[Application of PCR to DNA diagnosis and molecular epidemiology of varicella-zoster virus infection].
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Base Sequence↗
Biomedical subjects
S Inouye
Publications and source records attributed to S Inouye.
[A new counting method for airborne Japanese red cedar and grass pollen allergens by the immunoblotting technique].
We devised a new counting method of pollen allergen particles which improved the fluorescence immunoblotting technique by Schumacher et al (1988). And by which airborne pollen allergens became visible under 10X magnifier or naked eyes. Airborne pollen allergens collected on the Burkard's sampling tape were transferred onto nitrocellulose membrane and were reacted with anti Cry j I rabbit serum or anti Lol p I rabbit serum, and then treated with alkaline phosphatase conjugated F(ab')2 anti rabbit IgG. Finally, bluish purple spots were obtained by staining with BCIP/NBT phosphatase substrate system. This technique does not require any skillful morphological observation, and is more suitable to measure the amounts of airborne pollen allergen for given pollinosis patients because total pollen allergen particles with common antigenicity are measured. In Japanese red cedar pollen counts, we could not count the spots more than 400 grains per 0.16 cm2 of the sample trapping area due to many overlapping spots. In this case, we tried to calculate the value from the ratio of bluish purple coloured area to one pollen area. However, a more suitable method for estimating the content of pollinosis caused airborne allergens may be colorimetric quantitation using densitometry and displaying the value as allergen content.
Allergens↗
In vitro antimicrobial activity of a novel aminothiazolylglycylcephalosporin, MT0703S, compared with that of ceftazidime, cefoperazone and aztreonam.
The antibacterial activity of a novel aminothiazolylglycylcephalosporin, MT0703S, possessing a dihydroxypyridone moiety was compared in vitro with the activity of ceftazidime, cefoperazone, aztreonam and other beta-lactam antibiotics using seven bacterial species of a clinical origin. MT0703S showed the most potent activity against P. aeruginosa, including the ceftazidime-resistant strains, E. coli, K. pneumoniae and C. freundii. MT0703S was comparable to aztreonam but more active than ceftazidime and cefoperazone in its activity against K. oxytoca and E. cloacae, and comparable to ceftazidime against S. aureus and S. marcescens. MT0703S was more active than cefoperazone against S. marcescens but less active against S. aureus. The stability of MT0703S against various beta-lactamases appeared to be intermediate between the stability of ceftazidime and that of cefoperazone. The antimicrobial activity of MT0703S increased in a low-iron environment and decreased in a high ferric ion concentration.
Aztreonam↗
Histo-chemical studies on the anti-ulcer effect of bamboo grass in rats.
Oral administration of a hot-water extract (Folin) of bamboo grass (Sasa albomarginata Makino & Shibata) significantly reduced the incidence of water-immersion and restraint stress-, ethanol-induced and indomethacin-induced gastric ulcers in rats. Histological examination of the Folin-treated gastric mucosa showed that microscopic blood clots overlaid the superficial epithelium, maintaining the cellular integrity of gastric mucosa, especially against stress ulcer. In addition, Folin suppressed the incidence of hyperaemia and a decline of acid mucopolysaccharides in the ethanol-induced ulcer. Folin suppressed a release of histamine from rat mast cells, and stabilized erythrocytes and accelerated their agglutination under acid conditions. These results suggest that a microscopic haemostatic effect of Folin reinforced by a membrane-stabilizing effect might be responsible for the prevention of the gastric lesions.
Administration, Oral↗
Evaluation of countermeasures for reduction of mouse airborne allergens.
Three kinds of countermeasures for reduction of mouse airborne allergens were evaluated with use of an air sampler and immunochemical methods. Mouse cages and the sampler were placed inside a flexible-film isolator, and concentrations of mouse major allergens in the air were measured. The levels of the airborne allergens, prealbumin and albumin, generated by 10 males, were 3,050 and 492 pg/m3, respectively. Those by 10 females were lower, 317 and 270 pg/m3, respectively. When mouse cages were covered with a filter cap, the airborne allergens inside the isolator were decreased by 90%. When corncob was used as bedding in place of wood shavings, the airborne allergens were decreased by 57 and 77%, respectively. Therefore, for reduction of mouse airborne allergens, we recommend using female mice, covering the cages with filter caps, and using corncob bedding.
