hymecromone and 4-methylumbelliferyl-galactopyranoside

Xenoestrogens exert antiandrogenic effects on the human androgen receptor. In the analytical field, such antagonists block the detection of testosterone and falsify results obtained by sum parameter assays. Currently, such agonistic versus antagonistic effects are not differentiated in complex mixtures. Oppositely acting hormonal effects present in products of everyday use can only be differentiated after tedious fractionation and isolation of the individual compounds along with subjection of each fraction/compound to the status quo bioassay testing. However, such long-lasting procedures are not suited for routine. Hence, we developed a fast bioanalytical tool that figures out agonists versus antagonists directly in complex mixtures. Exemplarily, 8 cosmetics and 15 thermal papers were analyzed. The determined antagonistic potentials of active compounds found were comparable to the ones of known antagonists (in reference shown for bisphenol A, 4-n-nonylphenol and four parabens). Relevant biological/chromatographic parameters such as cell viability, culture conditions, dose response curves, limits of biological detection/quantification and working range (shown for testosterone, dihydrotestosterone, nandrolone and trenbolone) were investigated to obtain the best sensitivity of the biological detection. The developed and validated method was newly termed reversed phase high-performance thin-layer chromatography planar yeast ant-/agonistic androgen screen (RP-HPTLC-pYAAS bioassay). Results were also compared with the RP-HPTLC-Aliivibrio fischeri bioassay (applied on RP plates for the first time). As proof-of-concept, the transfer to another bioassay (RP-HPTLC-pYES) was successfully demonstrated, analogously termed RP-HPTLC-pYAES bioassay detecting anti-/estrogens (exemplarily shown for evaluation of 4 pharmaceuticals used in breast cancer treatment). The new imaging concept provides (1) detection and differentiation of individual agonistic versus antagonistic effects in the bioprofiles, (2) bioanalytical quantification of their activity potential by scanning densitometry and (3) characterization of unknown bioactive compound zones by hyphenation to high-resolution mass spectrometry. Depending on the hormonal bioassay, 15 samples were analyzed in parallel within 5 h or 6 h (calculated as 20 or 24 min per sample). For the first time, piezoelectric spraying of the yeast cells was successfully demonstrated for the planar yeast-based bioassays.

Topics: Aliivibrio fischeri; Androgen Receptor Antagonists; Androgens; Anti-Bacterial Agents; Benzhydryl Compounds; beta-Galactosidase; Biological Assay; Chromatography, Reverse-Phase; Chromatography, Thin Layer; Cosmetics; Endocrine Disruptors; Fluorescent Dyes; Galactosides; Humans; Hymecromone; Limit of Detection; Paper; Phenols; Proof of Concept Study; Receptors, Androgen; Saccharomyces cerevisiae

Reactive oxygen or nitrogen species are generated in the plant cell during the extreme stress condition, which produces toxic compounds after reacting with the organic molecules. The glutathione-S-transferase (GST) enzymes play a significant role to detoxify these toxins and help in excretion or sequestration of them. In the present study, we have cloned 1023 bp long promoter region of tau class GST from an extreme halophyte Salicornia brachiata and functionally characterized using the transgenic approach in tobacco. Computational analysis revealed the presence of abiotic stress responsive cis-elements like ABRE, MYB, MYC, GATA, GT1 etc., phytohormones, pathogen and wound responsive motifs. Three 5'-deletion constructs of 730 (GP2), 509 (GP3) and 348 bp (GP4) were made from 1023 (GP1) promoter fragment and used for tobacco transformation. The single event transgenic plants showed notable GUS reporter protein expression in the leaf tissues of control as well as treated plants. The expression level of the GUS gradually decreases from GP1 to GP4 in leaf tissues, whereas the highest level of expression was detected with the GP2 construct in root and stem under control condition. The GUS expression was found higher in leaves and stems of salinity or osmotic stress treated transgenic plants than that of the control plants, but, lower in roots. An efficient expression level of GUS in transgenic plants suggests that this promoter can be used for both constitutive as well as stress inducible expression of gene(s). And this property, make it as a potential candidate to be used as an alternative promoter for crop genetic engineering.

Topics: Base Sequence; Blotting, Southern; Chenopodiaceae; Cloning, Molecular; Computer Simulation; Galactosides; Genes, Plant; Genetic Vectors; Glucuronidase; Glutathione Transferase; Hymecromone; Molecular Sequence Data; Nicotiana; Nucleotide Motifs; Osmotic Pressure; Plants, Genetically Modified; Promoter Regions, Genetic; Salinity; Sequence Deletion; Sodium Chloride; Stress, Physiological

Gaucher disease (GD), caused by a defect of acid β-glucosidase (β-Glu), is one of the most common sphingolipidoses. Recently, ambroxol, an FDA-approved drug used to treat airway mucus hypersecretion and hyaline membrane disease in newborns, was identified as a chemical chaperone for GD. In the present study, we investigated the chaperone activity and toxicity of ambroxol on both cultured GD patient cells and normal mice. We found that ambroxol treatment significantly increased N370S, F213I, N188S/G193W and R120W mutant β-Glu activities in GD fibroblasts with low cytotoxicity. Additionally, we measured the β-Glu activity in the tissues of normal mice which received water containing increasing concentrations of ambroxol ad libitum for one week. No serious adverse effect was observed during this experiment. Ambroxol significantly increased the β-Glu activity in the spleen, heart and cerebellum of the mice. This result showed its oral availability and wide distribution and chaperone activity in the tissues, including the brain, and its lack of acute toxicity. These characteristics of ambroxol would make it a potential therapeutic chaperone in the treatment of GD with neurological manifestations.

Topics: Ambroxol; Animals; beta-Glucosidase; Body Weight; Cells, Cultured; Colorimetry; Dose-Response Relationship, Drug; Drinking; Expectorants; Fibroblasts; Fluorescent Dyes; Galactosides; Gaucher Disease; Gene Expression Regulation, Enzymologic; Humans; Hymecromone; Mice; Mice, Inbred C57BL; Molecular Chaperones; Mutation; Skin; Time Factors

A modified alpha-N-acetylgalactosaminidase (NAGA) with alpha-galactosidase A (GLA)-like substrate specificity was designed on the basis of structural studies and was produced in Chinese hamster ovary cells. The enzyme acquired the ability to catalyze the degradation of 4-methylumbelliferyl-alpha-D-galactopyranoside. It retained the original NAGA's stability in plasma and N-glycans containing many mannose 6-phosphate (M6P) residues, which are advantageous for uptake by cells via M6P receptors. There was no immunological cross-reactivity between the modified NAGA and GLA, and the modified NAGA did not react to serum from a patient with Fabry disease recurrently treated with a recombinant GLA. The enzyme cleaved globotriaosylceramide (Gb3) accumulated in cultured fibroblasts from a patient with Fabry disease. Furthermore, like recombinant GLA proteins presently used for enzyme replacement therapy (ERT) for Fabry disease, the enzyme intravenously injected into Fabry model mice prevented Gb3 storage in the liver, kidneys, and heart and improved the pathological changes in these organs. Because this modified NAGA is hardly expected to cause an allergic reaction in Fabry disease patients, it is highly promising as a new and safe enzyme for ERT for Fabry disease.

