Updated on 2021/12/26

写真a

 
MATSUSHITA Osamu
 
Organization
Faculty of Medicine, Dentistry and Pharmaceutical Sciences Professor
Position
Professor
External link

Degree

  • Doctor of Philosophy in Medical Science ( 1988.3   Okayama University )

Research Interests

  • Bacterial toxins

  • matrix-anchoring drugs

  • domain shuffling

Research Areas

  • Life Science / Bacteriology  / 毒素遺伝子の転写・翻訳レベルでの発現調節機構の解明

  • Life Science / Applied microbiology  / ドメイン・シャフリングによる新規薬物シーズの研究開発

Education

  • Okayama University   大学院医学研究科  

    1984.4 - 1988.3

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    Country: Japan

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  • Okayama University   医学部   医学科

    1978.4 - 1984.3

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    Country: Japan

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Research History

  • Okayama University   Graduate School of Medicine , Dentistry and Pharmaceutical Sciences   Professor

    2012.4

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    Country:Japan

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  • Kitasato University   School of Medicine   Professor

    2007.2 - 2012.3

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    Country:Japan

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  • Kagawa University   Faculty of Medicine   Associate Professor (as old post name)

    2003.10 - 2007.1

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    Country:Japan

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  • Kagawa Medical University   School of Medicine   Associate Professor (as old post name)

    1999.4 - 2003.9

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    Country:Japan

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  • Kagawa Medical University   School of Medicine   Research Assistant

    1992.4 - 1999.3

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    Country:Japan

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  • Okayama University   Medical School   Research Assistant

    1988.4 - 1992.3

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    Country:Japan

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Professional Memberships

Committee Memberships

  • 日本細菌学会   理事  

    2015.1 - 2018.12   

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    Committee type:Academic society

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  • 岡山県   備前地域感染症診査協議会 委員  

    2013.4   

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    Committee type:Municipal

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  • 岡山市   感染症診査協議会 委員(感染症部会長)  

    2013.4   

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    Committee type:Municipal

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Papers

  • Detection of in-frame mutation by IS30-family insertion sequence in the phospholipid phosphatidylglycerol synthase gene (pgsA2) of high-level daptomycin-resistant Corynebacterium striatum. Reviewed

    Kazuyoshi Gotoh, I. Putu Bayu Mayura, Yusaku Enomoto, Koji Iio, Osamu Matsushita, Fumio Otsuka, Hideharu Hagiya

    European Journal of Clinical Microbiology & Infectious Diseases   2021.10

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    Publishing type:Research paper (scientific journal)   Publisher:Springer Science and Business Media LLC  

    DOI: 10.1007/s10096-021-04369-1

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    Other Link: https://link.springer.com/article/10.1007/s10096-021-04369-1/fulltext.html

  • In vitro effectiveness of biapenem against IMP-producing Enterobacteriaceae. Reviewed

    Kazuyoshi Gotoh, Makoto Miyoshi, I Putu Bayu Mayura, Koji Iio, Osamu Matsushita, Fumio Otsuka, Hideharu Hagiya

    Journal of Medical Microbiology   70 ( 10 )   2021.10

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    Publishing type:Research paper (scientific journal)   Publisher:Microbiology Society  

    The options available for treating infections with carbapenemase-producing <italic>
    <named-content content-type="family">
    <ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://doi.org/10.1601/nm.3091" xlink:type="simple">Enterobacteriaceae</ext-link>
    </named-content>
    </italic> (CPE) are limited; with the increasing threat of these infections, new treatments are urgently needed. Biapenem (BIPM) is a carbapenem, and limited data confirming its <italic>in vitro</italic> killing effect against CPE are available. In this study, we examined the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of BIPM for 14 IMP-1-producing <italic>
    <named-content content-type="family">
    <ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://doi.org/10.1601/nm.3091" xlink:type="simple">Enterobacteriaceae</ext-link>
    </named-content>
    </italic> strains isolated from the Okayama region in Japan. The MICs against almost all the isolates were lower than 0.5 µg ml−1, indicating susceptibility to BIPM, while approximately half of the isolates were confirmed to be bacteriostatic to BIPM. However, initial killing to a 99.9 % reduction was observed in seven out of eight strains in a time–kill assay. Despite the small data set, we concluded that the <italic>in vitro</italic> efficacy of BIPM suggests that the drug could be a new therapeutic option against infection with IMP-producing CPE.

    DOI: 10.1099/jmm.0.001430

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  • Elizabethkingia anophelis, an emerging pathogen, inhibits RAW 264.7 macrophage function. Reviewed

    I. Putu Bayu Mayura, Kazuyoshi Gotoh, Hayato Nishimura, Erina Nakai, Takehiko Mima, Yumiko Yamamoto, Kenji Yokota, Osamu Matsushita

    Microbiology and Immunology   65 ( 8 )   317 - 324   2021.8

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    Authorship:Last author   Publishing type:Research paper (scientific journal)   Publisher:Wiley  

    DOI: 10.1111/1348-0421.12888

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    Other Link: https://onlinelibrary.wiley.com/doi/full-xml/10.1111/1348-0421.12888

  • Serodiagnosis and bacterial genome of Helicobacter pylori infection. Reviewed

    Aina Ichihara, Hinako Ojima, Kazuyoshi Gotoh, Osamu Matsushita, Susumu Take, Hiroyuki Okada, Akari Watanabe, Kenji Yokota

    Toxins   13 ( 7 )   467 - 467   2021.7

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    Publishing type:Research paper (scientific journal)   Publisher:MDPI AG  

    The infection caused by Helicobacter pylori is associated with several diseases, including gastric cancer. Several methods for the diagnosis of H. pylori infection exist, including endoscopy, the urea breath test, and the fecal antigen test, which is the serum antibody titer test that is often used since it is a simple and highly sensitive test. In this context, this study aims to find the association between different antibody reactivities and the organization of bacterial genomes. Next-generation sequences were performed to determine the genome sequences of four strains of antigens with different reactivity. The search was performed on the common genes, with the homology analysis conducted using a genome ring and dot plot analysis. The two antigens of the highly reactive strains showed a high gene homology, and Western blots for CagA and VacA also showed high expression levels of proteins. In the poorly responsive antigen strains, it was found that the inversion occurred around the vacA gene in the genome. The structure of bacterial genomes might contribute to the poor reactivity exhibited by the antibodies of patients. In the future, an accurate serodiagnosis could be performed by using a strain with few gene mutations of the antigen used for the antibody titer test of H. pylori.

    DOI: 10.3390/toxins13070467

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  • Antibacterial effects of disulfiram in Helicobacter pylori. Reviewed

    Tomomi Kobatake, Keiki Ogino, Hiroyuki Sakae, Kazuyoshi Gotoh, Akari Watanabe, Osamu Matsushita, Hiroyuki Okada, Kenji Yokota

    Infection and Drug Resistance   14   1757 - 1764   2021.5

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    Publishing type:Research paper (scientific journal)   Publisher:Informa UK Limited  

    DOI: 10.2147/idr.s299177

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  • Septic pulmonary emboli caused by Tsukamurella inchonensis: A case report. Reviewed

    Kazuyoshi Gotoh, I Putu Bayu Mayura, Hideharu Hagiya, Kyoichi Obata, Tatsuo Ogawa, Koji Iio, Takumi Fujimori, Fumio Otsuka, Osamu Matsushita

    Journal of Infection and Chemotherapy   27 ( 2 )   369 - 372   2021.2

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    Authorship:Last author   Publishing type:Research paper (scientific journal)   Publisher:Elsevier BV  

    DOI: 10.1016/j.jiac.2020.09.024

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  • Environmental survey of methicillin-resistant Staphylococci in a hospital in Japan. Reviewed

    AKARI WATANABE, TOKIKO WATANABE, SUSUMU KOKEGUCHI, YUMIKO YAMAMOTO, OSAMU MATSUSHITA, KENJI YOKOTA

    Biocontrol Science   26 ( 3 )   137 - 145   2021

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    Publishing type:Research paper (scientific journal)   Publisher:The Society for Antibacterial and Antifungal Agents, Japan  

    DOI: 10.4265/bio.26.137

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  • Enhancement of intestinal epithelial barrier function by Weissella confusa F213 and Lactobacillus rhamnosus FBB81 probiotic candidates in an in vitro model of hydrogen peroxide-induced inflammatory bowel disease. Reviewed

    Ni Nengah Dwi Fatmawati, Kazuyoshi Gotoh, I. Putu Bayu Mayura, Komang Ayu Nocianitri, Gede Ngurah Rsi Suwardana, Ni Luh Gede Yoni Komalasari, Yan Ramona, Masakiyo Sakaguchi, Osamu Matsushita, I. Nengah Sujaya

    BMC Research Notes   13 ( 1 )   489 - 489   2020.12

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    Publishing type:Research paper (scientific journal)   Publisher:Springer Science and Business Media LLC  

    <title>Abstract</title><sec>
    <title>Objective</title>
    <italic>Weissella confusa</italic> F213 (WCF213) and <italic>Lactobacillus rhamnosus</italic> FBB81 (LrFBB81) are two probiotic candidates isolated from humans in our previous study. Their functional activity on the mucosal barrier has not yet been adequately investigated. Therefore, the objective of this study was to investigate the effect of these strains on maintaining mucosal integrity in vitro. Caco-2 cell monolayers were pretreated with WCF213 and LrFBB81 before being exposed to hydrogen peroxide. The integrity of mucosal cells was evaluated by measuring the transepithelial resistance (TER), flux of FITC-labelled dextran, and ZO-1 protein distribution with the help of an immunofluorescence method.


    </sec><sec>
    <title>Results</title>
    WCF213 was found to significantly maintain the TER better than the control hydrogen peroxide-treated cells (<italic>p </italic>&lt; 0.001), followed by the strain combination, and LrFBB81 alone (<italic>p </italic>&lt; 0.05). The permeability of mucosa was also successfully maintained by the WCF213 strain. This was illustrated by the significant reduction in the flux of FITC-labelled dextran (<italic>p </italic>&lt; 0.05), which was larger than that exhibited by the other groups. The ZO-1 distribution of strain-treated cells showed less disruption than hydrogen peroxide-treated cells, consistent with the TER and FITC experimental results. These findings indicate that WCF213 and LrFBB81 plays important roles in the maintenance of mucosal integrity in a strain-dependent manner.


    </sec>

    DOI: 10.1186/s13104-020-05338-1

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    Other Link: https://link.springer.com/article/10.1186/s13104-020-05338-1/fulltext.html

  • Polyglycolic acid‐collagen tube combined with collagen‐binding basic fibroblast growth factor accelerates gait recovery in a rat sciatic nerve critical‐size defect model. Reviewed

    Hisako Fujimaki, Kentaro Uchida, Gen Inoue, Osamu Matsushita, Noriko Nemoto, Masayuki Miyagi, Kazuhide Inage, Shotaro Takano, Sumihisa Orita, Seiji Ohtori, Keisuke Tanaka, Hiroyuki Sekiguchi, Masashi Takaso

    Journal of Biomedical Materials Research Part B: Applied Biomaterials   108 ( 2 )   326 - 332   2020.2

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    Publishing type:Research paper (scientific journal)   Publisher:Wiley  

    DOI: 10.1002/jbm.b.34391

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    Other Link: https://onlinelibrary.wiley.com/doi/full-xml/10.1002/jbm.b.34391

  • Association of host immunity with Helicobacter pylori infection in recurrent gastric cancer. Reviewed

    Mayu Sato, Kou Miura, Chihiro Kageyama, Hiroyuki Sakae, Yuka Obayashi, Yoshiro Kawahara, Osamu Matsushita, Kenji Yokota, Hiroyuki Okada

    Infectious Agents and Cancer   14 ( 1 )   2019.12

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    Publishing type:Research paper (scientific journal)   Publisher:Springer Science and Business Media LLC  

    DOI: 10.1186/s13027-019-0221-1

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    Other Link: http://link.springer.com/article/10.1186/s13027-019-0221-1/fulltext.html

  • Acceleration of bone regeneration of horizontal bone defect in rats using collagen‐binding basic fibroblast growth factor combined with collagen scaffolds. Reviewed

    Shin Nakamura, Takashi Ito, Kentaro Okamoto, Takehiko Mima, Kentaro Uchida, Yasir D. Siddiqui, Masahiro Ito, Masako Tai, Keisuke Okubo, Keisuke Yamashiro, Kazuhiro Omori, Tadashi Yamamoto, Osamu Matsushita, Shogo Takashiba

    Journal of Periodontology   90 ( 9 )   1043 - 1052   2019.9

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    Publishing type:Research paper (scientific journal)   Publisher:Wiley  

    DOI: 10.1002/jper.18-0674

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    Other Link: https://onlinelibrary.wiley.com/doi/full-xml/10.1002/JPER.18-0674

  • Increase in antibiotic resistant Helicobacter pylori in a university hospital in Japan. Reviewed

    Chihiro Kageyama, Mayu Sato, Hiroyuki Sakae, Yuka Obayashi, Yoshiro Kawahara, Takehiko Mima, Osamu Matsushita, Kenji Yokota, Motowo Mizuno, Hiroyuki Okada

    Infection and Drug Resistance   Volume 12   597 - 602   2019.3

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    Publishing type:Research paper (scientific journal)   Publisher:Informa UK Limited  

    DOI: 10.2147/idr.s196452

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  • Ca2+‐induced orientation of tandem collagen binding domains from clostridial collagenase ColG permits two opposing functions of collagen fibril formation and retardation. Reviewed

    Perry Caviness, Ryan Bauer, Keisuke Tanaka, Katarzyna Janowska, Jeffrey Randall Roeser, Dawn Harter, Jes Sanders, Christopher Ruth, Osamu Matsushita, Joshua Sakon

    The FEBS Journal   285 ( 17 )   3254 - 3269   2018.9

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    Publishing type:Research paper (scientific journal)   Publisher:Wiley  

    DOI: 10.1111/febs.14611

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    Other Link: https://onlinelibrary.wiley.com/doi/full-xml/10.1111/febs.14611

  • Vibrio alginolyticus VepA induces lysosomal membrane permeability and cathepsin-independent cell death. Reviewed

    Agus Eka Darwinata, Kazuyoshi Gotoh, Takehiko Mima, Yumiko Yamamoto, Kenji Yokota, Osamu Matsushita

    Acta Medica Okayama   72 ( 3 )   231 - 239   2018.6

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    Authorship:Last author   Language:English   Publishing type:Research paper (scientific journal)  

    The bacterium Vibrio alginolyticus, an opportunistic pathogen in humans, has a type III secretion system (T3SS) that is responsible for its cytotoxicity toward eukaryotic cells. The effector of T3SS that is responsible for the cytotoxicity had not been identified. Here we demonstrate that VepA, a homolog of the T3SS effector in V. parahaemolyticus, is required for cytotoxicity in V. alginolyticus. VepA induces lysosomal membrane permeabilization, and it allows the leakage of only small molecules into the cytosol. Our findings revealed that VepA induces cathepsin-independent cell death in mammalian cells. The ferrous ion, one of the small molecules in the lysosome contents, appears to be involved in the cell death caused by V. alginolyticus VepA.

    DOI: 10.18926/AMO/56068

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  • DEC205 mediates local and systemic immune responses to Helicobacter pylori infection in humans. Reviewed

    Masahide Kita, Kenji Yokota, Chihiro Kageyama, Susumu Take, Kazuyoshi Goto, Yoshiro Kawahara, Osamu Matsushita, Hiroyuki Okada

    Oncotarget   9 ( 22 )   15828 - 15835   2018.3

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    Publishing type:Research paper (scientific journal)   Publisher:Impact Journals, LLC  

    DOI: 10.18632/oncotarget.24574

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  • Expression of collagenase is regulated by the VarS/VarA two-component regulatory system in Vibrio alginolyticus. Reviewed

    Takehiko Mima, Kazuyoshi Gotoh, Yumiko Yamamoto, Keiko Maeda, Taku Shirakawa, Shunsuke Matsui, Yumi Murata, Takaki Koide, Hiroshi Tokumitsu, Osamu Matsushita

    The Journal of Membrane Biology   251 ( 1 )   51 - 63   2018.2

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    DOI: 10.1007/s00232-017-9991-9

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    Other Link: http://link.springer.com/content/pdf/10.1007/s00232-017-9991-9.pdf

  • Basic fibroblast growth factor fused with tandem collagen-binding domains from Clostridium histolyticum collagenase ColG increases bone formation. Reviewed

    Hiroyuki Sekiguchi, Kentaro Uchida, Osamu Matsushita, Gen Inoue, Nozomu Nishi, Ryo Masuda, Nana Hamamoto, Takaki Koide, Shintaro Shoji, Masashi Takaso

    BioMed Research International   2018   1 - 8   2018

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    Publishing type:Research paper (scientific journal)   Publisher:Hindawi Limited  

    Basic fibroblast growth factor 2 (bFGF) accelerates bone formation during fracture healing. Because the efficacy of bFGF decreases rapidly following its diffusion from fracture sites, however, repeated dosing is required to ensure a sustained therapeutic effect. We previously developed a fusion protein comprising bFGF, a polycystic kidney disease domain (PKD; s2b), and collagen-binding domain (CBD; s3) sourced from the<italic> Clostridium histolyticum</italic> class II collagenase, ColH, and reported that the combination of this fusion protein with a collagen-like peptide, poly(Pro-Hyp-Gly)10, induced mesenchymal cell proliferation and callus formation at fracture sites. In addition,<italic> C. histolyticum</italic> produces class I collagenase (ColG) with tandem CBDs (s3a and s3b) at the C-terminus. We therefore hypothesized that a bFGF fusion protein containing ColG-derived tandem CBDs (s3a and s3b) would show enhanced collagen-binding activity, leading to improved bone formation. Here, we examined the binding affinity of four collagen anchors derived from the two clostridial collagenases to H-Gly-Pro-Arg-Gly-(Pro-Hyp-Gly)12-NH2, a collagenous peptide, by surface plasmon resonance and found that tandem CBDs (s3a-s3b) have the highest affinity for the collagenous peptide. We also constructed four fusion proteins consisting of bFGF and s3 (bFGF-s3), s2b-s3b (bFGF-s2b-s3), s3b (bFGF-s3b), and s3a-s3b (bFGF-s3a-s3b) and compared their biological activities to those of a previous fusion construct (bFGF-s2b-s3) using a cell proliferation assay in vitro and a mouse femoral fracture model in vivo. Among these CB-bFGFs, bFGF-s3a-s3b showed the highest capacity to induce mesenchymal cell proliferation and callus formation in the mice fracture model. The poly(Pro-Hyp-Gly)10/bFGF-s3a-s3b construct may therefore have the potential to promote bone formation in clinical settings.

    DOI: 10.1155/2018/8393194

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    Other Link: http://downloads.hindawi.com/journals/bmri/2018/8393194.xml

  • Effect of freeze-dried allograft bone with human basic fibroblast growth factor containing a collagen-binding domain from Clostridium histolyticum collagenase on bone formation after lumbar posterolateral fusion surgery in rats. Reviewed

    Gen Inoue, Kentaro Uchida, Osamu Matsushita, Hisako Fujimaki, Wataru Saito, Masayuki Miyagi, Hiroyuki Sekiguchi, Nozomu Nishi, Seiji Ohtori, Mizuki Yogoro, Masashi Takaso

    Spine   42 ( 17 )   E995 - E1001   2017.9

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    Publishing type:Research paper (scientific journal)   Publisher:Ovid Technologies (Wolters Kluwer Health)  

    DOI: 10.1097/brs.0000000000002074

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  • Enhancement of periosteal bone formation by basic fibroblast-derived growth factor containing polycystic kidney disease and collagen-binding domains from Clostridium histolyticum collagenase. Reviewed

    Kentaro Uchida, Osamu Matsushita, Nozomu Nishi, Gen Inoue, Kyosuke Horikawa, Masashi Takaso

    Journal of Tissue Engineering and Regenerative Medicine   11 ( 4 )   1165 - 1172   2017.4

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    Publishing type:Research paper (scientific journal)   Publisher:Wiley  

    DOI: 10.1002/term.2019

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  • Oriented collagen tubes combined with basic fibroblast growth factor promote peripheral nerve regeneration in a 15 mm sciatic nerve defect rat model. Reviewed

    Hisako Fujimaki, Kentaro Uchida, Gen Inoue, Masayuki Miyagi, Noriko Nemoto, Taro Saku, Yoshihiro Isobe, Kazuhide Inage, Osamu Matsushita, Saburo Yagishita, Jun Sato, Shotaro Takano, Yoshihiro Sakuma, Seiji Ohtori, Kazuhisa Takahashi, Masashi Takaso

    Journal of Biomedical Materials Research Part A   105 ( 1 )   8 - 14   2017.1

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    DOI: 10.1002/jbm.a.35866

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  • Basic fibroblast growth factor-anchored multilayered mesenchymal cell sheets accelerate periosteal bone formation. Reviewed

    Kentaro Uchida, Gen Inoue, Osamu Matsushita, Kyosuke Horikawa, Hiroyuki Sekiguchi, Wataru Saito, Shotaro Takano, Hisako Fujimaki, Masayuki Miyagi, Masashi Takaso

    BioMed Research International   2017   1 - 8   2017

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    Cell-based regenerative therapy has the potential to repair bone injuries or large defects that are recalcitrant to conventional treatment methods, including drugs and surgery. Here, we developed a multilayered cell-based bone formation system using cells coated with fibronectin-gelatin (FN-G) nanofilms. The multilayered mesenchymal cells (MLMCs) were formed after two days of culture and were shown to express higher levels of BMP-2 and VEGF compared to monolayer cultures of MCs. The MLMCs were used as a graft material in combination with a fusion protein consisting of basic fibroblast growth factor (bFGF), polycystic kidney disease (PKD) domain, and the collagen-binding domain (CBD) of<italic> Clostridium histolyticum</italic> collagenase. In femur sites grafted with the MLMCs, significantly higher levels of callus volume and bone mineral content were observed compared to the sham controls. The callus volume and bone mineral content were further increased in femur sites grafted with bFGF-PKD-CBD/MLMCs. Taken together, these results suggest that bFGF-PKD-CBD/MLMCs, which can be simply and rapidly generated in vitro, have the potential to promote bone repair when grafted into large defect sites.

