Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: KEGG:D02003 (NBT)
1,323 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Optimal conditions for the NBT-reduction test have been sought. Increasing heparin concentrations up to 100 units per ml and a delay in performance of the test, especially when blood specimens are kept at room temperature, resulted in higher values for the NBT index, which then sometimes exceeded the upper limit of normal in healthy people and in uninfected patients. The effect of pH, composition of the buffer, and dye concentration was also investigated. Phosphate-buffered saline pH 7-2 containing 0-1% NBT dye, without glucose, gave the most reliable results. In endotoxin-stimulated NBT tests, the following procedure is recommended: incubation of 0-1 ml whole blood with lyophilised endotoxin 20 mug per ml. for 15 min. in a 37 degree C water bath, followed by the standard test with a 0-2% NBT solution. By this technique, the leucocyte reaction to various types of lipopolysaccharides was of the same order of magnitude. Drug therapy having an effect on blood components lowered this reaction, whatever the source of endotoxin used as stimulant. The importance of NBT-reduction tests is discussed. Standard conditions of test performance are strictly requisite if comparable results are to be obtained and if data not corresponding with the apparent clinical and other laboratory findings are to be evaluated correctly. The stimulated NBT test, performed in parallel with the standard test, is useful in the interpretation of abnormal results and in the detection of factors with a temporary or permanent effect on the phagocytic activity of pmn leucocytes.
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PMID:Standardisation of the nitroblue-tetrazolium test. 23 38

A rapid method for qualitative and quantitative detection of specific oral microorganisms from subgingival dental plaque is described. Plaque samples were suspended in phosphate-buffered saline containing protease inhibitors and 0.5% formaldehyde, briefly sonicated to disperse bacterial aggregates, and applied to nitrocellulose membranes in a slot blot manifold. Subsequent incubations with species-specific rabbit antibody and anti-rabbit antibody-alkaline phosphatase conjugate and development with BCIP-NBT substrate resulted in an easily discernible, permanent stain being deposited at the sample application site. Comparison with known concentrations of pure, cultured microorganisms applied to the same membranes permitted qualitative or semiquantitative plaque characterization by visual inspection. Analysis of the blots with a computer-linked flatbed scanner provided quantitative data on microbial content. The reproducibility of the results (standard error of the mean, less than 10%) obtained with slot immunoblotting greatly exceeded that of the results obtained with immunofluorescence analysis (standard error of the mean, greater than 57%). Because it is versatile, rapid, sensitive, reproducible, permanent, and relatively inexpensive, slot immunoblotting lends itself to use in large-scale investigations for the detection and quantitation of specific microbial species.
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PMID:Slot immunoblot assay for detection and quantitation of periodontal disease-associated microorganisms in dental plaque. 166 11

We describe a new, simple, rapid, and sensitive nonradioactive technique for the analysis of genetic variations. Genomic DNA was amplified using polymerase chain reaction and amplified DNA was hybridized, with digoxigenin (DIG)-labeled sequence-specific oligonucleotides. High specificity and sensitivity was achieved when labeling the sequence-specific oligonucleotide at the 3' end with only one DIG using digoxigenin-11-2',3'-dideoxy-uridine-5'-triphosphate and DNA deoxynucleotidylexotransferase. The hybridized probes were detected using antidigoxigenin alkaline phosphatase, fab fragments, and X-phosphate/NBT for visualization. This method was applied to the analysis of HLA-DR4-DRB1 alleles in polymerase chain reaction-amplified genomic DNA and resulted in highly specific and sensitive hybridization signals discriminating even in cases of a one-base-pair mismatch. This technique is particularly suited for HLA oligotyping because it allows the use of tetramethylammonium chloride for the simplification of hybridization and washing conditions.
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PMID:Nonradioactive HLA class II typing using polymerase chain reaction and digoxigenin-11-2'-3'-dideoxy-uridinetriphosphate-labeled oligonucleotide probes. 167 54