Topics: alpha-N-Acetylgalactosaminidase; Amino Acid Substitution; Animals; Binding Sites; Catalysis; Cells, Cultured; CHO Cells; Cricetinae; Cricetulus; Culture Media, Conditioned; Disease Models, Animal; DNA, Complementary; Drug Stability; Enzyme Replacement Therapy; Fabry Disease; Fibroblasts; Fluorescent Dyes; Galactosides; Genetic Vectors; Humans; Hydrogen-Ion Concentration; Hymecromone; Immunohistochemistry; Kidney; Liver; Mice; Mice, Knockout; Models, Molecular; Molecular Weight; Myocardium; Recombinant Proteins; Retroviridae; Transfection; Trihexosylceramides

An enzyme-linked immunosorbent assay (ELISA) for prostate specific antigen (PSA) detection in human serum was developed based on the potentiometric detection of 6,8-difluoro-4-methylumbelliferone (DiFMU). The assays were carried out in anti-human PSA capture antibody modified microtiter plates (150 microL volume). After incubation in the PSA-containing serum samples, beta-galactosidase-labeled PSA tracer antibody was added. The beta-galactosidase label catalyzed the hydrolysis of 6,8-difluoro-4-methylumbelliferyl-beta-D-galactopyranoside (DiFMUG) and the resulting DiFMU(-) anion was detected by potentiometric microelectrodes with anion-exchanger membrane. The selectivity of the anion-exchanger electrode is governed by the lipophilicity of the anions in the sample. Since DiFMU(-) is much more lipophilic (log P = 1.83) than any of the inorganic anions normally present in the working buffers and occurs in its anionic form at the physiological pH (pK(a) = 4.19), it was chosen as the species to be detected. The potentiometric ELISA-based method detects PSA in serum with a linear concentration range of 0.1-50 ng/mL. These results confirm the applicability of potentiometric detection in diagnostic PSA assays. Owing to simple methodology and low cost, potentiometric immunoassays seem to offer a feasible alternative to the development of in vitro diagnostic platforms.

Topics: Anions; beta-Galactosidase; Enzyme-Linked Immunosorbent Assay; Galactosides; Humans; Hymecromone; Immunoassay; Male; Potentiometry; Prostate-Specific Antigen

A coliforms monitoring system in treated effluent of a wastewater treatment plant has been developed. In order to achieve rapid monitoring within 1 hour, an enzymatic fluorescence method without a culturing process was introduced to this system. It converts the increase rate of fluorescence intensity as enzymatic activity into the number of coliforms instead of converting fluorescence intensity itself. A flow injection analysis is used in this system for automatic measurement. Moreover, it is equipped with the pre-filtering unit to remove the interfering substances in the suspended solids causing deterioration in measurement precision. The good relationship (correlation coefficient of 0.90) between the obtained values using this system and the analysed values using the conventional direct counting method was observed in a test at an existing wastewater treatment plant.

Topics: beta-Galactosidase; Colony Count, Microbial; Enterobacteriaceae; Environmental Monitoring; Escherichia coli; Fluorescence; Galactosides; Hymecromone; Reproducibility of Results; Waste Disposal, Fluid

Trans-sialidase (TS; E.C. 3.2.1.18) catalyzes the transfer of preferably alpha2,3-linked sialic acid to another glycan or glycoconjugate, forming a new alpha2,3-linkage to galactose or N-acetylgalactosamine. In the absence of an appropriate acceptor, TS acts as a sialidase, hydrolytically releasing glycosidically linked sialic acid. Interest in TS has increased rapidly in recent years owing to its great relevance to the pathogenicity of trypanosomes and its possible application in the regiospecific synthesis of sialylated carbohydrates and glycoconjugates. Recently, the authors described a newly developed nonradioactive screening test for monitoring TS activity (1). In this highly sensitive and specific assay, 4-methylumbelliferyl-beta-D-galactoside is used as acceptor substrate and sialyllactose as donor to fluorimetrically detect enzyme activity in the low mU range (approximately 0.1-1 mU/mL possible). The test can be applied to screen a large number of samples quickly and reliably during enzyme purification, for testing inhibitors, and for monitoring TS activity during the production of monoclonal antibodies (2). This chapter focuses on the main steps of this assay and gives detailed instructions for performing a nonradioactive TS 96-well-plate fluorescence test. In addition, it describes the controls necessary when starting to monitor an unknown TS and facts to be considered when testing new substrates and inhibitors.

Topics: Animals; Biological Assay; Calibration; Enzyme Inhibitors; Fluorescence; Galactosides; Glycoproteins; Hymecromone; Neuraminidase; Sensitivity and Specificity; Trypanosoma congolense; Trypanosoma cruzi

Senescence-associated beta-galactosidase activity is a widely used biomarker for assessing replicative senescence in mammalian cells. This enzymatic activity has generally been measured by staining cells with the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside (X-gal) at pH 6.0, a reaction condition that suppresses lysosomal beta-galactosidase activity sufficiently to ensure that most nonsenescent cells will appear unstained. This article describes a quantitative method for measuring this activity and characterizes the method using extracts from senescent, quiescent, and presenescent human fibroblasts. The assay is capable of detecting relatively subtle changes in activity and confirms previous indications based on staining that confluency and contact inhibition of growth can cause a small increase in the expression of this biomarker. Investigation of the pH dependence of the activity in the cell extracts suggests that the senescent phenotype is correlated with an increase in total beta-galactosidase rather than with a shift in the pH optimum of the enzyme. This assay for measuring senescence-associated changes in beta-galactosidase is suitable for mechanistic studies of senescence regulation in which graduated changes in biomarker expression may be anticipated.

