Biomedical subjects

S Inouye

Publications and source records attributed to S Inouye.

At least 181 records · Page 10Linked to original sources

PubMed1995

[Lytic action of cefminox against slowly growing bacteria].

Lytic action of cefminox (CMNX) against slowly growing Escherichia coli (E. coli) K-12 JE1011 was compared with that of the related cephamycin cefmetazole (CMZ) or ampicillin (ABPC). The growth rate in doubling time was 30 min. at 37 degrees C. Minimal concentrations of CMNX, CMZ and ABPC showing clear lysis were 0.39, 3.13 and 12.5 micrograms/ml, respectively, under these conditions. When E. coli was cultured at 12 degrees C, the doubling time was 720 and 816 min. which were 24 and 27.2 fold as slow as those at 37 degrees C, respectively. In 12 degrees C culture, the minimal concentrations showing clear lysis were 0.78 (2 fold at 37 degrees C), 25 (8 fold) and 100 micrograms/ml and higher (8 fold or more) with CMNX, CMZ and ABPC, respectively. Therefore, it appeared that the lytic activity of CMNX was not significantly affected by bacterial growth rate and was demonstrated effectively against slowly growing bacteria at a low concentration. We determined release activity of cell wall peptidoglycan fragments by 5 beta-lactam antibiotics in addition to the above-mentioned 3 antibiotics. The results showed that the release activity was not proportional to the MIC and that of CMNX was the strongest among those antibiotics. The rapid and strong bactericidal action of CMNX against bacteria at the early stationary phase was suggested to result from the characteristic lytic action against slowly growing bacteria.

Ampicillin↗

PubMed1995

Adenovirus surveillance, 1982-1993, Japan. A report of the National Epidemiological Surveillance of Infectious Agents in Japan.

The Infectious Agents Surveillance Center, the National Institute of Health, Japan, received 17,265 reports from 1982 to 1993 on cases from whom adenovirus was isolated or detected; 85% from 57 public health institutes and the other 15% from two national hospitals and two commercial diagnostic laboratories. The followings were found. Three major diseases caused by adenovirus were upper respiratory tract infection, gastroenteritis, and conjunctivitis. Patients of upper respiratory tract infection numbered 6,837 (40% of all patients due to adenovirus), the identified serotypes being in order of frequency types 3, 2, 1, and 5. Those of gastroenteritis numbered 1,636 (9.5%). From 40% of the gastroenteritis patients, adenovirus was detected by electron microscopy or immunochemical methods without cultivation. From the remaining 60%, virus was isolated in tissue culture; the serotypes of the isolates resembled those causing upper respiratory tract infection. Patients of conjunctivitis numbered 3,437 (20%), the frequency being in order of types 3, 4, 8, 37, and 19. Conjunctivitis due to types 3 and 4 prevailed every summer; type 3 was isolated often from children with pharyngo-conjunctival fever and the other four types were mostly from adults with epidemic keratoconjunctivitis. Type 3 had a unique feature not seen in other types: it was most frequently isolated, causing upper respiratory tract infection, gastroenteritis, conjunctivitis, and pharyngo-conjunctival fever. Reports on isolation of type 7, which has been reported to cause severe pneumonia in many other countries, were as few as 28 (0.2%).

Adenoviridae Infections↗

PubMed1995

[Active transport of fosfomycin into cells of Escherichia coli, multidrug-resistant Pseudomonas aeruginosa and Staphylococcus aureus].

We examined effect of fosfomycin (FOM) on growth of Escherichia coli (E. coli) NIHJ JC-2, multidrug-resistant Pseudomonas aeruginosa (P. aeruginosa) PFS80 and Staphylococcus aureus (S. aureus) Smith. FOM inhibited the growth of these organisms at 0.1 approximately 0.5, 10 approximately 50 or 5 approximately 50 micrograms ml, respectively. In E. coli, FOM clearly lyzed the cells at 0.5 microgram/ml. These 3 bacteria incorporated radiolabeled FOM proportionately to incubation time. Intracellular concentration of FOM was estimated by considering the water content of E. coli cells. The ratio of intracellular to extracellular concentrations of FOM was larger than 1 after 5 approximately 10 min. incubation with FOM and reached about 7 after 40 min. FOM was incorporated actively into P. aeruginosa over extracellular concentration after 40-min. incubation on the basis of the above-mentioned water content. It was suggested that FOM was incorporated actively into various bacterial cells, and inhibited efficiently cell wall peptidoglycan synthesis, thus inhibited bacterial growth or lyzed the bacterial cells.

Anti-Bacterial Agents↗

PubMed1994

Stereospecificity of hydride transfer and substrate specificity for FMN-containing NAD(P)H-flavin oxidoreductase from the luminescent bacterium, Vibrio fischeri ATCC 7744.