We have developed a method that implies the use of a particular type of substrate which can be used in combination with alkaline phosphatase in detecting nucleic acid on filters. The method allows the detection of several different nucleic acid sequences on a single filter. In consecutive steps, the target DNA molecules are hybridized with different digoxigenin-labeled DNA probes. After each hybridization step, digoxigenin is detected with an antibody-alkaline phosphatase conjugate. This enzyme is subsequently visualized by a color reaction using different 2-hydroxy-3-naphthoic acid anilide (naphthol AS) phosphates as substrates in combination with varying diazonium salts. The multiple-staining procedure is based on the fact that the probe DNA-antibody complex can be removed while the color precipitate remains stably bound at its place on the filter. This allows several repeated hybridizations with other digoxigenin-labeled probes followed by antibody detection and color reaction with other naphthol AS phosphate-diazonium salt combinations. Aside from the ability to simultaneously visualize different target DNAs on a single filter, this new method provides several important features that are more powerful than the conventional 5-bromo-4-chloro-3-indolyl phosphate-nitro blue tetrazolium (BCIP-NBT) color reaction for alkaline phosphatase. The colors are more stable and brilliant than BCIP-NBT; their development is faster, the resolution of closely spaced bands is greater, and the background is much lower. The detection limit for alkaline phosphates is as good as with BCIP-NBT (0.1 pg of DNA). One major advantage is the simplicity of removing the colors by ethanol incubation. In this paper, the method is described using the example of Southern blotted DNA fragments.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A multiple-staining procedure for the detection of different DNA fragments on a single blot. 170 97

We have found that GM-CSF and DMSO have antagonistic effects on the proliferation but not maturation of asynchronously growing HL-60 cells such that growth in the presence of both more closely resembles normal hematopoiesis (Brennan et al., J. Cell Physiol. 132:246, 1987). Studies were undertaken to determine whether or not the agents affected the same mitogenic pathway and locus in the cell cycle. HL-60 populations containing at least 90% G1 cells were obtained by centrifugal elutriation, exposed to 100 u/ml recombinant human GM-CSF and/or 0-1.25% DMSO, and phosphoprotein changes quantified on autoradiograms of [32P]-orthophosphate-labeled cell proteins separated by giant 2-D gel electrophoresis. Results were correlated with 1) intracellular pH, determined by measurement of BCECF fluorescence; 2) [32P]-orthophosphate uptake; 3) cell cycle progression, determined by flow quantitation of DNA content in mithramycin or propidium iodide-stained cells; and 4) growth, determined by cell volume and concentration. GM-CSF stimulated and DMSO inhibited the GM-CSF-stimulated phosphorylation of 1 protein (approximately 65 kDa, p.i. 5.6) within 2 min of exposure. These effects were sustained through G1, not associated with changes in intracellular pH, and preceded similar antagonistic effects on phosphate uptake (15-30 minutes), cell volume change (16-24 hr), and cell concentration increase (28-32 hr). GM-CSF accelerated and DMSO inhibited G1 to S transit with the most marked antagonism observed in the second cycle following synchronization (28 to 40 hrs). Cell maturation (morphology, NBT reduction) was dominated by DMSO and not antagonized by GM-CSF. We have identified p65 as the nuclear intermediate filament protein, lamin B, on the basis of its locus on gels and its binding of a monoclonal antibody to intermediate filaments and antiserum to human lamin B on immunoblots. These studies suggest that at least part of the GM-CSF-DMSO antagonism is exerted through the same mitogenic pathway, that a major locus of cytokinetic effect is on G1 to S transit, and that nuclear envelope protein phosphorylation is an important early event.
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PMID:Interactions of dimethyl sulfoxide and granulocyte-macrophage colony-stimulating factor on the cell cycle kinetics and phosphoproteins of G1-enriched HL-60 cells: evidence of early effects on lamin B phosphorylation. 202 96

Mutations at positions beta IVS1-6, beta IVS1-110, and beta 39 of the beta globin gene are responsible for the three most common thalassemic genes in the Mediterranean population. The polymerase chain reaction (PCR) was employed to amplify a 536 base pair segment surrounding this region. Nonradioactive labelling of an oligonucleotide probe, specific for the beta IVS1-110 mutation, was achieved by incorporation of biotin-16-dUTP into a standard 3'-end labelling procedure. This probe was subsequently hybridized with the PCR amplification product and permitted detection of the mutant gene in a homozygous beta thalassemic child by a simple colour detection method using a streptavidin-alkaline phosphatase conjugate and NBT/BCIP (nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate) substrate. A known cloned mutant gene was similarly detected. Results could be obtained within 48 hr. These findings suggest that such an approach could provide a rapid and specific means for detection of beta thalassemic mutations without the need for radioactive probes.
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PMID:Direct detection of beta thalassemic mutations: use of biotin-labelled allele specific probes. 233 80