Topics: Aging; Animals; beta-Galactosidase; Biomarkers; Cell Count; Cell Extracts; Cells, Cultured; Cellular Senescence; Fibroblasts; Galactosides; Histocytochemistry; Humans; Hydrogen-Ion Concentration; Hymecromone; Mammals; Time Factors

Globoid cell leukodystrophy is an inherited metabolic disorder of the central nervous system caused by deficiency of the lysosomal enzyme galactocerebrosidase. Haematopoietic stem cell transplantation is the only available effective treatment. The engraftment from normal donors provides competent cells able to correct the metabolic defect. Umbilical cord blood cells have proved to significantly decrease complications and improve engraftment rate compared to adult marrow cells in haematopoietic stem cell transplantation. Umbilical cord blood cells must be of sufficient activity to provide central nervous system recovery after engraftment is obtained. Galactocerebrosidase activity is known to be affected by two polymorphic alleles found at nucleotides 502 and 1637 of the cDNA for this gene. This enzyme activity and the polymorphic alleles noted above were analysed in 83 random samples of umbilical cord blood. The activity, assayed with the fluorogenic substrate 6-hexadecanoylamino-4-methylumbelliferyl-beta-galactopyranoside, in those with neither polymorphic allele was 4.6 +/- 1.7 units (nmol/h per mg protein). This optimal choice of cord blood was found in only 24% of specimens. Homozygotes for 1637T > C with activity of only 1.5 +/- 0.4 units represented 16% of the samples. Those heterozygous for 1637T > C with slightly better activity (2.3 +/- 0.7 units) represented 52% of the samples. Choice of umbilical cord blood for haematopoietic stem cell transplantation, therefore, requires consideration not only of cell quantity and HLA compatibility but also selection for normal alleles to obtain maximal enzymatic activity for central nervous system correction.

Topics: Alleles; Central Nervous System; Cryopreservation; DNA, Complementary; Fetal Blood; Galactosides; Galactosylceramidase; Hematopoietic Stem Cell Transplantation; Heterozygote; HLA Antigens; Homozygote; Humans; Hymecromone; Leukodystrophy, Globoid Cell; Lysosomes; Mutation; Polymorphism, Genetic

Trypanosomes do not inhabit or grow in anopheles mosquitoes, the vector for the transmission of Plasmodium parasites the causative agent for malaria. The possession of lytic factors by the anopheline mosquito was thus considered. Head and midgut sections prepared in phosphate buffered saline were tested for trypanocidal action against T. congolense. While the head section was inactive towards the trypanosomes, the midgut extract at 0.2 mg ml(-1) diminished the motility of the parasites within 2 min of incubation; killing 50% of the population after 5 min. At 0.5 mg ml(-1) of the extract, about 90% of the parasites were killed within 2 min of incubation. The midgut fraction was subjected to a purification protocol involving successive chromatography on: octyl-sepharose, reactive brown agarose and fetuin-agarose columns. A final trypanocidal active fraction (gp45), which moved homogeneously during electrophoresis as a 45-kDa protein, was recovered from the fetuin-agarose column. The protein reacted positively with thiobarbituric acid, which suggests it is a sialoglycoprotein. Desialylation of the glycoprotein nullified its trypanocidal activity on T. congolense. Similarly, when the saccharides, lactose, methyl-beta-galactoside, lactulose, methyl-umbelliferyl-beta-galactoside (MU-Gal), were included in the culture medium, they inhibited the gp45 trypanocidal activity. Asialo-fetuin and asialo-RBC also inhibited the gp45-induced killing of T. congolense cells. The potential use of anopheline 45 kDa protein in the production of transgenic tsetse flies (Glossina spp.) in the control of trypanosomiasis is discussed.

Topics: Animals; Anopheles; Culicidae; Culture Media; Erythrocytes; Galactosides; Glycoproteins; Hymecromone; Lactose; Lactulose; Malaria; Methylgalactosides; N-Acetylneuraminic Acid; Neuraminidase; Polysaccharides; Thiobarbituric Acid Reactive Substances; Time Factors; Trypanocidal Agents; Trypanosoma

The interaction of different saccharides with the snake gourd (Trichosanthes anguina) seed lectin (SGSL) was investigated by fluorescence spectroscopy. Binding of 4-methylumbelliferyl-beta-D-galactopyranoside (MeUmb beta Gal) to SGSL resulted in a significant increase in the fluorescence emission intensity of the sugar at 376 nm, and this change was used to estimate the association constants for the binding interaction. Interestingly, the increase in emission intensity changed with a change in temperature, increasing from 19.2% at 20 degrees C to 80.2% at 40 degrees C. At 20 degrees C the association constant, K(a), for the MeUmb beta Gal-SGSL interaction was found by fluorescence titration to be 5.8 x 10(4) M(-1). From the temperature dependence of the association constants, the changes in enthalpy (Delta H) and entropy (Delta S) associated with binding of MeUmb beta Gal to SGSL were estimated to be -80.85 kJ.mol(-1) and -184.0 J.mol(-1).K(-1), respectively. Binding of unlabeled sugars was investigated by monitoring the decrease in fluorescence intensity when they were added to a mixture of SGSL and MeUmb beta Gal. The Ka values for different sugars were determined at several temperatures, and Delta H and Delta S were determined from the van't Hoff plots. Enthalpy-entropy compensation was noticed in all cases. The results indicate that saccharide binding to SGSL is enthalpy-driven and the negative contribution from entropy is, in general, quite high.

Topics: Agglutination; Binding, Competitive; Carbohydrates; Cucurbitaceae; Entropy; Galactosides; Hydrogen-Ion Concentration; Hymecromone; Lectins; Ligands; Plant Lectins; Spectrometry, Fluorescence; Spectrophotometry, Atomic; Spectrophotometry, Ultraviolet; Temperature; Thermodynamics

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

Topics: Animals; Chlorophenols; Erythrocytes; Fungicides, Industrial; Galactosides; Hymecromone; Oncorhynchus mykiss; Pyrimidines; Water Pollutants, Chemical

The essential trace element selenium (Se) is involved in the form of selenocysteine at the active site of several prokaryotic and eukaryotic proteins called selenoproteins. These proteins have recently attracted attention particularly in relation to their application to human health and new characteristics of the genetic code. We have recently described a selenium bioassay based on a recombinant DNA construct in which the expression of the lac' Z gene in Escherichia coli is proportionally and specifically driven by UGA-directed selenocysteine incorporation. Here we have further developed this bioassay for more rapid and sensitive detection and measurement of selenium that permits screening of the selenium status on agar plates. Again, the inclusion of selenium into the lac'Z-fusion product is reflected by the level of beta-galactosidase activity, which in turn is reflected by the intensity of fluorescence on agar plates. This fluorescing agent is a 4-methylumbelliferyl moiety which is released through the cleavage by the enzyme of 4-methylumbelliferyl-beta-D-galactoside. The intensity of the fluorescence is easily detected by uv irradiation and photographed by polaroid or video cameras.