    DOI: 10.1155/2017/4371460

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    Other Link: http://downloads.hindawi.com/journals/bmri/2017/4371460.xml

  • Acceleration of bone formation during fracture healing by poly(Pro-Hyp-Gly)10 and basic fibroblast growth factor containing polycystic kidney disease and collagen-binding domains from Clostridium histolyticum collagenase. Reviewed

    Hiroyuki Sekiguchi, Kentaro Uchida, Gen Inoue, Osamu Matsushita, Wataru Saito, Jun Aikawa, Keisuke Tanaka, Hisako Fujimaki, Masayuki Miyagi, Masashi Takaso

    Journal of Biomedical Materials Research Part A   104 ( 6 )   1372 - 1378   2016.6

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    DOI: 10.1002/jbm.a.35670

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  • Acceleration of callus formation during fracture healing using basic fibroblast growth factor-kidney disease domain-collagen-binding domain fusion protein combined with allogenic demineralized bone powder. Reviewed

    Wataru Saito, Kentaro Uchida, Osamu Matsushita, Gen Inoue, Hiroyuki Sekiguchi, Jun Aikawa, Hisako Fujimaki, Masashi Takaso

    Journal of Orthopaedic Surgery and Research   10 ( 1 )   2015.12

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    Publishing type:Research paper (scientific journal)   Publisher:Springer Science and Business Media LLC  

    DOI: 10.1186/s13018-015-0201-0

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    Other Link: http://link.springer.com/article/10.1186/s13018-015-0201-0/fulltext.html

  • Cross-excitation in peripheral sensory ganglia associated with pain transmission. Reviewed

    Katsuhiro Omoto, Kotaro Maruhama, Ryuji Terayama, Yumiko Yamamoto, Osamu Matsushita, Tomosada Sugimoto, Keiji Oguma, Yoshizo Matsuka

    Toxins   7 ( 8 )   2906 - 2917   2015.8

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    Publishing type:Research paper (scientific journal)   Publisher:MDPI AG  

    DOI: 10.3390/toxins7082906

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  • Structures of three polycystic kidney disease-like domains from Clostridium histolyticum collagenases ColG and ColH. Reviewed

    Ryan Bauer, Katarzyna Janowska, Kelly Taylor, Brad Jordan, Steve Gann, Tomasz Janowski, Ethan C. Latimer, Osamu Matsushita, Joshua Sakon

    Acta Crystallographica Section D Biological Crystallography   71 ( 3 )   565 - 577   2015.3

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    Publishing type:Research paper (scientific journal)   Publisher:International Union of Crystallography (IUCr)  

    <italic>Clostridium histolyticum</italic>collagenases ColG and ColH are segmental enzymes that are thought to be activated by Ca2+-triggered domain reorientation to cause extensive tissue destruction. The collagenases consist of a collagenase module (s1), a variable number of polycystic kidney disease-like (PKD-like) domains (s2a and s2b in ColH and s2 in ColG) and a variable number of collagen-binding domains (s3 in ColH and s3a and s3b in ColG). The X-ray crystal structures of Ca2+-bound holo s2b (1.4 Å resolution,<italic>R</italic>= 15.0%,<italic>R</italic>free= 19.1%) and holo s2a (1.9 Å resolution,<italic>R</italic>= 16.3%,<italic>R</italic>free= 20.7%), as well as of Ca2+-free apo s2a (1.8 Å resolution,<italic>R</italic>= 20.7%,<italic>R</italic>free= 27.2%) and two new forms of N-terminally truncated apo s2 (1.4 Å resolution,<italic>R</italic>= 16.9%,<italic>R</italic>free= 21.2%; 1.6 Å resolution,<italic>R</italic>= 16.2%,<italic>R</italic>free= 19.2%), are reported. The structurally similar PKD-like domains resemble the V-set Ig fold. In addition to a conserved β-bulge, the PKD-like domains feature a second bulge that also changes the allegiance of the subsequent β-strand. This β-bulge and the genesis of a Ca2+pocket in the archaeal PKD-like domain suggest a close kinship between bacterial and archaeal PKD-like domains. Different surface properties and indications of different dynamics suggest unique roles for the PKD-like domains in ColG and in ColH. Surface aromatic residues found on ColH s2a-s2b, but not on ColG s2, may provide the weak interaction in the biphasic collagen-binding mode previously found in s2b-s3.<italic>B</italic>-factor analyses suggest that in the presence of Ca2+the midsection of s2 becomes more flexible but the midsections of s2a and s2b stay rigid. The different surface properties and dynamics of the domains suggest that the PKD-like domains of M9B bacterial collagenase can be grouped into either a ColG subset or a ColH subset. The conserved properties of PKD-like domains in ColG and in ColH include Ca2+binding. Conserved residues not only interact with Ca2+, but also position the Ca2+-interacting water molecule. Ca2+aligns the N-terminal linker approximately parallel to the major axis of the domain. Ca2+binding also increases stability against heat and guanidine hydrochloride, and may improve the longevity in the extracellular matrix. The results of this study will further assist in developing collagen-targeting vehicles for various signal molecules.

    DOI: 10.1107/s1399004714027722

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  • [A study to determine the optimum antigens for the serodiagnosis of Helicobacter pylori infection in Japanese patients and the association with IgG subclass and gastric cancer]. Reviewed

    Masahide Kita, Susumu Take, Hiroyuki Okada, Osamu Matsushita, Kenji Yokota

    Rinsho byori. The Japanese Journal of Clinical Pathology   63 ( 2 )   180 - 186   2015.2

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    Atrophic gastritis is caused by Helicobacter pylori infection, and is involved in gastric cancer. In this study, we investigated the association with total IgG and IgG subclass antibodies using several strains isolated from Japanese in H. pylori positive and negative individuals, and gastric atrophy using measuring pepsinogen I and II levels. We found that total IgG antibody measurement using typical Japanese genotype as an antigen was available for diagnosis of H. pylori infection, whereas IgG1 and IgG2 antibodies were not for diagnosis. Furthermore, the IgG1/G2 ratio was elevated in a patient with gastric cancer. The accuracy of serodiagnosis of H. pylori infection may increase when the optimal antigens are used, and measurement IgG subclass may provide additional prediction of gastric cancer.

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  • Acceleration of bone formation during fracture healing by injectable collagen powder and human basic fibroblast growth factor containing a collagen-binding domain from Clostridium histolyticum collagenase. Reviewed

    Wataru Saito, Kentaro Uchida, Masaki Ueno, Osamu Matsushita, Gen Inoue, Nozomu Nishi, Takayuki Ogura, Shunji Hattori, Hisako Fujimaki, Keisuke Tanaka, Masashi Takaso

    Journal of Biomedical Materials Research Part A   102 ( 9 )   3049 - 3055   2014.9

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    DOI: 10.1002/jbm.a.34974

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  • Direct cytocidal effect of galectin-9 localized on collagen matrices on human immune cell lines. Reviewed

    Youko Fukata, Aiko Itoh, Yasuhiro Nonaka, Takashi Ogawa, Takanori Nakamura, Osamu Matsushita, Nozomu Nishi

    Biochimica et Biophysica Acta (BBA)   1840 ( 6 )   1892 - 1901   2014.6

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    DOI: 10.1016/j.bbagen.2014.01.019

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  • Acceleration of periosteal bone formation by human basic fibroblast growth factor containing a collagen‐binding domain from Clostridium histolyticum collagenase. Reviewed

    Kentaro Uchida, Osamu Matsushita, Kouji Naruse, Takehiko Mima, Nozomu Nishi, Shunji Hattori, Takayuki Ogura, Gen Inoue, Keisuke Tanaka, Masashi Takaso

    Journal of Biomedical Materials Research Part A   102 ( 6 )   1737 - 1743   2014.6

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    DOI: 10.1002/jbm.a.34841

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  • Acceleration of bone union after structural bone grafts with a collagen-binding basic fibroblast growth factor anchored-collagen sheet for critical-size bone defects. Reviewed

    Masaki Ueno, Kentaro Uchida, Wataru Saito, Osamu Matsushita, Mizuki Yogoro, Nozomu Nishi, Takayuki Ogura, Shunji Hattori, Gen Inoue, Keisuke Tanaka, Naonobu Takahira, Masashi Takaso

    Biomedical Materials   9 ( 3 )   035014 - 035014   2014.5

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    DOI: 10.1088/1748-6041/9/3/035014

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  • Treatment and prevention of chemotherapy-induced alopecia with PTH-CBD, a collagen-targeted parathyroid hormone analog, in a non-depilated mouse model. Reviewed

    Ranjitha Katikaneni, Tulasi Ponnapakkam, Osamu Matsushita, Joshua Sakon, Robert Gensure

    Anti-Cancer Drugs   25 ( 1 )   30 - 38   2014.1

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  • Cellular responses to Staphylococcus aureus alpha-toxin in chronic rhinosinusitis with nasal polyps. Reviewed

    Mitsuhiro Okano, Tazuko Fujiwara, Shin Kariya, Takaya Higaki, Takenori Haruna, Osamu Matsushita, Yohei Noda, Seiichiro Makihara, Kengo Kanai, Yasuyuki Noyama, Masami Taniguchi, Kazunori Nishizaki

    Allergology International   63 ( 4 )   563 - 573   2014

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  • The Genetic Diversity of Helicobacter pylori Virulence Genes Is Not Associated with Gastric Atrophy Progression Reviewed

    Kita Masahide, Yokota Kenji, Okada Hiroyuki, Take Susumu, Takenaka Ryuta, Kawahara Yoshiro, Oguma Keiji, Matsushita Osamu, Yamamoto Kazuhide

    Acta Medica Okayama   67 ( 2 )   93 - 98   2013.4

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    DOI: 10.18926/AMO/49667

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  • Effects of CB-VEGF-A injection in rat flap models for improved survival. Reviewed

    Minekatsu Akimoto, Akira Takeda, Osamu Matsushita, Joe Inoue, Keiko Sakamoto, Masakazu Hattori, Natsuko Kounoike, Eiju Uchinuma

    Plastic and Reconstructive Surgery   131 ( 4 )   717 - 725   2013.4

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  • Phospholipase C Produced by Clostridium botulinum Types C and D:Comparison of Gene, Enzymatic, and Biological Activities with Those of Clostridium perfringens Alpha-toxin Reviewed

    Fatmawati Ni Nengah Dwi, Sakaguchi Yoshihiko, Suzuki Tomonori, Oda Masataka, Shimizu Kenta, Yamamoto Yumiko, Sakurai Jun, Matsushita Osamu, Oguma Keiji

    Acta Medica Okayama   67 ( 1 )   9 - 18   2013.2

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    DOI: 10.18926/AMO/49252

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  • Structural comparison of ColH and ColG collagen-binding domains from Clostridium histolyticum. Reviewed

    Ryan Bauer, Jeffrey J. Wilson, Sagaya Theresa Leena Philominathan, Dan Davis, Osamu Matsushita, Joshua Sakon

    Journal of Bacteriology   195 ( 2 )   318 - 327   2013.1

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    <title>ABSTRACT</title>

    <named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">Clostridium histolyticum</named-content>
    secretes collagenases, ColG and ColH, that cause extensive tissue destruction in myonecrosis. The C-terminal collagen-binding domain (CBD) of collagenase is required for insoluble collagen fibril binding and subsequent collagenolysis. The high-resolution crystal structures of ColG-CBD (s3b) and ColH-CBD (s3) are reported in this paper. The new X-ray structure of s3 was solved at 2.0-Å resolution (
    <italic>R</italic>
    = 17.4%;
    <italic>R</italic>
    free
    = 23.3%), while the resolution of the previously determined s3b was extended to 1.4 Å (
    <italic>R</italic>
    = 17.9%;
    <italic>R</italic>
    free
    = 21.0%). Despite sharing only 30% sequence identity, the molecules resemble one another closely (root mean square deviation [RMSD] C
    α
    = 1.5 Å). All but one residue, whose side chain chelates with Ca
    2+
    , are conserved. The dual Ca
    2+
    binding site in s3 is completed by an unconserved aspartate. Differential scanning calorimetric measurements showed that s3 gains thermal stability, comparable to s3b, by binding to Ca
    2+
    (
    <italic>holo</italic>
    <italic>
    T
    m
    </italic>
    = 94.1°C;
    <italic>apo</italic>
    <italic>
    T
    m
    </italic>
    = 70.2°C).
    <italic>holo</italic>
    s3 is also stabilized against chemical denaturants urea and guanidine HCl. The three most critical residues for collagen interaction in s3b are conserved in s3. The general shape of the binding pocket is retained by altered loop structures and side chain positions. Small-angle X-ray scattering data revealed that s3 also binds asymmetrically to minicollagen. Besides the calcium-binding sites and the collagen-binding pocket, architecturally important hydrophobic residues and the hydrogen-bonding network around the
    <italic>cis</italic>
    -peptide bond are well conserved within the metallopeptidase subfamily M9B. CBDs were previously shown to bind to the extracellular matrix of various tissues. Compactness and extreme stability in physiological Ca
    2+
    concentration possibly make both CBDs suitable for targeted growth factor delivery.

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  • Bacterial collagen-binding domain targets undertwisted regions of collagen. Reviewed

    Sagaya Theresa Leena Philominathan, Takaki Koide, Osamu Matsushita, Joshua Sakon

    Protein Science   21 ( 10 )   1554 - 1565   2012.10

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  • Treatment for chemotherapy-induced alopecia in mice using parathyroid hormone agonists and antagonists linked to a collagen binding domain. Reviewed

    Ranjitha Katikaneni, Tulasi Ponnapakkam, Hirofumi Suda, Shigeru Miyata, Joshua Sakon, Osamu Matsushita, Robert C. Gensure

    International Journal of Cancer   131 ( 5 )   E813 - E821   2012.9

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  • A Single Injection of the anabolic bone agent, parathyroid hormone–collagen binding domain (PTH–CBD), results in sustained increases in bone mineral density for up to 12 months in normal female mice. Reviewed

    Tulasi Ponnapakkam, Ranjitha Katikaneni, Hirofumi Suda, Shigeru Miyata, Osamu Matsushita, Joshua Sakon, Robert C. Gensure

    Calcified Tissue International   91 ( 3 )   196 - 203   2012.9

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    DOI: 10.1007/s00223-012-9626-1

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  • Probing the 3-D structure, dynamics, and stability of bacterial collagenase collagen binding domain (apo- versus holo-) by limited proteolysis MALDI-TOF MS. Reviewed

    Cynthia R. Sides, Rohana Liyanage, Jackson O. Lay, Sagaya Theresa Leena Philominathan, Osamu Matsushita, Joshua Sakon

    Journal of the American Society for Mass Spectrometry   23 ( 3 )   505 - 519   2012.3

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  • Prevention of chemotherapy-induced osteoporosis by cyclophosphamide with a long-acting form of parathyroid hormone. Reviewed

    T Ponnapakkam, R Katikaneni, T Nichols, G Tobin, J Sakon, O Matsushita, R C Gensure

    Journal of endocrinological investigation   34 ( 11 )   e392 - e397   2011.12

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  • Identification of a novel virulence factor in Clostridium difficile that modulates toxin sensitivity of cultured epithelial cells. Reviewed International journal

    Masashi Miura, Haru Kato, Osamu Matsushita

    Infection and immunity   79 ( 9 )   3810 - 20   2011.9

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    Two glucosylating toxins named toxins A and B play a role in the pathogenesis of Clostridium Difficile infection. The interaction of the toxins with host cell factors proceeds to downstream stages of cytotoxic effects in cells, in which involvement of other C. difficile factors remains unknown. We utilized culture filtrate of C. difficile with a low dilution to characterize the influence of putative minor proteins on the organization of the actin cytoskeleton in cultured epithelial cells and found a previously uncharacterized F-actin aggregated structure, termed "actin aggregate," at the juxtanuclear region. We reasoned that formation of actin aggregate was due to an additional factor(s) in the culture filtrate rather than the glucosylating toxins, because treatment of purified toxins rarely caused actin aggregate in cells. We focused on a previously uncharacterized hypothetical protein harboring a KDEL-like sequence as a candidate. The product of the candidate gene was detected in culture filtrate of C. difficile ATCC 9689 and was renamed Srl. Purified glutathione S-transferase-tagged Srl triggered formation of actin aggregate in the cells in the presence of either toxin A or B and enhanced cytotoxicity of each of the two toxins, including decreases in both cell viability and transepithelial resistance of cultured epithelial monolayer, although the recombinant Srl alone did not show detectable cytotoxicity. Srl-neutralized culture filtrate partially inhibited morphological changes of the cells in parallel with decreased actin aggregate formation in the cells. Thus, Srl might contribute to the modulation of toxin sensitivity of intestinal epithelial cells by enhancing cytotoxicity of C. difficile toxins.

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  • Monthly administration of a novel PTH-collagen binding domain fusion protein is anabolic in mice. Reviewed

    Tulasi Ponnapakkam, R. Katikaneni, E. Miller, A. Ponnapakkam, S. Hirofumi, S. Miyata, L. J. Suva, J. Sakon, O. Matsushita, R. C. Gensure

    Calcified Tissue International   88 ( 6 )   511 - 520   2011.6

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  • Development of a high-throughput screening system for the compounds that inhibit collagen–protein interactions. Reviewed

    Hitomi Okano-Kosugi, Osamu Matsushita, Shinichi Asada, Andrew B. Herr, Kouki Kitagawa, Takaki Koide

    Analytical Biochemistry   394 ( 1 )   125 - 131   2009.11

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    DOI: 10.1016/j.ab.2009.07.017

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  • Ca2+-induced linker transformation leads to a compact and rigid collagen-binding domain of Clostridium histolyticum collagenase. Reviewed

    Sagaya T. L. Philominathan, Osamu Matsushita, Robert Gensure, Joshua Sakon

    FEBS Journal   276 ( 13 )   3589 - 3601   2009.6

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    DOI: 10.1111/j.1742-4658.2009.07078.x

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  • Unidirectional binding of clostridial collagenase to triple helical substrates. Reviewed

    Sagaya Theresa Leena Philominathan, Takaki Koide, Kentaro Hamada, Hiroyuki Yasui, Soenke Seifert, Osamu Matsushita, Joshua Sakon

    Journal of Biological Chemistry   284 ( 16 )   10868 - 10876   2009.4

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    DOI: 10.1074/jbc.m807684200

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  • 1H, 13C and 15N resonance assignments of Ca2+ bound collagen-binding domain derived from a clostridial collagenase. Reviewed

    Sagaya Theresa Leena Philominathan, Osamu Matsushita, John Brad Jordan, Joshua Sakon

    Biomolecular NMR Assignments   2 ( 2 )   127 - 129   2008.12

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  • [Behavioral approach to facilitate appropriate use of antibiotics].

    Osamu Matsushita, Kazuko Higuchi, Noriko Ishii, Kiyoshi Negayama, Tomohiko Taminato

    Rinsho byori. The Japanese journal of clinical pathology   56 ( 11 )   994 - 1006   2008.11

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    Many hospitals have infection control education programs to facilitate the appropriate use of antimicrobial agents. Even with these efforts, however, it is not rare to encounter irregular prescriptions. In order to solve this discrepancy between knowledge and actual behavior, we chose an alternative approach to improve the decision making process. Recent advances in information technology have made it possible to not only instantly integrate various bacterial examination results using a computer, but to simultaneously carry out the statistical analyses at a much lower cost. We employed a client-server system to accomplish these tasks in Kagawa University Hospital. By connecting CCD camera-equipped microscopes to the system directly, image uploading has become a single-clicking job. Various microbial examination data were automatically transferred to the system once they became available in analytical devices such as BacT/ALERT 3D, VITEK, and an MIC analyzer. These data were presented to hospital doctors in well-designed web windows without delay. By removing psychological barriers to access laboratory examination data, statistics, and relevant information, more doctors seemed to independently follow scientific processes to choose antimicrobial agents. The daily behavior of hospital doctors has also been influenced by the system, e. g., pasting the microscopic images onto clinical records, or starting Gram staining in their own wards. These subtle but fundamental changes will eventually alter the way they make prescription decisions. The computer system was also useful for the infection control team to monitor and detect nosocomial infections, which has become essential to carry out its daily activities.

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  • High-level expression of his-tagged clostridial collagenase in Clostridium perfringens. Reviewed International journal

    Eiji Tamai, Shigeru Miyata, Hiroaki Tanaka, Hirofumi Nariya, Motoo Suzuki, Osamu Matsushita, Naoya Hatano, Akinobu Okabe

    Applied microbiology and biotechnology   80 ( 4 )   627 - 35   2008.9

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    Clostridium histolyticum collagenase is used to isolate cells from various organs and tissues for tissue engineering, and also to treat destructive fibrosis; thus, the demand for high-grade enzyme preparations is increasing. In this study, we constructed a plasmid encoding C. histolyticum type II collagenase (ColH) with a C-terminal hexahistidine tag (ColH-his) to facilitate the purification of the enzyme through immobilized metal affinity chromatography (IMAC). When ColH-his was expressed in a protease-deficient mutant of Clostridium perfringens, it was produced in the culture supernatant more efficiently than the untagged ColH. ColH-his exhibited the same hydrolytic activity as ColH against 4-phenylazobenzyloxy-carbonyl-Pro-Leu-Gly-Pro-D-Arg (Pz peptide), a synthetic collagenase substrate. From 100 ml of the culture supernatant, approximately 1 mg of ColH-his was purified by ammonium sulfate precipitation, IMAC, and high-performance liquid chromatography on a MonoQ column. When IMAC was performed on chelating Sepharose charged with Zn(2+) instead of Ni(2+), a potential carcinogenic metal, the specific activities against Pz peptide and type I collagen decreased slightly. However, they were comparable to those reported for other recombinant ColHs and a commercial C. histolyticum collagenase preparation, suggesting that this expression system is useful for large-scale preparation of high-grade clostridial collagenases.

