Detection of one attomole of [Arg8]-vasopressin by novel noncompetitive enzyme immunoassay (hetero-two-site complex transfer enzyme immunoassay).
AuthorsHashida S, Tanaka K, Yamamoto N, Uno T, Yamaguchi K, Ishikawa E
JournalJ Biochem (Tokyo)
PubMed ID1778973
'One attomole of [Arg8]-vasopressin (AVP) was detected by a novel noncompetitive enzyme immunoassay (hetero-two-site complex transfer enzyme immunoassay). AVP was indirectly biotinylated using N-hydroxysuccinimidobiotin and trapped onto an anti-AVP IgG-coated polystyrene ball. After washing, biotinylated AVP was eluted from the polystyrene ball with HCl and was reacted with 2,4-dinitrophenyl-fluorescein disulfide-bovine ... More
Activation of cAMP-responsive genes by stimuli that produce long-term facilitation in Aplysia sensory neurons.
AuthorsKaang BK, Kandel ER, Grant SG
JournalNeuron
PubMed ID8384857
'One of the hallmarks of long-term memory in both vertebrates and invertebrates is the requirement for new protein synthesis. In sensitization of the gill-withdrawal reflex in Aplysia, this requirement can be studied on the cellular level. Here, long-term but not short-term facilitation of the monosynaptic connections between the sensory and ... More
Nonisotopic detection of RNA in an enzyme immunoassay using a monoclonal antibody against DNA-RNA hybrids.
AuthorsCoutlee F, Yolken RH, Viscidi RP
JournalAnal Biochem
PubMed ID2479292
'A sensitive nonisotopic solution hybridization assay for detection of RNA is described and characterized using a pSP65 plasmid model system. The assay procedure is based on a hybridization reaction in solution between a biotinylated DNA probe and a target RNA. The biotin-labeled hybrids are captured on a microtiter plate coated ... 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
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
Inhibition of in vitro endosomal vesicle fusion activity by aminoglycoside antibiotics.
AuthorsJones AT, Wessling-Resnick M
JournalJ Biol Chem
PubMed ID9737996
'The effects of two aminoglycoside antibiotics, neomycin and Geneticin, on the endocytic pathway were studied using a cell-free assay that reconstitutes endosome-endosome fusion. Both drugs inhibit the rate and extent of endosome fusion in a dose-dependent manner with IC50 values of approximately 45 microM and approximately 1 mM, respectively. Because ... More
Transcriptional properties of genomic transgene integration sites marked by electroporation or retroviral infection.
AuthorsBaer A, Schübeler D, Bode J
JournalBiochemistry
PubMed ID10852701
'As a possible consequence of their survival strategy, proviruses are predominantly found in transcription-promoting genomic sites. For certain applications, these findings have led to the preferential use of retroviral vectors for the stable integration of transgenes. This study demonstrates that transcription levels of single-copy proviruses, which have been established either ... More
Quantitative polymerase chain reaction by monitoring enzymatic activity of DNA polymerase.
AuthorsYang B, Yolken R, Viscidi R
JournalAnal Biochem
PubMed ID8434779
'Nucleic acid amplification by polymerase chain reaction (PCR) is a very powerful technique in terms of sensitivity but is limited in terms of ability to perform accurate quantitation. While there is a theoretical correlation between copies of input target sequence and those of PCR product, the quantitative nature of this ... More
Human lysosomal beta-galactosidase-cathepsin A complex: definition of the beta-galactosidase-binding interface on cathepsin A.
AuthorsPshezhetsky AV, Elsliger MA, Vinogradova MV, Potier M
JournalBiochemistry
PubMed ID7873522
'Human lysosomal beta-galactosidase is organized as a 680-kDa complex with cathepsin A (also named carboxypeptidase L and protective protein), which is necessary to protect beta-galactosidase from intralysosomal proteolysis. To understand the molecular mechanism of beta-galactosidase protection by cathepsin A, we defined the structural organization of their complex including the beta-galactosidase-binding ... 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
Comparison of Flavobacterium and Sphingobacterium species by enzyme profiles, with use of pattern recognition of two-dimensional fluorescence data.
