Rapid identification of Escherichia coli with a fluorogenic beta-glucuronidase assay.
AuthorsPapasian CJ, Hertlein G
JournalDiagn Microbiol Infect Dis
PubMed ID3329592
'We evaluated three fluorogenic methods (MUG; Remel, Lenexa, KS) for the rapid (less than 1 hr) identification of E. coli by detecting beta-glucuronidase activity. The methods included: direct disk inoculation test, tube test, and liquid spot reagent test. Fluorogenic tests were performed on pure cultures of lactose fermenters and compared ... More
Rapid characterisation and identification of mycobacteria using fluorogenic enzyme tests.
AuthorsHamid ME, Chun J, Magee JG, Minnikin DE, Goodfellow M
JournalZentralbl Bakteriol
PubMed ID8061408
'Sixty representatives of selected Mycobacterium and Nocardia species were examined for their ability to cleave 79 fluorogenic synthetic enzyme substrates based on the fluorophores 7-amino-4-methylcoumarin and 4-methylumbelliferone. The resultant data were analysed using the simple matching coefficient and clustering achieved using the unweighted pair group method with arithmetic averages algorithm. ... More
Single cell analysis and selection of living retrovirus vector-corrected mucopolysaccharidosis VII cells using a fluorescence-activated cell sorting-based assay for mammalian beta-glucuronidase enzymatic activity.
AuthorsLorincz MC, Parente MK, Roederer M, Nolan GP, Diwu Z, Martin DI, Herzenberg LA, Wolfe JH
JournalJ Biol Chem
PubMed ID9872999
'Mutations in the acid beta-glucuronidase gene lead to systemic accumulation of undegraded glycosaminoglycans in lysosomes and ultimately to clinical manifestations of mucopolysaccharidosis VII (Sly disease). Gene transfer by retrovirus vectors into murine mucopolysaccharidosis VII hematopoietic stem cells or fibroblasts ameliorates glycosaminoglycan accumulation in some affected tissues. The efficacy of gene ... More
Fibrinogen induces IL-8 synthesis in human neutrophils stimulated with formyl-methionyl-leucyl-phenylalanine or leukotriene B(4).
AuthorsKuhns DB, Nelson EL, Alvord WG, Gallin JI
JournalJ Immunol
PubMed ID11509634
'Human exudative neutrophils have greatly increased stores of the neutrophil chemoattractant IL-8 compared with peripheral blood cells, but the mechanism for the increase is not defined. In this report, we show that treatment of peripheral blood neutrophils with the chemotactic peptide fMLP or with leukotriene B(4) or fibrinogen results in ... More
Functional interactions of lanthanum and phospholipase D with the abscisic acid signaling effectors VP1 and ABI1-1 in rice protoplasts.
AuthorsGampala SS, Hagenbeek D, Rock CD
JournalJ Biol Chem
PubMed ID11139577
'cis,trans-Abscisic acid (ABA) plays an important role in plant growth and development, regulation of seed maturation, germination, and adaptation to environmental stresses. Knowledge of ABA mechanisms of action and the interactions of components required for ABA signal transduction is far from complete. Using transient gene expression in rice protoplasts, we ... More
Fluorescence histochemical detection of hydrolases in tissue sections and culture cells.
AuthorsGossrau R
JournalHistochemistry
PubMed ID6643137
'Fluorescence and dye histochemical methods are compared for the investigation of hydrolases in sections and culture cells. At present, only some of the synthetic substrates with fluorescent leaving groups may be used for the fluorescence localization of these enzymes in sections. This limitation is due to a reduced fluorescence intensity ... More
Measurement of active phagocytosis by polymorphonuclear leukocytes by fluorescence liberation from phagocytized microspheres.