Air Pollutants, Occupational↗
[Diagnosis of HIV infection by polymerase chain reaction with non-isotopic probes].
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Acquired Immunodeficiency Syndrome↗
[Serodiagnosis of virus infections].
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Antibodies, Viral↗
Cloning and sequence analysis of the ntrA (rpoN) gene of Pseudomonas putida.
The gene encoding a sigma factor NtrA (RpoN) was cloned from Pseudomonas putida by cross-hybridization with a probe containing a part of the corresponding Escherichia coli gene. The cloned gene complemented an ntrA mutation of E. coli in activation of xyl genes on the TOL plasmid. The predicted amino acid (aa) sequence of P. putida NtrA (497 aa; Mr 56,215) is highly homologous to NtrA proteins from Azotobacter vinelandii (81.7%), Klebsiella pneumoniae (52.6%), and Rhizobium meliloti (36.1%). There are two other open reading frames (ORF1, ORF3) upstream and downstream from the ntrA gene, respectively. These ntrA-flanking ORFs are also highly homologous to the corresponding ORFs of K. pneumoniae, A. vinelandii, and R. meliloti.
Amino Acid Sequence↗
Myxococcus xanthus msDNA.Mx162 exists as a complex with proteins.
Myxococcus xanthus, a myxobacterium, contains a peculiar branched RNA-linked DNA called msDNA. Reverse transcriptase has been shown to be required for the production of msDNA. Existence of proteins that bind to one of the two msDNAs in M. xanthus, msDNA.Mx162, was examined by gel retardation assays. Total cell-free extract yielded two distinct retarded bands. Both bands were sensitive to treatment with proteinase K, indicating that there is a protein(s) that is able to bind to msDNA. Further, the formation of the bands was inhibited by the addition of nonradioactive msDNA but not by a large excess of poly(dA) in the presence of a 5000-fold excess of poly(dI.dC).poly(dI.dC). In vivo footprinting using dimethyl sulfate revealed that the deoxynucleotide stretch from 60 to 161 is protected. When a M. xanthus cell lysate was centrifuged in a 16-30% glycerol gradient, msDNA was found to sediment in two peaks: a major peak corresponding in size to 14 S, and a minor one at 5 S. These results indicate that msDNA.Mx162 exists as a complex with specific proteins in the cell.
Base Sequence↗
Structural requirements of the RNA precursor for the biosynthesis of the branched RNA-linked multicopy single-stranded DNA of Myxococcus xanthus.
A precursor RNA molecule (pre-msdRNA) of approximately 375 bases is considered to form a stable secondary structure which serves as a primer as well as a template to synthesize the branched RNA-linked multicopy single-stranded DNA (msDNA) of Myxococcus xanthus. When 3-base mismatches were introduced into the stem structure immediately upstream of the branched rG residue to which msDNA is linked by a 2',5'-phosphodiester linkage, the production of msDNA was almost completely blocked. However, if additional 3-base substitutions were made on the other strand to resume the complementary base pairing, msDNA production was restored, being consistent with the proposed model of msDNA synthesis. We also found that the branched rG residue of pre-msdRNA could not be replaced with either rC or rA, while the 5' end (dC) of msDNA which is linked to the branched rG could be substituted with a dG residue. Together with several other mutations, the structural requirements of pre-msdRNA are discussed with respect to the mechanism of msDNA biosynthesis.
DNA, Bacterial↗
Binding of murine monoclonal antibodies to the active and inactive configurations of aequorin.