AuthorsPau CP, Patonay G, Moss CW, Hollis D, Carlone GM, Plikaytis BD, Warner IM
JournalClin Chem
PubMed ID3815801
'Enzyme profiles of eight Flavobacterium species and one Sphingobacterium species were compared after using a two-dimensional fluorescence technique. Enzyme contents and corresponding activities were rapidly determined for whole-cell preparations after incubation with a mixture of preselected fluorogenic substrates. A two-dimensional fluorescence spectrum of the resulting product mixture, measured with a ... More
Establishment of an enzyme release assay for cytotoxic T lymphocyte activity.
AuthorsOhmori H, Takai T, Tanigawa T, Honma Y
JournalJ Immunol Methods
PubMed ID1541835
'In the present report, we describe the establishment of a cell line that can be used as the target for measuring the activity of cytotoxic T lymphocytes (CTL) by an enzyme release assay. We transfected P3/NS1-Ag4-1 (NS-1), a myeloma cell line derived from BALB/c mice with Escherichia coli beta-galactosidase (beta-Gal) ... More
Evaluation of the use of chromogenic and fluorogenic substrates in solid-phase enzyme linked immunosorbent assays (ELISA).
AuthorsCrowther JR, Angarita L, Anderson J
JournalBiologicals
PubMed ID2178352
'Fluorogenic and chromogenic substrates were used in direct and trapping enzyme-linked immunosorbent assays (ELISA) for the detection of mouse IgG and foot-and-mouth disease virus (FMDV). The detection limits for both antigens were compared using different combinations of enzymes and substrates. Various times and concentrations of chemicals were used to obtain ... 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
A fluorescence staining method for the demonstration and measurement of lysosomal enzyme activities in single cells.
AuthorsLuyten GP, Hoogeveen AT, Galjaard H
JournalJ Histochem Cytochem
PubMed ID3926869
'A cytochemical fluorescence method is described that makes possible simple, rapid, and specific demonstration and measurement of the activities of a wide variety of lysosomal enzymes in single cells using 4-methylumbelliferyl derivatives as substrates. The validity of the method and a number of applications using normal and mutant human cells ... More
Properties of lysosomes in guinea pig heart: subcellular distribution and in vitro stability.
AuthorsWelman E, Peters TJ
JournalJ Mol Cell Cardiol
PubMed ID7679
Catalyzed reporter deposition, a novel method of signal amplification. Application to immunoassays.
AuthorsBobrow MN, Harris TD, Shaughnessy KJ, Litt GJ
JournalJ Immunol Methods
PubMed ID2558138
'A novel signal amplification method, catalyzed reporter deposition (CARD), and its application to immunoassays is described. The method involves utilizing an analyte-dependent reporter enzyme (ADRE) to catalyze the deposition of additional reporter on the surface in a solid-phase immunoassay. In the examples described, deposition of reporter is facilitated by using ... More
Fluorogenic substrates for differentiation of gram-negative nonfermentative and oxidase-positive fermentative bacteria.
AuthorsKämpfer P, Kulies I, Dott W
JournalJ Clin Microbiol
PubMed ID1624555
'A total of 803 strains of gram-negative nonfermentative and oxidase-positive fermentative bacteria (38 taxa) were investigated for their ability to hydrolyze 53 different fluorogenic 4-methylumbelliferyl- and beta-naphthylamide-linked substrates within 6 h of incubation. The hydrolysis of 16 fluorogenic substrates showed high separation index values among the tested taxa, was reproducible, ... More
Highly sensitive fluorimetric enzyme immunoassay for prostaglandin H synthase solubilized from cultured cells.
AuthorsRuan KH, Kulmacz RJ, Wilson A, Wu KK
JournalJ Immunol Methods
PubMed ID8509650
'A highly sensitive fluorimetric enzyme immunoassay was developed for prostaglandin H (PGH) synthase, an intrinsic membrane protein, using n-octyl beta-D-glucopyranoside for solubilization of the synthase from the endoplasmic reticulum membrane. An anti-PGH synthase IgG-coated polystyrene ball was reacted with a PGH synthase standard or crude detergent extract of cells, washed ... More
Fluorescence polarization as a tool to study lectin-sugar interaction. An investigation of the binding of 4-methylumbelliferyl beta-D-galactopyranoside to Abrus precatorious agglutinin.
