2024/03/29 更新

写真a

ミカミ ヨシカズ
三上 剛和
MIKAMI Yoshikazu
所属
教育研究院 医歯学系 医学系列 准教授
医歯学総合研究科 分子細胞医学専攻 細胞機能 准教授
職名
准教授
外部リンク

学位

  • 博士(理学) ( 2005年9月   総合研究大学院大学 )

研究キーワード

  • 細胞分化

  • 分子遺伝学 細胞生物学

  • 骨芽細胞

研究分野

  • ライフサイエンス / 解剖学

経歴(researchmap)

  • 新潟大学   医学部 顕微解剖学分野   准教授

    2016年 - 現在

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  • 日本大学   歯学部 病理学講座   助教

    2013年10月

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  • 日本大学   School of Dentistry

    2007年10月 - 2013年9月

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  • 日本大学   School of Dentistry

    2006年4月 - 2007年9月

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経歴

  • 新潟大学   医歯学総合研究科 分子細胞医学専攻 細胞機能   准教授

    2016年5月 - 現在

学歴

  • 総合研究大学院大学   生命科学研究科   遺伝学

    - 2003年

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所属学協会

 

論文

  • Inductive effect of SORT1 on odontoblastic differentiation of human dental pulp-derived stem cells

    Daisuke Omagari, Taku Toriumi, Hiromasa Tsuda, Manabu Hayatsu, Keisuke Watanabe, Yusuke Mizutami, Masaki Honda, Yoshikazu Mikami

    Differentiation   133   88 - 97   2023年9月

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    掲載種別:研究論文(学術雑誌)   出版者・発行元:Elsevier BV  

    DOI: 10.1016/j.diff.2023.08.001

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  • Rice peptide with amino acid substitution inhibits biofilm formation by Porphyromonas gingivalis and Fusobacterium nucleatum 査読 国際誌

    Aoi Matsugishi, Yukari Aoki-Nonaka, Mai Yokoji-Takeuchi, Miki Yamada-Hara, Yoshikazu Mikami, Manabu Hayatsu, Yutaka Terao, Hisanori Domon, Masayuki Taniguchi, Naoki Takahashi, Kazuhisa Yamazaki, Koichi Tabeta

    Archives of Oral Biology   121   104956 - 104956   2021年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Elsevier BV  

    OBJECTIVE: Rice peptide has antibacterial properties that have been tested in planktonic bacterial culture. However, bacteria form biofilm at disease sites and are resistant to antibacterial agents. The aim of this study was to clarify the mechanisms of action of rice peptide and its amino acid substitution against periodontopathic bacteria and their antibiofilm effects. DESIGN: Porphyromonas gingivalis and Fusobacterium nucleatum were treated with AmyI-1-18 rice peptide or its arginine-substituted analog, G12R, under anaerobic conditions. The amount of biofilm was evaluated by crystal violet staining. The integrity of the bacteria cytoplasmic membrane was studied in a propidium iodide (PI) stain assay and transmission electron microscopy (TEM). RESULTS: Both AmyI-1-18 and G12R inhibited biofilm formation of P. gingivalis and F. nucleatum; in particular, G12R inhibited F. nucleatum at lower concentrations. However, neither peptide eradicated established biofilms significantly. According to the minimum inhibitory concentration and minimum bactericidal concentration against P. gingivalis, AmyI-1-18 has bacteriostatic properties and G12R has bactericidal activity, and both peptides showed bactericidal activity against F. nucleatum. PI staining and TEM analysis indicated that membrane disruption by G12R was enhanced, which suggests that the replacement amino acid reinforced the electostatic interaction between the peptide and bacteria by increase of cationic charge and α-helix content. CONCLUSIONS: Rice peptide inhibited biofilm formation of P. gingivalis and F. nucleatum, and bactericidal activity via membrane destruction was enhanced by amino acid substitution.