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  • [Optimum preparation of levocarnitine chloride solution in the hospital pharmacy.] Reviewed

    Hiroaki TANAKA, Masato ASAKURA, Chiaki DOI, Noriyasu FUKUOKA, Eiji TAMAI, Shigeru MIYATA, Osamu MATSUSHITA, Akinobu OKABE, Kiyoshi NEGAYAMA, Hitoshi HOUCHI

    YAKUGAKU ZASSHI   126 ( 9 )   805 - 809   2006.9

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  • Changes in ganglioside content affect the binding of Clostridium perfringens epsilon-toxin to detergent-resistant membranes of Madin-Darby canine kidney cells. Reviewed

    Seiko Shimamoto, Eiji Tamai, Osamu Matsushita, Junzaburo Minami, Akinobu Okabe, Shigeru Miyata

    Microbiology and Immunology   49 ( 3 )   245 - 253   2005.3

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  • High-level expression of clostridial sialidase using a ferredoxin gene promoter-based plasmid. Reviewed International journal

    A Takamizawa, S Miyata, O Matsushita, M Kaji, Y Taniguchi, E Tamai, S Shimamoto, A Okabe

    Protein Expression And Purification   36 ( 1 )   70 - 75   2004.7

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    A "large" sialidase isozyme (NanI) from Clostridium perfringens is a representative microbial sialidase with broad substrate specificity, being used for the analysis of sialoglycoconjugates. It is also a possible virulence factor. However, purification of the native enzyme in a large quantity is not practical due to its low productivity. To obtain the enzyme in a satisfactory yield, a gene encoding the NanI was transcriptionally fused to the fdx gene promoter (P-fdx) in a shuttle-vector, pFF, and transformed into C perfringens 13. The resultant strain released the enzyme into the culture medium, as the original strain does. The enzyme activity increased during the first 6 h of culture and thereafter remained at maximal levels. The maximal activity was approximately 3000-fold compared with that of the original strain, and 15-fold compared with that of recombinant Escherichia coli, which possesses extra copies of the tRNA gene for selected rare codons. This suggests the usefulness of a P-fdx-based plasmid for expressing AT-rich genes in C perfringens. The enzyme was successfully purified by two-step procedure with a specific activity of 2860 U/mg using 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid and a yield of 1.69 mg of NanI per 100 ml of culture. The method described here can facilitate purification of NanI in enough quality and quantity to analyze the role of sialoglycoconjugates in cells and the pathogenic importance of NanI sialidase. (C) 2004 Elsevier Inc. All rights reserved.

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  • A novel type of DNA curvature present in a Clostridium perfringens ferredoxin gene: characterization and role in gene expression. Reviewed International journal

    Masato Kaji, Osamu Matsushita, Eiji Tamai, Shigeru Miyata, Yuki Taniguchi, Seiko Shimamoto, Seiichi Katayama, Shushi Morita, Akinobu Okabe

    Microbiology (Reading, England)   149 ( Pt 11 )   3083 - 3091   2003.11

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    This study has revealed that a Clostridium perfringens ferredoxin gene (per-fdx) possesses a novel type of DNA curvature, which is formed by five phased A-tracts extending from upstream to downstream of the -35 region. The three A-tracts upstream of the promoter and the two within the promoter are located at the positions corresponding to A-tracts present in a C. perfringens phospholipase C gene (plc) and a Clostridium pasteurianum ferredoxin gene (pas-fdx), respectively. DNA fragments of the per-fdx, pas-fdx and plc genes (nucleotide positions -69 to +1 relative to the transcription initiation site) were fused to a chloramphenicol acetyltransferase reporter gene on a plasmid, pPSV, and their in vivo promoter activities were examined by assaying the chloramphenicol acetyltransferase activity of each C. perfringens transformant. Comparison of the three constructs showed that the order of promoter activity is, in descending order, per-fdx, pas-fdx and plc. Deletion of the three upstream A-tracts of the per-fdx gene drastically decreased the promoter activity, as demonstrated previously for the plc promoter. Substitution of the most downstream A-tract decreased the promoter activities of the per-fdx and pas-fdx genes. These results indicate that not only the phased A-tracts upstream of the promoter but also those within the promoter stimulate the promoter activity, and suggest that the high activity of the per-fdx promoter is due to the combined effects of these two types of A-tracts.

    DOI: 10.1099/mic.0.26503-0

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  • Accumulation of Clostridium perfringens epsilon-toxin in the mouse kidney and its possible biological significance. Reviewed International journal

    Eiji Tamai, Tetsuya Ishida, Shigeru Miyata, Osamu Matsushita, Hirofumi Suda, Shoji Kobayashi, Hiroshi Sonobe, Akinobu Okabe

    Infection and immunity   71 ( 9 )   5371 - 5   2003.9

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    In this paper we show that Clostridium perfringens epsilon-toxin accumulates predominantly in the mouse kidney, where it is distributed mainly in glomeruli, capillaries, and collecting ducts. Although some pycnotic and exfoliated epithelial cells were observed in distal tubuli and collecting ducts, there were no findings indicative of severe renal injury. Bilateral nephrectomy increased the mouse lethality of the toxin, suggesting that the kidney contributes to the host defense against the lethal toxicity of epsilon-toxin.

    DOI: 10.1128/IAI.71.9.5371-5375.2003

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  • A bacterial collagen-binding domain with novel calcium-binding motif controls domain orientation. Reviewed

    Wilson JJ, Matsushita O, Okabe A, Sakon J

    The EMBO Journal   22 ( 8 )   1743 - 1752   2003.4

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  • Dual-site recognition of different extracellular matrix components by anti-angiogenic/neurotrophic serpin, PEDF. Reviewed

    Norihisa Yasui, Terumi Mori, Daisuke Morito, Osamu Matsushita, Hiroki Kourai, Kazuhiro Nagata, Takaki Koide

    Biochemistry   42 ( 11 )   3160 - 3167   2003.3

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  • Clostridium perfringens ε-toxin forms a heptameric pore within the detergent-insoluble microdomains of Madin-Darby canine kidney cells and rat synaptosomes. Reviewed

    Shigeru Miyata, Junzaburo Minami, Eiji Tamai, Osamu Matsushita, Seiko Shimamoto, Akinobu Okabe

    Journal of Biological Chemistry   277 ( 42 )   39463 - 39468   2002.10

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    DOI: 10.1074/jbc.m206731200

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  • Phased A-tracts bind to the α subunit of RNA polymerase with increased affinity at low temperature. Reviewed

    Seiichi Katayama, Osamu Matsushita, Eiji Tamai, Shigeru Miyata, Akinobu Okabe

    FEBS Letters   509 ( 2 )   235 - 238   2001.12

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    DOI: 10.1016/s0014-5793(01)03148-9

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  • Cleavage of a C-terminal peptide Is essential for heptamerization of Clostridium perfringens ε-Toxin in the synaptosomal membrane. Reviewed

    Shigeru Miyata, Osamu Matsushita, Junzaburo Minami, Seiichi Katayama, Seiko Shimamoto, Akinobu Okabe

    Journal of Biological Chemistry   276 ( 17 )   13778 - 13783   2001.4

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    DOI: 10.1074/jbc.m011527200

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  • Substrate Recognition by the Collagen-binding Domain of Clostridium histolyticum Class I Collagenase Reviewed

    Osamu Matsushita, Takaki Koide, Ryoji Kobayashi, Kazuhiro Nagata, Akinobu Okabe

    Journal of Biological Chemistry   276 ( 12 )   8761 - 8770   2001.3

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    DOI: 10.1074/jbc.m003450200

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  • Collagen-binding domain of a Clostridium histolyticum collagenase exhibits a broad substrate spectrum both in vitro and in vivo. Reviewed

    T Toyoshima, O Matsushita, J Minami, N Nishi, A Okabe, T Itano

    Connective Tissue Research   42 ( 4 )   281 - +   2001

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    The substrate spectrum of the tandem collagen-binding domain (CBD) of Clostridium histolyticumclass I collagenase (ColG) was examined both in vitro and in vivo. CBD bound to insoluble type I. II, III and IV collagens in vitro, and to skin, aorta, tendon, kidney, trachea and corneal tissues containing various types of collagen fibrils or sheets. CBD bound to all kinds of collagen fibrils regardless of their diameters and also bound to sheet-forming collagen in the glomerular basal lamina or Descemet's membrane of the cornea. This wide substrate spectrum expands possible applications of the drug delivery system we proposed previously (PNAS 95:7018-7023, 1998). Therapeutic agents fused with CBD will bind not only to subcutaneous tissues, but also to other tissues containing non-type I collagen.

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  • Construction and virulence testing of a collagenase mutant of Clostridium perfringens. Reviewed

    Milena M Awad, Darren M Ellemor, Amy E Bryant, Osamu Matsushita, Richard L Boyd, Dennis L Stevens, John J Emmins, Julian I Rood

    Microbial Pathogenesis   28 ( 2 )   107 - 117   2000.2

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    DOI: 10.1006/mpat.1999.0328

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  • Analysis of genes involved in nitrate reduction in Clostridium perfringens. Reviewed

    Katsuyo Fujinaga, Yuki Taniguchi, Yezhou Sun, Seiichi Katayama, Junzaburo Minami, Osamu Matsushita, Akinobu Okabe

    Microbiology   145 ( 12 )   3377 - 3387   1999.12

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    DOI: 10.1099/00221287-145-12-3377

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  • The hydA gene encoding the H2-evolving hydrogenase of Clostridium perfringens: molecular characterization and expression of the gene. Reviewed

    Masato Kaji, Yuki Taniguchi, Osamu Matsushita, Seiichi Katayama, Shigeru Miyata, Shushi Morita, Akinobu Okabe

    FEMS Microbiology Letters   181 ( 2 )   329 - 336   1999.12

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    DOI: 10.1111/j.1574-6968.1999.tb08863.x

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  • A Clostridium perfringens hem gene cluster contains a cysGB homologue that is Involved in cobalamin biosynthesis. Reviewed

    Michio Koyama, Seiichi Katayama, Masato Kaji, Yuki Taniguchi, Osamu Matsushita, Junzaburo Minami, Shushi Morita, Akinobu Okabe

    Microbiology and Immunology   43 ( 10 )   947 - 957   1999.10

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    DOI: 10.1111/j.1348-0421.1999.tb03355.x

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  • Promoter upstream bent DNA activates the transcription of the Clostridium perfringens phospholipase C gene in a low temperature-dependent manner. Reviewed

    Katayama S, Matsushita O, Jung CM, Minami J, Okabe A

    The EMBO Journal   18 ( 12 )   3442 - 3450   1999.6

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    DOI: 10.1093/emboj/18.12.3442

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  • Purification of bovine S100A12 from recombinant Escherichia coli. Reviewed

    Kayoko Yamashita, Yuhta Oyama, Tsuyoshi Shishibori, Osamu Matsushita, Akinobu Okabe, Ryoji Kobayashi

    Protein Expression and Purification   16 ( 1 )   47 - 52   1999.6

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    DOI: 10.1006/prep.1998.1026

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  • Identification of metal ligands in the Clostridium histolyticum ColH collagenase. Reviewed

    Chang-Min Jung, Osamu Matsushita, Seiichi Katayama, Junzaburo Minami, Jun Sakurai, Akinobu Okabe

    Journal of Bacteriology   181 ( 9 )   2816 - 2822   1999.5

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    <title>ABSTRACT</title>

    A
    <italic>Clostridium histolyticum</italic>
    116-kDa collagenase has an H
    415
    EXXH motif but not the third zinc ligand, as found in already characterized zinc metalloproteinases. To identify its catalytic site, we mutated the codons corresponding to the three conserved residues in the motif to other amino acid residues. The mutation affecting His
    415
    or His
    419
    abolished catalytic activity and zinc binding, while that affecting Glu
    416
    did the former but not the latter. These results suggest that the motif forms the catalytic site. We also mutated the codons corresponding to other amino acid residues that are likely zinc ligands. The mutation affecting Glu
    447
    decreased markedly both the enzymatic activity and the zinc content, while that affecting Glu
    446
    or Glu
    451
    had smaller effects on activity and zinc binding. These mutations caused a decrease in
    <italic>k</italic>
    cat
    but no significant change in
    <italic>
    K
    m
    </italic>
    . These results are consistent with the hypothesis that Glu
    447
    is the third zinc ligand. The spacing of the three zinc ligands is the same in all known clostridial collagenases but not in other known gluzincins, indicating that they form a new gluzincin subfamily. The effects of mutations affecting Glu
    446
    and Glu
    451
    suggest that the two residues are also involved in catalysis, possibly through an interaction with the two zinc-binding histidine residues.

    DOI: 10.1128/jb.181.9.2816-2822.1999

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  • Three distinct anti-allergic drugs, amlexanox, cromolyn and tranilast, bind to S100A12 and S100A13 of the S100 protein family. Reviewed

    Shishibori T, Oyama Y, Matsushita O, Yamashita K, Furuichi H, Okabe A, Maeta H, Hata Y, Kobayashi R

    Biochemistry Journal   338 ( Pt 3 )   583 - 589   1999.3

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  • Gene duplication and multiplicity of collagenases in Clostridium histolyticum. Reviewed

    Osamu Matsushita, Chang-Min Jung, Seiichi Katayama, Junzaburo Minami, Yukie Takahashi, Akinobu Okabe

    Journal of Bacteriology   181 ( 3 )   923 - 933   1999.2

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    <title>ABSTRACT</title>

    <italic>Clostridium histolyticum</italic>
    collagenase contains a number of different active components. Previously we have shown that
    <italic>colH</italic>
    encodes a 116-kDa collagenase (ColH) and a 98-kDa gelatinase. We purified a different 116-kDa collagenase (ColG) from the culture supernatant and sequenced its gene (
    <italic>colG</italic>
    ). We also identified four other gelatinases (105, 82, 78, and 67 kDa) and determined their N-terminal amino acid sequences, all of which coincided with that of either ColG or ColH. Hybridization experiments showed that each gene is present in a single copy and each gene is transcribed into a single mRNA. These results suggest that all the gelatinases are produced from the respective full-length collagenase by the proteolytic removal of C-terminal fragments. The substrate specificities of the enzymes suggest that
    <italic>colG</italic>
    and
    <italic>colH</italic>
    encode class I and class II enzymes, respectively. Analysis of their DNA locations by pulsed-field gel electrophoresis and nucleotide sequencing of their surrounding regions revealed that the two genes are located in different sites on the chromosome.
    <italic>C. histolyticum colG</italic>
    is more similar to
    <italic>C. perfringens colA</italic>
    than to
    <italic>colH</italic>
    in terms of domain structure. Both
    <italic>colG</italic>
    and
    <italic>colA</italic>
    have a homologous gene,
    <italic>mscL</italic>
    , at their 3′ ends. These results suggest that gene duplication and segment duplication have occurred in an ancestor cell common to
    <italic>C. histolyticum</italic>
    and
    <italic>C. perfringens</italic>
    and that further divergence of the parent gene produced
    <italic>colG</italic>
    and
    <italic>colA</italic>
    .

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  • Collagen-binding growth factors: Production and characterization of functional fusion proteins having a collagen-binding domain. Reviewed

    N. Nishi, O. Matsushita, K. Yuube, H. Miyanaka, A. Okabe, F. Wada

    Proceedings of the National Academy of Sciences   95 ( 12 )   7018 - 7023   1998.6

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    DOI: 10.1073/pnas.95.12.7018

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  • A study of the collagen-binding domain of a 116-kDa Clostridium histolyticum collagenase. Reviewed

    Osamu Matsushita, Chang-Min Jung, Junzaburo Minami, Seiichi Katayama, Nozomu Nishi, Akinobu Okabe

    Journal of Biological Chemistry   273 ( 6 )   3643 - 3648   1998.2

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    DOI: 10.1074/jbc.273.6.3643

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  • Lambda-toxin of Clostridium perfringens activates the precursor of epsilon-toxin by releasing its N- and C-terminal peptides. Reviewed

    Junzaburo Minami, Seiichi Katayama, Osamu Matsushita, Chieko Matsushita, Akinobu Okabe

    Microbiology and Immunology   41 ( 7 )   527 - 535   1997.7

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    DOI: 10.1111/j.1348-0421.1997.tb01888.x

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  • Expression of the colH gene encoding Clostridium histolyticum collagenase in Bacillus subtilis and Its application to enzyme purification. Reviewed

    Chang-Min Jung, Osamu Matsushita, Seiichi Katayama, Junzaburo Minami, Lichiro Ohhira, Akinobu Okabe

    Microbiology and Immunology   40 ( 12 )   923 - 929   1996.12

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    DOI: 10.1111/j.1348-0421.1996.tb01161.x

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  • An upstream activating sequence containing curved DNA involved in activation of the Clostridium perfringens plc promoter. Reviewed

    C. Matsushita, O. Matsushita, S. Katayama, J. Minami, K. Takai, A. Okabe

    Microbiology   142 ( 9 )   2561 - 2566   1996.9

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    DOI: 10.1099/00221287-142-9-2561

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  • Analysis of the phospholipase C gene of Clostridium perfringens KZ1340 isolated from antarctic soil. Reviewed

    Kohtaro Kameyama, Osamu Matsushita, Seiichi Katayama, Junzaburo Minami, Masazumi Maeda, Shinichi Nakamura, Akinobu Okabe

    Microbiology and Immunology   40 ( 4 )   255 - 263   1996.4

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    DOI: 10.1111/j.1348-0421.1996.tb03344.x

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  • Purification, characterization, and primary structure of Clostridium perfringens lambda-toxin, a thermolysin-like metalloprotease. Reviewed

    F Jin, O Matsushita, S Katayama, S Jin, C Matsushita, J Minami, A Okabe

    Infection and Immunity   64 ( 1 )   230 - 237   1996.1

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    The lambda-toxin of Clostridium perfringens type B NCIB10691 was purified by ammonium sulfate precipitation, followed by size exclusion, anion-exchange, and hydrophobic interaction chromatography. The purified toxin had an apparent molecular mass of 36 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The toxin possessed casein-hydrolyzing activity, which was inhibited specifically by metal chelators, indicating that the toxin is a metalloprotease. The gene encoding the lambda-toxin (lam), which was shown by Southern analysis to be located on a 70-kb plasmid, was cloned into Escherichia coli cells. Nucleotide and N-terminal amino acid sequencing revealed that the lam gene encodes a 553-amino-acid protein, which is processed into a mature form, the molecular mass of which was calculated to be 35,722 Da. The deduced amino acid sequence of the mature enzyme contains an HEXXH motif characteristic of zinc metalloproteases and is homologous to other known enzymes belonging to the thermolysin family. The purified toxin degraded various biologically important substances, such as collagen, fibronectin, fibrinogen, immunoglobulin A, and the complement C3 component. It caused an increase in vascular permeability and hemorrhagic edema on injection into the dorsal skin of mice. These results suggest that the toxin contributes to the pathogenesis of histolytic infection by lambda-toxin-producing C. perfringens.

    DOI: 10.1128/iai.64.1.230-237.1996

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  • Phylogenetic analysis of phospholipase C genes from Clostridium perfringens types A to E and Clostridium novyi. Reviewed

    K Tsutsui, J Minami, O Matsushita, S Katayama, Y Taniguchi, S Nakamura, M Nishioka, A Okabe

    Journal of Bacteriology   177 ( 24 )   7164 - 7170   1995.12

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    The phylogenetic interrelationships between strains of 5 toxin types (A to E) of Clostridium perfringens were examined by analysis of differences in the nucleotide sequences of phospholipase C genes (plc genes) among 10 strains, including 3 strains for which the plc gene sequences have been previously reported. A plc gene was also cloned from a Clostridium novyi type A strain and sequenced to analyze the interspecies diversity of plc genes. Phylogenetic trees constructed by the neighbor-joining method revealed that the phylogeny of C. perfringens strains is not related to toxin typing, in agreement with the results of a comparative genome mapping study by Canard et al. (B. Canard, B. Saint-Joanis, and S. T. Cole, Mol. Microbiol. 6:1421-1429, 1992). Various C. perfringens phospholipase C enzymes were purified from cultures of Escherichia coli cells into which the encoding plc genes had been cloned. All of the enzymes showed the same specific activity. On the other hand, the level of plc transcripts differed greatly (up to 40-fold) from one C. perfringens strain to another. No significant difference in the nucleotide sequence of the plc promoter region was observed for any of the plc genes. These results suggest that the variation in phospholipase C activity among different strains is not due to mutation in the plc coding region but to that in an extragenic region. The evolution of C. perfringens phospholipase C is discussed on the basis of similarities and differences between clostridial plc genes.

    DOI: 10.1128/jb.177.24.7164-7170.1995

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  • A Method for purification of Clostridium perfringens phospholipase C from recombinant Bacillus subtilis cells. Reviewed

    Y Hirata, J Minami, M Koyama, O Matsushita, S Katayama, F Jin, H Maeta, A Okabe

    Applied and Environmental Microbiology   61 ( 11 )   4114 - 4115   1995.11

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    We developed a method to purify Clostridium perfringens phospholipase C from a culture of recombinant Bacillus subtilis cells. This method consists of three purification steps, and it allowed us to obtain 6.2 mg of pure phospholipase C from 800 ml of culture.