AuthorsKhan MI, Surolia N, Mathew MK, Balaram P, Surolia A
JournalEur J Biochem
PubMed ID7227364
'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 ... More
Enzyme immunoassay techniques. An overview.
AuthorsPorstmann T, Kiessig ST
JournalJ Immunol Methods
PubMed ID1613258
'In spite of the great variety of enzyme immunoassays (EIA) they can be classified into two groups 'analyte-observed' and 'reagent-observed' assays, depending on their reaction principle. The latter are favored by use of monoclonal antibodies and are characterized by a greater sensitivity, a larger measuring range, a lower susceptibility to ... More
A rapid and sensitive ELISA for serum ferritin employing a fluorogenic substrate.
AuthorsKonijn AM, Levy R, Link G, Hershko C
JournalJ Immunol Methods
PubMed ID6816855
'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 ... More
Fluorescence probe studies of the state of tropomyosin in reconstituted muscle thin filaments.
AuthorsIshii Y, Lehrer SS
JournalBiochemistry
PubMed ID3663633
'The monomer fluorescence of N-(1-pyrenyl)maleimide-labeled tropomyosin bound to F-actin (PTm-actin) increases when myosin subfragment 1 (S1) binds to actin and is half complete when only approximately 1 S1 is bound to 7 actin subunits [Ishii, Y., & Lehrer, S. S. (1985) Biochemistry 24, 6631-6638]. Similar studies of the binding of ... 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
Quantification of picogram levels of specific DNA immobilized in microtiter wells.
AuthorsNagata Y, Yokota H, Kosuda O, Yokoo K, Takemura K, Kikuchi T
JournalFEBS Lett
PubMed ID3157598
'A non-radioisotopic and sensitive method for quantification of specific DNA immobilized in microtiter wells has been developed. This method is based upon the immobilization of DNA in microtiter wells and hybridization with biotinylated DNA probe which is followed by complexing with avidin-beta-galactosidase. By measuring fluorescence emitted from the hydrolyzed product ... More
Improved FACS-Gal: flow cytometric analysis and sorting of viable eukaryotic cells expressing reporter gene constructs.
AuthorsFiering SN, Roederer M, Nolan GP, Micklem DR, Parks DR, Herzenberg LA
JournalCytometry
PubMed ID1905992
'The previously reported FACS-Gal assay (Nolan et al., Proc Natl Acad Sci USA 85:2603-2607, 1988) measures E. coli lacZ-encoded beta-galactosidase activity in individual viable eukaryotic cells for a variety of molecular and cellular biological applications. Enzyme activity is measured by flow cytometry, using a fluorogenic substrate, which is hydrolyzed and ... More
A trial to compare the effects of pH, buffer concentration, and NaCl, on one fluorescent and two bioluminescent bacterial tests for acute toxicity.
AuthorsMariscal A, Carnero M, Gómez-Aracena J, Fernández-Crehuet J
JournalMicrobios
PubMed ID9569662
'Of all the bioassays to determine acute toxicity described in the literature, those that employ bacteria as indicator organisms are usually the most rapid and the most economic, although alone they cannot predict the possible toxic effect of any type of substance. When bioassays are employed to test the toxicity ... More
Selective inactivation of eukaryotic beta-galactosidase in assays for inhibitors of HIV-1 TAT using bacterial beta-galactosidase as a reporter enzyme.
AuthorsYoung DC, Kingsley SD, Ryan KA, Dutko FJ
JournalAnal Biochem
PubMed ID8297011
'Bacterial beta-galactosidase is one of several reporter enzymes used in studying the transcriptional activity of eukaryotic promoters. Although it is one of the easiest and least expensive enzymes to assay, its use has been limited because of its low sensitivity, which is due in part to endogenous levels of beta-galactosidase ... More
New algal enzyme bioassay for the rapid assessment of aquatic toxicity.
AuthorsPeterson SM, Stauber JL
JournalBull Environ Contam Toxicol
PubMed ID8661858
Fluorigenic glycosidase substrates: their use in the identification of some slow growing mycobacteria.