'Phagocytosis by polymorphonuclear leukocytes (PMN) was determined by a newly developed technique based on measurement of liberation of a fluorescence substance from PMN phagosomes; 4-methylumbelliferyl-beta-D-glucuronide (4MUGL), which is a substrate of beta-glucuronidase in lysosome, was conjugated with a microsphere, and 4-methylumbelliferone (4MU) liberated from phagocytized 4MUGL-microspheres was measured. The microspheres ... More
De novo activation of the beta-phaseolin promoter by phosphatase or protein synthesis inhibitors.
AuthorsLi G, Bishop KJ, Hall TC
JournalJ Biol Chem
PubMed ID11031270
'The promoter for the phaseolin (phas) bean seed protein gene adopts an inactive chromatin structure in leaves of transgenic tobacco. This repressive architecture, which confers stringent spatial regulation, is disrupted upon transcriptional activation during embryogenesis in a process that requires the presence of both a transcription factor (PvALF) and abscisic ... More
Quantitative fluorometric assay for rapid enzymatic characterization of Bifidobacterium longum and related bifidobacteria.
AuthorsO'Brien M, Mitsuoka T
JournalMicrobiol Immunol
PubMed ID1808458
'The quantitative, semi-automated assay described here is an alternative characterization method allowing for highly sensitive and specific detection of bifidobacterial enzymes. Twenty strains of Bifidobacterium longum, including the type strain ATCC 15707, and type strains of 15 other Bifidobacterium species were enzymatically characterized using 20 4-methylumbelliferyl conjugated substrates. Enzyme activities ... More
GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.
AuthorsJefferson RA, Kavanagh TA, Bevan MW
JournalEMBO J
PubMed ID3327686
'We have used the Escherichia coli beta-glucuronidase gene (GUS) as a gene fusion marker for analysis of gene expression in transformed plants. Higher plants tested lack intrinsic beta-glucuronidase activity, thus enhancing the sensitivity with which measurements can be made. We have constructed gene fusions using the cauliflower mosaic virus (CaMV) ... More
Methylumbelliferyl-beta-D-glucuronide-based medium for rapid isolation and identification of Escherichia coli.
AuthorsTrepeta RW, Edberg SC
JournalJ Clin Microbiol
PubMed ID6365956
'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 ... More
Fluorogenic and chromogenic substrates used in bacterial diagnostics.
AuthorsManafi M, Kneifel W, Bascomb S
JournalMicrobiol Rev
PubMed ID1943991
'Methods based on the application of chromogenic and fluorogenic substrates enable specific and rapid detection of a variety of bacterial enzymatic activities. By using these techniques, enzymatic reactions can be examined simultaneously or individually, either directly on the isolation plate or in cell suspensions. For this purpose, various testing principles ... More
Properties of lysosomes in guinea pig heart: subcellular distribution and in vitro stability.
AuthorsWelman E, Peters TJ
JournalJ Mol Cell Cardiol
PubMed ID7679
Spectrofluorometric assay for rapid detection of total and fecal coliforms from surface water.
AuthorsPark SJ, Lee EJ, Lee DH, Lee SH, Kim SJ
JournalAppl Environ Microbiol
PubMed ID7646043
'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 ... More
New toxicity determination method that uses fluorescent assay of Escherichia coli.
AuthorsMariscal A, García A, Carnero M, Gómez E, Fernández-Crehuet J
JournalBiotechniques
PubMed ID8068344
'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 ... More
Methods for Escherichia coli identification in food, water and clinical samples based on beta-glucuronidase detection.
AuthorsFrampton EW, Restaino L
JournalJ Appl Bacteriol
PubMed ID8468256
Arachidonate and other fatty acids mobilize Ca2+ ions and stimulate beta-glucuronidase release in a Ca(2+)-dependent fashion from undifferentiated HL-60 cells.