    DOI: 10.1128/aem.61.11.4114-4115.1995

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  • Genetic and phenotypic analysis of Borrelia miyamotoi sp. nov., isolated from the ixodid tick Ixodes persulcatus, the vector for Lyme disease in Japan. Reviewed

    M. Fukunaga, Y. Takahashi, Y. Tsuruta, O. Matsushita, D. Ralph, M. McClelland, M. Nakao

    International Journal of Systematic Bacteriology   45 ( 4 )   804 - 810   1995.10

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    DOI: 10.1099/00207713-45-4-804

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  • Identification of the gene encoding a mechanosensitive channel MscL homologue in Clostridium perfringens. Reviewed

    Osamu Matsushita, Chang-Min Jung, Akinobu Okabe

    Gene   165 ( 1 )   147 - 148   1995.1

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    DOI: 10.1016/0378-1119(95)00490-w

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  • Role of alpha-toxin in Clostridium perfringens infection determined by using recombinants of C. perfringens and Bacillus subtilis. Reviewed

    M Ninomiya, O Matsushita, J Minami, H Sakamoto, M Nakano, A Okabe

    Infection and Immunity   62 ( 11 )   5032 - 5039   1994.11

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    Clostridium perfringens type A strains which differed in alpha-toxin (phospholipase C [PLC]) productivity were inoculated intraperitoneally or intravenously into mice, and then their 50% mouse lethal doses (LD50) were determined. Strain NCTC 8237 produced ninefold higher PLC activity than strain 13. The mean LD50 for the former was 1 log unit lower than that for the latter. Two isogenic strains were constructed from strain 13: strain 13(pJIR418 alpha) (pJIR418 alpha contains the plc gene), which produced ninefold higher PLC activity than strain 13; and strain 13 PLC-, which showed no PLC productivity at all because of transformation-mediated gene disruption. The mean LD50 for strain 13(pJIR418 alpha) was 1 log unit lower than those for strain 13 PLC- and strain 13. These results indicate that PLC functions as a virulence-determining factor when it is produced in a sufficient amount. Such a difference in LD50 was also observed between Bacillus subtilis with and without the cloned plc gene. Inoculation of B. subtilis PLC+ intravenously into mice caused marked thrombocytopenia and leukocytosis. Mice inoculated with B. subtilis at 2 LD50 died because of circulatory collapse. Histological examination revealed that intravascular coagulation and vascular congestion occurred most prominently in the lungs. These results suggest that PLC plays a key role in the systemic intoxication of clostridial myonecrosis, probably by affecting the functions of platelets and phagocytes.

    DOI: 10.1128/iai.62.11.5032-5039.1994

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  • Cloning and nucleotide sequence analysis of the colH gene from Clostridium histolyticum encoding a collagenase and a gelatinase. Reviewed

    K Yoshihara, O Matsushita, J Minami, A Okabe

    Journal of Bacteriology   176 ( 21 )   6489 - 6496   1994.11

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    The colH gene encoding a collagenase was cloned from Clostridium histolyticum JCM 1403. Nucleotide sequencing showed a major open reading frame encoding a 116-kDa protein of 1,021 amino acid residues. The deduced amino acid sequence contains a putative signal sequence and a zinc metalloprotease consensus sequence, HEXXH. A 116-kDa collagenase and a 98-kDa gelatinase were copurified from culture supernatants of C. histolyticum. While the former degraded both native and denatured collagen, the latter degraded only denatured collagen. Peptide mapping with V8 protease showed that all peptide fragments, except a few minor ones, liberated from the two enzymes coincided with each other. Analysis of the N-terminal amino acid sequence of the two enzymes revealed that their first 24 amino acid residues were identical and coincided with those deduced from the nucleotide sequence. These results indicate that the 98-kDa gelatinase is generated from the 116-kDa collagenase by cleaving off the C-terminal region, which could be responsible for binding or increasing the accessibility of the collagenase to native collagen fibers. The role of the C-terminal region in the functional and evolutional aspects of the collagenase was further studied by comparing the amino acid sequence of the C. histolyticum collagenase with those of three homologous enzymes: the collagenases from Clostridium perfringens and Vibrio alginolyticus and Achromobacter lyticus protease I.

    DOI: 10.1128/jb.176.21.6489-6496.1994

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  • Comparison of the virulence of methicillin-resistant and methicillin-sensitive Staphylococcus aureus. Reviewed

    Sadao Mizobuchi, Junzaburo Minami, Fu Jin, Osamu Matsushita, Akinobu Okabe

    Microbiology and Immunology   38 ( 8 )   599 - 605   1994.8

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    DOI: 10.1111/j.1348-0421.1994.tb01829.x

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  • A Clostridium perfringens vector for the selection of promoters. Reviewed

    Chieko Matsushita, Osamu Matsushita, Michio Koyama, Akinobu Okabe

    Plasmid   31 ( 3 )   317 - 319   1994.5

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    DOI: 10.1006/plas.1994.1035

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  • Enterotoxic activity of Klebsiella oxytoca cytotoxin in rabbit intestinal loops. Reviewed

    J Minami, S Katayama, O Matsushita, H Sakamoto, A Okabe

    Infection and Immunity   62 ( 1 )   172 - 177   1994.1

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    We examined the enterotoxicity of a Klebsiella oxytoca cytotoxin which is produced by K. oxytoca OK-1, a strain from a patient with antibiotic-associated hemorrhagic colitis. Injection of the cytotoxin into ligated ileal and colonic loops in rabbits caused the accumulation of fluid in the loops. The fluid was bloody in the ileal loops but not in the colonic ones. Histological examination revealed intense mucosal hemorrhage with erosion in the ileum, whereas no microscopic change was noted in the colon. The fluid accumulation was shown to be a dose-dependent response in both ileal and colonic loops. The amounts of the cytotoxin required for maximal fluid accumulation in ileal and colonic loops were 60 and 10 micrograms, respectively. Fluid accumulation was first noticeable in ileal loops 12 h and in colonic ones 5 h after the injection of these doses of the cytotoxin and then proceeded with time. When K. oxytoca OK-1, a cytotoxin-producing strain, was inoculated into the loops at doses of 1 x 10(8) and 5 x 10(9) CFU, similar fluid accumulation was observed. However, inoculation of K. oxytoca ATCC 13182, a non-cytotoxin-producing strain, at the same doses did not cause any change. These results suggest that the cytotoxin-producing strain of K. oxytoca is the causative organism of antibiotic-associated hemorrhagic colitis and that the toxin is the factor responsible for pathogenesis.

    DOI: 10.1128/iai.62.1.172-177.1994

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  • Purification and characterization of Clostridium perfringens 120-kilodalton collagenase and nucleotide sequence of the corresponding gene. Reviewed

    O Matsushita, K Yoshihara, S Katayama, J Minami, A Okabe

    Journal of Bacteriology   176 ( 1 )   149 - 156   1994.1

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    Clostridium perfringens type C NCIB 10662 produced various gelatinolytic enzymes with molecular masses ranging from approximately 120 to approximately 80 kDa. A 120-kDa gelatinolytic enzyme was present in the largest quantity in the culture supernatant, and this enzyme was purified to homogeneity on the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme was identified as the major collagenase of the organism, and it cleaved typical collagenase substrates such as azocoll, a synthetic substrate (4-phenylazobenzyloxy-carbonyl-Pro-Leu-Gly-Pro-D-Arg [Pz peptide]), and a type I collagen fibril. In addition, a gene (colA) encoding a 120-kDa collagenase was cloned in Escherichia coli. Nested deletions were used to define the coding region of colA, and this region was sequenced; from the nucleotide sequence, this gene encodes a protein of 1,104 amino acids (M(r), 125,966). Furthermore, from the N-terminal amino acid sequence of the purified enzyme which was found in this reading frame, the molecular mass of the mature enzyme was calculated to be 116,339 Da. Analysis of the primary structure of the gene product showed that the enzyme was produced with a stretch of 86 amino acids containing a putative signal sequence. Within this stretch was found PLGP, the amino acid sequence constituting the Pz peptide. This sequence may be implicated in self-processing of the collagenase. A consensus zinc-binding sequence (HEXXH) suggested for vertebrate Zn collagenases is present in this bacterial collagenase. Vibrio alginolyticus collagenase and Achromobacter lyticus protease I showed significant homology with the 120-kDa collagenase of C. perfringens, suggesting that these three enzymes are evolutionarily related.

    DOI: 10.1128/jb.176.1.149-156.1994

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  • Comparison of the alpha-toxin genes of Clostridium perfringens type A and C strains: evidence for extragenic regulation of transcription. Reviewed

    S Katayama, O Matsushita, J Minami, S Mizobuchi, A Okabe

    Infection and Immunity   61 ( 2 )   457 - 463   1993.2

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    The Clostridium perfringens plc gene encoding phospholipase C (alpha-toxin) was cloned from type C NCIB 10662, a strain which produces low levels of phospholipase C activity. The nucleotide sequence of a cloned 3.1-kb HindIII fragment was determined. The same fragment was also cloned from type A NCTC 8237, a phospholipase C-overproducing strain. In this case, an open reading frame (ORF2) truncated in the previously cloned 2-kb fragment was also sequenced. Comparison of the nucleotide sequence between the 3.1-kb fragments of the two type strains shows some differences both in the plc gene and in ORF2. However, when the 3.1-kb fragment was cloned into plasmid pUC19 and expressed in Escherichia coli, the plc genes from both type strains were similarly expressed and the toxins produced showed similar levels of activity. Northern blot analysis revealed that the type A strain produced 16 to 23 times more plc mRNA than the type C strain. These results indicate that in C. perfringens the two plc genes are transcribed at different rates, probably because of a difference in a locus lying outside of the cloned fragments. Gel retardation analysis showed that the type A strain possessed two different proteins that bound different regions of the plc gene. However, one of these proteins, which binds within the plc coding region, was not found in the type C strain, suggesting that it plays a role in the regulation of the plc gene expression.

    DOI: 10.1128/iai.61.2.457-463.1993

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  • Melibiose transport system in Lactobacillus plantarum. Reviewed

    Chiyuki Tamura, Osamu Matsushita

    Microbiology and Immunology   36 ( 11 )   1119 - 1128   1992.11

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    DOI: 10.1111/j.1348-0421.1992.tb02116.x

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  • Properties of a genetically reconstructed Prevotella ruminicola endoglucanase. Reviewed

    G Maglione, O Matsushita, J B Russell, D B Wilson

    Applied and Environmental Microbiology   58 ( 11 )   3593 - 3597   1992.11

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    A pUC19-derived plasmid was constructed that coded for a hybrid cellulase with the Thermomonospora fusca E2 cellulose-binding domain at its C terminus joined to the Prevotella ruminicola 40.5-kDa carboxymethyl cellulase (CMCase). The hybrid enzyme was purified and characterized enzymatically. It bound tightly to cellulose, and its specific activities on carboxymethyl cellulose, amorphous cellulose, and ball-milled cellulose were 1.5, 10, and 8 times that of the 40.5-kDa CMCase, respectively. Furthermore, the modified enzyme gave synergism with an exocellulase in the degradation of filter paper, while the 40.5-kDa CMCase did not.

    DOI: 10.1128/aem.58.11.3593-3597.1992

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  • Role of the upstream region containing an intrinsic DNA curvature in the negative regulation of the phospholipase C gene of Clostridium perfringens. Reviewed

    Tatsuo Toyonaga, Osamu Matsushita, Sei-ichi Katayama, Junzaburo Minami, Akinobu Okabe

    Microbiology and Immunology   36 ( 6 )   603 - 613   1992.6

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    DOI: 10.1111/j.1348-0421.1992.tb02060.x

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  • Membrane lipids of Mycoplasma orale: lipid composition and synthesis of phospholipids. Reviewed

    Hirai Y, Kukida S, Matsushita O, Nagamachi E, Tomochika K, Kanemasa Y

    Physiological Chemistry and Physics and Medical NMR   24 ( 1 )   21 - 27   1992

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  • Adaptational changes of fatty acid composition and the physical state of membrane lipids following the change of growth temperature in Yersinia enterocolitica. Reviewed

    Eiko Nagamachi, Sei-ichiro Shibuya, Yoshikazu Hirai, Osamu Matsushita, Ken-ichi Tomochika, Yasuhiro Kanemasa

    Microbiology and Immunology   35 ( 12 )   1085 - 1093   1991.12

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    Publishing type:Research paper (scientific journal)   Publisher:Wiley  

    DOI: 10.1111/j.1348-0421.1991.tb01630.x

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  • A Bacteroides ruminicola 1,4-beta-D-endoglucanase is encoded in two reading frames. Reviewed

    O Matsushita, J B Russell, D B Wilson

    Journal of Bacteriology   173 ( 21 )   6919 - 6926   1991.11

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    Authorship:Lead author   Publishing type:Research paper (scientific journal)   Publisher:American Society for Microbiology  

    Escherichia coli transformed with a plasmid containing a Bacteroides ruminicola endoglucanase (carboxymethyl cellulase [CMCase]) gene produced three immunologically cross-reacting CMCases which had molecular weights of 40,500, 84,000, and 88,000, while B. ruminicola produced CMCases with molecular weights of 82,000 and 88,000. The two B. ruminicola enzymes (purified from culture supernatants) had different N-terminal amino acid sequences, but each enzyme was encoded by the same gene (three independent clones had the same DNA sequence). The 88,000-molecular-weight CMCase (88K CMCase) gene appeared to contain two open reading frames which overlapped for 18 bp and were -1 out of frame, and each open reading frame contained several stop codons near the overlap region. The two 88K CMCase open reading frames had enough DNA to produce a protein of 106K, but the mobility of the enzyme in sodium dodecyl sulfate gels gave a value which was 20% lower. On the basis of the -1 frame shift and the large deviation in theoretical versus actual size, it appears that an unusual event (e.g., ribosomal hopping or RNA splicing) is involved in either the translation or the transcription of the 88K B. ruminicola CMCase gene. The 82K CMCase was completely encoded in the second reading frame, and its size was in agreement with the DNA sequence.

    DOI: 10.1128/jb.173.21.6919-6926.1991

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  • An indirect immunofluorescence method for detection of Mycoplasma hominis in vaginal smears. Reviewed

    Yoshikazu Hirai, Tomohisa Kanatani, Masako Ono, Osamu Matsushita, Yasuhiro Kanemasa

    Microbiology and Immunology   35 ( 10 )   831 - 839   1991.10

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    Publishing type:Research paper (scientific journal)   Publisher:Wiley  

    DOI: 10.1111/j.1348-0421.1991.tb02023.x

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  • Cloning and sequencing of a Bacteroides ruminicola B14 endoglucanase gene. Reviewed

    O Matsushita, J B Russell, D B Wilson

    Journal of Bacteriology   172 ( 7 )   3620 - 3630   1990.7

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    Authorship:Lead author   Publishing type:Research paper (scientific journal)   Publisher:American Society for Microbiology  

    Bacteroides ruminicola B(1)4, a noncellulolytic rumen bacterium, produces an endoglucanase (carboxymethylcellulase [CMCase]) that is excreted into the culture supernatant. Cultures grown on glucose, fructose, maltose, mannose, and cellobiose had high specific activities of CMCase (greater than 3 mmol of reducing sugar per mg of protein per min), but its synthesis was repressed by sucrose. B. rumincola did not grow on either ball-milled or acid-swollen cellulose even though the CMCase could hydrolyze swollen cellulose. The CMCase gene was cloned into Escherichia coli, and its nucleotide sequence contained a single open reading frame coding for a protein of 40,481 daltons. The enzyme was overproduced in E. coli under the control of the tac promoter and purified to homogeneity. The N-terminal sequence, amino acid composition, and molecular weight of the purified enzyme were similar to the values predicted from the open reading frame of the DNA sequence. However, the CMCase present in B. ruminicola was found to have a monomer molecular weight of 88,000 by Western immunoblotting. This discrepancy appeared to have resulted from our having cloned only part of the CMCase gene into E. coli. The amino acid sequence of the CMCase showed homology to sequences of beta-glucanases from Ruminococcus albus and Clostridium thermocellum.

    DOI: 10.1128/jb.172.7.3620-3630.1990

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  • Lincomycin increases the half-life of beta-lactamase mRNA. Reviewed

    O Matsushita, A Okabe, H Hayashi, Y Kanemasa

    Antimicrobial Agents and Chemotherapy   33 ( 6 )   805 - 809   1989.6

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    Authorship:Lead author   Publishing type:Research paper (scientific journal)   Publisher:American Society for Microbiology  

    Escherichia coli K-12 strains isolates carrying plasmid pBR322 were grown in the presence of subinhibitory concentrations of lincomycin, which stimulated beta-lactamase synthesis about 2.5-fold, and the effects of the drug on the synthesis and degradation of bla mRNA were studied. The bla mRNA levels determined by 1-min pulse-labeling with [3H]uridine were significantly higher in a lincomycin-containing culture than in the control culture, indicating that stimulation of beta-lactamase synthesis is caused by an increase in the amount of bla mRNA. The enhancing effect of lincomycin was observed in strains harboring pBR322 delta P1 and pBR322 delta P3, which lacked the P1 or P3 promoter, respectively, as well as in the strain harboring pBR322. S1 nuclease analysis showed that the half-life of bla mRNA increased about 2.7-fold when lincomycin was present. These results indicate that the increase in beta-lactamase synthesis caused by lincomycin is due to an increase in the stability of bla mRNA rather than activation of its synthesis.

    DOI: 10.1128/aac.33.6.805

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  • Effects of lincomycin on synthesis of TEM beta-lactamase by Escherichia coli. Reviewed

    AKINOBU OKABE, OSAMU MATSUSHITA, HIDEO HAYASHI

    The Journal of Antibiotics   41 ( 5 )   667 - 674   1988

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    Publishing type:Research paper (scientific journal)   Publisher:Japan Antibiotics Research Association  

    DOI: 10.7164/antibiotics.41.667

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  • Lincomycin stimulates synthesis of TEM-2 beta-lactamase by Escherichia coli. Reviewed

    A Okabe, O Matsushita, S Katayama, H Hayashi

    Antimicrobial Agents and Chemotherapy   30 ( 1 )   82 - 87   1986.7

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    Publishing type:Research paper (scientific journal)   Publisher:American Society for Microbiology  

    Lincomycin increased the TEM-2 beta-lactamase activity of Escherichia coli K-12 cells carrying plasmid RP4 at a concentration which slightly inhibited cell growth. In a control culture beta-lactamase activity reached its maximal level in late log phase, whereas when lincomycin was present beta-lactamase activity continued to increase into the stationary phase. Lincomycin (100 micrograms/ml) inhibited both cell growth and protein synthesis by about 35% but stimulated beta-lactamase activity 2.5-fold per ml of culture and about 4-fold per cell after 20 h of growth. The amount of beta-lactamase produced in each culture was also compared by densitophotometry of a stained sodium dodecyl sulfate-polyacrylamide gel. The relative values were in good agreement with the relative enzyme activities, indicating that the stimulatory effect of lincomycin was due to an increase in the amount of beta-lactamase protein. Inactivation of beta-lactamase appeared to be faster when lincomycin was present. This was determined by measuring the decrease in beta-lactamase activity when phenethyl alcohol was present to prevent maturation of the enzyme. There was no significant difference in plasmid copy number between the cells grown in the presence or absence of lincomycin. These results indicate that lincomycin stimulates transcription, translation, or translocation of beta-lactamase.

    DOI: 10.1128/aac.30.1.82

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Books

  • コンパクト微生物学

    監修 小熊, 恵二, 堀田, 博, 編集 林, 俊治, 石戸, 聡(第9章 臓器感染症 消化器系 泌尿生殖系 神経系)

    南江堂  2021.3  ( ISBN:9784524226368

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    Total pages:xix, 294p   Language:Japanese

    CiNii Books

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  • シンプル微生物学

    編集 小熊, 恵二, 堀田, 博, 若宮, 伸隆(感染症成立の要因; クロストリジウム属)

    南江堂  2018.3  ( ISBN:9784524254835

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    Total pages:xvi, 457p   Language:Japanese

    CiNii Books

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MISC

  • New Helicobacters other than H. pylori.

    Yokota K, Kita M, Okada H, Matsushita O, Oguma K

    Nihon Rinsho   71 ( 8 )   1374 - 1379   2013.8

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    Language:Japanese  

    Since discovery of Helicobacter pylori, more than 30 species non-H. pylori Helicobacter spp. (NHPH) have been reported. Those NHPH were now classified into gastric Helicobacter spp. and enterohepatic Helicobacter spp.(EHS). Gastric NHPH show tight spiral and long shape in the gastric mucosa, and we can distinguish from H. pylori by light microscope. Some gastric NHPH may be zoonosis and cause gastritis in human. H. hepaticus and H. cinaedi belongs in EHS were detected in human diseases. H. hepaticus may be associated with hepatobiliary diseases in humans. Surprisingly, it was reported that H. cinaedi infection was associated with atrial arrhythmias and atherosclerosis. Many NHPH will be recognized as human pathogen in the future.