The stereospecificity of the hydride transfer in NAD(P)H-flavin reductase reaction of V. fischeri ATCC 7744 was determined by 1H-NMR spectroscopy using stereospecifically labeled reduced beta-nicotinamide adenine dinucleotide (beta-NADH). The recombinant flavoenzyme, purified from E. coli cells, selectively transferred the pro-R hydrogen at the C-4 position of the nicotinamide ring to flavin and is therefore classified as an A-side specific enzyme. Lumiflavin was used for the reductase reaction, but lumichrome and alpha-NADH were not utilized as electron acceptor and donor, respectively.

FMN Reductase↗

PubMed1994

Circadian rhythms of adenosine triphosphate contents in the suprachiasmatic nucleus, anterior hypothalamic area and caudate putamen of the rat--negative correlation with electrical activity.

Contents of adenosine triphosphate (ATP) in the suprachiasmatic nucleus (SCN), anterior hypothalamic area (AH) and caudate putamen (CPu) were determined every 4 h in rats kept under constant dark conditions. All tested regions of the brain exhibited circadian rhythms in ATP content. Among these, the amplitude of the rhythm was largest in the SCN. Peaks of the ATP content were observed at circadian time 18 in the SCN, 6 in the AH and 10 in the CPu. In view of the observation that at around these peak times electrical or glucose uptake activity of each brain region was lowest, the present result suggests that ATP content is negatively correlated with electrical or glucose uptake activity.

Adenosine Triphosphate↗

PubMed1994

Evidence for redox forms of the Aequorea green fluorescent protein.

Highly purified recombinant Aequorea green fluorescent protein is able to undergo a reversible oxidation-reduction reaction in the presence of molecular oxygen. In the oxidized form in near UV light, the protein is highly fluorescent, but when reduced with sodium dithionite, it becomes completely non-fluorescent. On exposure to molecular oxygen the reduced, non-fluorescent protein reverts to its original fluorescent state.

Amino Acid Sequence↗

PubMed1994

NAD(P)H-flavin oxidoreductase from the bioluminescent bacterium, Vibrio fischeri ATCC 7744, is a flavoprotein.

The NAD(P)H-flavin oxidoreductase gene from the bioluminescent bacterium, Vibrio fischeri ATCC 7744, was expressed in Escherichia coli, and the enzyme purified using Cibacron Blue 3G-A affinity column chromatography from crude extracts in a single step. The purified enzyme had a typical flavoprotein absorption spectrum and flavin mononucleotide (FMN) was identified as a prosthetic group, non-covalently bound in a molar ratio of 1:1. The enzyme catalyzed the electron transfer from NADH via FMNH2 to various other electron acceptors. Reduced flavin produced by flavin reductase participated non-enzymatically in the following reactions: H2O2-forming NADH oxidase-like, oxygen-insensitive nitroreductase-like, diaphorase (quinone reductase)-like and bacterial luciferase reactions.

Amino Acid Sequence↗

PubMed1994

Circadian rhythms of norepinephrine in the rat suprachiasmatic nucleus.

Norepinephrine (NE) contents in the suprachiasmatic nucleus (SCN) in rats kept under light-dark (LD) or constant dark (DD) conditions displayed significant variations over one day with a peak during the subjective day and a trough during the subjective night. These results indicate that rhythmic variation of NE in the SCN is driven by an endogenous pacemaker and independent of external light. In agreement with these observations, immunocytochemical study localized tyrosine hydroxylase immunopositive fibers in the medial part of the SCN. Unlike serotonin, histamine and neuropeptide Y projections to the ventrolateral SCN, NE innervation may represent the general activity level of the brainstem, from where these fibers arise.

Animals↗

PubMed1994

Requirements of the secondary structures in the primary transcript for multicopy single-stranded DNA synthesis by reverse transcriptase from bacterial retron-Ec107.

Multicopy single-stranded DNA (msDNA) is produced by bacterial retroelements called retrons. It consists of single-stranded DNA that is linked to an internal G residue of an RNA molecule by a 2',5'-phosphodiester linkage. It has been demonstrated that specific primary sequences, as well as the secondary structures immediately downstream of the G residue, are essential for the cDNA priming reaction (Shimamoto, T., Hsu, M.-Y., Inouye, S., and Inouye, M. (1993) J. Biol. Chem. 268, 2684-2692). We have now examined the requirement of the structures in the region corresponding to DNA for msDNA synthesis. The upper stem region consisting of 71 bases of msDNA-Ec107 was found not to be essential, and this region could be deleted to efficiently produce a truncated msDNA containing only a 36-base single-stranded DNA. Various mutations including base replacements, deletions, and insertions were constructed in the lower stem region. It was found that any mutations resulting in more stable secondary structures caused reduction in msDNA synthesis. The results indicated that reverse transcriptase requires a loose secondary structure in the template RNA near the cDNA priming site for cDNA elongation.

