Showing papers by "Hideo Hayashi published in 2000"

Identification of novel VirR/VirS-regulated genes in Clostridium perfringens

Abstract: Novel genes that are regulated in Clostridium perfringens by the two-component regulatory system, VirR/VirS, were identified using a differential display method. A plasmid library was constructed from C. perfringens chromosomal DNA, and the plasmids were hybridized with cDNA probes prepared from total RNA of wild-type strain 13 and its virR mutant derivative TS133. Three clones were identified that carry newly identified VirR/VirS-regulated genes, two of which were positively regulated and one of which was negatively regulated. Genes located on the identified clones were deduced by nucleotide sequencing, and the target genes of the VirR/VirS system were identified with a set of Northern hybridizations. A 4.9 kb mRNA transcribing the metB (cystathionine gamma-synthase), cysK (cysteine synthase) and ygaG (hypothetical protein) genes was negatively regulated, whereas 1.6 and 6.0 kb transcripts encoding ptp (protein tyrosine phosphatase) and cpd (2',3'-cyclic nucleotide 2'-phosphodiesterase) respectively, were shown to be positively regulated by the VirR/VirS system. The other gene, hyp7, whose transcript was positively regulated by the VirR/VirS system, was shown to activate the transcription of the colA (kappa-toxin) and plc (alpha-toxin) genes, but not the pfoA (theta-toxin) gene in C. perfringens. These results suggested that the global regulatory system VirR/VirS could regulate various genes, other than toxin genes, both positively and negatively and that the hyp7 gene might encode a novel regulatory factor for toxin production in C. perfringens.

Genetic analysis of the ycgJ-metB-cysK-ygaG operon negatively regulated by the VirR/VirS system in Clostridium perfringens.

Abstract: The 5'-flanking region of the metB-cysK-ygaG operon, whose expression is negatively regulated by the VirR/VirS system in C. perfringens, was analyzed. The region contained the ycgJ, mscL, and colA genes encoding a hypothetical protein, a large conductance mechanosensitive channel protein, and kappa-toxin (collagenase), respectively. Northern analysis revealed that the ycgJ gene was transcribed as a 4.9-kb operon together with the metB-cysK-ygaG genes and that this operon was negatively regulated by the VirR/VirS system. It is indicated that the pfoA (theta-toxin or perfringolysin O), colA, and ycgJ-metB-cysK-ygaG genes that belong to the VirR/VirS regulon are situated very close together in a 26.5-kb region of the chromosome, but do not form a pathogenic island.

Epidemiological analysis of methicillin resistant Staphylococcus aureus in Thailand.

Abstract: The geographical distribution of 65 clinical isolates of methicillin resistant Staphylococcus aureus (MRSA) recovered from 7 hospitals in Thailand was investigated. The presence of mecA gene in MRSA was determined by specific PCR with the use of primers 5´-GTAGTTGTCGGGTTTGGT-3´ and 5´- GGTATCATCTTGTACCCA-3´. Chromosomal DNA restriction analysis with SmaI was resolved by pulsed- field gel electrophoresis (PFGE) compared with antibiotype analysis and phage type analysis. All 65 strains carried mecA gene. They all were resistant to penicillin, tetracycline, erythromycin, amoxicillin/clavulanic acid and variably resistant to gentamicin, ofloxacin, trimethoprim-sulfamethoxazole, chloramphenicol, fosfomycin and clindamycin; and all isolates were susceptible to vancomycin. A total of 19 PFGE patterns designated

Trichinella spiralis-specific monoclonal antibodies and affinity-purified antigen-based diagnosis.

Abstract: Hybridomas secreting monoclonal antibodies (MAbs) to Trichinella spiralis were produced. Myeloma cells were fused with splenocytes of a mouse immunized with excretory-secretory (E-S) antigen of infective larvae. A large percentage of growing hybrids secreted antibodies cross-reactive to many of 23 heterologous parasites tested. Only 6 monoclones (designated 3F2, 5D1, 10F6, 11E4, 13D6 and 14D11) secreted MAbs specific to the E-S antigen and/or a crude extract (CE) of T. spiralis infective larvae. The 6 monoclones secreted IgM, IgG3, IgM, IgG3, IgG3 and IgG3, respectively. Clone 5D1 was selected to mass produce MAbs which were then coupled to CNBr-activated Sepharose CL-4B to prepare an affinity-purified antigen. Dot-blot ELISA with either purified antigen or CE was evaluated. There were 17 patients with acute trichinellosis and 76 individuals convalescing from T. spiralis infection (group 1). Controls were 170 patients with parasitic infections other than trichinellosis (group 2) and 35 healthy parasite-free controls (group 3). CE-ELISA was positive in all group 1 patients. However, sera from many group 2 patients also were reactive (opisthorchiasis-44.2%, schistosomiasis-44%, gnathostomiasis-30%, paragonimiasis-28.6%, taeniasis-27.3%, strongyloidiasis-23.1% and hookworm infections-20%). Affinity-purified antigen was 100% specific, all sera from group 2 and group 3 individuals tested negative. Although 74 of 76 patients (97.4%) with convalescing trichinellosis tested positive, sera from only 3 of 17 patients (17.6%) with acute T. spiralis were reactive. Thus, CE antigen is appropriate when sensitivity is needed, while purified antigen should be used when specificity is required. Dot-blot ELISA is easier to perform, more rapid and less expensive than indirect ELISA. Many samples can be assayed simultaneously, special equipment is not required, and results can be preserved for retrospective analysis. Dot-blot ELISA is therefore the method of choice for the rapid diagnosis of trichinellosis, particularly when more complex laboratory tests are unavailable.