    PubMed

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  • Clostridial hydrolytic enzymes degrading extracellular components. Reviewed

    Osamu Matsushita, Akinobu Okabe

    Toxicon   39 ( 11 )   1769 - 1780   2001.11

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    Authorship:Lead author   Publisher:Elsevier BV  

    DOI: 10.1016/s0041-0101(01)00163-5

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  • Studies on the Clostridial Collagenases. Invited Reviewed

    MATSUSHITA Osamu

    Nippon Saikingaku Zasshi   54 ( 4 )   753 - 761   1999.11

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    Authorship:Lead author, Last author, Corresponding author  

    DOI: 10.3412/jsb.54.753

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  • Bacterial metalloproteases. Reviewed

    OKABE Akinobu, MATSUSHITA Osamu, MINAMI Junzaburo

    Nippon Saikingaku Zasshi   50 ( 4 )   971 - 989   1995.10

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Industrial property rights

  • Fusion proteins of collagen-binding domain and parathyroid hormone

    Robert C. Gensure, Joshua Sakon, Osamu Matsushita, Tulasi Ponnapakkam

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    Applicant:THE BOARD OF TRUSTEES OF THE UNIVERSITY OF ARKANSAS

    Application no:US 16/517,98  Date applied:2019.7.22

    Announcement no:US 2019/0338010 A1  Date announced:2019.11.7

    Patent/Registration no:US 10,519,213 B2  Date registered:2019.12.31 

    Rights holder:THE BOARD OF TRUSTEES OF THE UNIVERSITY OF ARKANSAS;OCHSNER CLINIC FOUNDATION;NATIONAL UNIVERSITY CORPORATION KAGAWA UNIVERSITY

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  • Fusion proteins of collagen-binding domain and parathyroid hormone

    Robert C. Gensure, Joshua Sakon, Osamu Matsushita, Tulasi Ponnapakkam

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    Applicant:The Board of Trustees of the University of Arkansas

    Application no:US 16/249,540  Date applied:2019.1.16

    Announcement no:US 2019/0135890 A1  Date announced:2019.5.9

    Patent/Registration no:US 10,358,471 B2  Date registered:2019.7.23 

    Rights holder:The Board of Trustees of the University of Arkansas;Ochsner Clinic Foundation;National University Corporation Kagawa University

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  • コラーゲン結合タンパク質による治療剤の送達

    サコン ジョシュア, フィロミネイサン サガヤ テレサ レーナ, ポンナパッカム トゥラシ, カティカネニ ランジータ, 小出 隆規, 松下 治, ジェンシュア ロバート シー, 西 望

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    Applicant:ザ ボード オブ トラスティーズ オブ ザ ユニバーシティ オブ アーカンソー

    Application no:特願2018-038513  Date applied:2018.3.5

    Announcement no:特開2018-104461  Date announced:2018.7.5

    Patent/Registration no:特許第6697019号  Date registered:2020.4.27 

    J-GLOBAL

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  • Delivery of therapeutic agents by a collagen binding protein

    Tulasi Ponnapakkam, Sagaya Theresa Leena Philominathan, Joshua Sakon, Ranjitha Katikaneni, Takaki Koide, Osamu Matsushita, Robert C. Gensure, Nozomu Nishi

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    Applicant:The Board of Trustes of the University of Arkansas

    Application no:US 15/407,589  Date applied:2017.1.17

    Announcement no:US 2017/0204390 A1  Date announced:2017.7.20

    Patent/Registration no:US 11,001,820 B2  Date registered:2021.5.11 

    Rights holder:The Board of Trustes of the University of Arkansas;Montefiore Medical CenterThe Kitasato Institute;National University Corporation Kagawa University

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  • Fusion proteins of collagen-binding domain and parathyroid hormone

    Robert C. Gensure, Joshua Sakon, Osamu Matsushita, Tulasi Ponnapakkam

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    Applicant:THE BOARD OF TRUSTEES OF THE UNIVERSITY OF ARKANSAS

    Application no:US 15/387,817  Date applied:2016.12.22

    Announcement no:US 2017/0101457 A1  Date announced:2017.4.13

    Patent/Registration no:US 10,202,434 B2  Date registered:2019.2.12 

    Rights holder:THE BOARD OF TRUSTEES OF THE UNIVERSITY OF ARKANSAS;OCHSNER CLINIC FOUNDATION;NATIONAL UNIVERSITY CORPORATION KAGAWA UNIVERSITY

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  • Delivery of therapeutic agents by a collagen binding protein

    Tulasi Ponnapakkam, Sagaya Theresa Leena Philominathan, Joshua Sakon, Ranjitha Katikaneni, Takaki Koide, Osamu Matsushita, Robert C. Gensure

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    Applicant:The Board of Trustes of the University of Arkansas

    Application no:US 15/386,626  Date applied:2016.12.21

    Announcement no:US 2017/0106093 A1  Date announced:2017.4.20

    Patent/Registration no:US 10,213,488 B2  Date registered:2019.2.26 

    Rights holder:The Board of Trustes of the University of Arkansas;The Kitasato Institute;Montefiore Medical Center

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  • コラーゲン結合領域と副甲状腺ホルモンとの融合タンパク

    松下 治, ジェンシュア, ロバート シー, サコン ジョシュア, ポンナパッカム トゥラシ

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    Applicant:ザ ボード オブ トラスティーズ オブ ザ ユニバーシティ オブ アーカンソー

    Application no:特願2016-157345  Date applied:2016.8.10

    Announcement no:特開2017-25069  Date announced:2017.2.2

    Patent/Registration no:特許第6366653号  Date registered:2018.7.13  Date issued:2018.8.1

    Rights holder:ザ ボード オブ トラスティーズ オブ ザ ユニバーシティ オブ アーカンソー;国立大学法人 香川大学;オクスナー クリニック ファウンデイション

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  • 神経再生用移植材料、神経再生用移植材料の製造方法、及び神経再生用移植材料製造用キット

    内田 健太郎, 井上 玄, 藤巻 寿子, 高相 晶士, 佐久 太郎, 礒部 仁博, 松下 治, 美間 健彦, 西 望, 服部 俊治, 田中 啓友, 小倉 孝之

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    Applicant:学校法人北里研究所

    Application no:特願2016-554137  Date applied:2015.10.16

    Publication no:WO2016/060252  Date published:2016421

    Patent/Registration no:特許第6699821号  Date registered:2020.5.7  Date issued:2020.5.27

    Rights holder:学校法人北里研究所;株式会社アトリー;国立大学法人 岡山大学;国立大学法人 香川大学;株式会社ニッピ

    J-GLOBAL

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  • コラーゲン結合領域と副甲状腺ホルモンとの融合タンパク

    松下 治, ジェンシュア ロバート シー, サコン ジョシュア, ポンナパッカム トゥラシ

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    Applicant:ザ ボード オブ トラスティーズ オブ ザ ユニバーシティ オブ アーカンソー

    Application no:特願2015-173678  Date applied:2015.9.3

    Announcement no:特開2016-094385  Date announced:2016.5.26

    Patent/Registration no:特許第5989876号  Date registered:2016.8.19 

    J-GLOBAL

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  • Fusion proteins of collagen-binding domain and parathyroid hormone

    Robert C. Gensure, Joshua Sakon, Osamu Matsushita, Tulasi Ponnapakkam

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    Applicant:Ochsner Clinic Foundation

    Application no:US 14/743,629  Date applied:2015.6.18

    Announcement no:US 2015/0284701 A1  Date announced:2015.10.8

    Patent/Registration no:US 9,528,099 B2  Date registered:2016.12.27 

    Rights holder:Ochsner Clinic Foundation;National University Corporation Kagawa University;The Board of Trustees of the University of Arkansas

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  • コラーゲン結合領域と副甲状腺ホルモンとの融合タンパク

    松下 治, ジェンシュア ロバート シー, サコン ジョシュア, ポンナパッカム トゥラシ

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    Applicant:ザ ボード オブ トラスティーズ オブ ザ ユニバーシティ オブ アーカンソー

    Application no:特願2013-194484  Date applied:2013.9.19

    Announcement no:特開2014-040431  Date announced:2014.3.6

    Patent/Registration no:特許第5821066号  Date registered:2015.10.16 

    J-GLOBAL

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  • Fusion proteins of collagen-binding domain and parathyroid hormone

    Robert C. Gensure, Joshua Sakon, Osamu Matsushita, Tulasi Ponnapakkam

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    Applicant:National University Corporation Kagawa University

    Application no:US 13/898,058  Date applied:2013.5.20

    Announcement no:US 2013/0337017 A1  Date announced:2013.12.19

    Patent/Registration no:US 9,062,300 B2  Date registered:2015.5.23 

    Rights holder:National University Corporation Kagawa University;Ochsner Clinic Foundation;The Board of Trustees of the University of Arkansas

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  • Delivery of therapeutic agents by a collagen binding protein

    Tulasi Ponnapakkam, Sagaya Theresa Leena Philominathan, Joshua Sakon, Ranjitha Katikaneni, Takaki Koide, Osamu Matsushita, Robert C. Gensure

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    Applicant:The Kitasato Institute

    Application no:US 14/378,067  Date applied:2013.2.11

    Announcement no:US 2015/0038423 A1  Date announced:2015.2.5

    Publication no:WO2013/120060  Date published:2013815

    Patent/Registration no:US 9,526,765 B2  Date registered:2016.12.27 

    Rights holder:The Kitasato Institute;Montefiore Medical Center;Board of Trustees of the University of Arkansas

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  • コラーゲン結合タンパク質による治療剤の送達

    サコン ジョシュア, フィロミネイサン サガヤ テレサ レーナ, ポンナパッカム トゥラシ, カティカネニ ランジータ, 小出 隆規, 松下 治, ジェンシュア ロバート シー, 西 望

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    Applicant:ザ ボード オブ トラスティーズ オブ ザ ユニバーシティ オブ アーカンソー

    Application no:特願2014-547503  Date applied:2012.12.14

    Publication no:特表2015-513312  Date published:201557

    Patent/Registration no:特許第6366507号  Date registered:2018.7.13  Date issued:2018.8.1

    Rights holder:ザ ボード オブ トラスティーズ オブ ザ ユニバーシティ オブ アーカンソー;学校法人 北里研究所;モンテフィオーレ メディカル センター;国立大学法人 香川大学

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  • Delivery of therapeutic agents by a collagen binding protein

    Joshua Sakon, Sagaya Theresa Leena Philominathan, Ranjitha Katikaneni, Osamu Matsushita, Tulasi Ponnapakkam, Takaki Koide, Robert C Gensure, Nozomu Nishi

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    Applicant:The Board of Trustees of the University of Arkansas

    Application no:EP 18173596.0 A  Date applied:2012.12.14

    Announcement no:EP 3391899 A1  Date announced:2018.10.24

    Publication no:12857691.5/2 790 717  Date published:20181024

    Patent/Registration no:EP 3 391 899 B1  Date registered:2020.7.15 

    Rights holder:The Board of Trustees of the University of Arkansas;The Kitasato Institute;Montefiore Medical Center;National University Corporation Kagawa University

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  • Delivery of therapeutic agents by a collagen binding protein

    Tulasi Ponnapakkam, Sagaya Theresa Leena Philominathan, Joshua Sakon, Ranjitha Katikaneni, Takaki Koide, Osamu Matsushita, Robert C. Gensure, Nozomu Nishi

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    Applicant:The Kitasato Institute

    Application no:US 14/365,226  Date applied:2012.12.14

    Announcement no:US 2014/0377215 A1  Date announced:2014.12.25

    Publication no:WO013/090770  Date published:2013620

    Patent/Registration no:US 9,579.273 B2  Date registered:2017.2.28 

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  • Delivery of therapeutic agents by a collagen binding protein

    Joshua Sakon, Sagaya Theresa Leena Philominathan, Ranjitha Katikaneni, Osamu Matsushita, Tulasi Ponnapakkam, Takaki Koide, Robert C Gensure, Nozomu Nishi

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    Applicant:The Board of Trustees of the University of Arkansas

    Application no:EP 12857691.5 A  Date applied:2012.12.14

    Announcement no:EP 2790717 A4  Date announced:2016.3.9

    Publication no:WO2013/090770  Date published:2013620

    Patent/Registration no:EP 2 790 717 B1  Date registered:2018.5.30 

    Rights holder:The Board of Trustees of the University of Arkansas;The Kitasato Institute;Montefiore Medical Center;National University Corporation Kagawa University

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  • Delivery of therapeutic agents using bacterial collagen-binding polypeptide segments

    Joshua Sakon, Sagaya Theresa Leena Philominathan, Ranjitha Katikaneni, Osamu Matsushita, Tulasi Ponnapakkam, Takaki Koide, Robert C. Gensure, Nozomu Nishi

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    Applicant:THE BOARD OF TRUSTEES OF THE UNIVERSITY OF ARKANSAS

    Application no:US 2012/069831  Date applied:2012.12.14

    Publication no:WO2013/090770  Date published:2013620

    Patent/Registration no:CA 2 859 412 C  Date registered:2021.5.25 

    Rights holder:THE BOARD OF TRUSTEES OF THE UNIVERSITY OF ARKANSAS;MONTEFIORE MEDICAL CENTER;THE KITASATO INSTITUTE

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  • 成長因子アンカーリング型骨移植材料および成長因子アンカーリング型骨移植材料製造用キット

    内田 健太郎, 成瀬 康治, 高相 晶士, 美間 健彦, 松下 治, 原口 高志, 西 望

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    Applicant:学校法人北里研究所

    Application no:特願2013-515034  Date applied:2012.3.26

    Patent/Registration no:特許第5512887号  Date registered:2014.4.4 

    J-GLOBAL

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  • Growth factor anchoring type bone graft material, method for producing growth factor anchoring type bone graft material, kit for producing growth factor anchoring type bone graft material, and method for forming bone

    Kentaro Uchida, Koji Naruse, Masashi Takaso, Takehiko Mima, Osamu Matsushita, Takashi Haraguchi, Nozomu Nishi

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    Applicant:SCHOOL JURIDICAL PERSON KITASATO INSTITUTE

    Application no:US 14/117,599  Date applied:2012.3.26

    Announcement no:US 2014/0335146 A1  Date announced:2014.11.13

    Publication no:WO2012/157339  Date published:20121122

    Patent/Registration no:US 9,248,164 B2  Date registered:2016.2.2 

    Rights holder:SCHOOL JURIDICAL PERSON KITASATO INSTITUTE;NATIONAL UNIVERSITY CORPORATION KAGAWA UNIVERSITY

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  • Growth factor anchoring type bone graft material, method for producing growth factor anchoring type bone graft material, kit for producing growth factor anchoring type bone graft material, and method for forming bone

    Kentaro Uchida, Koji Naruse, Masashi Takaso, Takehiko Mima, Osamu Matsushita, Takashi Haraguchi, Nozomu Nishi

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    Applicant:School Juridical Person Kitasato Institute

    Application no:EP 12785014.7 A  Date applied:2012.3.26

    Announcement no:EP 2708246 A4  Date announced:2014.11.19

    Publication no:WO2012/157339  Date published:20121122

    Patent/Registration no:EP 2 708 246 B1  Date registered:2017.11.1 

    Rights holder:School Juridical Person Kitasato Institute;National University Corporation Kagawa University

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  • コラーゲン結合領域と副甲状腺ホルモンとの融合タンパク

    松下 治, ジェンシュア ロバート シー, サコン ジョシュア, ポンナパッカム トゥラシ

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    Applicant:ザ ボード オブ トラスティーズ オブ ザ ユニバーシティ オブ アーカンソー

    Application no:特願2010-503048  Date applied:2008.4.9

    Patent/Registration no:特許第5520811号  Date registered:2014.4.11 

    J-GLOBAL

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  • Fusion proteins of collagen-binding domain and parathyroid hormone

    Joshua Sakon, Osamu Matsushita, Robert C. Gensure, Tulasi Ponnapakkam

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    Applicant:The Board of Trustees of The University of Arkansas

    Application no:EP 16169069.8 A  Date applied:2008.4.9

    Announcement no:EP 3091075 A1  Date announced:2016.11.9

    Patent/Registration no:EP 3 091 075 B1  Date registered:2018.6.13 

    Rights holder:THE BOARD OF TRUSTEES OF THE UNIVERSITY OF ARKANSAS;National University Corporation Kagawa University;Ochsner Clinic Foundation

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  • Fusion proteins of collagen-binding domain and parathyroid hormone

    Joshua Sakon, Robert C. Gensure, Osamu Matsushita

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    Applicant:The Board of Trustees of the University of Arkansas

    Application no:US 12/594,547  Date applied:2008.4.9

    Announcement no:US 2010/0129341 A1  Date announced:2010.5.27

    Patent/Registration no:US 8.450,273 B2  Date registered:2013.5.28 

    Rights holder:The Board of Trustees of the University of Arkansas;Ochsner Clinic Foundation;National University Corporation Kagawa University

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  • Fusion proteins of collagen-binding domain and parathyroid hormone

    Joshua Sakon, Osamu Matsushita, Robert C. Gensure, Tulasi Ponnapakkam

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    Applicant:THE BOARD OF TRUSTEES OF THE UNIVERSITY OF ARKANSAS

    Application no:EP 08742686.2 A  Date applied:2008.4.9

    Announcement no:EP 2155874 A4  Date announced:2010.9.1

    Publication no:WO2008/124166  Date published:20081016

    Patent/Registration no:EP 2 155 874 B1  Date registered:2016.5.11 

    Rights holder:THE BOARD OF TRUSTEES OF THE UNIVERSITY OF ARKANSAS;National University Corporation Kagawa University;Ochsner Clinic Foundation

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  • Fusion proteins of collagen-binding domain and parathyroid hormone

    Osamu Matsushita, Robert C. Gensure, Joshua Sakon, Tulasi Ponnapakkam

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    Applicant:The Board of Trustees of the University of Arkansas; National University Corporation Kagawa University; Ochsner Clinic Foundation

    Application no:2 683 862  Date applied:2008.4.9

    Date announced:2008.10.16

    Patent/Registration no:CA 2 930 681 C  Date registered:2019.10.15 

    Rights holder:The Board of Trustees of the University of Arkansas;Ochsner Clinic Foundation;National University Corporation Kagawa University

  • Fusion protein of collagen-binding domain and parathyroid hormone

    Osamu Matsushita, Robert C. Gensure, Joshua Sakon, Tulasi Ponnapakkam

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    Applicant:THE BOARD OF TRUSTEES OF THE UNIVERSITY OF ARKANSAS

    Application no:2 683 862  Date applied:2008.4.9

    Date announced:2008.10.16

    Patent/Registration no:CA 2 930 681 C  Date registered:2019.10.15 

    Rights holder:THE BOARD OF TRUSTEES OF THE UNIVERSITY OF ARKANSAS;NATIONAL UNIVERSITY CORPORATION KAGAWA UNIVERSITY;OCHSNER CLINIC FOUNDATION

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  • Fusion protein of collagen-binding domain and parathyroid hormone

    Joshua Sakon, Osamu Matsushita, Robert C. Gensure, Tulasi Ponnapakkam

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    Applicant:THE BOARD OF TRUSTEES OF THE UNIVERSITY OF ARKANSAS

    Application no:US2008/004589  Date applied:2008.4.9

    Publication no:WO2008/124166 A3  Date published:20081016

    Patent/Registration no:CA 2 683 862 C  Date registered:2016.8.2 

    Rights holder:THE BOARD OF TRUSTEES OF THE UNIVERSITY OF ARKANSAS;NATIONAL UNIVERSITY CORPORATION KAGAWA UNIVERSITY;OCHSNER CLINIC FOUNDATION

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  • Fusion proteins of collagen-binding domain and parathyroid hormone

    Joshua Sakon, Osamu Matsushita, Robert C. Gensure, Tulasi Ponnapakkam

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    Applicant:The Board of Trustees of the University of Arkansas; National University Corporation Kagawa University; Ochsner Clinic Foundation

    Application no:US2008/004589  Date applied:2008.4.9

    Publication no:WO2008/124166 A3  Date published:20081016

    Patent/Registration no:CA 2 683 862 C  Date registered:2016.8.2 

    Rights holder:The Board of Trustees of the University of Arkansas;National University Corporation Kagawa University;Ochsner Clinic Foundation

  • 新規酵素活性ポリペプチド及びそれを用いる融合タンパク質切断キット

    松下 治, 岡部 昭延, 鄭 昌敏

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    Applicant:生化学工業株式会社

    Application no:特願平9-310887  Date applied:1997.11.12

    Announcement no:特開平11-137256  Date announced:1999.5.25

    Patent/Registration no:特許第4484971号  Date registered:2010.4.2 

    J-GLOBAL

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Awards

  • 黒屋奨学賞受賞

    1999.4   日本細菌学会   ガス壊疽菌群のコラゲナーゼに関する研究

    松下 治

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Research Projects

  • 絞扼性神経障害に関与するT細胞サブセットの同定と神経ペプチド による治療法の開発

    Grant number:21K09308  2021.04 - 2024.03

    日本学術振興会  科学研究費助成事業 基盤研究(C)  基盤研究(C)

    高相 晶士, 松下 治, 廣澤 直也, 佐藤 雅, 宮城 正行

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    Grant amount:\4160000 ( Direct expense: \3200000 、 Indirect expense:\960000 )

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  • バイオミメティック Veing Wrapping による末梢神経障害治療法の確立

    Grant number:20K09415  2020.04 - 2023.03

    日本学術振興会  科学研究費助成事業 基盤研究(C)  基盤研究(C)

    井上 玄, 内田 健太郎, 松下 治, 大鳥 精司, 馬渕 洋, 宮城 正行

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    Grant amount:\4290000 ( Direct expense: \3300000 、 Indirect expense:\990000 )

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  • Development of periodontal tissue regeneration therapy for horizontal alveolar bone resorption with collagen-binding FGF-2

    Grant number:19H03831  2019.04 - 2023.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)  Grant-in-Aid for Scientific Research (B)

    高柴 正悟, 松下 治, 平山 晴子, 山本 直史, 美間 健彦, 伊東 孝

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    Grant amount:\17030000 ( Direct expense: \13100000 、 Indirect expense:\3930000 )