Base Sequence↗

PubMed1994

Structural similarity of a developmentally regulated bacterial spore coat protein to beta gamma-crystallins of the vertebrate eye lens.

The solution structure of Ca(2+)-loaded protein S (M(r) 18,792) from the Gram-negative soil bacterium Myxococcus xanthus has been determined by multidimensional heteronuclear NMR spectroscopy. Protein S consists of four internally homologous motifs, arranged to produce two domains with a pseudo-twofold symmetry axis, overall resembling a triangular prism. Each domain consists of two topologically inequivalent "Greek keys": the second and fourth motifs form standard Greek keys, whereas the first and third motifs each contain a regular alpha-helix in addition to the usual four beta-strands. The structure of protein S is similar to those of the vertebrate eye lens beta gamma-crystallins, which are thought to be evolutionarily related to protein S. Both protein S and the beta gamma-crystallins function by forming stable multimolecular assemblies. However, protein S possesses distinctive motif organization and domain packing, indicating a different mode of oligomerization and a divergent evolutionary pathway from the beta gamma-crystallins.

Animals↗

PubMed1994

Aequorea green fluorescent protein. Expression of the gene and fluorescence characteristics of the recombinant protein.

Expression of the cDNA for Aequorea green fluorescent protein in E. coli yielded a fused protein with fluorescence excitation and emission spectra virtually identical to those of the native green fluorescent protein. Further, a solution of the protein, when mixed with aequorin and calcium ion, emitted a greenish luminescence characteristic of the in vivo luminescence of the animal, indicating a radiationless energy transfer to the protein.

Amino Acid Sequence↗

PubMed1994

Unusual helix-containing greek keys in development-specific Ca(2+)-binding protein S. 1H, 15N, and 13C assignments and secondary structure determined with the use of multidimensional double and triple resonance heteronuclear NMR spectroscopy.

Multidimensional heteronuclear NMR spectroscopy has been used to determine almost complete backbone and side-chain 1H, 15N, and 13C resonance assignments of calcium loaded Myxococcus xanthus protein S (173 residues). Of the range of constant-time triple resonance experiments recorded, HNCACB and CBCA(CO)NH, which correlate C alpha and C beta with backbone amide resonances of the same and the succeeding residue respectively, proved particularly useful in resolving assignment ambiguities created by the 4-fold internal homology of the protein S amino acid sequence. Extensive side-chain 1H and 13C assignments have been obtained by analysis of HCCH-TOCSY and 15N-edited TOCSY-HMQC spectra. A combination of NOE, backbone amide proton exchange, 3JNH alpha coupling constant, and chemical shift data has been used to show that each of the protein S repeat units consists of four beta-strands in a Greek key arrangement. Two of the Greek keys contain a regular alpha-helix between the third and fourth strands, resulting in an unusual and possibly unique variation on this common folding motif. Despite similarity between two nine-residue stretches in the first and third domains of protein S and one of the Ca(2+)-binding sequences in bovine brain calmodulin [Inouye, S., Franceschini, T., & Inouye, M. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6829-6833], the protein S topology in these regions is incompatible with an EF-hand calmodulin-type Ca(2+)-binding site.

Amino Acid Sequence↗

PubMed1994

NMR-derived three-dimensional solution structure of protein S complexed with calcium.

BACKGROUND: Protein S is a developmentally-regulated Ca(2+)-binding protein of the soil bacterium Myxococcus xanthus. It functions by forming protective, multilayer spore surface assemblies which may additionally act as a cell-cell adhesive. Protein S is evolutionarily related to vertebrate lens beta gamma-crystallins. RESULTS: The three-dimensional solution structure of Ca(2+)-loaded protein S has been determined using multi-dimensional heteronuclear NMR spectroscopy. (Sixty structures were calculated, from which thirty were selected with a root mean square difference from the mean of 0.38 A for backbone atoms and 1.22 A for all non-hydrogen atoms.) The structure was analyzed and compared in detail with X-ray crystallographic structures of beta gamma-crystallins. The two internally homologous domains of protein S were compared, and hydrophobic cores, domain interfaces, surface ion pairing, amino-aromatic interactions and potential modes of multimerization are discussed. CONCLUSIONS: Structural features of protein S described here help to explain its overall thermostability, as well as the higher stability and Ca2+ affinity of the amino-terminal domain relative to the carboxy-terminal domain. Two potential modes of multimerization are proposed involving cross-linking of protein S molecules through surface Ca(2+)-binding sites and formation of the intramolecular protein S or gamma B-crystallin interdomain interface in an intermolecular content. This structural analysis may also have implications for Ca(2+)-dependent cell-cell interactions mediated by the vertebrate cadherins and Dictyostelium discoideum protein gp24.

Amino Acid Sequence↗

PubMed1994

Specificity of priming reaction of HIV-1 reverse transcriptase, 2'-OH or 3'-OH.