Topics: Agar; beta-Galactosidase; Biological Assay; Cloning, Molecular; Escherichia coli; Fluorescent Dyes; Galactosides; Hymecromone; Selenium; Sensitivity and Specificity

A fluorometric assay of lectin binding to yeast cells is reported. The relative amount of biotinylated lectins bound to the yeast cells was estimated by enzyme activity using 4-methylumbelliferyl-beta-D-galactoside as a substrate for the lectin-bound beta-galactosidase through biotin-avidin interaction. Binding properties of 4 mannose-specific and 3 glucose/mannose-specific lectins to 22 different species of yeast cells were studied. The binding reaction of biotinylated lectins to the yeast cells was rapid and became constant within 10 min. Each lectin showed its characteristic binding specificity to each yeast species. The relative fluorescent intensities observed for 4-methylumbelliferone released by the action of bound beta-galactosidase were good indicators for the classification of yeast cells in quantitative base. We found that the yeast cells of the Saccharomyces genus can be classified into three groups, and those of Pichia were grouped into two groups. The present method can examine many samples simultaneously and be completed within 3 h.

Topics: Agglutination; Avidin; beta-Galactosidase; Biotin; Biotinylation; Candida; Concanavalin A; Galactosides; Glucose; Hymecromone; Kinetics; Lectins; Mannose; Membrane Glycoproteins; Pichia; Protein Binding; Saccharomyces; Spectrometry, Fluorescence; Yeasts

Drinking water regulations under the Final Coliform Rule require that total coliform-positive drinking water samples be examined for the presence of Escherichia coli or fecal coliforms. The current U.S. Environmental Protection Agency-approved membrane filter (MF) method for E. coli requires two media, an MF transfer, and a total incubation time of 28 h. A newly developed MF method, the MI agar method, containing indoxyl-beta-D-glucuronide and 4-methylumbelliferyl-beta-D-galactopyranoside for the simultaneous detection of E. coli and total coliforms, respectively, by means of their specific enzyme reactions, was compared with the approved method by the use of wastewater-spiked tap water samples. Overall, weighted analysis of variance (significance level, 0.05) showed that the new medium recoveries of total coliforms and E. coli were significantly higher than those of mEndo agar and nutrient agar plus MUG (4-methylumbelliferyl-beta-D-glucuronide), respectively, and the background counts were significantly lower than those of mEndo agar (< 5%). Generally, the tap water source, overall chlorine level, wastewater source, granular activated carbon treatment of the tap water, and method of grouping data by E. coli count for statistical analysis did not affect the performance of the new medium.

Topics: Agar; Analysis of Variance; Chlorine; Colony Count, Microbial; Enterobacteriaceae; Escherichia coli; Galactosides; Glucuronates; Hymecromone; Indoles; Micropore Filters; United States; United States Environmental Protection Agency; Waste Disposal, Fluid; Water Microbiology; Water Supply

Topics: Animals; Biological Assay; Copper; Copper Sulfate; Culture Media; Daphnia; Fluorescent Dyes; Galactosides; Guidelines as Topic; Hymecromone; Lethal Dose 50; Observer Variation; Reference Standards; Reproduction; Toxicity Tests; United States; United States Environmental Protection Agency

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

A method is described for the detection of Escherichia coli beta-galactosidase-expressing leukemic cells in ex vivo bone marrow samples. 4-Methylumbelliferyl-beta-D-galactopyranoside is used as a substrate in a kinetic assay. D-Galactose is used to suppress endogenous lysosomal beta-galactosidase activity, yielding a sixfold increase in sensitivity. With this assay, the detection limit is one leukemic cell per 10(4) normal bone marrow cells.

Topics: Animals; beta-Galactosidase; Disease Models, Animal; Escherichia coli; Galactose; Galactosides; Genetic Markers; Hydrogen-Ion Concentration; Hymecromone; In Vitro Techniques; Kinetics; Leukemia, Myeloid, Acute; Lysosomes; Rats; Rats, Inbred BN; Sensitivity and Specificity; Substrate Specificity

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

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

The binding of winged bean basic agglutinin (WBA I) to 4-methylumbelliferyl (MeUmb) galactosides was examined by extrinsic fluorescence titration and stopped-flow spectrofluorimetry. Upon binding to WBA I, MeUmb alpha-galactosides show quenching in fluorescence intensity, decrease in UV absorbance with a concomitant blue shift, and decrease in fluorescence excited-state lifetimes. However, their beta-analogues show enhancement in fluorescence intensity, increase in UV absorbance with a red shift, and an increase in fluorescence excited-state lifetimes. This implies that the umbelliferyl groups of alpha- and beta-galactosides experience non-polar and polar microenvironments, respectively, upon binding to WBA I. Replacement of the anomeric hydroxyl group of galactose by 4-methylumbelliferyl moiety increases the affinity of resulting saccharides. Substitution of C-2 hydroxyl of galactose by an acetamido group leads to increased affinity due to a favorable entropy change. This suggests that acetamido group of MeUmb-alpha/beta-GalNAc binds to a relatively non-polar subsite of WBA I. Most interestingly, this substitution also reduces the association rate constants dramatically. Inspection of the activation parameters reveals that the enthalpy of activation is the limiting factor for the differences in the forward rate constants for these saccharides and the entropic contribution to the activation energy is small.

Topics: Acetylgalactosamine; Fabaceae; Fluorescent Dyes; Galactosides; Hymecromone; Kinetics; Lectins; Plant Lectins; Plants, Medicinal; Regression Analysis; Spectrometry, Fluorescence; Thermodynamics

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

The fluorescence of 4-methylumbelliferyl beta-D-galactopyranoside (MeUmbGalp) was quenched in the presence of cold agglutinin, showing that there was binding between MeUmbGalp and cold agglutinin. That binding was saccharide-specific. By using this quenching phenomenon, the association constants (Ka) of the binding of cold agglutinin at different temperatures (10 degrees C and 15 degrees C) to MeUmbGalp and also the number of binding sites were calculated. The Ka values were found to be 2.63 x 10(3) M-1 at 10 degrees C and 1.58 x 10(3) M-1 at 15 degrees C. Though there is a change in Ka values, the number of binding sites was calculated to be six at both temperatures (10 degrees C and 15 degrees C). From the Ka values the thermodynamic parameters (free energy, enthalpy and entropy) of the binding were derived, and analysis of the data indicated that the binding is spontaneous, exothermic and hydrophobic in nature.