AuthorsPackham DE, Jiang L, Conigrave AD
JournalCell Calcium
PubMed ID8521454
'Arachidonate (1-300 microM) mobilized Ca2+ ions from an intracellular store and stimulated the entry of Ca2+ ions from the extracellular fluid in undifferentiated HL-60 cells that had been loaded with Fura-2. The integrated response was biphasic in form: arachidonate liberated Ca2+ ions from the intracellular store first, resulting in a ... More
Determination of 4-methylumbelliferone after separation from its conjugates by high-performance liquid chromatography. Application to lysosomal enzyme activity assays.
AuthorsSandman R
JournalJ Chromatogr
PubMed ID6841548
'A high-performance liquid chromatographic method is described for the separation and estimation of 4-methylumbelliferone in the presence of its conjugates. The technique utilizes a simple, isocratic eluent and fluorometric detection. Percentages of 4-methylumbelliferone in the conjugates and fluorescences of the conjugates are reported. 4-Methylumbelliferone, liberated under currently used conditions for ... More
Non-age related differences in thrombin responses by platelets from male patients with advanced Alzheimer's disease.
AuthorsDavies TA, Fine RE, Johnson RJ, Levesque CA, Rathbun WH, Seetoo KF, Smith SJ, Strohmeier G, Volicer L, Delva L
JournalBiochem Biophys Res Commun
PubMed ID8333868
'Alzheimer''s Disease(AD), characterized by a deposition of beta-amyloid peptide (beta/A4) in the brain and in the cerebral microvasculature of affected individuals, is derived from its precursor protein (beta APP) via proteolytic processing by enzyme(s) which have not yet been characterized or localized. Since platelets carry APP in one of their ... More
Mucopolysaccharidosis: secondarily induced abnormal distribution of lysosomal isoenzymes.
AuthorsKint JA, Dacremont G, Carton D, Orye E, Hooft C
JournalScience
PubMed ID4268963
'Total activities of acid hydrolases in liver of two patients with mucopolysaccharidosis are decreased for beta-galactosidase, alpha-galactosidase, and arylsulfatase A; total activities of four other hydrolases are normal or increased. The isoenzyme distribution of five hydrolases (beta-glucuronidase, alpha-glucosidase, beta- galactosidase, N-acetyl-beta-glucosaminidase, and alpha-galactosidase) is ábnormal in that the isoelectric points ... More
Histochemical analysis of CaMV 35S promoter-beta-glucuronidase gene expression in transgenic rice plants.
AuthorsBattraw MJ, Hall TC
JournalPlant Mol Biol
PubMed ID2102372
'The cauliflower mosaic virus promoter is commonly used to drive transcription of chimeric genes in transgenic plants, including the cereals. To determine the tissue and cell types of cereal plants that the promoter functions in, transgenic rice plants containing a CaMV 35S promoter/GUS chimeric gene were analyzed for GUS activity. ... More
Manipulation of beta-glucuronidase for use as a reporter in vacuolar targeting studies.
AuthorsFarrell LB, Beachy RN
JournalPlant Mol Biol
PubMed ID2103475
'It has been documented that when furnished with an endomembrane signal sequence for the endoplasmic reticulum, beta-glucuronidase (GUS) is N-glycosylated, resulting in the nearly complete loss of enzymatic activity. To enable use of beta-glucuronidase as a reporter protein in secretory and vacuolar targeting studies, one of the two putative N-linked ... More
In vitro fusion of endosomes following receptor-mediated endocytosis.
AuthorsDiaz R, Mayorga L, Stahl P
JournalJ Biol Chem
PubMed ID3360775
'Receptor-mediated endocytosis and receptor recycling involve a series of intracellular membrane fusion events that appear to play an important role in the regulation of the overall rate and efficiency of the process. An endosome-endosome fusion assay is described using two ligands that (i) rapidly and efficiently enter the endosomal compartment ... More
Proportion of beta-D-glucuronidase-negative Escherichia coli in human fecal samples.