It has not been unambiguously demonstrated whether the priming reaction of human immunodeficiency virus, type 1 (HIV-1) cDNA synthesis initiates with either the 2'-OH or 3'-OH group of the 3'-terminal adenosine residue of tRNA(Lys-3). In this report, we synthesized tRNA(Lys-3) of which the 3'-terminal adenosine residue lacks either a 2'-OH or 3'-OH. These tRNA molecules were used for the HIV-1 cDNA-priming reaction in a cell-free system consisting of a 141-base RNA template and purified HIV-1 reverse transcriptase. It was found that under the conditions used, the tRNA containing the 2'-deoxyadenosine was able to initiate the cDNA synthesis, while the tRNA with the 3'-deoxyadenosine was not. The results show that retroviral reverse transcriptase specifically primes cDNA synthesis from the 3'-OH group. This is in contrast to bacterial reverse transcriptase, which initiates cDNA synthesis from the 2'-OH group of an internal guanosine residue of a template RNA.

Base Sequence↗

PubMed1994

Identification of the Myxococcus xanthus 59-kDa membrane-associated GTP-binding protein as a proton-translocating ATPase.

Five GTP-binding proteins have been detected in Myxococcus xanthus by photoaffinity cross-linking with azido-GTP [Muñoz-Dorado et al., J. Biol. Chem. 265 (1990a) 2702-2706]. One of them, the 59-kDa membrane-associated GTP-binding protein, has been purified. The N-terminal sequence of a 10-kDa fragment from the protease V8 digestion of the purified protein has been determined and degenerate oligodeoxyribonucleotides based on that sequence have been used to isolate and clone the gene that encodes the GTP-binding protein. The gene was sequenced and further analysis of the sequence revealed that the protein encoded by this gene shows very high homology with the alpha subunit of proton-translocating ATPases.

Amino Acid Sequence↗

PubMed1994

Production of single-stranded DNA in mammalian cells by means of a bacterial retron.

msDNA-Ec67, a peculiar multicopy single-stranded DNA of a specific sequence was produced in NIH3T3 mouse cells. Retron-Ec67, a retroelement from Escherichia coli, was introduced under the T7 polymerase promoter and the non-translated 5'-region of the encephalomyocarditis virus. The construct was then transfected into the NIH3T3 cells constitutively producing T7 RNA polymerase. Forty-eight hours after transfection, msDNA-Ec67 was detected in the cells by means of Southern blot hybridization and reverse transcriptase extension assay. The potential use of bacterial retrons as a vector for single-stranded DNA production in mammalian cells is discussed.

3T3 Cells↗

PubMed1994

Day-night variation of preprosomatostatin messenger RNA level in the suprachiasmatic nucleus.

Day-night variation of the preprosomatostatin mRNA level in the suprachiasmatic nucleus (SCN), the site of the circadian pacemaker, was determined in rats maintained under light-dark cycles by semiquantitative Northern blot hybridization of micropunched tissues. Preprosomatostatin mRNA abundance in the SCN showed significant variation during a day with a peak level at around light on and a trough at around 4 h after light off. This time course was essentially identical with that previously found in blinded rats, indicating that environmental light did not produce an immediate effect on the preprosomatostatin mRNA level in the SCN. Moreover, this rhythm in the preprosomatostatin mRNA appeared specific to the SCN, since levels in the cortex and anterior hypothalamus did not oscillate even under light-dark conditions.

Animals↗

PubMed1994

3'-terminal sequence of a small round structured virus (SRSV) in Japan.

We determined the nucleotide sequence of about 1,000 bases from the 3'-terminus of a small round structured virus (SRSV), which caused a gastroenteritis outbreak in Chiba Prefecture, Japan, in 1987. The sequence was compared with the corresponding sequence region of Norwalk virus; it consisted of a part of the open reading frame 2 (ORF2), whole ORF3, and 3'-noncoding region (NCR). The 624-base-long ORF3 had sequence homology of 68% with the corresponding region of Norwalk virus. (The amino acid sequence homology was 74%.) The 94-base-long NCR had 65% homology with Norwalk virus. We then selected two consensus-sequence portions in the above sequence between Chiba and Norwalk viruses for primers in the reverse transcriptase-polymerase chain reaction (RT-PCR). Using this primer set, we detected 669-bp bands in agarose gel electrophoresis of RT-PCR products from feces containing Chiba or Norwalk viruses. Furthermore, in Southern hybridization with Chiba probes which were labeled with digoxigenin-dUTP in PCR, the bands of the two viruses were clearly stained under a low stringency condition. Since both Chiba and Norwalk viruses were detected by the above primer set although they are geographically and chronologically different viruses, our primer-pair may be useful for detection of a broad range of SRSVs which cause gastroenteritis in different areas.

Base Sequence↗