Topics: Agglutinins; Binding Sites; Cryoglobulins; Fluorescent Dyes; Galactosides; Glycosides; Hymecromone; Mathematics; Spectrometry, Fluorescence; Thermodynamics; Umbelliferones

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

We have previously established a non-competitive solid-phase enzyme-linked immunosorbent assay (ELISA) specific for interleukin-1 alpha (IL-1 alpha) using a combination of polyclonal antibody as the immobilized antibody, biotinylated monoclonal antibody as the second antibody and avidin-peroxidase. The level of detection of that ELISA was 200-500 pg/ml. In order to improve its sensitivity, we have used streptavidin-beta-D-galactosidase and the fluorogenic substance 4-methylumbelliferyl-D-galactopyranoside as enzyme substrate. With this system IL-1 alpha could be detected at concentrations as low as 10-50 pg/ml, which was about 10-20 times more sensitive than conventional mouse thymocyte co-stimulator assays. Furthermore, the assay system was specific for IL-1 alpha in that neither IL-1 beta nor interleukin-2 (IL-2) interfered.

Topics: Antibodies, Monoclonal; Biological Assay; Biotin; Enzyme-Linked Immunosorbent Assay; Galactosides; Humans; Hymecromone; Interleukin-1; Nitrophenylgalactosides; Recombinant Proteins

Schwann cells synthesize two heparan sulfate proteoglycans, one that is a component of the Schwann cell basement membrane and a smaller one that is an integral component of the Schwann cell plasma membrane. To determine the functions of these molecules, Schwann cell-nerve cell cultures were grown in medium containing a specific inhibitor of proteoglycan biosynthesis, 4-methylumbelliferyl-beta-D-xyloside. Treatment with 1 mM beta-D-xyloside caused a 90% reduction in the accumulation of 35SO4-labeled proteoglycans in the cell layer of the cultures. Gel filtration analysis revealed that both the basement membrane and plasma membrane proteoglycans were affected. Inhibition of proteoglycan biosynthesis was accompanied by an inhibition of laminin deposition into extracellular matrix as determined by immunostaining of cultures and by immunoblotting of cell-associated proteins. This occurred even though there was no decrease in the amount of laminin detected in the medium of beta-D-xyloside-treated cultures. Deposition of collagen type IV was similarly affected. In addition, there was no myelin produced in beta-D-xyloside treated cultures. However, when beta-xyloside-treated cultures were supplied with exogenous basement membrane, Schwann cells produced numerous myelin segments. These results indicate that Schwann cell proteoglycans play an essential role in basement membrane assembly, and that the integral plasma membrane proteoglycan is not required for the basement membrane to exert its effects on Schwann cell differentiation.

Topics: Animals; Cell Differentiation; Cells, Cultured; Fluorescent Antibody Technique; Galactosides; Ganglia, Spinal; Glycosides; Hymecromone; Laminin; Methylglycosides; Myelin Sheath; Proteoglycans; Rats; Schwann Cells; Umbelliferones

To enable shorter and more convenient testing, the Phadezym RAST and Phadezym IgE PRIST procedures for the determination of specific and total IgE were modified in three ways: (i) allergen-coupled paper discs were tested in microtitre wells; (ii) the incubation times were reduced to 1 hr with serum and 2 hr with the anti-IgE by shaking the plates at room temperature; and (iii) the fluorogenic substrate used reduced the development time to 15 min. Determination of IgE antibody specific for fifteen inhalation allergens by the modified fluorescence test (FEIA) and by the conventional Phadezym RAST (EIA) was performed on the serum of thirty-two patients suffering from asthma/rhinitis: correlation studies for these sera showed that 96.1% of the results fell in the same class. In these patients, both FEIA and EIA detected the same proportion of skin-prick tests (SPT) positive results (67%). With the FEIA, 4/165 (2.4%) class 1 results were found in eleven non-atopic subjects (symptom free, fifteen negative SPT, total IgE lower than 80 kU/l), compared to 1/165 (0.6%) with the EIA. In twenty cord sera, both FEIA and EIA found 4/300 (1.3%) class 1 results. For the determination of total serum IgE, the microtitre FEIA showed a detection limit of 0.5 kU/l and an excellent correlation with Phadezym IgE PRIST (n = 66 serum, r = 0.99). These data indicate that the adaptation of Phadezym RAST and Phadezym IgE PRIST to microtitre plates and fluorescence technology has resulted in a time-saving and easy to perform within-day assay which provided results as reproducible, sensitive and specific as those of the conventional procedure.

Topics: Adolescent; Adult; Asthma; Child; Evaluation Studies as Topic; Female; Fluorescent Dyes; Galactosides; Humans; Hymecromone; Immunoglobulin E; Male; Middle Aged; Radioallergosorbent Test; Radioimmunoassay; Radioimmunosorbent Test; Rhinitis; Skin Tests

A total of 78 strains, representing 21 Vibrio species, were examined by using taurocholate-tellurite-gelatin agar (TTGA) medium and modified TTGA medium containing 4-methylumbelliferyl-beta-D-galactoside (150 micrograms/ml). Modified TTGA medium allowed for simple and direct detection of beta-D-galactosidase (beta-gal) activity. This feature, in conjunction with other differential characteristics of TTGA medium, gave improved differentiation of the vibrios tested. The modified TTGA medium allowed for easy differentiation of Vibrio cholerae (beta-gal+) from Vibrio parahaemolyticus (beta-gal-). The 4-methylumbelliferyl-beta-D-galactoside substrate is inexpensive and very stable. Incorporation into the agar did not affect the performance of TTGA as a differential medium. The assay for beta-gal activity with this substrate was specific and sensitive.