    DOI: 10.1016/j.archoralbio.2020.104956

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  • Reactive oxygen species-dependent release of damage-associated molecular patterns from human gingival epithelial Ca9-22 cells during butyrate or propionate exposure 査読

    Yui Fujiwara, Takahisa Murofushi, Ryosuke Koshi, Yoshikazu Mikami, Hiromasa Tsuda

    Journal of Oral Science   63 ( 2 )   195 - 197   2021年

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    掲載種別:研究論文(学術雑誌)   出版者・発行元:Nihon University School of Dentistry  

    DOI: 10.2334/josnusd.20-0411

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  • Gap junction with MLO-A5 osteoblast-like cell line induces ALP and BSP transcription of 3T3-L1 pre-adipocyte like cell line via Hspb1 while retaining adipogenic differentiation ability 査読 国際誌

    Daisuke Omagari, Manabu Hayatsu, Kiyofumi Yamamoto, Masayuki Kobayashi, Naruchika Tsukano, Masaaki Nameta, Yoshikazu Mikami

    Bone   141   115596 - 115596   2020年12月

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    担当区分:最終著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Elsevier BV  

    In bone tissues, gap junctions form direct links between the cytoplasm of an osteocyte and another adjacent osteocyte or osteoblast, which underlie both bone formation and bone resorption. We have previously demonstrated that alkaline phosphatase (ALP) and bone sialoprotein (BSP), which are osteoblast markers, were induced in mesenchymal stem cells (MSCs) co-cultured with osteoblast-like cell line. However, the molecular mechanism of this process has not been fully addressed. Furthermore, few advances have been made toward elucidating the communication networks that link the status of committed cells such as (pre-) adipocytes that differentiated from MSCs as well as osteoblasts. Therefore, the objective of the present study was to investigate the mechanism underlying the communication network between pre-adipocytes and osteoblasts. We evaluated the effect of co-culture with osteoblast on the cell status of pre-adipocytes using murine osteoblast-like cell line, MLO-A5, and pre-adipocyte-like cell line, 3T3-L1, respectively. The results presented here demonstrated that osteoblasts and pre-adipocytes communicate via gap junctions, and the ensuing drastic increase in ALP and BSP transcription in co-cultured pre-adipocytes was induced, at least partly, via heat shock protein family B member 1 (Hspb1). In addition, terminal differentiation into adipocytes was suppressed in pre-adipocytes during co-culture with osteoblast without loss of adipogenic differentiation ability. Interestingly, after co-culture with osteoblasts, isolated co-cultured pre-adipocytes were able to differentiate to adipocytes as well as original pre-adipocytes. These results suggest that gap junctional communication with osteoblasts suppressed adipogenic differentiation of pre-adipocytes without loss of adipogenic differentiation ability.

    DOI: 10.1016/j.bone.2020.115596

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  • コメペプチドとそのアミノ酸置換体はPorphyromonas gingivalis、Fusobacterium nucleatumのバイオフィルム形成を阻害する

    松岸 葵, 野中 由香莉, 竹内 麻衣, 原 実生, 早津 学, 三上 剛和, 牛木 辰男, 土門 久哲, 山崎 和久, 多部田 康一

    日本歯周病学会会誌   62 ( 春季特別 )   133 - 133   2020年5月

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    記述言語:日本語   出版者・発行元:(NPO)日本歯周病学会  

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  • コメペプチドとそのアミノ酸置換体はPorphyromonas gingivalis、Fusobacterium nucleatumのバイオフィルム形成を阻害する

    松岸 葵, 野中 由香莉, 竹内 麻衣, 原 実生, 早津 学, 三上 剛和, 牛木 辰男, 土門 久哲, 山崎 和久, 多部田 康一

    日本歯周病学会会誌   62 ( 春季特別 )   133 - 133   2020年5月

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    記述言語:日本語   出版者・発行元:(NPO)日本歯周病学会  

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  • SLPI facilitates cell migration by regulating lamellipodia/ruffles and desmosomes, in which Galectin4 plays an important role 査読 国際誌

    Yusuke Mizutani, Daisuke Omagari, Manabu Hayatsu, Masaaki Nameta, Kazuo Komiyama, Yoshikazu Mikami, Tatsuo Ushiki