AuthorsChang GW, Brill J, Lum R
JournalAppl Environ Microbiol
PubMed ID2655534
'Convenient assays and reports that almost all clinical isolates of Escherichia coli produce beta-D-glucuronidase (GUR) have led to great interest in the use of the enzyme for the rapid detection of the bacterium in water, food, and environmental samples. In these materials, E. coli serves as an indicator of possible ... More
Continuous spectrophotometric assay for beta-glucuronidase.
AuthorsAich S, Delbaere LT, Chen R
JournalBiotechniques
PubMed ID11314267
'A continuous spectrophotometric assay has been developed for detecting beta-glucuronidase activity. In the assay, Para-nitrophenyl beta-D-glucuronide is cleaved to yield a chromophoric product. With the commercial E. coli enzyme, it is demonstrated that the reactions can be continuously monitored by the increase of absorbance at 405 nm. The method is ... More
AuthorsLorincz M, Roederer M, Diwu Z, Herzenberg LA, Nolan GP
JournalCytometry
PubMed ID8866216
'We report the development of a new fluorescence-activated cell sorter (FACS)-based reporter gene system utilizing the enzymatic activity of the E. coli beta-glucuronidase (gus) gene. When loaded with the Gus substrate fluorescein-di-beta-D-glucuronide (FDGlcu), individual mammalian cells expressing and translating gus mRNA liberate sufficient levels of intracellular fluorescein for quantitative analysis ... More
Rapid characterization of periodontal bacterial isolates by using fluorogenic substrate tests.
AuthorsMaiden MF, Tanner A, Macuch PJ
JournalJ Clin Microbiol
PubMed ID8789019
'Eighty-nine species of subgingival bacteria, represented by 121 reference strains and 892 patient isolates, including gram-negative, gram-positive, aerobic, facultatively anaerobic, microaerophilic, and anaerobic species, were characterized with a panel of fluorogenic, 4-methylumbelliferyl-linked substrate tests. Identifications of all patient isolates were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell ... More
Sensitive method for the quantification of beta-glucuronidase activity in human urine using capillary electrophoresis with fluorescence detection.
AuthorsWu X, Loganathan D, Linhardt RJ
JournalJ Chromatogr B Biomed Sci Appl
PubMed ID9653947
'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 ... More
Glycosidases in the nervous system. 3. Separation, purification, and substrate specificities of beta-galactosidases and beta-glucuronidase from brain.
AuthorsJungalwala FB, Robins E
JournalJ Biol Chem
PubMed ID5679961
Plasma arylsulfatase and beta-glucuronidase in acute alcoholism.
AuthorsGeokas MC, Rinderknecht H
JournalClin Chim Acta
PubMed ID4732886
Nondestructive assay for beta-glucuronidase in culture media of plant tissue cultures.
AuthorsGould J
JournalMethods Enzymol
PubMed ID1479907
A quantitative assay for beta-D-glucuronidase (GUS) using microtiter plates.
AuthorsRao AG, Flynn P
JournalBiotechniques
PubMed ID2322451
Glycosidases in the nervous system. I. Assay, some properties, and distribution of beta-galactosidase, beta-glucoronidase, and beta-glucosidase.
AuthorsRobins E, Hirsch HE, Emmons SS
JournalJ Biol Chem
PubMed ID4971343
Acid hydrolases in uncultured amniotic fluid cells.
Authorsden Tandt WR
JournalClin Chim Acta
PubMed ID5056629
Glycosidases in the nervous system. II. Localization of beta-galactosidase, beta-glucuronidase, and beta-glucosidase in individual nerve cell bodies.
AuthorsRobins E, Hirsch HE
JournalJ Biol Chem
PubMed ID4971344
Purification and properties of beta-glucuronidase from human placenta.
AuthorsBrot FE, Bell CE, Sly WS
JournalBiochemistry
PubMed ID413568
Lysosomal enzymes of cultured amniotic fluid cells.
AuthorsButterworth J, Sutherland GR, Broadhead DM, Bain AD
JournalClin Chim Acta
PubMed ID4694473
A rapid method for identifying bacterial enzymes.