Topics: Agar; beta-Galactosidase; Galactosides; Gelatin; Humans; Hymecromone; Species Specificity; Taurocholic Acid; Tellurium; Vibrio

The conformation and saccharide-binding properties of peanut agglutinin (PNA) depend on pH as studied by analytical ultracentrifugation, fluorescence, circular dichroism, equilibrium dialysis, and absorption spectroscopy. PNA is tetrameric in neutral solution and dissociates reversibly into dimers below pH 5.1. Below pH 3.4, the lectin is totally dimeric. Lowering of the pH induces reversible changes in the tertiary and secondary structures of PNA. Binding of saturating amounts of lactose to tetrameric (pH 6.9) or dimeric (pH 3.2) PNA resulted in identical ultraviolet difference spectra. Fluorescence studies of PNA as a function of pH in the presence of lactose indicated that tryptophanyl residues, present at or near the saccharide binding site, are more accessible to the ligand in dimeric than in tetrameric PNA. For solutions of dimeric PNA, containing only minor amounts of tetramers (pH 3.6), equilibrium dialysis with MeUmb-beta Gal beta(1----3)GalNac showed that the binding capacity of PNA was the same as for tetrameric PNA (one binding site per protomer) but the apparent association constant was one order of magnitude lower than for tetrameric PNA. The enhancement of MeUmb-beta Gal beta(1----3)GalNac fluorescence upon binding to PNA was pH dependent: 50% at neutrality, 16% at pH 3.7, and unobservable at pH 3.0, suggesting that the microenvironment of this PNA-bound chromophore changed progressively with pH and was dependent on ionization of an acidic amino acid residue.

Topics: Carbohydrate Metabolism; Dialysis; Disaccharides; Galactosides; Hydrogen-Ion Concentration; Hymecromone; Lectins; Macromolecular Substances; Mathematics; Peanut Agglutinin; Polymers; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet

Topics: Animals; beta-Galactosidase; Dose-Response Relationship, Radiation; Galactosides; Hematoporphyrins; Hymecromone; L Cells; Lysosomes; Spectrometry, Fluorescence

The properties and distribution of beta-galactosidase were studied in the mouse brain using the artificial substrate methylumbelliferyl-beta-galactoside. Enzyme activities were compared between an audiogenic seizure-susceptible mouse strain (DBA/2) and three non-susceptible strains of mice (BALB/c, C3H/He and Swiss A2G). At all ages, DBA/2 mice have significantly lower beta-galactosidase activity compared with the three other mouse strains: this is attributed to the different alleles present at the Bgs locus. The low activity of beta-galactosidase is also evident when the natural substrate GMl-ganglioside is hydrolyzed. In contrast to this low GMl-ganglioside-beta-galactosidase activity, there is no difference in the activity of the second form of acid beta-galactosidase, galactosylceramidase, in DBA/2 mice at 7 and 14 days. However, at 21 and 28 days the activity is significantly lower in DBA/2 mice compared with the other strains of mice. These results on beta-galactosidase activity in the brain of seizure-susceptible and non-susceptible mice are discussed in relation to published levels of GMl-ganglioside and galactosylceramide present in the developing mouse brain.

Topics: Acoustic Stimulation; Animals; beta-Galactosidase; Disease Susceptibility; Galactosidases; Galactosides; Galactosylceramides; Hymecromone; Lactosylceramides; Mice; Mice, Inbred A; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Inbred DBA; Seizures; Species Specificity; Subcellular Fractions; Substrate Specificity

Microspectrofluorometry of L and WI-38 cells reveals chemical/structural changes due to quiescence or senescence, i.e., lipid peroxidation, spontaneous or photosensitized by hematoporphyrin. Cells treated with hematoporphyrin and a lysosomal umbelliferone probe show a fast-rising umbelliferone emission, plus a fluorescent photoproduct. Studies in rapidly growing versus quiescent L, early passage/late passage WI-38 cells, suggest accumulation of fluorescence Schiff bases (i.e., their association with granular regions of cells in stationary phase, spectral properties, fast increase in photosensitized cells) and a possible lysosomal membrane permeabilization in quiescent or senescent cells.

Topics: Animals; Cell Division; Cell Line; Galactosides; Hematoporphyrins; Hymecromone; Kinetics; L Cells; Mice; Spectrometry, Fluorescence

Of 44 fluorogenic substrates tested for their ability to differentiate species of fecal streptococci, four yielded species-differentiating reactions. The remaining substrates either yielded uniformly positive, negative, or variable strain-dependent reactions. One substrate, 4-methylumbelliferone-alpha-D-galactoside, was hydrolyzed by Streptococcus bovis and S. faecium and its biotypes. 4-Methylumbelliferone-alpha-D-galactoside and a colorimetric starch substrate were incorporated into the fecal streptococcal selective medium of Donnelly and Hartman (Appl. Environ. Microbiol. 35:576-581, 1978). Three phenotypic groups were identifiable on the new fluorescent gentamicin-thallous-carbonate agar: (i) starch hydrolysis and fluorescence (S. bovis), (ii) no starch hydrolysis but fluorescence (S. faecium and its biotypes), and (iii) no starch hydrolysis or fluorescence (S. faecalis, S. avium, S. equinus, S. mitis, and S. salivarius). Of the presumptive identifications from sewage, swine, and bovine samples, 86% were confirmed as being correct. The new medium has potential application in water, food, environmental, and possibly clinical microbiology.

Topics: Animals; Cattle; Culture Media; Feces; Fluorescence; Fluorescent Dyes; Galactosides; Glycosides; Humans; Hymecromone; Sewage; Starch; Streptococcus; Swine; Umbelliferones

Enzyme immunoassay techniques are widely use to quantify various antigens and antibodies. The final step of these techniques (i.e. enzyme reaction) may be carried out in several ways (e.g. chromogenic, fluorogenic, or radioactive substrate and thermometric measurement). This paper compares the effectiveness of the chromogenic and the fluorogenic substrates in the beta-galactosidase immunoassay. Using microtitration plates (150 microliter samples) coated with anti-human IgE, and anti-human IgE labeled with E. coli beta-galactosidase, the lowest concentrations of IgE that one could detect employing either the chromogenic (o-nitrophenyl-beta-D-galactopyranoside) or the fluorogenic (4-methyl-umbelliferyl-beta-D-galactopyranoside) substrate were determined. It was found that both substrates were almost equally effective in measuring the lowest concentration of IgE (0.075-0.13 IU/ml) under the optimal conditions. But, using fluorogenic substrate and suitable apparatuses the enzyme immunoassay can be miniaturized. Thus by using decreasing volumes of reagents, progressively smaller amounts of antigen were quantified: as the sample volumes were reduced from 150 to 10 microliter and finally to 0.3 microliter a progressive decrease from 7 x 107 molecules of IgE to 2.9 x 107 molecules and to 1.5 x 106 molecules was observed. The corresponding lowest detection limits were 0.075 IU/ml, 0.46 IU/ml and 0.8 IU/ml.