    Cell Adhesion & Migration   14 ( 1 )   195 - 203   2020年1月

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    担当区分:責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Informa UK Limited  

    To elucidate the underlying mechanism of secretory leukocyte protease inhibitor (SLPI)-induced cell migration, we compared SLPI-deleted human gingival carcinoma Ca9-22 (ΔSLPI) cells and original (wild-type: wt) Ca9-22 cells using several microscopic imaging methods and gene expression analysis. Our results indicated reduced migration of ΔSLPI cells compared to wtCa9-22 cells. The lamellipodia/dorsal ruffles were smaller and moved slower in ΔSLPI cells compared to wtCa9-22 cells. Furthermore, well-developed intermediate filament bundles were observed at the desmosome junction of ΔSLPI cells. In addition, Galectin4 was strongly expressed in ΔSLPI cells, and its forced expression suppressed migration of wtCa9-22 cells. Taken together, SLPI facilitates cell migration by regulating lamellipodia/ruffles and desmosomes, in which Galectin4 plays an important role.

    DOI: 10.1080/19336918.2020.1829264

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  • Dual effect of polyphosphate on mineralization of rat osteoblast ROS17/2.8 cells in a dose-dependent manner 査読

    Yoshikazu Mikami, Daisuke Omagari, Yusuke Mizutani, Manabu Hayatsu, Tatsuo Ushiki, Hiromasa Tsuda

    Journal of Pharmacological Sciences   138 ( 3 )   209 - 213   2018年11月

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    担当区分:筆頭著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Elsevier BV  

    Inorganic polyphosphate (polyP), a linear polymer of orthophosphate, is found at high concentrations in osteoblasts. We demonstrated the effects of various polyP concentrations on the mineralization of rat osteoblast ROS17/2.8 cells. Mineralization of ROS17/2.8 was induced by a high polyP concentration (1 mg/mL), which was accompanied by an upregulation of the bone sialoprotein and osteocalcin. In contrast, a low polyP concentration (1 × 10-2 mg/mL) reduced mineralization without affecting the osteogenic gene expression. Furthermore, gene expression profiling and forced expression analysis indicated that phosphodiesterase 11a could be a candidate involved in the dose-dependent effect of polyP on osteoblast mineralization.

    DOI: 10.1016/j.jphs.2018.10.002

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  • A bacterial metabolite ameliorates periodontal pathogen-induced gingival epithelial barrier disruption via GPR40 signaling 査読

    Yamada Miki, Takahashi Naoki, Matsuda Yumi, Sato Keisuke, Yokoji Mai, Sulijaya Benso, Maekawa Tomoki, Ushiki Tatsuo, Mikami Yoshikazu, Hayatsu Manabu, Mizutani Yusuke, Kishino Shigenobu, Ogawa Jun, Arita Makoto, Tabeta Koichi, Maeda Takeyasu, Yamazaki Kazuhisa

    SCIENTIFIC REPORTS   8 ( 1 )   9008   2018年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1038/s41598-018-27408-y

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  • Combined effects of starvation and butyrate on autophagy-dependent gingival epithelial cell death 査読

    M. Evans, T. Murofushi, H. Tsuda, Y. Mikami, N. Zhao, K. Ochiai, T. Kurita-Ochiai, M. Yamamoto, K. Otsuka, N. Suzuki

    Journal of Periodontal Research   52 ( 3 )   522 - 531   2017年6月

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    掲載種別:研究論文(学術雑誌)   出版者・発行元:Wiley  

    DOI: 10.1111/jre.12418

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  • CAY10591, a SIRT1 activator, suppresses cell growth, invasion, and migration in gingival epithelial carcinoma cells 査読

    Murofushi Takahisa, Tsuda Hiromasa, Mikami Yoshikazu, Yamaguchi Yoko, Suzuki Naoto

    Journal of Oral Science   59 ( 3 )   415 - 423   2017年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:日本大学歯学部  