AuthorsGrange JM, McIntyre G
JournalJ Appl Bacteriol
PubMed ID541300
Identity of beta-glucosidase, beta-xylosidase and one of the beta-galactosidase activities in human liver when assayed with 4-methylumbelliferyl-beta-D-glycosides studies in cases of Gaucher's disease.
AuthorsOckerman PA
JournalBiochim Biophys Acta
PubMed ID5672844
The use of 4-methylumbelliferyl glycosides in binding studies with the lectins BS I-A4, BS I-B4 and BS II from Bandeiraea (Griffonia) simplicifolia.
Authorsde Boeck H, Loontiens FG, Delmotte FM, de Bruyne CK
JournalFEBS Lett
PubMed ID7238871
Genetic heterogeneity in GM1-gangliosidosis.
AuthorsGaljaard H, Hoogeveen A, de Wit-Verbeek HA, Reuser AJ, Ho MW, Robinson D
JournalNature
PubMed ID808736
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
The fluorimetric estimation of N-acetyl-beta-glucosaminidase and beta-galactosidase in blood plasma.
AuthorsWoollen JW, Walker PG
JournalClin Chim Acta
PubMed ID5880046
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
The abnormalities of lysosomal enzymes in mucopolysacc- haridoses.
AuthorsVan Hoof F, Hers HG
JournalEur J Biochem
PubMed ID4236776
A rapid method for identifying bacterial enzymes.
AuthorsMaddocks JL, Greenan MJ
JournalJ Clin Pathol
PubMed ID810496
A microplate fluorimetric assay for transfection of the beta-galactosidase reporter gene.
AuthorsRakhmanova VA, MacDonald RC
JournalAnal Biochem
PubMed ID9514778
A microspectrofluorometric study of porphyrin-photosensitized single living cells--I. Membrane alterations.
Determination of acid hydrolases in human platelets.
AuthorsDangelmaier CA, Holmsen H
JournalAnal Biochem
PubMed ID6247939
Amplification systems in immunoenzymatic techniques.
AuthorsAvrameas S
JournalJ Immunol Methods
PubMed ID1613256
Fluorescent method for testing the enzymic activity of mycobacteria.
AuthorsSlosárek M
JournalFolia Microbiol (Praha)
PubMed ID6776020
The possibility of utilizing derivatives of 4-methylumbelliferone (4-MUBF) for the determination of activity of enzymes from the group of hydrolyses was tested in 20 strains of 12 species of mycobacteria. The method proved to be suitable especially for determination of arylsulphatase, beta-D-galactose and acid phosphatase. ... 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
A chemiluminescent assay for quantitation of beta-galactosidase in the femtogram range: application to quantitation of beta-galactosidase in lacZ-transfected cells.
AuthorsJain VK, Magrath IT
JournalAnal Biochem
PubMed ID1807155
An optimized chemiluminescent assay for beta-galactosidase using a chemiluminescent substrate AMPGD (3-(4-methoxyspiro[1,2-dioxetane-3,2'-tricyclo-[3.3.1. 1(3,7)]decan]-4- yl)phenyl-beta-D-galactopyranoside) is described. This assay is rapid and sensitive and can detect as little as 2 fg of beta-galactosidase. Its use for the quantitation of beta-galactosidase in cells transfected with lacZ-expressing vectors is described. It is possible ... More
Systemic delivery of human growth hormone by injection of genetically engineered myoblasts.
A recombinant gene encoding human growth hormone (hGH) was stably introduced into cultured myoblasts with a retroviral vector. After injection of genetically engineered myoblasts into mouse muscle, hGH could be detected in serum for 3 months. The fate of injected myoblasts was assessed by coinfecting the cells with two retroviral ... More
Enzyme-linked fluorescence immunoassays using beta-galactosidase and antibodies covalently bound to polystyrene plates.
AuthorsNeurath AR, Strick N
JournalJ Virol Methods
PubMed ID6795220
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 ... More
Fluorescence sandwich enzyme-linked immunosorbent assay for detecting human interleukin-2 receptors.