AuthorsMaddocks JL, Greenan MJ
JournalJ Clin Pathol
PubMed ID810496
Reporter genes and transient assays for plants.
AuthorsMatthews BF, Saunders JA, Gebhardt JS, Lin JJ, Koehler SM
JournalMethods Mol Biol
PubMed ID8528416
Reporter genes.
AuthorsSuter-Crazzolara C, Klemm M, Reiss B
JournalMethods Cell Biol
PubMed ID8531814
Determination of conjugated glucuronic acid by combining enzymatic hydrolysis with lucigenin chemiluminescence.
AuthorsKlopf LL, Nieman TA
JournalAnal Chem
PubMed ID3985340
Determination of acid hydrolases in human platelets.
AuthorsDangelmaier CA, Holmsen H
JournalAnal Biochem
PubMed ID6247939
The GUS reporter gene system.
AuthorsJefferson RA
JournalNature
PubMed ID2689886
The GUS reporter gene system is already a powerful tool for the assessment of gene activity in transgenic plants. Further developments may lead to routine in vivo analysis and fusion genetics. ... More
Rapid simultaneous assessment of neutrophil superoxide generation and lysosomal enzyme release.
AuthorsMandell BF, Ohliger D, Rella J
JournalJ Immunol Methods
PubMed ID3036950
A rapid method for the simultaneous measurement of neutrophil superoxide generation and beta-glucuronidase release is described. Assay of beta-glucuronidase using a fluorescent substrate is shown to be valid in the presence of reduced or unreduced ferricytochrome C, a prerequisite for the simultaneous assessment of this enzyme activity and O-2 generation. ... More
Simplified procedure for transient transformation of plant protoplasts using polyethylene glycol treatment.
AuthorsLyznik LA, Peng JY, Hodges TK
JournalBiotechniques
PubMed ID2064765
A modification of the polyethylene glycol-mediated transformation procedure which eliminates the manual polyethylene glycol dilution step is presented. A transformation mixture of protoplasts, DNA and polyethylene glycol was plated directly onto agarose blocks after incubation. The procedure was simple and fast, thereby suitable for screening the gene activity of large ... More
Study of influence of sex and age on human serum lysosomal enzymes by using 4-methylumbelliferyl substrates.
AuthorsAnnunziata P, Dimatteo G
JournalClin Chim Acta
PubMed ID719894
The sex and age dependence of activity of eight glycosidases and acid phosphatase was assayed in serum samples using the 4-methylumbelliferyl substrates. The activity of these enzymes does not change in relation to sex and to age except for acid phosphatase and beta-galactosidase which show significantly higher values in children ... More
Detection of coliform bacteria and Escherichia coli by multiplex polymerase chain reaction: comparison with defined substrate and plating methods for water quality monitoring.
AuthorsBej AK, McCarty SC, Atlas RM
JournalAppl Environ Microbiol
PubMed ID1768116
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 ... More
High-performance liquid chromatographic quantification of 4-methylumbelliferyl-beta-D-glucuronide as a probe for human beta-glucuronidase activity in tissue homogenates.
AuthorsSperker B, Schick M, Kroemer HK
JournalJ Chromatogr B Biomed Appl
PubMed ID8930768
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 ... More
Evaluation of a fluorogenic assay for detection of Escherichia coli in foods.
AuthorsRobison BJ
JournalAppl Environ Microbiol
PubMed ID6385845
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 ... More
An evaluation of the use of 4-methylumbelliferyl-beta-D-glucuronide (MUG) in different solid media for the detection and enumeration of Escherichia coli in foods.
AuthorsVillari P, Iannuzzo M, Torre I
JournalLett Appl Microbiol
PubMed ID9134776
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). ... More
Fluorogenic assay for rapid detection of Escherichia coli in chilled and frozen foods: collaborative study.