    本研究は,水平性骨吸収に対する歯周組織再生療法を実現するために,既に歯科臨床で応用されている塩基性線維芽細胞増殖因子(bFGF)と細菌由来のコラーゲン結合ドメイン(CBD)を組み合わせた融合タンパク質(CBFGF)を用いた研究である。本研究の目的は,CBFGFの最適化とイヌを用いた実験モデルによる非臨床試験データの取得である。2019年度は,認可済みのbFGF製剤に合わせて,CBFGFを組換融合型から架橋型へ変更するための実験とイヌの骨欠損モデルを用いた実験を実施した。まず,CBFGFの最適化について,架橋反応の比率・濃度などの反応条件を決定するために,多量のbFGFとCBDを要した。そこで,大腸菌生産系を用いてbFGFとCBDを精製し,実験効率の改善を図った。現在は,CBDとbFGFを架橋する適切な条件を探索しているところである。また,イヌを用いた実験モデルでは,歯周組織再生療法の適応症である垂直性骨欠損(2壁性)で組換融合型CBFGFの有効性を実証し,水平性骨欠損および垂直性骨欠損(1壁性)を作製して,組換融合型CBFGFを投与した。現在,動物へのタンパク質の投与は終了し,一部のサンプルはCT撮影まで行っている。
    また,組換融合型CBFGFについてこれまでに得られたデータについては,研究発表(Takashiba S, International Academy of Periodontology, 2019;Nakamura S, et al, International Association of Dental Research, General Session & Exhibition, 2019;岡本ら,日本歯科保存学会,2019;高柴,BioJapan 2019)を行って,今後の研究の進め方やCBFGFの製剤化に関して,様々な研究者や企業と情報交換を行った。

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  • ピロリ菌除菌療法における腸内エコシステム破綻のメカニズムと制御

    Grant number:19K08395  2019.04 - 2022.03

    日本学術振興会  科学研究費助成事業 基盤研究(C)  基盤研究(C)

    岡田 裕之, 後藤 和義, 横田 憲治, 松下 治, 田中 健大, 岡上 昇太郎

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    Grant amount:\4160000 ( Direct expense: \3200000 、 Indirect expense:\960000 )

    大学新入生において標準的なピロリ菌除菌レジメンの下で下痢・軟便を主とした副作用を起こす患者の腸内細菌叢に共通するファクターを見出すことを目的とする。さらにはメタゲノムデータと常在細菌叢の抗菌薬感受性を融合させることで、なぜ特定の菌叢を持つ(または持たない)ことでDysbiosisが起こるのか、そのメカニズムを説明する。さらに内視鏡的な胃炎、組織学的胃炎評価も行い、最終的に腸内細菌叢解析データと融合する。平成31年度(2019年度)から3年間にわたり岡山大学医学部・歯学部新入生(医学科・保健学科・歯学部)に対してピロリ菌検診を例年通り実施し、H.pylori-IgG抗体陽性例を本研究の対象とする。
    2019年度は新入生314人中17人が抗体陽性であった。抗体陽性者14人に内視鏡検査を行い、内視鏡的胃炎、組織学的胃炎の評価および菌株培養を行った。組織学的陽性例は現感染と診断した。組織学的胃炎、菌株培養陰性例に対しては、さらに尿素呼気試験も行い、それら3検査とも陰性の場合は抗体検査が偽陽性と判断し、未感染と診断した。その結果、現感染11名、未感染3名であった。現感染者には除菌治療を行い、除菌前、除菌1週後、2ケ月後の糞便採取を行うとともに、除菌前後2週間の排便回数も含めた消化管症状についてアンケートを実施した。未感染者に対しても1回糞便採取とアンケートを実施した。
    得られた糞便の核酸抽出を実施した。
    2020年度、2021年度新入生に対しても同様に進めていく予定である。

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  • Elucidation of substrate recognition mechanism of bacterial collagenases to develop angiogenic drug seeds

    Grant number:18K07111  2018.04 - 2021.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    松下 治, 美間 健彦, 内田 健太郎

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    Grant amount:\4420000 ( Direct expense: \3400000 、 Indirect expense:\1020000 )

    細菌性コラゲナーゼは、触媒モジュールと基質アンカー・モジュールよりなる。塩基性線維芽細胞増殖因子(bFGF)を基質アンカー・モジュールと融合(CB-bFGF)して、bFGFをコラーゲン基剤に結合し、bFGFの組織修復能を局所で長期間発揮させることができた。本複合剤を整形外科領域で骨移植や重症骨折後の骨新生誘導に応用できた。
    本課題では、歯周病による歯槽骨の水平欠損に対する本複合剤の有用性をラット・モデルにより検討した。CB-bFGF固相化コラーゲンを骨欠損部に充填することで、有意な骨形成を誘導できた。オステオカルシン、proliferating cell nuclear antigen、オステオポンチン陽性細胞が増加することを示した(論文1)。生体内でのCB-bFGFの薬物動態と治療効果を明らかにするため、イヌを用いた前臨床研究を実施し解析を進めている(論文準備中)。
    CB-bFGFを用いた神経再生を試みた。「ナーブリッジ」は、我が国で開発された神経再生用医療機器である。ポリグリコール酸製の外鞘の内腔にコラーゲンを充填した構造を有し、神経欠損部に固定して中枢側から伸長する自己神経を末梢側へ誘導する。CB-bFGFにより神経再生に必要な血管などの支持組織を効果的に誘導できれば、本機器による神経再生を促進できる。15mmにわたり坐骨神経を欠損したラット・モデルを作成し、CB-bFGFを固相化したナーブリッジによる神経再生を組織学的、行動学的に検討した。8週後のミエリン繊維の再生は、CB-bFGF群ラットでは7/8 (87.5%)、bFGF群では3/8 (37.5%) 、PBS群では1/8 (12.5%)に認められた。また4週後の歩行機能を足跡面積を用いて評価したところ、CB-bFGF群ラットは、bFGF群、PBS群より優位に広範囲の歩行が認められた(論文2)。

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  • 腸管スキャフォールドとコラーゲン結合型成長因子を用いた拍動性グラフトの創成

    Grant number:18K08759  2018.04 - 2021.03

    日本学術振興会  科学研究費助成事業 基盤研究(C)  基盤研究(C)

    笠原 真悟, 松下 治, 王 英正, 美間 健彦

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    Grant amount:\4030000 ( Direct expense: \3100000 、 Indirect expense:\930000 )

    組織工学を用いた臓器再生研究の分野で、脱細胞化した心臓に心筋細胞を移植する
    ことで拍動が得られることが報告された。また、脱細胞化した腸管を組織工学の鋳型として利用できることも報告された。これまでに我々は、細菌性コラゲナーゼのコラーゲン・アンカー部を用いて結合組織やコラーゲン基剤に成長因子をアンカリングすることで、その効果を持続的に発揮させられることを示した。
    2019年度は、ラット小腸を腸間膜動静脈を含め脱細胞化して足場とし、心筋細胞の増殖と血管新生を誘導する種々の成長因子をアンカリングしつつ、ラット新生児の心臓由来細胞を播種することで、栄養血管を備えた拍動する心筋の筒を作製しようと考えていた。
    まずラット小腸の脱細胞化を試みた。生体から小腸を採取し、動静脈にカニュレーションし、界面活性剤を24から48時間程度還流することにより脱細胞化できることを確認した。続いて、脱細胞化した小腸の細胞外マトリックスを足場として、心筋細胞を生着させようと試みた。従来我々が用いていた解放空間における灌流装置で、脱細胞化小腸にラット新生児心筋細胞を付着させ培養液を還流したが、良好な成育は認められなかった。心筋の破砕の程度、還流流量などを変化させたが、十分な生着といえる成果は得られなかった。そこで、温度、湿度、CO2濃度を管理できるインキュベータ内で培養すべく、小型の還流装置を試作した。しかしながら最適な還流量の確立が困難で、現在足場素材、大きさの再検討、それに対する流量の検討を進めている。

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  • 注入型局所硬化ゲルを用いた幹細胞・成長因子送達による新規難治性骨折治療法の開発

    Grant number:18K09079  2018.04 - 2021.03

    日本学術振興会  科学研究費助成事業 基盤研究(C)  基盤研究(C)

    内田 健太郎, 松下 治, 馬渕 洋

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    Grant amount:\4420000 ( Direct expense: \3400000 、 Indirect expense:\1020000 )

    移植型、局所投与型骨形成促進剤の利点を併せ持つ新規骨形成促進材料を開発すべく検討を行った。昨年度までにデキストラン(Dex)を主骨格とする局所硬化ゲル(Dexゲル)にbFGFを含有させることでマウス骨折モデルにおける骨形成を促進できることを示した。しかし、難治性骨折モデルに対する効果は十分ではなかった。本年度はヒアルロン酸(HA)を主骨格とする局所硬化ゲルとBMP2の併用による骨形成促進効果を検討した。マウス難治性骨折モデルを作製後、骨折群(control群)、局所硬化ヒアルロン酸ゲル投与群(HA群)、BMP-2含有局所硬化ヒアルロン酸ゲル投与群(HA+BMP-2群)を作成した。骨折後4週で屠殺して大腿骨を採取後micro CTを撮影し、骨折部における新生骨量(BV)、骨塩量(BMC)を測定した。また、骨折部の骨癒合率を評価した。また、新生仮骨を採取し、real time PCR法にてalkaline phosphatase、osterix、osteocalcinの発現量を検討したほか、組織学的にも評価した。control群、HA群と比較し、HA+BMP-2群で4週におけるBV、BMCの高値を認めた。HA+BMP-2群では、micro CT画像および組織切片標本において新生仮骨による骨折部の架橋を認め、骨癒合率も有意に高かった。新生仮骨におけるアルカリホスファターゼ、オステリックス、オステオカルシンの発現量もHA+BMP-2群で有意に高かった。BMP-2含有局所硬化ヒアルロン酸ゲル剤は、難治性骨折モデルにおいて骨癒合を促進することが可能であった。本方法はBMP-2の局所送達による新規難治性骨折治療法として有用である可能性が示唆された。

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  • Development of functional multilayered mesenchymal stem cells sheet for lumbar fusion surgery

    Grant number:17K10982  2017.04 - 2020.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    Inoue Gen

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    Grant amount:\4680000 ( Direct expense: \3600000 、 Indirect expense:\1080000 )

    Cell-based regenerative therapy has the potential to promote bone formation in spinal fusion. Here, we developed a multilayered cell-based bone formation system using cells coated with fibronectin-gelatin (FN-G) nanofilms. The multilayered human mesenchymal stem cells (hMSCs) were formed after two days of culture and were shown to express higher levels of BMP-2, TGF-β, bFGF and VEGF compared to monolayer cultures of hMSCs. The MLMCs were used as a graft material in combination with a collagen-binding bFGF and BMP-2. In grafted with the hMSCs, significantly higher levels of callus volume and bone mineral content were observed compared to the sham controls. The callus volume and bone mineral content were further increased in situ grafted with bFGF-PKD-CBD/BMP-2/hMSCs. Taken together, these results suggest that bFGF-PKD-CBD/BMP-2/hMSC may offer novel therapeutic approaches to augment spinal fusion.

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  • Clarification of molecular mechanisms underlying the analgesic effect of botulinum toxin and applied study

    Grant number:26350499  2014.04 - 2017.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    YAMAMOTO YUMIKO, MATSUSHITA Osamu, OGUMA Keiji

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    Grant amount:\4810000 ( Direct expense: \3700000 、 Indirect expense:\1110000 )

    We developed an assay to measure the proteolytic activity of botulinum toxin (BoNT), and attempted to synthesize fluorescently labeled full-length BoNT, in order to study molecular mechanisms for the analgesic effect of BoNT. We also generated four kinds of anti-peptide antibodies against SNAP-25, and showed that BoNT/A or BoNT/E cleaves SNAP-25 in cultured trigeminal ganglion neurons. This suggests that peripherally administered BoNT decreases the release of neurotransmitters via the proteolysis of SNARE proteins in sensory ganglion neurons and relieves neuropathic pain.

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  • Structure-function analysis of bacterial toxins in tissue destruction to apply their functional domains to regenerative medicine

    Grant number:26460527  2014.04 - 2017.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    MATSUSHITA Osamu, SAKON Joshua

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    Grant amount:\4940000 ( Direct expense: \3800000 、 Indirect expense:\1140000 )

    Flesh-eating bacteria produce collagenases to degrade collagen fibrils and to destruct the host tissue. The enzymes consist of a catalytic domain, PKD domain(s) (PKD), and collagen-binding domain(s) (CBD). Since PKD enhanced the binding of CBD to insoluble collagen, PKD-CBD region derived from a collagenase was used to anchor growth factors to collagenous matrix, e.g. high-density collagen sheet or demineralized bone matrix. These composite materials were utilized to induce osteogenesis at bone defect sites. 3D-structure of PKD was determined by X-ray crystallography, which gave an insight into the molecular basis of the collagen anchoring. Furthermore, dimeric CBD was shown to be an appropriate anchor for a synthetic collagenous matrix.

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  • Establishment of novel osteochondral allografting combined with growth factor- collagen-binding domain fusion technology

    Grant number:26462277  2014.04 - 2017.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    Onuma Kenji, MATSUSHITA Osamu

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    Grant amount:\4810000 ( Direct expense: \3700000 、 Indirect expense:\1110000 )

    We investigated study for improved osteochondral allografting combined with basic fibroblast growth factor (bFGF)- collagen-binding domain (CBD) fusion protein. In the preliminary study, optimal reaction time for binding bFGF-CBD to rat osteochondral tissue (OCT) samples was 15 minutes. Cylindrical OCTs were harvested from rabbit femoral condyles and then preserved for 1 week in the preservation solutions at 4 ℃. Then OCTs were reacted to bind bFGF-CBD for 15 minutes in the solution containing bFGF-CBD and then implanted to rabbit osteochondral defect models. While in the control samples, OCTs were not bound with bFGF-CBD. At 4 and 8 weeks after implantation, condyles were harvested and then samples were accessed using micro-CT and histological samples stained with HE and safranin-o. We could not observed significant difference between OCT samples with bFGF-CBD and control samples. These results concluded that bFGF-CBD might not improve osteochondral allografting of rabbit model.

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  • Development of collagen binding VEGF-C for lymphangiogenesis

    Grant number:26462731  2014.04 - 2017.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    Matsumoto Hiroshi

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    Grant amount:\4810000 ( Direct expense: \3700000 、 Indirect expense:\1110000 )

    We tried to make collagen binding VEGF-C and to verify its effect for developing a new treatment for lymphedema. A fusion protein of the collagen binding domain which is part of the gas gangrene toxin and the lymphangiogenic factor VEGF - C was designed and a fusion protein production system using insect cells was established. Collagen binding ability was confirmed by collagen adhesion test. However, proliferative activity against lymphatic endothelial cells could not be confirmed and phosphorylation signals could not be detected by MAPK signaling test. It is considered that there is a possibility that it has no physiological activity because it is not following the secretory pathway as a cause. We tried to design and produce a protein with signal peptide, but it did not lead to production.

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  • Musculoskeletal regeneration using Growth factor-anchored materials

    Grant number:26293341  2014.04 - 2017.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)  Grant-in-Aid for Scientific Research (B)

    Masashi Takaso

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    Grant amount:\11960000 ( Direct expense: \9200000 、 Indirect expense:\2760000 )

    We performed codon optimization for the protein purification toward the clinical setting. Codon optimization improved the protein production without loss of bFGF activity. To attempt to apply the bone regeneration seeds to the sciatic nerve regeneration and cartilage regeneration, we developed a new collagen scaffold materials, oriented collagen tubes (OCT) for peripheral nerve regeneration and high density collagen scaffold (HDCS) for cartilage repair. bFGF-anchored OCTs promoted pheripheral nerve tissue repair and motor function. HDCS had higher collagen density, mechanical property, and ability to promote cartilage repair compared to existing collagen materials. These materials are promising agent for promoting musculoskeletal repair in the clinical setting.

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  • Development of novel fracture healing methods using artificical perioteum and collagen-binding growth factor containing a collagen-binding domain from Clostridium histolyticum collagenase

    Grant number:25670659  2013.04 - 2015.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research  Grant-in-Aid for Challenging Exploratory Research

    TAKASO Masashi, UCHIDA Kentaro, MATSUSHITA Osamu, MABUCHI Yo, INOUE Gen

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    Grant amount:\3770000 ( Direct expense: \2900000 、 Indirect expense:\870000 )

    We attempted to develop a functional artificial periosteum using mesenchymal stem cells (MSCs) and a fusion protein (collagen binding-bFGF, CB-bFGF) consisting of bFGF and the collagen-binding domain (CBD) of Clostridium histolyticum collagenase for bone repair. Artificial periosteum had higher expression of trophic factots than monolayer cultured MSCs. Moreover, the combination of the collage CB-bFGF with artificial periosteum induced bone formation compared to artificial periosteum alone in rat models. Taken together, these properties suggest that the CB-bFGF/artificial periosteum composite is a promising material for bone repair in the clinical setting.

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  • Preclinical study to elucidate molecular mechanism of matrix anchoring using bacterial proteins

    Grant number:23590516  2011 - 2013

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    MATSUSHITA Osamu, ADACHI Eijiro, INOUE Jo, MORI Nozomu, UCHIDA Kentaro, MIMA Takehiko, MIYASHITA Takenori

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    Grant amount:\5330000 ( Direct expense: \4100000 、 Indirect expense:\1230000 )

    Flesh-eating bacteria produce collagenases, enzymes to degrade tissue collagen fibrils. The enzyme possesses a collagen-binding domain(s). Although its molecular size is small, the domain forms sandwich-like structure to bind to untwisted region of triple-helical collagen molecule. Meanwhile, human growth factor accelerates cell growth. Various growth factors were fused with the domain to anchor them on tissue collagen fibrils, demineralized bone matrix, or collagen sheet/particles. Using these novel materials, osteogenesis or angiogenesis was successfully induced.

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  • Generating artificial parenchymal and hollow organs fabricated with collagen fibrils,various cells, and cytokines using a bioreactor that we have developed

    Grant number:22300167  2010 - 2012

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)  Grant-in-Aid for Scientific Research (B)

    ADACHI Eijiro, AKAIKE Toshihiro, TAGAWA Youichi, NISHIYAMA Toshio, MATSUSHITA Osamu, HATTORI Masakazu

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    Grant amount:\17290000 ( Direct expense: \13300000 、 Indirect expense:\3990000 )

    We present a novel approach to generate parenchymal organs, such as liver and hollow tissues, blood vessels, based on the use of cultured cells, atelo collagen I and cytokines. Dense connective tissues were generated simultaneously by accumulating collagen fibrils and fibroblasts on a sheet of polylactic acid mesh or artificial blood vessels of synthetic polymers simultaneously using a bioreactor system that we designed by ourselves. For artificial liver tissues, hepatic cells were introduced into the reactor following the perfusion of another medium containing atelo collagen I. In order to make artificial vessels, cultured endothelial cells were circulated the bioreactor. Embedded hepatic cells can retain their specific functions, such as the albumin and the urea production. The two-layered artificial vessels were well-defined i.e. the layer of collagen fibrils and the endothelial layer. These results suggest that this novel approach can produce biological tissues mechanically solid for surgical operation.

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  • Basic science technology study of new revision total hip arthroplasty using a fusion protein and the nanowire processing

    Grant number:21591923  2009 - 2011

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    NARUSE Kouji, ITOMAN Moritoshi, URABE Ken, MATSUSHITA Osamu

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    Grant amount:\4680000 ( Direct expense: \3600000 、 Indirect expense:\1080000 )

    In this study we aimed to develop a new reconstruction technique for widespread bone defects that occur during revision and second revision total hip arthroplasty. We strived to significantly improve joint function restoration techniques that are currently based on existing bone allograft. prosthesis composite materials by using hormone-collagen binding domain and growth factor. collagen binding domain as molecular biological tools, as well as metal. plated nanowire processing technology as a medical engineering tool. Our study revealed that basic fibroblast growth factors. collagen binding domain were useful in the development of the new reconstruction technique.

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  • Translational research on a drug delivery system using substrate binding domain derived from bacterial collagenases

    Grant number:20590452  2008 - 2010

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    MATSUSHITA Osamu, KOIDE Takaki, TAMAI Eiji, MIYATA Shigeru, MIMAMI Junzaburo

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    Grant amount:\4810000 ( Direct expense: \3700000 、 Indirect expense:\1110000 )

    'Flesh-eating bacteria' produce collagenases to hydrolyze collagen fibrils in tissue. These enzymes possess domain(s) to bind to this insoluble substrate (CBD). We identified amino acid residues responsible for the binding. We also showed that CBD binds preferentially to the under-wound region in the collagen molecule, and that the binding is unidirectional. Ca^<2+> binds to the linker region of CBD. We showed that the binding modifies CBD into a more stable and more efficient conformation. Finally, we showed the potential benefits of matrix anchoring of various bioactivity substances using CBD, which are applicable to various therapies in regenerative medicine.

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  • A study on a mechanism for the transcriptional regulation of an epsilon-toxin gene by a novel type of bent DNA

    Grant number:18590428  2006 - 2007

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    OKABE Akinobu, MIYATA Shigeru, NARIYA Hirofumi, MATSUSHITA Osamu, TAMAI Eigi

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    Grant amount:\3860000 ( Direct expense: \3500000 、 Indirect expense:\360000 )

    An epsilon-toxin gene of Clostridium perfringens has bent DNA in a promoter region. It has been shown to possess another weakly-bent DNA in the coding region, which regulates epsilon-toxin gene expression along with the upstream bent DNA. When a DNA fragment coveting the two bent DNA regions were PCR amplified and cloned into an E coli plasmid, A-tract consisting of 8 adenine residues located at the downstream bent DNA lost the 8th adenine. This suggests that cloning of the fragment causes plasmid instability. We constructed plasmids expressing C. perfringens LrpC with and without His' by using our plasmid vector pFF. Unfortunately, transformats of C. perfringens strains 13 and SM101 carrying these plasmids failed to produce large amounts of recombinant LrpC proteins. Therefore, we failed to purify LrpC from these cultures. One likely reason for the difficulty of purification is possible proteolytic breakdown of the recombinant product during purification. We constructed a clostripain-like protease-deficient mutant, since this is the most potent thiol-protease among proteases produced by the organism and probably decreases the yield of LrpC products. We are currently attempting to purify LrpC by using this mutant To assess whether or not his-tagged LrpC functions normally, we examined biological properties of both transformants carrying plasmids containing LrpC and LrpC-his genes. The result indicates LrpC and LrpC-his are involved in the onset of spore-formation, as demonstrated for Bacillus subtilis, proving that LrpC-his is fractional in C perfringens. We prepared cell lysate from Strain 13 with and without a plasmid containing the LrpC gene and also PCR-products corresponding to the fragment containing the two bent DNA regions of the epsilon-toxin gene. The gel retardation assay using these did not show the specific interaction between the DNA and LrpC probably because of impurity of protein samples or the presence of LrpC in the wild type as well as in the transformant. To solve theseproblems, we are currently purifying LrpC-his by using Ni-chelating Sepharose from large-scale culture of the C. perfringens transformant We have also attempted an alternative method involving an E coli expression system as reported for purification of B. subtilis LrpC. Since we constructed a LrpC gene-disrupted mutant, we have undertaken the gel retardation assay by using lysates from a wild-type strain and its isogenic LrpC(-)mntant.