Topics: Animals; Antigen-Antibody Reactions; beta-Galactosidase; Cattle; Chromogenic Compounds; Dose-Response Relationship, Drug; Electronics; Fluorescent Dyes; Galactosidases; Galactosides; Glycosides; Humans; Hydrogen-Ion Concentration; Hymecromone; Immunoenzyme Techniques; Miniaturization; Nitrophenylgalactosides; Sheep; Spectrometry, Fluorescence; Umbelliferones

A fluorescent enzyme-linked immunosorbent assay is described for the rapid measurement of serum ferritin. Increased sensitivity was achieved by using 4-methyl-umbelliferyl-beta-D-galactopyranoside as the substrate for beta-galactosidase coupled to the purified antiferritin antibody. Further enhancement of the specific antigen-antibody reaction was attained by the addition of 4% polyethylene glycol 6000 to the antiferritin-beta-galactosidase conjugate. The procedure is performed in microELISA plates. These modifications of the method permit the measurement of serum ferritin at concentrations ranging from 0.25 to 50 microgram/liter with a coefficient of variation of 8% or less. The entire procedure is performed at ambient temperature and is completed within one working day. The cost of the assay is less than 10% of the immunoradiometric assay for serum ferritin.

Topics: Animals; beta-Galactosidase; Enzyme-Linked Immunosorbent Assay; Female; Ferritins; Galactosides; Humans; Hymecromone; Immune Sera; Male; Polyethylene Glycols; Rabbits; Spectrometry, Fluorescence; Spleen; Thalassemia

The binding of 4-methylumbelliferyl galactose and 4-methylumbelliferyl N-acetylgalactosamine to ricin, Ricinus communis agglutinin, and ricin B chain was studied by fluorescence polarization and equilibrium dialysis. The binding of [3H]galactose to ricin was also studied by equilibrium dialysis. The results were consistent with binding of 2 mol of ligand to ricin (which contains 1 A chain and 1 B chain) and ricin B chain and 4 mol of ligand to the agglutinin (which contains 2 A chains and 2 B chains). There was no evidence for interaction between the 2 sites on any of the B chains.

Topics: Carbohydrates; Galactosides; Glycosides; Hymecromone; Kinetics; Lectins; Ligands; Plant Lectins; Plant Proteins; Protein Binding; Ricin; Spectrometry, Fluorescence; Umbelliferones

Topics: Arachis; Galactosides; Glycosides; Hymecromone; Kinetics; Lectins; Peanut Agglutinin; Plant Lectins; Spectrometry, Fluorescence; Structure-Activity Relationship; Umbelliferones

A simple and sensitive fluorometric method has been described for the differential determination of the activity of lysosomal alpha-galactosidase A and alpha-galactosidase B. The procedure employs 4-methylumbelliferyl-alpha-D-galactopyranoside as substrate and N-acetylgalactosamine as an inhibitor of alpha-galactosidase B, but not of alpha-galactosidase A to differentiate the two activities. This method was shown to be applicable in the differentiation of the two enzyme activities in human tissues and in the diagnosis of the heterozygous and hemizygous genotypes for Fabry's disease in cultured skin fibroblasts.

Topics: Acetylgalactosamine; Adult; alpha-Galactosidase; alpha-N-Acetylgalactosaminidase; Cells, Cultured; Fabry Disease; Female; Fibroblasts; Galactosidases; Galactosides; Genetic Carrier Screening; Hexosaminidases; Humans; Hymecromone; Lysosomes; Male

A solid-phase enzyme-linked immunoassay using a fluorogenic substrate (4-methylumbelliferyl-beta-D-galactopyranoside) was developed. Antibodies were covalently linked to glutaraldehyde-activated 96-well aminopolystyrene plates. Antigens from test samples were adsorbed to the solid phase and detected using antibodies conjugated with E. coli beta-galactosidase. Glutaraldehyde, N-succinimidyl-3-(2-pyridyldithio)-propionate or N-succinimidyl-6-(4-azido-2-nitrophenylamino)-hexanoate were used as linkers between antibodies and the enzyme. The measurement of fluorescence can be automated for rapid screening of many specimens. The sensitivity limit of the test for HBsAg is about 5-10 pg.

Topics: Animals; beta-Galactosidase; Enzyme-Linked Immunosorbent Assay; Fluorescence; Galactosides; Hepatitis B Antibodies; Hepatitis B Antigens; Hepatitis B e Antigens; Hepatitis B Surface Antigens; Humans; Hymecromone; Immunoenzyme Techniques; Marmota; Polystyrenes

A sensitive enzyme immunoassay for plasma betamethasone was developed using betamethasone-3-(O-carboxymethyl)oxime-beta-D-galactosidase conjugate as a labelled antigen and 4-methylumbelliferyl-beta-D-galactoside as a fluorescence substrate. The performances of the enzyme immunoassay were compared with that of a radioimmunoassay using 3H-betamethasone and the same antiserum. The minimal detectable level for the enzyme immunoassay was 0.15 pg/tube or 0.15 ng/ml of plasma, which was remarkably more sensitive than the radioimmunoassay level of 10 pg/tube or 2 ng/ml of plasma. The specificity was sufficient, in particular, the cross reactivity of cortisol as 0.008%. However, the precision of the enzyme immunoassay was inferior to that of the radioimmunoassay.

Topics: Betamethasone; Cross Reactions; Galactosides; Humans; Hymecromone; Immunoenzyme Techniques; Microchemistry; Radioimmunoassay; Radioisotope Dilution Technique; Tritium

The binding of 4-methylumbelliferyl beta-D-galactopyranoside (MeUmb-Galp), to Mormordica charantia lectin was studied by equilibrium dialysis and quenching of ligand fluorescence. The fluorescence of MeUmb-Galp decreases as a function of solvent polarity. On binding to M. charantia lectin, its fluorescence was nearly 100% quenched, showing that the binding of the glycoside takes place in hydrophobic environment. The binding of the fluorescent sugar was saccharide-specific as evidenced by reversal of MeUmb-Galp fluorescence quenching by lactose. The association constant is independent of the experimental method used and at 25 degrees C the value is (1.96 +/- 0.05) X 10(4) M-1. The number of binding sites as determined by equilibrium dialysis and fluorescence quenching agree very well with each other; n being equal to 1.98 +/- 0.02. The Ka value for the glycoside was also determined by competition studies employing reversal of fluorescence quenching of MeUmb-Galp by lactose. The value of ka obtained for lactose is 1.21 X 10(4) M-1 at 30 degrees C. The internal consistency of the association constant and number of binding site values at low and high saturation indicates the absence of additional subsite on M. charantia lectin. The thermodynamic parameters do not differ greatly with change in temperature; the values of - delta H degrees and - delta S degrees are equal to 30 +/- 9.63 kJ mol-1 and 21 +/- 0.3 J mol-1 K-1 respectively in the range of 15--35 degrees C indicting that the binding of M. charantia lectin to saccharide is exothermic in nature.

