungs were perfused in a recirculating system for 2 hrs, 4-methylumbelliferone from the perfusion medium was conjugated in perfused lungs with endogenous UDP glucuronic acid to 4-methyl-umbelliferylglucuronide. When hamsters were exposed to cigarette smoke during one hour in an inhalation chamber 20 hrs before the perfusion, the rate of glucuronide conjugation was slightly increased. When isolated lungs were ventilated intermittently with cigarette smoke during the perfusion, the rate of glucuronide conjugation was decreased.

Topics: Animals; Biotransformation; Cricetinae; Glucuronates; Hymecromone; In Vitro Techniques; Lung; Male; Mesocricetus; Perfusion; Smoking; Uridine Diphosphate Glucuronic Acid