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  • Interdisciplinary study on the calcium-dependent conformational change of collagen-binding domain from clostridial collagenases

    Grant number:18590429  2006 - 2007

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    MATSUSHITA Osamu, MIYATA Shigeru

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    Grant amount:\3930000 ( Direct expense: \3600000 、 Indirect expense:\330000 )

    Histotoxic clostridia produce collagneases responsible for extensive tissue destruction in gas gangrene. C-terminal collagen-binding domain (CBD) of these enzymes is the minimal segment responsible to bind to collagen fibril. We have shown that CBD can be applied to anchor growth factors in local tissue. Three orientations of tropocollagen were proposed to interact with 'hot spot' residues in CBD.
    CBD was isotopically enriched and one peak is observed for each residue in the 'H-'5N HSQC spectrum. The spectrum with well dispersed cross peaks also indicated that CBD was properly folded. NMR titration study with a tropocollagen analog narrowed the binding interface to a cleft. NMR titrations with spin-labeled analog of collagenous peptide unambiguously identified the orientation of tropocollagen on CBD. The saddle like binding cleft could bend a tropocollagen. CBD may aid in hydrolysis by 1) binding to collagenous peptide in one orientation to facilitate disbanding of the fibril and 2) by bending the collagenous triple helix to assist unwinding of triple helix in such a way that the scissile peptide bond would be exposed. Pararthyroid hormone (PTH) is used for the treatment of osteoporosis, but it is so quickly metabolized that it must be given by daily injection. To prolong its duration of action, we have synthesized a fusion protein (PTH-CBD) of PTH and CBD. PTH-CBD retained its ability to bind collagen in vitro, and stimulated cAMP accumulation with similar potency and efficacy to human PTH in LL-CPK cells stably transfected with the PTH receptor. Weekly injections of PTH-CBD in normal young female mice for 8 weeks resulted in a significant increase in spinal bone mineral density. There were no side effects observed. This novel fusion protein represents an application of a new concept in drug design, combining individual protein domains to create an agent with unique properties

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  • A study on the molecular mechanism of substrate recognition by Clostridial collagenases

    Grant number:16590363  2004 - 2005

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    MATSUSHITA Osamu, KOIDE Takaki

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    Grant amount:\3600000 ( Direct expense: \3600000 )

    Three distinct domains [metallopeptidase catalytic domain (CD), PKD domain (PKD), and collagen-binding domain (CBD)] are present in clostridial collagenases. The variation in the number of PKDs and CBDs found in Clostridiun histolyticum collagenases class I (CD+PKD+2CBDs) and class II (CD+2PKDs+CBD) led us to investigate their evolutionary histories. In this study, facilitated by a novel purification method and universal PCR primers, we obtained six new sequences and carried out a phylogenetic analysis.
    Sequences homologous to clostridial collagenases were divided into two subfamilies, M9A and M9B. The M9A sequences were found in proteobacteria such as Burkholderia and Vibrio with one exception Streptomyces coelicolor of Actinobacteria, while the M9B sequences were in the firmicutes Clostridium and Bacillus. S.coelicolor might have acquired the M9A enzyme by horizontal gene transfer. The M9A enzymes possess PPC domains (PPC) instead of CBDs at their C-termini. Although PPCs showed sequence similarity with CBDs, the calcium-binding and substrate-binding sites identified in CBDs were not conserved in the PPCs. CBD may have evolved from PPCs by acquiring these sites. Among M9B enzymes, the numbers of PKDs and CBDs varied from none to two and one to three, respectively.
    Inference on the loss and gain of PKDs and CBDs, carried out by taking into account the phylogenetic relationships of the clostridial species, indicated their frequent loss and gain in Clostridial lineages.
    Clostridia may have modulated the number of C-terminal domains frequently to adapt themselves to their specific pathogenic/saprophytic life styles.

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  • Studies on the law temperature-dependent enhancement of transcription by a bent DNA

    Grant number:15590404  2003 - 2004

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    KATAYAMA Seiichi, MATSUSHITA Osamu

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    Grant amount:\3600000 ( Direct expense: \3600000 )

    We would like to make clear the structure how the bent DNA (phased A-tracts), upstream the promoter of plc gane encoding the phospholpase C in Clostridium perfringens, binds to the RNA polymerase (RNAP)α subunit (315 aa) through its C-terminai domain (α CTD, 79 aa). To determine the structrue, the co-crystallization of the phased A-tracts DNA (33 bp) and RNAP α subunit or α CTD is necessary rpaA gene encoding the RNAP α subunit and the grace encoding the α CTD were cloned into pET16b. Each gene was overexpreessed in E.coli BL21(DE3)pLysS. Then both His, tagged proteins were purified as much as available for crystallization. But the amounts of the purified proteins were only a few mg. They did not reads at 50 mg enough for crystallization. Dr.Joshua Sakon (University of Arkansas, U.S.A.) pointed out that the stability of the complex of the phased A-tracts and the α subunit or the α CTD at 25℃ (a certain low temperature) was very important, and should be examined. Since we have not examined the stability and the method by which the overproduced proteins were purled in a large scale have been not established yet, the co-crystallization of the phased A-tracts DNA and the RNAP α subunit or the α CTD has not been tied.
    In parallel, we tried to establish the system of the reconstitution of the RNAP(α2, β, β', σ) to identify the residues of amino-acids involved in the interaction between the phased A-tracts and the α CTD. The β, β', and σ subunits were overproduced in appropriate E.coli strains, and became inclusion bodies. They were solubilized in 6 M urea or guanidine hydrochloride. These solubilized proteins and the His- tagged α subunit or its N-terminal domain (α NTD, 228 aa) were mixed in the reconstitution of the RNAP. The both reconstituted RNAPs, containing the His-α subunit or the His-α NTD, have slight activities at the same level. This suggested that we night introduce the mutations in the α CTD without any influence on the activity, and that the reconstitution of the RNAP would contribute greatly to the studies on the regulation of transcription through the α CTD.

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  • A study on the mechanism underlying the action of Clostridium perfringens alpha- and epsilon-toxins to membrane lipid rafts

    Grant number:15390144  2003 - 2004

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)  Grant-in-Aid for Scientific Research (B)

    OKABE Akinobu, MATSUSHITA Osamu, MIYATA Shigeru, TAMAI Eiji, KOBAYASHI Ryoji, TOKUDA Masaaki, TOKUMITSU Hiroshi

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    Grant amount:\15400000 ( Direct expense: \15400000 )

    Epsilon- and alpha-toxins produced by Clostridium perfringens are major toxins most responsible for enterotoxeamia in domestic animals and human gas gangrene, respectively. In an attempt to elucidate the molecular mechanism by which the two toxins exhibit their toxicity, we have undertaken this research. Epsilon-Toxin has been shown to exhibit high affinity to lipid rafts of neuronal and kidney cell membranes and to permealize the membranes through heptamerization of the toxin. We examined how a membrane lipid environment affects the binding of the toxin to lipid rafts and its heptamerization. The depletion and bio synthesis inhibition of cholesterol, a major lipid component of MDCK cell lipid rafts, inhibited the heptamerization of the toxin, while the bio synthesis inhibition of sphingolipid, another one, by the treatment with fumonisin B1 increased the sensitivity of MDCK cells against epsilon-toxin. Similar results were observed with PDMP, an inhibitor of glycosphigolipid bio synthesis. When the bio synthesis of sphingomyelin, a representative sphingolipid of lipid rafts, was inhibited, their sensitivity was decreased. The exogenous addition of ganglioside G_<M1> to MDCK cell cultures caused decreases in the binding and heptamerization of the toxin and also in the sensitivity of MDCK cells to the toxin. Prolongation of culture time of MDCK cells caused an increase in ganglioside contents but did a decrease in their sensitivity. When MDCK cells, which had been cultured for a prolonged time, was treated with C. perfringens sialidase, their sensitivity to the toxin was increased. Based on these results, we concluded that an increase in ganglioside contents leading to an increase in sialic acid results in the inhibition of epsilon-toxin-binding to lipid rafts. We have also examined relationship between rafts and platelet aggregation induced by alpha-toxin, which is regarded as being most responsible for the pathogenesis of gas gangrene. Alpha-Toxin, which possesses both phospholipase C and sphingomyelinase activities showed high affinity to the lipid rafts of platelets. It degraded sphingomyelin locating preferentially in lipid rafts, generating ceramide. A mutant alpha-toxin lacking enzymatic activity bound preferentially to lipid rafts but did not aggregate platelets. We also showed that alpha-toxin aggregates lipid rafts and this ability was inhibited by the addition of anti-ceramide monoclonal antibody. These results led us to conclude that ceramide created by sphingomyelinase activity of alpha-toxin induces clustering of lipid rafts, leading to triggering of signal pathways involved in platelet aggregation.

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  • 特殊ベント・プロモーターを利用した新規高発現ベクターの構築

    Grant number:14657069  2002 - 2003

    日本学術振興会  科学研究費助成事業 萌芽研究  萌芽研究

    岡部 昭延, 玉井 栄治, 宮田 茂, 松下 治

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    Grant amount:\3400000 ( Direct expense: \3400000 )

    ウエルシュ菌(Clostridium perfringens)のフェレドキシン遺伝子のプロモーターの上流域からプロモーター内部にかけて存在する5つの連続するPhased A-tractsは、新しいタイプのBent DNAであり、その折れ曲がり角度は110°と強い。上流3つのA-tractsと下流2つのA-tractsの相乗作用によりフェレドキシンのプロモーターを活性化することを明らかにした。この特殊なベント・プロモーターをC.perfringens/Escherichia coliのシャトル・ベクター兼プロモーター選択ベクターであるpPSVにクローン化し、高発現ベクターpFFを構築した。C.perfringens/pFF系の有用性を調べるために、C.perfringensの75kDaのシアリダーゼ(NanI)の構造遺伝子とpFFをTranscriptional fusionし、pFFNを構築した。対照として、nanI遺伝子の全領域を組み込んだベクターpPSVNを構築した。さらにAT-rich遺伝子の高発現系として開発されたE.coli BL21(DE3)CodonPlus-RILにおけるNanI産生能と比較するために、pUC19にnanIをクローン化した。C.perfringes/pFFNは、C.perfringens/pPSVNの60倍、大腸菌系の約15倍の産生を示した。培養液100mlから2段階精製法で約3mgの高純度のNanIが精製された。以上のことから、C.perfringens/pFF系はシアリダーゼの高発現系に有用であることが明らかとなった。pFFの有用性について、120kDaのコラゲナーゼ遺伝子についても同様に検討したところ、発現量の改善は見られたが、C.perfringens菌そのものを培養した時と同様に、分解産物も認められた。コラゲナーゼ産生のためには、今後Protease欠損の変異株の利用や培養条件などについて検討する必要があることが明らかとなった。

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  • Basic research on the development of novel vaccines with collagen-anchoring potency.

    Grant number:14570239  2002 - 2003

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    MATSUSHITA Osamu

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    Grant amount:\3600000 ( Direct expense: \3600000 )

    The crystal structure of a collagen-binding domain with an N-terminal domain linker from Clostridium histolyticum class I collagenase was determined in the absence and presence of calcium. The mature enzyme is composed of four domains, a catalytic domain, a spacing domain (PKD), and two collagen-binding domains (CBDs). The CBD monomer reveals a beta-sheet sandwich fold. Extensive mutagenesis of conserved surface residues and collagen-binding studies allow us to identify the protein's collagen-binding surface and propose likely collagen-protein binding models. A twelve-residue-long linker is found at the N-terminus of each CBD. In the absence of calcium, the linker adopts an alpha helix. The addition of calcium unwinds the linker and anchors it to the distal side of the sandwich as a new beta-strand. The conformational change of the linker upon calcium binding is confirmed by changes in the Stokes and hydrodynamic radii as measured by size exclusion chromatography and by dynamic light scattering with and without calcium. The domain becomes more rigid and efficient for collagen-binding in the presence of calcium.
    In addition, various collagenases were purified from other Clostridial species. Their structural genes were sequenced to show that they possess variable domain organizations. These implies reiterated domain-duplication events during gene evolution. In order to reveal molecular basis to hydrolyze triple-helical peptide substrates, we have produced recombinant catalytic domains derived from these enzymes. We started their structural analysis by X-ray crystallography. At the moment, crystals were obtained from C. histolyticum class I collagenase, which are under the X-ray analysis.

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  • Study on structure biology and molecular pathology of Clostridium perfringens epsilon-toxin

    Grant number:13470060  2001 - 2002

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)  Grant-in-Aid for Scientific Research (B)

    OKABE akinobu, KOBAYASHI ryoji, MIYATA shigeru, MATSUSHITA osamu, TOKUDA masaki, TOKUMITSU hiroshi

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    Grant amount:\10300000 ( Direct expense: \10300000 )

    Clostridium perfringens epsilon-protoxin, in which His6 was N-terminally tagged and a factor Xa cleavage site was generated to cleave an N-terminal propeptide, was replaced with Se-methionine. The Se-methionine protoxin was purified, and then the N-terminal propeptide was cleaved off with factor Xa, followed by crystallization. Although the resulting crystal was shown to be twined, we are now attempting to solve the three-dimensional structure of the protoxin by computer analysis.
    We showed that epsilon-toxin (e-toxin) assembles to a heptameric pore within the lipid rafts of the rat synaptosome and Madin-Darby canine kidney (MDCK) cell membranes. To assess how physicochemical properties of the lipid rafts affect e-toxin assembly, we change major lipid constituents, cholesterol and gangliosides of MDCK cells. The heptamerization of e-toxin and its cytotoxicity towards MDCKcells was decreased by depletion of cholesterol, and was adversely stimulated by inhibition of gangliosides synthesis, suggesting that alteration in a lipid rafts environment modulates the assembly and/or the insertion of the toxin therein.
    In an attempt to study the molecular pathology of e-toxin enterotoxeamia, we examined the distribution of e-toxin by whole body autoradiography involving mice injected intravenously with 35S-labeled e-toxin. The toxin was most prominently distributed in the kidneys, and fairly abundantly in the brain, spinal cord, and nasal turbinates. Immunostaining of the kidneys showed that the toxin was detected mainly in the glomeruli and capillaries, and that it was also detectable in the distal tubules and collecting ducts. Although histological examination showed some pathological changes, e.g. shrinkage of glomeruli and degeneration of epithelial cells in the distal tubules and collecting ducts, they were not so severe as those found in the brain such as neuronal cell damage and perivascular edema. The biological relevance of the toxin accumulation in the kidneys was approached by examining an effect of nephrectomy on the lethal toxicity of e-toxin against mice. The nephrectomy shortened the time required for the toxin to kill mice. When mice was intoxicated with botulinus toxin or C. perfringens alpha-toxin, such an effect of nephrectomy was not observed. Based on these results, we propose that the kidneys contribute to the host defense by accumulating circulating e-toxin and thereby protecting the brain from a lethal damage.
    A cDNA clone encoding for a portion of a putative e-toxin receptor has been isolated by a yeast two-hybrid system. Studies are currently under way to identify the corresponding whole receptor protein and also to characterize molecular mechanism of e-toxin cytotoxicity involving e-toxin-resistant clones isolated fromMDCK cells

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  • 水素ガス産生用ウエルシュ菌バイオリアクター開発のための基礎研究

    Grant number:12877048  2000 - 2001

    日本学術振興会  科学研究費助成事業 萌芽的研究  萌芽的研究

    岡部 昭延, 宮田 茂, 松下 治, 片山 誠一

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    Grant amount:\1800000 ( Direct expense: \1800000 )

    ウエルシュ菌(Clostridium perfringens)を用いた水素ガス産生用バイオリアクター開発のための基礎研究として、培養条件とヒドロゲナーゼ関連の遺伝子改変の影響について検討した。C. perfringens NCTC8237株をTYG培地で培養し、水素ガス産生に対する培地のpHの影響を調べた。pHを調整しない場合、増殖とともにpHは低下し、水素ガスは60%であるのに対し、pHを7.4に維持した場合、90%に改善された。ガス成分比では大きく改善されたが、水素ガス量は改善されなかった。pH未調整の場合も、培養の初期においては水素ガスの成分比は高く、培養が進むにしたがって低下した。C. perfringens strain 13からクローニングしたビドロゲナーゼ遺伝子(hydA)をpJIR418をベクターとして遺伝子投与し、水素ガス産生性を調べた。ガス成分比、水素ガスの産生量において、非投与菌との間に有意な差は見られなかった。hydAの発現量は増加していることから、ヒドロゲナーゼの活性が水素ガス産生の律速ではないことが明らかとなった。本菌の主要な発酵経路である酢酸発酵、酪酸発酵はヒドロゲナーゼへの電子の供給と競合すると考えられるが、上記のpH調製時の場合と同様、遺伝子投与の場合もこれら発酵産物の産生は影響を受けなかった。hydA遺伝子の下流に位置するbutylate kinase(buk)の遺伝子破壊により酪酸発酵への電子の供給を遮断することを試みた。しかし変異菌は得られず、本遺伝子は必須と考えられた。ヒドロゲナーゼに電子を供給するフェレドキシン(Fdx)の菌体内の濃度を高めることにより、水素ガスの産生が増加することを期待し、すでに決定されたゲノムの塩基配列を基に、fdx遺伝子のクローニングを行った。本遺伝子には独特のベント構造と鉄制御に関係する配列を有していることが明らかとなった。本遺伝子の投与と鉄添加により水素ガス産生能の改善の可能性が示唆された。

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  • Drug design based on the three-dimensional structure of collagen-binding domain, and its application

    Grant number:12670258  2000 - 2001

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    MATSUSHITA Osamu, NISHI Nozomu

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    Grant amount:\3300000 ( Direct expense: \3300000 )

    Tandem collagen-binding domains (CBD's) are present at the C-terminus of Clostridium histicum class I collagenase. Three-dimesional structure of the domain was determined in the presence and abscence of Ca^<2+> ion. Addition of the ion altered conformation of the N-terminal linker peptide from an alpha-helix to beta-sheets, which stabilizes the beta-sandwich domain structure and increases the substrate affinity. (Joint project with Dr. Joshua Sakon et al. University of Arkansas, U. S. A.) in order to investigate the mode of substrate binding, mutated CBD's were constructed, where various surface-oriented amino acid residues are altered. By surface plasmon resonance using a sensor chip with an immobilized collagenous peptide, G(POG)_8, we determined their binding constants against this artificial substrate. This expriment showed that a hydrophobic surface of the sandwitch plays a key role for the substrate binding.
    Binding of CBD against various types of collagen was studied by immunohistochemistry. Light and electron microscopic observation was performed after allowing CBD to bind to prefixed collagen-rich tissues, i.e. kidney, cartilage and aorta. CBD bound to all these tissues, but with no periodicity. CBD also bound to various types of collagen in vitro. These results suggested that CBD recognizes its triple helical confomation.
    We purified collagenases from three gelatinolytic Clostridia, and cloned their structural genes. Comparison of the deduced sequences revealed that they possess unique segmental structure. We could observe the dynamic rearrangements of the enzyme strucure by comparing the primary sequence of various enzymes.

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  • Neuron specific calcium-binding Protein, calbrain, is involved in memory formation.

    Grant number:11694284  1999 - 2000

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B).  Grant-in-Aid for Scientific Research (B).

    TOKUDA Masaaki, OKABE Akinobu, SUGIMOTO Katsuyoshi, KOBAYASHI Ryoji, TAKEUCHI Yoshiki

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    Grant amount:\3800000 ( Direct expense: \3800000 )

    A novel Ca2+-binding protein, termed calbrain, was isolated from a human brain cDNA library. The analysis of deduced amino acid sequence revealed that calbrain contains two putative EF-hand motifs that show significantly high homology to those of the calmodulin(CaM)family rather than two EF-hand protein families. Studies in vitro revealed that calbrain competitively inhibited CaM binding to Ca2+/calmodulin-dependent kinase II(Ki = 129 nM)and reduced its kinase activily and autophosphorylation.
    Ca2+/calmodulin-dependent protein kinase I(CaM-kinase I)in rat retina was shown to be present in rat retina. Developmental studies revealed that CaM-kinase I expression increased in accordance with postnatal development. Expression of CaM-kinase I in the retinas of rats raised in the complete darkness markedly decreased.
    CaMK-I was shown to mainly localize in the cytoplasm of the control and LTP-induced neurons, and a significant increase of immunoreactivity was observed in the latter neurons. A part of CaMK-I was found to translocate to the nuclei of LTP-induced hippocampal CA1 neurons.
    To investigate the mechanism of chronic cell death following postischemic hypothermia, the change of N-methyl-D-aspartate receptor(NMDAR)was examined in the CA1 subfield of the gerbil hippocampus. At 1 week following postischemic hypothermia(32 degrees Cx4 h), all CA1 neurons survived ; however, immunoreactivity of NMDAR1 increased in neuronal perikarya whereas decreased in dendrites in the CA1 neurons. LTP was also significantly depressed at 1 week after hypothermia.
    Morphological changes of the suprachiasmatic nucleus(SCN)of the hypothalamus were investigated in mice exhibiting intoxication signs of stages 2 or 3 after a short application term of 6% ethanol. Alterations in glial cells and neurons were examined using anti-glial fibrillary acidic protein(GFAP)and short-term ethanol exposure led to strong expression of GFAP-immunoreactivily(GFAP-IR)in the dorsomedial part of the SCN.