AuthorsMoberg LJ, Wagner MK, Kellen LA
JournalJ Assoc Off Anal Chem
PubMed ID3292512
A collaborative study was conducted to compare a proposed LST-MUG method with the AOAC official method for Escherichia coli detection. E. coli produces an enzyme, beta-glucuronidase, which cleaves the substrate, 4-methyl-umbelliferyl-beta-D-glucuronide (MUG), to yield a fluorescent end product. Incorporation of the MUG substrate into lauryl tryptose broth (LST) enables a ... More
Detection of Escherichia coli by the nutrient agar plus 4-methylumbelliferyl beta-D-glucuronide (MUG) membrane filter method.
AuthorsShadix LC, Dunnigan ME, Rice EW
JournalCan J Microbiol
PubMed ID8306208
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 ... More
Glycosaminoglycan storage in cultured neonatal murine mucopolysaccharidosis type VII neuroglial cells and correction by beta-glucuronidase gene transfer.
AuthorsTaylor RM, Wolfe JH
JournalJ Neurochem
PubMed ID9109535
The inherited deficiency of beta-glucuronidase activity causes the lysosomal storage disorder mucopolysaccharidosis (MPS) type VII (Sly disease). The sequential catabolism of glycosaminoglycans in lysosomes is blocked, and undegraded substrates accumulate in cells of many tissues, including neurons and glia in the brain. To evaluate the deficient metabolic pathway, primary cultures ... More
Rapid identification of Enterobacteriaceae with microbial enzyme activity profiles.
AuthorsGodsey JH, Matteo MR, Shen D, Tolman G, Gohlke JR
JournalJ Clin Microbiol
PubMed ID7016897
A total of 539 clinical isolates belonging to 10 species of the Enterobacteriaceae family were identified by enzyme activity profiles within 30 min of test inoculation. Each isolate was grown at 37 degrees C for 18 h on Mueller-Hinton agar and suspended to an optical density of 200 Klett units ... More
Intracellular thiols regulate activation of nuclear factor kappa B and transcription of human immunodeficiency virus.
AuthorsStaal FJ, Roederer M, Herzenberg LA, Herzenberg LA
JournalProc Natl Acad Sci U S A
PubMed ID2263644
The activation of nuclear factor kappa B (NF-kappa B) has been implicated in the regulation of transcription of a variety of genes and has been shown to be essential for the expression of genes controlled by the long terminal repeat of human immunodeficiency virus (HIV LTR). We show here that ... More
Calmodulin regulates endosome fusion.
AuthorsColombo MI, Beron W, Stahl PD
JournalJ Biol Chem
PubMed ID9065429
Calmodulin (CaM) has previously been implicated in regulated exocytosis, transcytosis, and receptor recycling. We have investigated the role of CaM in endocytic transport by examining the effects of several CaM antagonists in intact cells. We present evidence indicating that the mixing of sequentially internalized ligands is inhibited by CaM antagonists, ... More
Fluorogenic assay for rapid detection of Escherichia coli in food.
AuthorsMoberg LJ
JournalAppl Environ Microbiol
PubMed ID3911904
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 ... More
A fluorogenic substrate for beta-glucuronidase: applications in fluorometric, polyacrylamide gel and histochemical assays.
AuthorsZhou M, Upson RH, Diwu Z, Haugland RP
JournalJ Biochem Biophys Methods
PubMed ID9029263
We have developed a fluorogenic substrate, ELF-97 beta-D-glucuronide, that provides significant advantages over existing substrates in detecting beta-glucuronidase activity. ELF-97 beta-D-glucuronide allows the detection of enzymatic activity in situ, yielding a hydrolytic product that exhibits maximal fluorescence within the physiological pH range. This substrate yields a hydrolytic product that demonstrates ... More
Release of azurophilic granule contents in fMLP-stimulated neutrophils requires two activation signals, one of which is a rise in cytosolic free Ca2+.