Topics: Binding Sites; Galactosides; Glycosides; Hymecromone; Lectins; Plant Lectins; Plants; Spectrometry, Fluorescence; Umbelliferones

Polarization of ligand fluorescence was used to study the binding of 4-methylumbelliferyl beta-D-galactopyranoside (MeUmb-Galp) to Abrus precatorious agglutinin. The binding of the fluorescent sugar to the lectin led to considerable polarization of the MeUmb-Galp fluorescence, which was also quenched by about 30% on binding to the lectin. The binding of the fluorescent sugar was carbohydrate-specific, as evidenced by inhibition of both fluorescence polarization and quenching when lectin was preincubated with lactose. The association constant as determined by fluorescence polarization is 1.42 x 10(4) M-1 at 25 degrees C and is in excellent agreement with those determined by fluorescence quenching (Ka = 1.51 x 10(4) M-1) and equilibrium dialysis (Ka = 1.62 x 10(4) M-1) at 25 degrees C. The numbers of binding sites as determined by fluorescence polarization, quenching and equilibrium dialysis agree very well with one another, n being equal to 2.0 +/- 0.05. The consistency between the association constant value determined by fluorescence polarization, quenching and equilibrium dialysis shows the validity of this approach to study lectin-sugar interaction.

Topics: Binding Sites; Dialysis; Fluorescence Polarization; Galactosides; Glycosides; Hymecromone; Lectins; Spectrometry, Fluorescence; Umbelliferones

1. A method is described for following continuously the action of beta-galactosidase on 4-methylumbelliferyl beta-D-galactoside at pH 4.5, in which 4-methylumbelliferone production is measured at fluorescence excitation and emission wavelengths of 324 and 444nm respectively. 2. Initial-rate studies show that the presence of salt activates beta-galactosidase up to 100 mM, but is inhibitory above that concentration. The enzyme is very unstable at 37 degrees C and low ionic strength, but stability increases with ionic strength. 3. The stability of the enzyme at 37 degrees C decreases markedly with rising pH in the range 5.9--8.0. 4. Gel-filtration patterns demonstrate that there is a marked tendency to polymerization with increasing ionic strength. The gel-filtration pattern shows decreasing amounts of dimer with increasing pH. 5. The correlation between activity, stability and molecular form of beta-galactosidase is discussed. It is suggested that the dimeric form of the enzyme is the most stable and active form. The implications of this finding for the assay of beta-galactosidase under physiological conditions for prenatal diagnosis are discussed. 6. Evidence for the possible occurrence of a 36 000-mol.wt. from of beta-galactosidase is presented. 7. A computer program for the calculation of initial rates has been deposited as Supplementary Publication SUP 50114 (4 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1981) 193, 5.

Topics: Chromatography, Gel; Galactosidases; Galactosides; Glycosides; Humans; Hymecromone; Kinetics; Liver; Macromolecular Substances; Osmolar Concentration; Spectrometry, Fluorescence; Umbelliferones

Leukocytes were isolated from 14 patients (7 males and 7 females ) with Gaucher disease of the Norrbottnian type (Type 3), 32 obligate heterozygotes (16 males and 16 females) for this disease and 20 controls (10 males and 10 females). After collection, the cells were transported in dry ice to the laboratory, where they were assayed. The assays were repeated after the cells had been stored for 12 months. beta-Glucosidase activity was assayed with D-[glucose-U-14C]glucosylceramide at pH 5.8 with Cutscum-Na-cholate as a detergent and 4-methylumbelliferyl-beta-glucoside at pH 4.1 with Triton-Na-taurocholate as a detergent. The activities of two marker enzymes, 4-methylumbelliferyl-beta-galactosidase and N-acetyl-beta-glucosaminidase, were assayed in aliquots of the same leukocyte samples. The activity of beta-galactosidase remained constant during storage, N-acetyl-beta-glucosaminidase increased, while beta-glucosidase decreased as assayed with the natural as well as with the artificial substrate. beta-Glucosidase activity was significantly lower in the female than in male controls and heterozygotes. When assayed with natural substrate beta-glucosidase activity in leukocytes from the male patients was 6--12% of the control mean value and 10--15% in those from the female patients. The corresponding figures found when the artificial substrate was used were 15--30% and 22--45%. The values for the heterozygotes were respectively 42--68% and 34--79% with the natural substrate, and 33--82% and 51--109% with the artificial substrate. No correlation was found between the age of the patient and the beta-glucosidase activity.

Topics: Acetylglucosamine; Acetylglucosaminidase; Adolescent; Adult; Age Factors; Aged; beta-Galactosidase; beta-Glucosidase; Child; Child, Preschool; Drug Stability; Female; Galactosides; Gaucher Disease; Glucosamine; Glucosidases; Glucosides; Glucosylceramidase; Heterozygote; Homozygote; Humans; Hymecromone; Leukocytes; Male; Middle Aged; Sex Factors

The binding of Ricinus communis (castor-bean) agglutinin 1 to saccharides was studied by equilibrium dialysis and fluorescence polarization by using the fluorescently labelled sugar 4-methylumbelliferyl beta-D-galactopyranoside. No appreciable change in ligand fluorescence of 4-methylumbelliferyl beta-D-galactopyranoside was considerably polarized on its binding to the lectin. The association constants obtained by Scatchard analysis of equilibrium-dialysis and fluorescence-polarization data do not differ much from each other, and at 25 degrees C, Ka = 2.4 (+/- 0.2) X 10(4)M-1. These values agree reasonably well with that reported in the literature for Ricinus agglutinin 1. The number of binding sites obtained by the different experimental procedures is 1.94 +/- 0.1 per molecule of 120 000 daltons and is equal to the reported value of 2. The consistency in the values of Ka and number of binding sites indicate the absence of additional subsites on Ricinus agglutinin 1 for its specific sugars. In addition, the excellent agreement between the binding parameters obtained by equilibrium dialysis and fluorescence polarization indicate the potential of ligand-fluorescence-polarization measurements in the investigation of lectin-sugar interactions.

Topics: Binding Sites; Chemical Phenomena; Chemistry; Fluorescence Polarization; Galactosides; Glycosides; Hymecromone; Kinetics; Lectins; Plant Lectins; Plants, Toxic; Ricinus communis; Umbelliferones