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Hiroshi Yoshikura

Publications and source records attributed to Hiroshi Yoshikura.

At least 19 recordsLinked to original sources
PubMed2006

Outbreaks of multidrug-resistant Pseudomonas aeruginosa in community hospitals in Japan.

We previously reported an outbreak in a neurosurgery ward of catheter-associated urinary tract infection with multidrug-resistant (MDR) Pseudomonas aeruginosa strain IMCJ2.S1, carrying the 6'-N-aminoglycoside acetyltransferase gene [aac(6')-Iae]. For further epidemiologic studies, 214 clinical isolates of MDR P. aeruginosa showing resistance to imipenem (MIC >or= 16 microg/ml), amikacin (MIC >or= 64 microg/ml), and ciprofloxacin (MIC >or= 4 microg/ml) were collected from 13 hospitals in the same prefecture in Japan. We also collected 70 clinical isolates of P. aeruginosa that were sensitive to one or more of these antibiotics and compared their characteristics with those of the MDR P. aeruginosa isolates. Of the 214 MDR P. aeruginosa isolates, 212 (99%) were serotype O11. We developed a loop-mediated isothermal amplification (LAMP) assay and a slide agglutination test for detection of the aac(6')-Iae gene and the AAC(6')-Iae protein, respectively. Of the 212 MDR P. aeruginosa isolates, 212 (100%) and 207 (98%) were positive in the LAMP assay and in the agglutination test, respectively. Mutations of gyrA and parC genes resulting in amino acid substitutions were detected in 213 of the 214 MDR P. aeruginosa isolates (99%). Of the 214 MDR P. aeruginosa isolates, 212 showed pulsed-field gel electrophoresis patterns with >or=70% similarity to that of IMCJ2.S1 and 83 showed a pattern identical to that of IMCJ2.S1, indicating that clonal expansion of MDR P. aeruginosa occurred in community hospitals in this area. The methods developed in this study to detect aac(6')-Iae were rapid and effective in diagnosing infections caused by various MDR P. aeruginosa clones.

Acetyltransferases↗
PubMed2006

Emergence of rifampicin resistance in methicillin-resistant Staphylococcus aureus in tuberculosis wards.

To assess whether the occurrence of rifampicin (RFP) resistance in methicillin-resistant Staphylococcus aureus (MRSA) is related to treatment of tuberculosis, we determined the RFP susceptibility of MRSA isolates obtained from tuberculosis patients and screened for mutation(s) in the rpoB gene of these isolates. The MICs of RFP for 84 MRSA isolates obtained from two hospitals in Japan were determined. DNA was sequenced in the region 1318-1602 nucleotides (nt) of the rpoB gene, which includes RFP resistance-determining clusters I (1384-1464 nt, 462-488 amino acids). The majority of MRSA isolates from tuberculosis wards, i.e., 48 of 51 (94%) [33 of 34 in a Tokyo hospital (97%) and 15 of 17 in a Chubu hospital (88%)], were resistant to RFP. Meanwhile, no isolates of 33 from the other wards were resistant to RFP. All RFP-resistant MRSA isolates had a mutation(s), including novel mutation(s) such as Val453-->AEPhe, Asp471-->AEAsn, and Ile527-->AELeu, in rpoB. An emergence of RFP-resistant MRSAs in tuberculosis wards in Japan was strongly suggested.

Amino Acid Sequence↗
PubMed2005

Immunological detection of severe acute respiratory syndrome coronavirus by monoclonal antibodies.