We compared the sensitivity of a chemiluminescent substrate 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl- 1,2-dioxetane (AMPPD) and a chromogenic substrate 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) for detection of an alkaline phosphatase label in a hepatitis B virus "core antigen" DNA (HBVc) probe hybridization assay. Chemiluminescent signal obtained from AMPPD hydrolysis by alkaline phosphatase was detected with Polaroid Instant Black and White Type 612 film. The chemiluminescent assay detected 1.18 x 10(6) copies of HBVc plasmid DNA in 30 min. By comparison, 9.8 x 10(7) copies of DNA could be measured using chromogenic BCIP/NBT substrate within the same incubation time. After further development, the chemiluminescent endpoint permitted detection of 4.39 x 10(4) copies of HBVc plasmid DNA in 2 h.
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PMID:A comparison of chemiluminescent and colorimetric substrates in a hepatitis B virus DNA hybridization assay. 281 49

The effect of EDTA and H2O2 on iron autoxidation in Mops buffer depends on the pH of the solution. At acidic pH, EDTA caused the oxidation of a stoichiometric amount of iron. At neutral and alkaline pH, EDTA and H2O2 not only oxidizes a stoichiometric amount of iron but also causes the oxidation of the Fe2+ exceeding the concentration of these compounds. In the presence of EDTA, oxidation of Fe2+ in exceeding the concentration of these compounds has a shorter lag phase and an increased rate compared with that in the absence. The solution develops a yellow colour whose intensity is proportional to the amount of Fe2+ exceeding the concentration of these compounds in solution. When the reaction is conducted in the presence of NBT, formazan formation is greatly reduced compared to the control without EDTA and H2O2. The Fe3+-EDTA complex and Fe3+ affected iron oxidation, development of the yellow colour and NBT reduction in a similar fashion. In all these experimental conditions, iron oxidation is greatly reduced in the presence of mannitol, sorbitol and catalase. In phosphate buffer, EDTA oxidized a stoichiometric amount of iron without affecting free Fe2+ oxidation. Fe3+ has no effect on iron oxidation in this buffer.
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PMID:Iron oxidation in Mops buffer. Effect of EDTA, hydrogen peroxide and FeCl3. 314 95

A number of immunocytochemical detection systems for determining the chromosomal localization of specific nucleic acid sequences by non-radioactive in situ hybridization have been compared. The procedures were: 1. the peroxidase/diaminobenzidine (PO/DAB) combination, either or not gold/silver intensificated; 2. alkaline phosphatase marking using the nitro-blue tetrazolium plus bromochloro-indolyl phosphate substrate combination (AP/NBT + BCIP); and 3. immunogold with or without silver enhancement. The procedures were first tested and optimized in dot blot experiments and then applied to in situ hybridization. As hybridization probes, both a middle-repetitive and a unique sequence (modified with 2-acetylaminofluorene (AAF] were used. The advantages and disadvantages of the various methods for reflection contrast (RC) or transmission electron microscopic (TEM) visualization of hybrids are discussed.
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PMID:Non-radioactive in situ hybridization. A comparison of several immunocytochemical detection systems using reflection-contrast and electron microscopy. 361 Jun 73

Using a digoxygenin-labelled DNA probe derived from the porcine repeat element PRE-1, we have developed a protocol for the detection of transplanted porcine islets and hepatocytes against a background of murine host tissue. Analysis of this probe by Southern blotting indicated that PRE-1 hybridizes to pig genomic DNA but not to human or mouse DNA. On tissue sections, hybridizing probe was detected using alkaline phosphatase-conjugated antidigoxygenin antibody visualized with 5-bromo-4-chloro-3-indolyl-phosphate/4-nitro-blue tetrazolium chloride (BCIP/NBT) substrate. We have demonstrated sensitive and highly specific staining of porcine nuclei in fixed, paraffin embedded tissue sections, and have applied the technique to detect porcine pancreatic islets and hepatocytes transplanted into murine kidney and spleen. Application of this technique include detection of transplanted cells or organs across the variety of xenogeneic barriers.
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PMID:Porcine repeat element DNA: in situ detection of xenotransplanted cells. 777 59


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