Murine monoclonal IgG1 antibodies (MAb), designated Aq-11 and Aq-12, were prepared against the photoprotein aequorin from jelly fish. Aequorin is a calcium-sensitive photoprotein which consists of a single polypeptide chain, apoaequorin, and a functional chromophore, coelenterazine. Native aequorin consists of two species with molecular masses of 25 and 23.5 kDa. MAb Aq-12 was found by immunoblot analysis to bind specifically to the 25 kDa species, while MAb Aq-11 reacted with the 23.5 kDa protein. Activation of apoaequorin with coelenterazine was associated with a shift of the 23.5 kDa molecule to the 25 kDa species. In contrast, treatment with calcium ions induced a shift back to the 23.5 kDa form. These changes between the active and inactive forms were identified by reactivity with MAbs Aq-11 and Aq-12. The results thus indicate that these MAbs should be useful in monitoring activation of this photoprotein.
Aequorin↗
Factors affecting the efficiency of protein synthesis in Escherichia coli. Production of a polypeptide of more than 6000 amino acid residues.
Factors affecting the efficiency of protein synthesis were analyzed in Escherichia coli. For this purpose the lacZ gene was fused to produce polypeptides from a dimer (molecular weight 229,957) to a hexamer (molecular weight 684,924) of beta-galactosidase. From pulse-chase experiments it was found that only 45% of the ribosomes which reached to the end of the first monomer were able to complete the second monomer unit. Similarly, for every addition of a monomer unit to synthesize the multimers from the trimer to the hexamer approximately half of the ribosomes failed to complete the synthesis of the added unit. Furthermore, the stability of the polypeptides decreased as their sizes increased. As a result, the overall efficiency of the production of the beta-galactosidase polymers dropped by a factor of approximately 3 on a weight basis for each addition of a monomer unit.
Amino Acids↗
Reverse transcriptase with concomitant ribonuclease H activity in the cell-free synthesis of branched RNA-linked msDNA of Myxococcus xanthus.
msDNA is a peculiar molecule consisting of a branched RNA linked to single-stranded DNA via a 2',5' phosphodiester bond. A cell-free system, utilizing cells permeabilized with phenethyl alcohol, was established to study the synthesis of msDNA in M. xanthus. Permeablized cells labeled with [alpha-32P]dCTP in the presence of ddGTP, ddATP, or ddTTP produce a band that migrates at the same position as the full-sized msDNA in an polyacrylamide gel. However, when this band is treated with ribonuclease A prior to gel electrophoresis, it results in many different-sized bands. This indicates that during the labeling, intermediates are produced in which single-stranded DNAs of various lengths are associated with a compensatory length of RNA such that the total length for each intermediate is identical. These results provide evidence for the previously proposed model in which msDNA is synthesized by reverse transcriptase using a folded RNA precursor as a primer as well as a template. Furthermore, we found that there is a precise coupling mechanism of reverse transcriptase and ribonuclease H.
Cell-Free System↗
Reverse transcriptase associated with the biosynthesis of the branched RNA-linked msDNA in Myxococcus xanthus.
Myxobacteria have been shown to produce a peculiar RNA-DNA complex called msDNA, in which a single-stranded DNA is branched out from a RNA molecule (msdRNA) by a 2',5' phosphodiester linkage. It has been predicted that reverse transcriptase is required for msDNA biosynthesis. We identified a gene for reverse transcriptase in M. xanthus in the region that has been demonstrated to code for a cis- or transacting element for msDNA synthesis. This gene is located immediately downstream of the msdRNA coding region, and codes for a polypeptide of 485 amino acid residues. The polypeptide shows sequence similarity with retroviral reverse transcriptases. This fact, together with the mode of msDNA synthesis, suggests a possible relationship between retroviruses and the msDNA system. The analysis of the gene and the distribution of the msDNA system in independent isolates of M. xanthus indicate that the element is as old as other essential genes in M. xanthus and that it was not recently acquired into the genome.
Amino Acid Sequence↗
Reverse transcriptase in a clinical strain of Escherichia coli: production of branched RNA-linked msDNA.