AuthorsHonda M, Nagao S, Yamamoto N, Tanaka Y, Tozawa H, Tokunaga T
JournalJ Immunol Methods
PubMed ID2453583
A very sensitive fluorescence sandwich enzyme-linked immunosorbent assay (FS-ELISA) for the detection of soluble and cell-associated human interleukin-2 receptors (IL-2R) has been developed. For use in this assay system, two anti-human IL-2R monoclonal antibodies, H 48 and biotinylated HA 26, were selected from six monoclonal antibodies directed against different epitopes ... More
Isolation of mutant T lymphocytes with defects in capacitative calcium entry.
AuthorsSerafini AT, Lewis RS, Clipstone NA, Bram RJ, Fanger C, Fiering S, Herzenberg LA, Crabtree GR
JournalImmunity
PubMed ID7648396
Calcium and calcium-binding proteins play important roles in the signaling cascade leading from the initial engagement of TCRs on T cells to the fully activated state. To undertake a molecular dissection of this cascade, we first isolated a Jurkat T cell line derivative containing the NF-AT promoter element driving transcription ... More
Identification of candidate active site residues in lysosomal beta- hexosaminidase A.
AuthorsFernandes MJ, Yew S, Leclerc D, Henrissat B, Vorgias CE, Gravel RA, Hechtman P, Kaplan F
JournalJ Biol Chem
PubMed ID8995368
The beta-hexosaminidases (Hex) catalyze the cleavage of terminal amino sugars on a broad spectrum of glycoconjugates. The major Hex isozymes in humans, Hex A, a heterodimer of alpha and beta subunits (alphabeta), and Hex B, a homodimer of beta subunits (betabeta), have different substrate specificities. The beta subunit (HEXB gene ... More
Comparison of assays for detection of agents causing membrane damage in Staphylococcus aureus.
AuthorsO'Neill AJ, Miller K, Oliva B, Chopra I,
JournalJ Antimicrob Chemother
PubMed ID15531595
OBJECTIVES: To develop a novel beta-galactosidase leakage assay for Staphylococcus aureus and to evaluate this alongside other simple methods for detection of agents that cause membrane damage in staphylococci. METHODS: Using a PCR-based approach, a derivative of S. aureus RN4220 was constructed carrying the Escherichia coli lacZ gene under the ... More
Physicochemical studies of binding of 4-methylumbelliferyl beta-D-galactopyranoside to cold agglutinin.
AuthorsMitra D, Sarkar M
JournalBiochem J
PubMed ID2818572
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 ... More
Measurement of antigen-specific mouse IgE by a fluorometric reverse (IgE-capture) ELISA.
AuthorsSakaguchi M, Inouye S, Miyazawa H, Tamura S
JournalJ Immunol Methods
PubMed ID2783445
A reverse, or IgE-capture, enzyme-linked immunosorbent assay (ELISA) for measuring ovalbumin-specific IgE antibody in the serum of immunized mice has been developed. Microplate wells were first coated with a commercial anti-mouse IgE rat monoclonal antibody, and then incubated with two-fold serial dilutions of test sera with 10% normal mouse serum ... 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
Lineage analysis in the vertebrate nervous system by retrovirus-mediated gene transfer.
AuthorsPrice J, Turner D, Cepko C
JournalProc Natl Acad Sci U S A
PubMed ID3099292
We describe a cell-lineage marking system applicable to the vertebrate nervous system. The basis of the technique is gene transfer using the retroviral vector system. We used Escherichia coli beta-galactosidase as a marker gene and demonstrate a high level of expression of this marker from the viral long terminal repeat ... More
Spectrophotometric and fluorimetric assays of galactocerebrosidase activity, their use in the diagnosis of Krabbe's disease.
AuthorsBesley GT, Gatt S
JournalClin Chim Acta
PubMed ID7214711
Derivatives of galactocerebroside were prepared containing coloured (w-2,4,6-trinitrophenylaminolauric acid) or fluorescent (11-(9-anthroyloxy) undecanoic acid) fatty acid moieties.. These cerebrosides were used as substrates for galactocerebrosidase activity. By overcoming problems associated with the radioactively labelled substrates normally used, yet retaining good enzyme-substrate specificity, these derivatives provided useful and reliable alternative substrates ... More
The separation and characterization of the methylumbelliferyl beta-galactosidases of human liver.