AuthorsNiessen HW, Kuijpers TW, Roos D, Verhoeven AJ
JournalCell Signal
PubMed ID1786209
We have used a continuous spectrofluorimetric method to analyse the role of cytosolic free Ca2+ ([Ca2+]i) in the lysosomal enzyme release from the azurophilic granules in human neutrophils stimulated with f-Met-Leu-Phe (fMLP) in the presence of cytochalasin B. Measurements were performed with the beta-glucuronidase substrate 4-methylumbelliferyl-beta-D-glucuronide. We found that the ... More
Comparative study of commercial 4-methylumbelliferyl-beta-D-glucuronide preparations with the Standard Methods membrane filtration fecal coliform test for the detection of Escherichia coli in water samples.
AuthorsClark DL, Milner BB, Stewart MH, Wolfe RL, Olson BH
JournalAppl Environ Microbiol
PubMed ID1854205
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 ... More
Lumenal location of the microsomal beta-glucuronidase-egasyn complex.
Mouse liver beta-glucuronidase is stabilized within microsomal vesicles by complexation with the accessory protein egasyn. The location of the beta-glucuronidase-egasyn complex and free egasyn within microsomal vesicles was investigated. Surprisingly, it was found that neither the complex nor free egasyn are intrinsic membrane components. Rather, both are either free within ... More
Localized transient expression of GUS in leaf discs following cocultivation with Agrobacterium.
AuthorsJanssen BJ, Gardner RC
JournalPlant Mol Biol
PubMed ID2101312
A chimaeric gene has been constructed that expresses beta-D-glucuronidase (GUS) in transformed plant tissues, but not in bacterial cells. This gene has proved extremely useful for monitoring transformation during the period immediately following gene transfer from Agrobacterium tumefaciens. GUS expression was detectable 2 days after inoculation, peaked at 3-4 days ... More
Targeting a foreign protein to chloroplasts using fusions to the transit peptide of a chlorophyll a/b protein.
AuthorsKavanagh TA, Jefferson RA, Bevan MW
JournalMol Gen Genet
PubMed ID3071742
We have constructed chimaeric genes consisting of sequences encoding the transit peptide and 4, 16, 24, 53 or 126 amino-terminal residues of the mature chlorophyll a/b binding (Cab) apoprotein fused to the Escherichia coli gene encoding beta-glucuronidase (GUS). These genes were introduced into tobacco plants and the fate of the ... More
Deglucuronidation of a flavonoid, luteolin monoglucuronide, during inflammation.
AuthorsShimoi K, Saka N, Nozawa R, Sato M, Amano I, Nakayama T, Kinae N
JournalDrug Metab Dispos
PubMed ID11717168
In this study, we investigated whether luteolin monoglucuronide was converted to free aglycone during inflammation using human neutrophils stimulated with ionomycin/cytochalasin B and rats treated with lipopolysaccharide (LPS). beta-Glucuronidase activity was assayed using 4-methylumbelliferyl-glucuronide and methanol extracts of rat plasma containing luteolin monoglucuronide. The released 4-methylumbelliferone, a fluorescent molecule, was ... More
Immunoliposome sandwich assay for the detection of Escherichia coli O157:H7.
AuthorsPark S, Durst RA
JournalAnal Biochem
PubMed ID10805533
We describe the development of a field-portable colorimetric immunoassay for the detection of Escherichia coli O157:H7, using antibody-directed liposomes (immunoliposomes) encapsulating dye as an analytical reagent. Antibodies (anti-E. coli O157:H7) thiolated by 2-iminothiolane were coupled to malemide-tagged liposomes encapsulating the marker dye, sulforhodamine B. Transmission electron microscopy showed that the ... More
The role of glycosylation and phosphorylation in the expression of active human beta-glucuronidase.