In order to establish immunological detection methods for severe acute respiratory syndrome coronavirus (SARS-CoV), we established monoclonal antibodies directed against structural components of the virus. B cell hybridomas were generated from mice that were hyper-immunized with inactivated SARS-CoV virion. By screening 2,880 generated hybridomas, we established three hybridoma clones that secreted antibodies specific for nucleocapsid protein (N) and 27 clones that secreted antibodies specific for spike protein (S). Among these, four S-protein specific antibodies had in vitro neutralization activity against SARS-CoV infection. These monoclonal antibodies enabled the immunological detection of SARS-CoV by immunofluorescence staining, Western blot or immunohistology. Furthermore, a combination of monoclonal antibodies with different specificities allowed the establishment of a highly sensitive antigen-capture sandwich ELISA system. These monoclonal antibodies would be a useful tool for rapid and specific diagnosis of SARS and also for possible antibody-based treatment of the disease.

Animals↗
PubMed2005

International symposium on infectious agent transmission model building--focusing on assessment of risk to communities.

The Susceptible Infected and Recovery (SIR) Model proposed by Robert May in the UK is the basis of the present mathematical model building of infectious disease epidemics. Need for model building incorporating more social and other relevant factors has been recognized. An important example is the introduction of idea of the scale-free distribution of links among the people. More refined models by taking into account the nature of a pathogen, geo-sociological factors, lifestyles of the people, etc., have been developed. For example, Koopman proposed a model for prediction of epidemic expansion based on actual epidemiological data. Eubank proposed a model assessing the bio-terror attack using a model city where every day activity is going on. The present workshop, participated by experts from the US, the UK and Japan, is the first meeting of the proposed series of conference on this issue.

Communicable Diseases↗
PubMed2004

A subcutaneously injected UV-inactivated SARS coronavirus vaccine elicits systemic humoral immunity in mice.

The recent emergence of severe acute respiratory syndrome (SARS) was caused by a novel coronavirus, SARS-CoV. It spread rapidly to many countries and developing a SARS vaccine is now urgently required. In order to study the immunogenicity of UV-inactivated purified SARS-CoV virion as a vaccine candidate, we subcutaneously immunized mice with UV-inactivated SARS-CoV with or without an adjuvant. We chose aluminum hydroxide gel (alum) as an adjuvant, because of its long safety history for human use. We observed that the UV-inactivated SARS-CoV virion elicited a high level of humoral immunity, resulting in the generation of long-term antibody secreting and memory B cells. With the addition of alum to the vaccine formula, serum IgG production was augmented and reached a level similar to that found in hyper-immunized mice, though it was still insufficient to elicit serum IgA antibodies. Notably, the SARS-CoV virion itself was able to induce long-term antibody production even without an adjuvant. Anti-SARS-CoV antibodies elicited in mice recognized both the spike and nucleocapsid proteins of the virus and were able to neutralize the virus. Furthermore, the UV-inactivated virion induced regional lymph node T-cell proliferation and significant levels of cytokine production (IL-2, IL-4, IL-5, IFN-gamma and TNF-alpha) upon restimulation with inactivated SARS-CoV virion in vitro. Thus, a whole killed virion could serve as a candidate antigen for a SARS vaccine to elicit both humoral and cellular immunity.

Adjuvants, Immunologic↗
PubMed2004

Oligo(dA-dT)-dependent signal amplification for the detection of proteins in cells.

An ultrasensitive protein detection system in situ named the ImmunoAT-tailing method was developed. It consists of three elementary processes: (i) detection of a protein by a primary antibody and a biotinylated secondary antibody; (ii) linking of biotinylated 15-base oligo(dA-dT) to the biotinylated immunocomplex via streptavidin; and (iii) self-priming elongation of oligo(dA-dT) by the Klenow fragment, 3' to 5' exo-. After the elongation reaction in the presence of dATP, dTTP, and dye-labeled dUTP, the protein was labeled with a large number of the dye molecules. The poly(dA-dT) elongated without the labeled nucleotides was detected by 4',6-diamidino-2-phenylindole (DAPI) staining. By combining the different labelings, double staining was possible. This ImmunoAT-tailing method has a specificity and sensitivity higher than that of tyramide signal amplification.

Animals↗
PubMed2003

Statistics of trinucleotides in coding sequences and evolution.