AuthorsCheetham PS, Dance NE
JournalBiochem J
PubMed ID962854
1. A previously uncharacterized form of human liver acid beta-galactosidase (EC 3.2.1.23), possibly a dimer of molecular weight 160 000, was resolved by gel filtration. It has the same ability to hydrolyse GM1 ganglioside as the two other acid beta-galactosidase forms. 2. The low-molecular-weight forms of acid beta-galactosidase undergo salt-dependent ... More
4-Methylumbelliferyl-glycosides as fluorescence probes of sugar-binding sites on lectin molecules: spectral properties and dependence of fluorescence on polarity and viscosity.
AuthorsDecastel M, Vincent M, Matta KL, Frénoy JP
JournalArch Biochem Biophys
PubMed ID6465890
The spectral properties of 4-methylumbelliferyl-glycosides (MeUmb-glycosides) were investigated in order to assess their usefulness as probes of the microenvironment of sugar binding sites on lectin molecules. It was shown that the abnormally high values for fluorescence polarization of free MeUmb-glycosides (from 0.07 to 0.251) were due neither to their molecular ... 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
The use of N-[beta-(4-diazophenyl)ethyl]maleimide as a coupling agent in the preparation of enzyme-antibody conjugates.
AuthorsFujiwara K, Saita T, Kitagawa T
JournalJ Immunol Methods
PubMed ID3131437
The present study was undertaken to develop a novel method for the enzyme labeling of antibodies. Goat anti-rabbit IgG was used as a prototype and coupled to beta-D-galactosidase (Gal) with a new heterobifunctional cross-linking agent N-[beta-(4-diazophenyl)ethyl]maleimide (DPEM). The antibody was first azo-coupled with DPEM to introduce the maleimide groups into ... More
A rapid ELISA for measuring insulin in a large number of research samples.
AuthorsMacDonald MJ, Gapinski JP
JournalMetabolism
PubMed ID2657325
An enzyme-linked immunosorbent assay (ELISA) for insulin was developed. Anti-insulin antibody was bound to the bottom of 96-well microtiter plates. Insulin conjugated to beta-galactosidase was used as a label and methyl umbilliferyl beta-D galactoside was used as an enzyme substrate. To estimate insulin, relative fluorescence was measured with a fluorescent ... More
A rapid fluorescence bioassay for the determination of selenium on agar plates.
AuthorsLiu Z, Reches M, Engelberg-Kulka H
JournalAnal Biochem
PubMed ID9025905
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 ... More
A reverse-sandwich ELISA for IgG antibody to a pollen allergen.
AuthorsMiyazawa H, Inouye S, Sakaguchi M, Koizumi K
JournalJ Allergy Clin Immunol
PubMed ID3170988
We have developed a reverse-type sandwich ELISA for measurement of IgG (+IgA) antibody to a major allergen of Sugi (Japanese cedar) pollens. In this assay, microplate wells were coated with the allergen proteins that had been dispersed in the presence of 0.5 mol/L NaCl and 20 micrograms/ml of bovine serum ... More
Quantitative assay of senescence-associated beta-galactosidase activity in mammalian cell extracts.
AuthorsGary RK, Kindell SM
JournalAnal Biochem
PubMed ID16004951
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 ... More
Miniaturization of beta-galactosidase immunoassays using chromogenic and fluorogenic substrates.
AuthorsLabrousse H, Guesdon JL, Ragimbeau J, Avrameas S
JournalJ Immunol Methods
PubMed ID6799580
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 ... More
An enzyme-linked procedure for the detection and estimation of surface receptors on cells.
AuthorsCameron DJ, Erlanger BF
JournalJ Immunol
PubMed ID774980
An enzyme immunoassay procedure has been developed for the visualization and estimation of lymphocyte surface receptors. beta-Galactosidase (beta-gal'ase) was covalently linked to sheep anti-rabbit immunoglobulin (SARIg), to ovalbumin (OA), and to adenosine (A). Exposure of rabbit peripheral lymphocytes to SARIg-beta-gal'ase and subsequent incubation with the fluorogenic substrate fluorescein-beta-digalactopyranoside allowed visualization ... More
Which of the commonly used marker enzymes gives the best results in colorimetric and fluorimetric enzyme immunoassays: horseradish peroxidase, alkaline phosphatase or beta-galactosidase?