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  • Studies on neurotropism of Clostridium perfringens epsilon-toxin and molecular mechanism of its toxicity toward neuronal cells

    Grant number:11470069  1999 - 2000

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B).  Grant-in-Aid for Scientific Research (B).

    OKABE Akinobu, MIYATA Shigeru, KATAYAMA Seiichi, MATSUSHITA Osamu, TOKUDA Masaaki, KOBAYASHI Ryoji

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    Grant amount:\14300000 ( Direct expense: \14300000 )

    In order to examine distribution of Clostridium perfringens epsilon-toxin (ε-toxin) in tissues of intoxicated animals, ^<125>I-labelled ε-toxin was injected intravenously into a mouse, and then subjected to whole body autoradiography. Although the toxin was detected mainly in the brain and kidneys, it was also in the thyroids, stomach, and salivary glands. Since iodolysis might have occurred after the intoxication, the whole body autoradiography was performed using ^<14>C-labelled ε-toxin. The labelled toxin was detected exclusively in the brain, spinal cord and kidneys. Analysis by immunohistochemistry revealed that the toxin was distributed mainly in the hippocampus and the glomerulus. However, other areas were also stained, and proteins in these tissues were shown to be cross-reactive with the antibody. An attempt has been made to increase the specificity of the immunoreactivity by using pre-adsorbed polyclonal or monoclonal antibodies. The action of ε-toxin on the rat synaptosomal membrane was investigated by using ^<125>I-labelled ε-toxin. While the ε-toxin monomer was initially detected in the membrane, it formed a large complex with incubation time. The large complex was shown to consist of a heptamer of ε-toxin. When the toxin and inactive protoxin were incubated with the membrane, the heptamerization of the monomer but not its association with the membrane was inhibited dose-dependently by the protoxin. These results suggest that a C-terminal propeptide, which was cleaved upon activation by tryptic digestion, does not affect the binding of ε-toxin to its receptor, while it masks a region necessary for the heptamerization. Several cultured neuronal cells were tested for ε-toxin cell toxicity. Either undifferentiated or differentiated cells were not sensitive to the toxin. A method for the isolation and identification of an ε-toxin binding receptor from the membrane fraction of the calf brain have been established : preparation of large quantities of the synaptosomal membrane, differential solubilization, immunological detection such as slot blotting and western blotting, and affinity chromatography. A few proteins including predominant 45 K protein, which seemed to possess a high affinity to the toxin, were isolated from the calf brain cell membrane and also from the calf kidney cell membrane. Identification and purification of these proteins are now in progress.

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  • Function of promoter upstream bent DNA of the Clostridium perfringens phospholipase C gene

    Grant number:10670260  1998 - 1999

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    KATAYAMA Seiichi, MATSUSHITA Osamu, OKABE Akinobu

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    Grant amount:\3200000 ( Direct expense: \3200000 )

    The Clostridium perfringens phospholipase C gene (plc) possesses three phased A-tracts upstream of the promoter (-66 to 40). The A-tracts form bent DNA, facilitate the formation of the RNA polymerase (RNAP)-plc promoter complex through extension of the contact region, and activate the transcription in a low temperature-dependent manner. In order to clarify the mechanism of the transcriptional regulation by the A-tracts, it seems important to elucidate specific subunit/domain of RNAP and the A-tracts responsible for the extended contact.
    We cloned rpoA encoding α subunit of the C. perfringens RNAP. The deduced amino acid sequence was identical to the N-terminal sequence of purified RNAP α subunit. The coding region of rpoA was cloned into an expression vector to overproduce and purify the α subunit (α-WT, 315 aa). N-terminal (α-NTD, 228 aa) and C-terminal (α-CTD, 79 aa) domains of the subunit were also purified in the same manner.
    The gel retardation assays showed that α-WT and α-CTD bind to a DNA fragment containing three phased A-tracts and plc promoter (3Ap), but that α-NTD does not. From this result, a possibility was raised that α-CTD binds to the A-tracts. We are trying to show the contact region between 3Ap and α-CTD by hydroxyl radical footprinting at the moment.

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  • Analysis of the activation and mode of action of Clostridium perfringens ε-toxin

    Grant number:09670286  1997 - 1999

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    MATSUSHITA Osamu, KATAYAMA Seiichi, OKABE Akinobu

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    Grant amount:\3300000 ( Direct expense: \3300000 )

    1. The activation of Clostridium perfringens epsilon-prototoxin (ε-prototoxin) by λ-toxin, trypsin and chymotrypsin was examined. The mouse lethality test showed that the 50% lethal doses (LDィイD250ィエD2) of the prototoxin with and without λ-toxin treatment were 110 and 70,000 ng/kg of body weight, respectively. LDィイD250ィエD2 of the prototoxin treated with trypsin and trypsin plus chymotrypsin were 320 and 65 ng/kg of body weight, respectively. Determination of the N-terminal amino acid sequence of each activated 8-prototoxin revealed that λ-toxin cleaved between the 10th and 11th amino acid residues from the N-terminus of the prototoxin, while trypsin and trypsin plus chymotrypsin did so between the 13th and 14th amino acid residues. The C-terminus deduced from the molecular weight is located at the 23th or 30th amino acid residue from the C-terminus of the prototoxin, suggesting that removal of not only N- but also C-terminal peptides is responsible for the activation of the prototoxin.
    2. The neurotoxicity of ε-toxin was examined by histological examination of the rat brain. Injection of ε-toxin at a sublethal dose, 50 ng/kg, caused neuronal damage predominantly in the hippocampus: pyramidal cells in the hippocampus showed marked shrinkage and karyopyknosis, and the cells lost the immunoreactivity to microtubule-associated protein 2 (MAP-2). Timm's zinc staining revealed that zinc ions were depleted in the mossy layers of the CA3 subfield containing glutamate as a synaptic transmitter. Prior injection of either a glutamate-release inhibitor or glutamate-receptor antagonist protected the hippocampus from the neuronal damage caused by 8-toxin. These results suggest that 8-toxin acts on the glutamatergic system and evokes excessive release of glutamate, leading to neuronal damage.

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  • コラーゲン結合ドメインによるガス壊疸の発症予防

    Grant number:09770184  1997 - 1998

    日本学術振興会  科学研究費助成事業 奨励研究(A)  奨励研究(A)

    松下 治

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    Grant amount:\1900000 ( Direct expense: \1900000 )

    ガス壊疸菌群によるガス壊疸感染巣の成立と周辺組織への浸潤には、起因菌の産生するコラゲナーゼが重要な役割を演じると考えられる。本酵素の構造活性相関を明らかにし、その知見を予防に応用することが本研究の課題である。
    平成9年度は、本酵素群のうちの一つ(C.histolyticum ColH,クラスI酵素)が、N末側の水解活性ドメインとC末側のコラーゲン結合ドメイン(CBD)により構成されていることを明らかにした(Matsushita et al.,J.Biol.Chem.273:3643-3648,1998)。ついで2種類の酵素(C.histolyticum ColGおよびColH)から種々の長さのC末端領域を生産し、そのコラーゲン結合能を測定したところ、クラスI酵素(CoIG)ではデュプリケートして、クラスII酵素(ColH)では単独で存在するC末端領域(約120アミノ酸残基)がCBDの最小構成単位であることが明らかとなった。
    平成10年度は、まず、これらの酵素の遺伝子がC.histolyticumの染色体上に独立した転写単位として存在していること、本菌はこれら以外にはコラゲナーゼ遺伝子を有していないことを明らかにした(Matsushita et al.,J.Bacteriol.181:923-933,1999)。これらの基礎的知見に基づき、クラスI酵素(ColG)のC末側のCBDを精製し、家兎を免役して抗体画分を精製した。現在、得られた抗体をもちいてマウスを受動免役し、これがC.histolyticumのマウス致死活性に影響を与えるか否かを検討しているところである。さらに、この領域でよく保存されているアミノ酸残基を順次アラニンに部位特異的に置換し、変異CBDを有する融合タンパク質を生産した。現在、これらの変異ペプチドの基質結合能を測定し、基質と直接相互作用している部位の特定を試みている。

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  • Study on function of phospholipase C gene binding protein from Clostridium perfringens

    Grant number:08670308  1996 - 1997

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    OKABE Akinobu, KATAYAMA Seiichi, MATSUSHITA Osamu, MINAMI Junzaburo

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    Grant amount:\2500000 ( Direct expense: \2500000 )

    Phospholipase C (PLC) is one of the major virulence factors of Clostridium perfringens. The expression of a PLC-encoding gene (plc) is regulated by bent DNA locating upstream of a plc promoter and also by plc-binding protein, which can bind to the coding region of the gene. The plc-binding protein was partially purified from C.perfringens NCTC 8237, a PLC high-producing strain. One polypeptide, of which molecular mass was estimated to be 56 kDa by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) , bound to a fragment within the plc coding region. Its N-terminal amino acid sequence was determined and PCR product was generated using degenerate primers corresponding to the sequence. A 6-kb EcoRI fragment from the NCTC 8237 chromosome, which hybridized with the PCR product, was cloned into pUC19. Nucleotide sequencing of this fragment and similarity search of the predicted amino acid sequence revealed that the gene encoding for the 56 K polypeptide is hemD-ctyGC and other open reading frames found in the fragment are all related to the enzymes for heme biosynthesis. When the 6-kb fragment was cloned into a shuttle-vector pJIR418 and C.perfringens strain 13 was transformed with the plasmid, othe hemD-cysGC-encoding gene was expressed in the transformant. However, extract from the transformant did not show a positive gel retardation to the plc gene. It may be possible that the plc-binding protein comigrates with the 56 K polypeptide on SDS-PAGE.Study was also conducted on a role of the bent DNA locating upstream of the plc promoter. It has been shown to play a role in the regulation of the plc gene expression in response to alteration of temperature.

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  • ウェルシュ菌MSCLチャンネルによる病原遺伝子発現制御に関する研究

    Grant number:08770193  1996

    日本学術振興会  科学研究費助成事業 奨励研究(A)  奨励研究(A)

    松下 治

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    Grant amount:\1100000 ( Direct expense: \1100000 )

    mscL遺伝子の前後で相同組換えを行うことにより、mscLウエルシュ菌の作製を試みた。
    まず、mscL遺伝子が薬剤耐性遺伝子により分断された組換え用DNA断片を作製した。colAから下流約5kbを含むpKY3134プラスミドにコードされたmscL遺伝子内のHincII部位に、pJIR418シャトル・ベクターより単離したクロラムフェニコール耐性遺伝子(Cm^r)断片(1331 bp)の挿入を試みたが、大腸菌中で組換え体を得ることができなかった。そこで、mscL下流側の約0.3kb断片を含むpKY3136プラスミドにCm^rとmscL下流側の約1.8kb断片を順次連結し、組換え用DNA断片を含むpKJ3プラスミドを作製した。各段階の正確な連結は、塩基配列を決定して確認した。
    次に、pKJ3プラスミドをSmaI切断し、10μgの線状DANを用いてC.perfringens strain 13の形質転換を試みたところ、3株のクロラムフェニコール耐性を得ることができた。これらの株の染色体DNAを調製し、クロラムフェニコール耐性遺伝子断片をプローブとしてサザン・ハイブリダイゼーションを行ったところ、予想された長さの断片一つのみが検出された。ところが、pKJ3プラスミドのベクター・プラスミドであるpUCプラスミドをプローブとしたハイブリダイゼーションでもシグナルが検出された。このことから、これらの株は菌体内で再環状化したpKJ3プラスミドが、一回の相同組換えにより染色体上に挿入された変異株であると考えられた。
    そこで、pKJ3プラスミドを二つの異なる制限酵素(SacI,SphI)で切断し、再環状化不可能な線状の組換え用DNAを得た。現在、このDNAによるstrain13の相同組換えを試みている。

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  • ガス壊疽菌プロテアーゼのプロ領域ペプチドの機能解析

    Grant number:06770202  1994

    日本学術振興会  科学研究費助成事業 奨励研究(A)  奨励研究(A)

    松下 治

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    Grant amount:\900000 ( Direct expense: \900000 )

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  • Function of protein (s) which can bind to an alpha-toxin gene of Clostridium perfringens

    Grant number:05670259  1993 - 1994

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for General Scientific Research (C)  Grant-in-Aid for General Scientific Research (C)

    OKABE Akinobu, MATSUSHITA Osamu, MINAMI Junzaburo

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    Grant amount:\2200000 ( Direct expense: \2200000 )

    A study on a mechanism of regulation of an alpha-toxin gene (plc) in Clostridium perfringens has been made. We attempted to clone a gene encoding for a DNA binding protein which can bind to the plc gene. First, we cloned the plc gene from strain 13 into a plasmid pUC19. A region of chloramphenicol acetyl transferase gene (catP) starting from ribosome binding sequence to transcriptional terminator was inserted within a coding region of the plc gene. The resulting plasmid was introduced into C.perfringens strain 13. Thus obtained chloramphenicol-resistant strain was shown to have a chromosomal plc gene fused to a catP gene mediated by homologous recombination. This mutant strain formed a tiny colony on agar containing 100 mug/ml of chloramphenicol under the condition for transformation. After DNA library of type ANCTC8247 chromosomal DNA was constructed by using pJIR418 and the mutant strain, we tried to select transformants on agar containing 200 mug/ml of chloramphenicol based on the assumption that trans-acting factor bound to the plc gene can stimulate expression of the catP gene and thereby resistance of the strain increases resistance against the drug. However, we failed to obtain such clone. This could be either due to rearrangement of catP gene occurring in the presence of high concentration of the drug or due to possible toxicity displayd by a cloned gene into a high copy number of plasmid.
    We purified RNA plymerase from C.perfringens and also partially purified DNA binding proteins from Plc high producer, type A NCTC8237 of C.perfringens. Thus we established in vitro transcription system for examining the expression of plc gene in the presence or absence of DNA binding protein (s). Another important finding with respect to the plc gene expression is that static bent DNA present in the upstream DNA binding region is a cis-element stimulating the transcription from the gene and its stimulatory effect is prominent at low temperature.

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  • ガス壊疽菌コラゲナーゼの反応機構の解析

    Grant number:05770193  1993

    日本学術振興会  科学研究費助成事業 奨励研究(A)  奨励研究(A)

    松下 治

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    Grant amount:\900000 ( Direct expense: \900000 )

    1.性状の解析
    本酵素の性状をアゾコール分解活性を指標に解析した。活性はpH4.5〜9.0の広い範囲で認められ、至適pHはホウ酸バッファーで7.2、リン酸バッファーで7.0であった。至適温度は42°Cであった。10mM CaCl_2存在下で1mMのo-phenanthrolineの添加により完全に阻害された。これらの結果から、本酵素はNeutral metalloproteaseである事が示唆された。酵素標品中の2価金属イオンを、5mM過剰のEDTA添加とSephadex G-10カラムによって除去した後、種々の2価金属イオンを再添加して、金属イオン要求性を調べた。30muMnoZn^<2+>添加によって酵素活性が最も回復したのにたいし、Ca^<2+>では1mM以上の濃度が活性の回復(90%)に必要であった。また、Mg^<2+>では約30%の回復しか得られなかった。以上より、本酵素はZn-proteaseであると結論された。
    2.産生系の確立
    本酵素の金属結合部位を特定するためには、部位特異適突然変異導入による解析が有効である。その前段階として、本酵素の大腸菌中での発現系を得る事を試みた。本酵素構造遺伝子をプラスミド上でT3 promorterの下流に連結し、え.coli BL21(pTG119)にtransformした。本菌では、IPTG添加によりT3 RNA polymeraseの発現が誘導され、その結果collagenaseの大量生産が可能なはずである。ところが実際には、IPTGを添加すると菌の増殖が停止し、collagenase活性の誘導は僅かであった。ペリプラスム画分からImmunoblottingによりcollagenase関連抗原を検出を試みると、authentic proteinと同じ分子量の抗原は僅かであり、分子量の小さい抗原が多種多量に観察された。転写または翻訳レベルでの酵素産生の中絶が示唆された。
    以上の問題を解決するため、本来の宿主であるC.perfringensの持つcollagenase遺伝子(colA)をgene targettingにより除去し、colAを持つplasmidをC.perfringens colA変異株に形質転換する事により、本酵素の構造活性相関に関する詳細な解析を進める予定である。

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  • 分子生物学的手法による酸耐性セルロース分解菌の作成と,反芻獣肥育事故防止への応用

    Grant number:03770237  1991

    日本学術振興会  科学研究費助成事業 奨励研究(A)  奨励研究(A)

    松下 治

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    Grant amount:\800000 ( Direct expense: \800000 )

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  • Developmental Studies on the Laboratory diagnosis at an early stage of Mycoplasam pneumoniae-infection (mycoplasmal pneumonia)

    Grant number:02557023  1990 - 1991

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Developmental Scientific Research (B)  Grant-in-Aid for Developmental Scientific Research (B)

    KANEMASA Yasuhiro, FUJII Mari, MATSUSHITA Osamu, KOTANI Nobuyuki, KUNITOMI Taiji, HIRAI Yoshikazu

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    Grant amount:\5700000 ( Direct expense: \5700000 )

    We developed an indirect immunofluorescence test (IF) and a latex agglutination test (LAT) for detection of Mycoplasma pneumonias in respiratory exudated as rapid diagnosis at an early stage of M. pneumoniae-infection. Further, IF and LAT were compared with DNA-probe test (DP) which was the only commercially available test for the rapid detection of the organism.
    Firstly, we prepared polyclonal antibody specific to M. Pneumoniae. The antibody cross-react with many species of mycoplasmas, but not with normal human serum or with respiratory exudates from healthy person. Cross-reactivity of the antibody with species of mycoplasmas other than M. Renitalium was fully diminished when absorbed with horse serum and yeast extract. components of culture medium. -M. genitalium showed a cross-reaction in LAT with using the absorbed antibody and also in DP. However, the cross-reactivity with M. Renitalium, is unlikely to cause significant diagnostic confusion because the titer of M. genitalium was significantly lower than., that of M. pneumonia, and M. genitalium is exclusively a genital tract colonizer. Therefore, IF and LAT with using the absorbed antibody, and DP are specific enough to be used for the detection of M. pneumonia in respiratory exudates.
    The detection limit of IF was about 2 x 10^5 cfu/ml, that-, of LAT was 2 x 10^5 cfu/ml and that cif DP was 5 x 10^4 cfu/ml in vitro. DP had the highest sensitivity among three methods.
    Among clinical specimens(throat smears)from patients with serologically confirmed M. pneumoniae-infection, 85.7% gave a positive test in IF. Among clinical specimens in which M. pneumoniae was detected by culture method, positive rate in IF was 73.3%, that in LAT was 63.3% and that in DP was 26.6%.
    A effect of the incubation of M. pneumonia suspension was examined. The suspension and the mixture (M. Pneumoniae suspension and respiratory exudates) were incubated at 37 ゚C. An aliquot was removed with time course, and examinedin LAT and DP. The samples showed the same titer within three days in LAT. The titer (rate of sample-cpm to negativp- control-cpm) of each sample decreased remarkably within 5 h. It was considered that target SS'olecules in LAT and IF were accumulated in the pharyngeal portion. However, target molecule in DP (ribosomal RNA) was destructed much sooner, and the accumulation could not be expected. The reason f. or the low positive rate of DP in clinical'specimens may be due to the fast breakdown of target molecule.
    Consequently, IF and LAT must be applicable to the detection of M. pneumoniae in respiratory exudates from the patients in clinical laboratories. Especially, LAT was recommended because the procedure of LAT can be completed within a half hour without complicated manner. though the positive rate of LAT in clinical specimens was slightly lower than that of IF. Now we are examinating a process of treating clinical samples for rising the detection limit of LAT.

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Class subject in charge

  • Medical Tutorial (2021academic year) 1st semester  - 火2~3

  • Medical Tutorial (2021academic year) 1st semester  - 火2~3

  • Introduction to Medical and Dental Sciences (2021academic year) Concentration  - その他

  • Project-based Learning in Molecular Pathogenesis (2021academic year) special  - その他

  • Lecture: Myocardial Infarction (2021academic year) special  - その他

  • Combat against Infectious Diseases (2021academic year) Second semester  - 木1~2

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  • Research Projects and Practicals: Bacteriology I (2021academic year) special  - その他

  • Lecture and Research Projects: Bacteriology I (2021academic year) special  - その他

  • Research Projects and Practicals: Bacteriology II (2021academic year) special  - その他

  • Lecture and Research Projects: Bacteriology II (2021academic year) special  - その他

  • Social Medicine and Dentistry (2021academic year) Concentration  - その他

  • Microbiology (2021academic year) special  - その他

  • Practice in Microbiology (2021academic year) special  - その他

  • Medical Tutorial (2020academic year) 1st semester  - 火2,火3

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  • Research Projects (2020academic year) Year-round  - その他

  • Project-based Learning in Molecular Pathogenesis (2020academic year) special  - その他

  • Lecture: Myocardial Infarction (2020academic year) special  - その他

  • Combat against Infectious Diseases (2020academic year) Second semester  - 木1,木2

  • Practicals (2020academic year) Year-round  - その他

  • Research in Dental Sciences I (2020academic year) Year-round  - その他

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  • Research Projects (2020academic year) Year-round  - その他

  • Research Presentation in Preventive Medicine (2020academic year) Year-round  - その他

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  • Research Projects and Practicals: Bacteriology I (2020academic year) special  - その他

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Academic Activities

  • 第94回日本細菌学会総会

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    日本細菌学会  2021.3.23 - 2021.3.25

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  • 第68回日本細菌学会中国・四国支部総会

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    日本細菌学会中国・四国支部  2015.10.3 - 2015.10.4

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