AuthorsShipley JM, Grubb JH, Sly WS
JournalJ Biol Chem
PubMed ID8505339
Phosphorylation of mannose residues on N-linked oligosaccharide side chains of lysosomal enzymes targets them to lysosomes. We used site-directed mutagenesis to observe the effect of eliminating selective glycosylation sites from human beta-glucuronidase on enzyme sorting. Expression studies allowed us to determine which of four potential sites were glycosylated, preferentially phosphorylated, ... More
Enzyme capture assay for rapid identification of Escherichia coli in blood cultures.
AuthorsHuang SW, Wu JJ, Chang TC
JournalJ Clin Microbiol
PubMed ID8077387
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 ... More
Coagglutination and enzyme capture tests for detection of Escherichia coli beta-galactosidase, beta-glucuronidase, and glutamate decarboxylase.
AuthorsKaspar CW, Hartman PA, Benson AK
JournalAppl Environ Microbiol
PubMed ID3111364
Polyclonal antibodies to Escherichia coli beta-galactosidase, beta-glucuronidase, and glutamate decarboxylase were used in coagglutination tests for identification of these three enzymes in cell lysates. Enzyme capture assays were also developed for the detection of E. coli beta-galactosidase and beta-glucuronidase. The enzymes were released by using a gentle lysis procedure that ... More
Isolation and characterization of recombinant human cathepsin E expressed in Chinese hamster ovary cells.
AuthorsTsukuba T, Hori H, Azuma T, Takahashi T, Taggart RT, Akamine A, Ezaki M, Nakanishi H, Sakai H, Yamamoto K
JournalJ Biol Chem
PubMed ID8463263
The cDNA sequence encoding precursor forms of human cathepsin E (CE), an intracellular aspartic proteinase, was expressed in Chinese hamster ovary cells using an SV40 promotor-driven expression vector. By immunoelectron microscopic studies using an anti-human CE antibody and by Percoll density gradient fractionation, the expressed CE was found to be ... More
Confirmational identification of Escherichia coli, a comparison of genotypic and phenotypic assays for glutamate decarboxylase and beta-D-glucuronidase.
AuthorsMcDaniels AE, Rice EW, Reyes AL, Johnson CH, Haugland RA, Stelma GN
JournalAppl Environ Microbiol
PubMed ID8795225
Genotypic and phenotypic assays for glutamate decarboxylase (GAD) and beta-D-glucuronidase (GUD) were compared for their abilities to detect various strains of Escherichia coli and to discriminate among other bacterial species. Test strains included nonpathogenic E. coli, three major groups of diarrheagenic E. coli, three other non-coli Escherichia species, and various ... More
Fluorogenic assays for immediate confirmation of Escherichia coli.
AuthorsFeng PC, Hartman PA
JournalAppl Environ Microbiol
PubMed ID7049088
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 ... More
New medium for the simultaneous detection of total coliforms and Escherichia coli in water.
A new membrane filter agar medium (MI agar) containing a chromogen, indoxyl-beta-D-glucuronide, and a fluorogen, 4-methylumbelliferyl-beta-D-galactopyranoside, was developed to simultaneously detect and enumerate Escherichia coli and total coliforms (TC) in water samples on the basis of their enzyme activities. TC produced beta-galactosidase, which cleaved 4-methylumbelliferyl-beta-D-galactopyranoside to form 4-methylumbelliferone, a compound ... More
Critical elements of oligosaccharide acceptor substrates for the Pasteurella multocida hyaluronan synthase.
AuthorsWilliams KJ, Halkes KM, Kamerling JP, DeAngelis PL
JournalJ Biol Chem
PubMed ID16361253
Three-dimensional structures are not available for polysaccharide synthases and only minimal information on the molecular basis for catalysis is known. The Pasteurella multocida hyaluronan synthase (PmHAS) catalyzes the polymerization of the alternating beta1,3-N-acetylglucosamine-beta1,4-glucuronic acid sugar chain by the sequential addition of single monosaccharides to the non-reducing terminus. Therefore, PmHAS possesses ... More