AuthorsPorstmann B, Porstmann T, Nugel E, Evers U
JournalJ Immunol Methods
PubMed ID3923120
Comparing the marker enzymes horseradish peroxidase (HRP), alkaline phosphatase (AP) and beta-galactosidase (beta Gal) in IgG-coupled form with respect to their temperature-dependent kinetics over a period of 22 h the temperature of 37 degrees C warrants highest substrate turnover for all enzymes at all reaction times using fluorogens. Also applying ... More
Optimisation of an enzymatic method for beta-galactosidase.
AuthorsMcGuire JB, James TJ, Imber CJ, St Peter SD, Friend PJ, Taylor RP
JournalClin Chim Acta
PubMed ID12417103
BACKGROUND: The enzyme beta-galactosidase present in the Kupffer cells of the liver has potential as a marker of liver dysfunction prior to transplantation. Spectrophotometric methods have insufficient sensitivity. METHODS: Fluorimetric methods have the required sensitivity and we have optimised such a method in a microtitre plate format to improve its ... More
Fluorogenic substrates based on fluorinated umbelliferones for continuous assays of phosphatases and beta-galactosidases.
AuthorsGee KR, Sun WC, Bhalgat MK, Upson RH, Klaubert DH, Latham KA, Haugland RP
JournalAnal Biochem
PubMed ID10452797
Fluorogenic substrates based on 4-methylumbelliferone (4-MU) have been widely used for the detection of phosphatase and glycosidase activities. One disadvantage of these substrates, however, is that maximum fluorescence of the reaction product requires an alkaline pH, since 4-MU has a pK(a) approximately 8. In an initial screening of five phosphatase ... More
Construction and expression of an adenosine deaminase::lacZ fusion gene.
AuthorsShen Q, van Beusechem VW, Einerhand MP, Hendrikx PJ, Valerio D
JournalGene
PubMed ID1901822
A eukaryotic expression vector was constructed in which the coding nucleotide sequences (ADA) of human adenosine deaminase (ADA) were fused in frame with the coding sequences of the bacterial gene lacZ encoding beta-galactosidase (beta Gal). This ADA::lacZ fusion gene was anticipated to encode a hybrid protein that has retained the ... More
Binding of 4-methylumbelliferyl beta-D-galactopyranoside to Momordica charantia lectin: fluorescence-quenching studies.
AuthorsKhan MI, Mazumder T, Pain D, Gaur N, Surolia A
JournalEur J Biochem
PubMed ID7215338
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 ... More
Characterization of T cell mutants with defects in capacitative calcium entry: genetic evidence for the physiological roles of CRAC channels.
AuthorsFanger CM, Hoth M, Crabtree GR, Lewis RS
JournalJ Cell Biol
PubMed ID7593187
Prolonged Ca2+ influx is an essential signal for the activation of T lymphocytes by antigen. This influx is thought to occur through highly selective Ca2+ release-activated Ca2+ (CRAC) channels that are activated by the depletion of intracellular Ca2+ stores. We have isolated mutants of the Jurkat human T cell line ... More
The fate of cholesterol exiting lysosomes.
AuthorsLange Y, Ye J, Chin J
JournalJ Biol Chem
PubMed ID9202016
Cholesterol released from ingested low density lipoproteins in lysosomes moves both to the plasma membrane and to the endoplasmic reticulum (ER) where it is re-esterified. Whether cholesterol can move directly from lysosomes to ER or first must traverse the plasma membrane has not been established. To examine this question, the ... More
Controlling programmed cell death with a cyclophilin-cyclosporin-based chemical inducer of dimerization.
BACKGROUND: Cell death can occur either from physical damage (necrosis) or cellular suicide (apoptosis). Apoptosis is essential for the development of multicellular organisms and disregulated apoptosis underlies many human diseases. The Fas receptor (Fas) is a membrane signaling protein that mediates a death signal following its aggregation by the Fas ... 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