Branched RNA-linked multicopy single-stranded DNA (msDNA) originally detected in myxobacteria has now been found in a clinical isolate of Escherichia coli. Although lacking homology in the primary structure, the E. coli msDNA is similar in secondary structure to the myxobacterial msDNA's, including the 2',5'-phosphodiester linkage between RNA and DNA. A chromosomal DNA fragment responsible for the production of msDNA was cloned in an E. coli K12 strain; its DNA sequence revealed an open reading frame (ORF) of 586 amino acid residues. The ORF shows sequence similarity with retroviral reverse transcriptases and ribonuclease H. Disruption of the ORF blocked msDNA production, indicating that this gene is essential for msDNA synthesis.
Amino Acid Sequence↗
Measurement of antigen-specific mouse IgE by a fluorometric reverse (IgE-capture) ELISA.
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 as diluent for the capturing of only IgE class molecules. Biotinylated ovalbumin and then beta-D-galactosidase-conjugated streptavidin were added and, finally, 4-methylumbelliferyl-beta-D-galactoside was used as the enzyme substrate. The fluorescence intensity of the reaction product (4-methylumbelliferone) was determined on a microplate fluorescence reader. The sensitivity of this assay was equal to that of passive cutaneous anaphylaxis (PCA). In contrast to indirect ELISAs this IgE-capture assay is free from competition by non-IgE antibodies. Furthermore, it requires much less antigen than the PCA assay.
Animals↗
Central administration of muscimol phase-shifts the mammalian circadian clock.
The suprachiasmatic nucleus (SCN) of the hypothalamus contains a neural oscillatory system which regulates many circadian rhythms in mammals. Immunohistochemical evidence indicates that a relatively high density of GABAergic neurons exist in the suprachiasmatic region. Since intraperitoneal injections of the benzodiazepine, triazolam, have been shown to induce phase shifts in the free-running circadian rhythm of locomotor activity in the golden hamster, the extent to which microinjections of muscimol, a specific agonist for gamma-aminobutyric acid (GABA), may cause phase-shifts in hamster activity rhythms was investigated. Stereotaxically implanted guide cannulae aimed at the region of the SCN were used to deliver repeated microinjections in individual animals. A phase-response curve (PRC) generated from microinjections of muscimol revealed that the magnitude and direction of permanent phase-shifts in the activity rhythm were associated with the time of administration. The PRC generated for muscimol was characterized by maximal phase-advances induced 6 h before activity onset and by maximal phase-delays which occurred 6 h after activity onset. The PRC for muscimol had a shape similar to a PRC previously generated for the short-acting benzodiazepine, triazolam. Single microinjections of different doses of muscimol given 6 h before activity onset induced phase-advances in a dose-dependent fashion. Histological analysis revealed that phase shifts induced by the administration of muscimol were associated with the proximity of the injection site to the SCN area. These data indicate that a GABAergic system may exist within the suprachiasmatic region as part of a central biological clock responsible for the regulation of the circadian rhythm of locomotor activity in the golden hamster.
Animals↗
Bioluminescence of the Ca2+-binding photoprotein aequorin after cysteine modification.
Aequorin is a monomeric Ca2+-binding protein (Mr, 21,400) that emits light upon reacting with Ca2+. The protein has three Ca2+-binding sites, three cysteine residues, and a noncovalently bound chromophore that consists of coelenterazine and molecular oxygen. Light is emitted via an intramolecular reaction in which coelenterazine is oxidized by the bound oxygen. After light emission, aequorin may be regenerated by incubating the protein with coelenterazine, dissolved oxygen, EDTA, and 2-mercaptoethanol. To understand structure-function relationships in this protein, we used the technique of site-specific mutagenesis to replace the three cysteine residues with serine. Six of the seven modified aequorins had reduced luminescence activity, whereas the seventh with all three cysteines replaced by serine had luminescence activity equal to or greater than that of the wild-type aequorin. Further, the time required for the regeneration of the triply substituted aequorin was substantially increased compared to the time required for the regeneration of the wild-type aequorin. The results suggest that cysteine plays an important role in the regeneration of aequorin but not in its catalytic activity.
Aequorin↗