The aim of this paper is to give measurements indicative of evolutional stages of the species. Two types of statistics of trinucleotides in coding regions are analysed for 27 species. The first one is the codon space, the nucleotide ratio for each of the three codon positions. We apply principal component analysis on this space and extract two principal components faithfully describing the original distribution of the codon space. The first principal component corresponds to the GC content. The second principal component classifies the species into three evolutional groups, Archaea, Bacteria and Eukaryota. The second statistics is the real and theoretical frequency of amino acids. The real frequency of an amino acid in a coding sequence is its frequency in the translated protein. The theoretical frequency is the expected frequency calculated from the ratio of nucleotides. We introduce the discrepancy between these two frequencies as an index of non-randomness of nucleotides in the sequence. This index of non-randomness divides the species into two groups: eukaryotes having smaller non-randomness (i.e. being more random) and prokaryotes having higher non-randomness.

Animals↗
PubMed2003

An Escherichia coli strain, BJ5183, that shows highly efficient conservative (two-progeny) DNA double-strand break repair of restriction breaks.

We examined the mode of recombination in an Escherichia coli strain, BJ5183, which has been frequently used in recovery and cloning of eukaryotic DNA. One of the important criteria in characterizing a homologous recombination mechanism is whether it produces two recombinant DNA molecules or only one recombinant DNA molecule out of two parental DNA molecules. Our previous work transferring plasmid molecules with a restriction break into Escherichia coli cells distinguished two modes in recombination stimulated by a double-strand break. In a recBC sbcA mutant strain, where recET genes on the Rac prophage are responsible for recombination (RecE pathway), recombination is often conservative, in the sense that it generates two recombinants out of two parental DNAs. In a recBC sbcBC mutant strain, in which recA and recF genes are responsible (RecF pathway), recombination is non-conservative, in the sense that it generates only one recombinant out of two parental DNAs. Unexpectedly, BJ5183, described as recBC sbcBC, showed very efficient conservative (two-progeny) double-strand break repair. Moreover, this recombination was not eliminated by disruption of its recA gene, which is essential to the RecF pathway. Our polymerase chain reaction analysis detected a recET gene homologue in this strain. This region was easily replaced by a RECT::Tn10 through general transduction and the resulting recT-negative derivative was defective in the conservative double-strand break repair. These results led us to conclude that, in strain BJ5183, the action of recET homologue is responsible for the conservative double-strand break repair as in the RecE pathway. BJ5183 carries a mutation in the endA gene, which codes for Endonuclease I. An endA mutation conferred a higher double-strand break-repair activity to a recBC sbcA mutant strain.

Bacterial Proteins↗
PubMed2003

In situ hybridization AT-tailing with catalyzed signal amplification for sensitive and specific in situ detection of human immunodeficiency virus-1 mRNA in formalin-fixed and paraffin-embedded tissues.

In situ hybridization is one of the most important techniques to visualize gene expression at the cellular level in various tissues. The in situ hybridization-AT tailing (ISH-AT) method uses a specially designed and synthesized oligonucleotide probe that has (AT)10 on the 3' side. This (AT)10 of the probe is elongated by DeltaTth DNA polymerase in the presence of dATP, dTTP, and labeled dUTP in the tissue after hybridization. Through this process the target is labeled with many hapten molecules. In this study, we detected human immunodeficiency virus type 1 RNA in formalin-fixed and paraffin-embedded tissues obtained from autopsied patients with acquired immunodeficiency syndrome by combining ISH-AT with the catalyzed signal amplification (CSA) system (ISH-AT-CSA), although we failed to detect signals from the same samples by conventional in situ hybridization using RNA probes (RISH) with CSA (RISH-CSA). We demonstrated that the ISH-AT-CSA method was superior to RISH-CSA in terms of both sensitivity and specificity, and that it was applicable to fluorescence in situ hybridization and double staining with immunohistochemistry for the characterization of cell phenotypes.

Adenine↗