研究者業績

越智 定幸

ochi sadayuki

基本情報

所属
藤田保健衛生大学 医学部 医学科 微生物学 准教授
学位
博士(薬学)

J-GLOBAL ID
201501019788646061
researchmap会員ID
7000012700

MISC

 7
  • Kentaro Tsukamoto, Hideyuki Arimitsu, Sadayuki Ochi, Keiji Nakamura, Yoshikazu Tanaka, Nipawan Nuemket, Koki Taniguchi, Shunji Kozaki, Takao Tsuji
    MICROBIOLOGY AND IMMUNOLOGY 56(10) 664-672 2012年10月  査読有り
    Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release at peripheral nerve terminals. They are serologically classified from A to G, C/D and D/C mosaic neurotoxins forming further subtypes of serotypes C and D. Cultured primary neurons, as well as neuronal cell lines such as PC12 and Neuro-2a, are often utilized in cell-based experiments on the toxic action of botulinum toxins. However, there are very few reports of the use of neural cell lines for studying BoNTs/C and D. In addition, the differentiated P19 neuronal cell line, which possesses cholinergic properties, has yet to be tested for its susceptibility to BoNTs. Here, the responsiveness of differentiated P19 cells to BoNT/C and BoNT/DC is reported. Both BoNT/C and BoNT/DC were shown to effectively bind to, and be internalized by, neurons derived from P19 cells. Subsequently, the intracellular substrates for BoNT/C and BoNT/DC were cleaved by treatment of the cells with the toxins in a ganglioside-dependent manner. Moreover, P19 neurons exhibited high sensitivity to BoNT/C and BoNT/DC, to the same extent as cultured primary neurons. These findings suggest that differentiated P19 cells possess full sensitivity to BoNT/C and BoNT/DC, thus making them a novel susceptible cell line for research into BoNTs.
  • Masataka Oda, Manabu Hashimoto, Masaya Takahashi, Yuka Ohmae, Soshi Seike, Ryoko Kato, Aoi Fujita, Hideaki Tsuge, Masahiro Nagahama, Sadayuki Ochi, Teppei Sasahara, Shunji Hayashi, Yoshikazu Hirai, Jun Sakurai
    PLOS ONE 7(6) e38054 2012年6月  査読有り
    Bacillus cereus (B. cereus) is a pathogen in opportunistic infections. Here we show that Bacillus cereus sphingomyelinase (Bc-SMase) is a virulence factor for septicemia. Clinical isolates produced large amounts of Bc-SMase, grew in vivo, and caused death among mice, but ATCC strains isolated from soil did not. A transformant of the ATCC strain carrying a recombinant plasmid containing the Bc-SMase gene grew in vivo, but that with the gene for E53A, which has little enzymatic activity, did not. Administration of an anti-Bc-SMase antibody and immunization against Bc-SMase prevented death caused by the clinical isolates, showing that Bc-SMase plays an important role in the diseases caused by B. cereus. Treatment of mouse macrophages with Bc-SMase resulted in a reduction in the generation of H2O2 and phagocytosis of macrophages induced by peptidoglycan (PGN), but no effect on the release of TNF-alpha and little release of LDH under our experimental conditions. Confocal laser microscopy showed that the treatment of mouse macrophages with Bc-SMase resulted in the formation of ceramide-rich domains. A photobleaching analysis suggested that the cells treated with Bc-SMase exhibited a reduction in membrane fluidity. The results suggest that Bc-SMase is essential for the hydrolysis of SM in membranes, leading to a reduction in phagocytosis.
  • Nagahama M, Oda M, Ochi S, Kobayashi K, Sakurai J
    J Glycom Lipidom S3 001 2012年  査読有り
  • Nagahama M, Oda M, Ochi S, Kobayashi K, Sakurai J
    Res Adv Infect Immun 1 1-12 2011年  査読有り
  • Hideyuki Arimitsu, Kentaro Tsukamoto, Sadayuki Ochi, Keiko Sasaki, Michio Kato, Koki Taniguchi, Keiji Oguma, Takao Tsuji
    PROTEIN EXPRESSION AND PURIFICATION 67(2) 96-103 2009年10月  査読有り
    Cholera toxin (CT) B subunit (CTB) was overproduced using a novel expression system in Escherichia coli. An expression plasmid was constructed by inserting the gene encoding the full-length CTB and the Shine-Dalgarno (SD) sequence derived from CTB or from the heat-labile enterotoxin B subunit (LTB) of enterotoxigenic E. coli into the lacZ alpha gene fragment in the pBluescript SK(+) vector. The E. coli strain MV1184 was transformed with each plasmid and then cultured in CAYE broth containing lincomycin. Recombinant CTB (rCTB) was purified from each cell extract. rCTB was overproduced in both transformants without obvious toxicity and was structurally and biologically identical to that of CT purified from Vibrio cholerae, indicating that the original SD and CTB signal sequences were also sufficient to express rCTB in E. coli. Lincomycin-induced rCTB expression was inhibited by mutating the lac promoter, suggesting that lincomycin affects the lactose operon. Based on these findings, we constructed a plasmid that contained the wild-type CT operon and successfully overproduced CT (rCT) using the same procedure for rCTB. Although rCT had an intact A subunit, the amino-terminal modi. cations and biological properties of the A and B subunits of rCT were identical to those of CT. These results suggest that this novel rCTB over-expression system would also be useful to generate both wild-type and mutant CT proteins that will facilitate further studies on the characteristics of CT, such as mucosal adjuvant activity. (C) 2009 Elsevier Inc. All rights reserved.

講演・口頭発表等

 29

所属学協会

 1

教育内容・方法の工夫(授業評価等を含む)

 1
  • 件名
    パワーポイントと板書による講義
    開始年月日
    2009
    終了年月日
    2013
    概要
    M2「微生物学」においてパワーポイントと板書を用いた講義を行い、学生の理解度を確認し講義レベルの調整に努めた。