    <p>SIRT1 is a NAD-dependent histone deacetylase that is important in a wide variety of physiological and pathophysiological processes. Although many studies have examined the relationship between SIRT1 and cancer, the role of SIRT1 in tumor malignancy is controversial. Here, we examined the effects of the SIRT1 activator CAY10591 in gingival epithelial carcinoma Ca9-22 cells. CAY10591 treatment dose- and time-dependently increased SIRT1 level and activity. The treatment decreased cell growth and induced cell-cycle repressor p21 levels. In addition, dimethyl sulfoxide significantly reduced cellular invasion and migration, and CAY10591 enhanced this decrease. Quantitative PCR analysis showed that CAY10591 decreased expression of several invasion/migration promoter genes and induced repressor genes. Our findings suggest that CAY10591 suppresses cell growth and invasion/migration activity in gingival squamous cell carcinoma Ca9-22 cells.</p>

    DOI: 10.2334/josnusd.16-0696

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  • Comparison of gene expression profiles of gingival carcinoma Ca9-22 cells and colorectal adenocarcinoma HT-29 cells to identify potentially important mediators of SLPI-induced cell migration 査読

    Tsuyoshi Takamura, Hisashi Suguro, Yoshikazu Mikami, Takashi Iwase, Yusuke Komiyama, Kayo Kuyama, Kazuo Komiyama, Hiderou Oki

    Journal of Oral Science   59 ( 2 )   279 - 287   2017年

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    担当区分:責任著者   掲載種別:研究論文(学術雑誌)   出版者・発行元:Nihon University School of Dentistry  

    DOI: 10.2334/josnusd.16-0534

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  • Alkaline phosphatase determines polyphosphate-induced mineralization in a cell-type independent manner 査読

    Yoshikazu Mikami, Hiromasa Tsuda, Yuko Akiyama, Masaki Honda, Noriyoshi Shimizu, Naoto Suzuki, Kazuo Komiyama

    Journal of Bone and Mineral Metabolism   34 ( 6 )   627 - 637   2016年11月

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    担当区分:筆頭著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Springer Science and Business Media LLC  

    Polyphosphate [Poly(P)] has positive effects on osteoblast mineralization; however, the underlying mechanism remains unclear. In addition, it is unknown whether Poly(P) promotes mineralization in soft tissues. We investigated this by using various cells. Poly(P) concentrations of 1 and 0.5 mg/mL yielded high levels of mineralization in ROS17/2.8 osteoblast cells. Similarly, Poly(P) induced mineralization in cell types expressing alkaline phosphatase (ALP), namely, ATDC5 and MC3T3-E1, but not in CHO, C3H10T1/2, C2C12, and 3T3-L1 cells. Furthermore, forced expression of ALP caused Poly(P)-induced mineralization in CHO cells. These results suggest that ALP determines Poly(P)-induced mineralization in a cell-type independent manner.

    DOI: 10.1007/s00774-015-0719-6

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    その他リンク: http://link.springer.com/article/10.1007/s00774-015-0719-6/fulltext.html

  • Human uterus myoma and gene expression profiling: A novel in vitro model for studying secretory leukocyte protease inhibitor-mediated tumor invasion 査読

    Yoshikazu Mikami, Atsushi Fukushima, Yusuke Komiyama, Takashi Iwase, Hiromasa Tsuda, Yasuhiko Higuchi, Satoshi Hayakawa, Kayo Kuyama, Kazuo Komiyama

    Cancer Letters   379 ( 1 )   84 - 93   2016年8月

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    担当区分:筆頭著者, 責任著者   掲載種別:研究論文(学術雑誌)   出版者・発行元:Elsevier BV  

    DOI: 10.1016/j.canlet.2016.05.028

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  • Loop-mediated isothermal amplification combined with PCR and immunohistochemistry for detecting <i>Porphyromonas gingivalis</i> in periapical periodontitis 査読

    Taiichi Kitano, Yoshikazu Mikami, Takashi Iwase, Masatake Asano, Kazuo Komiyama

    Journal of Oral Science   58 ( 2 )   163 - 169   2016年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Nihon University School of Dentistry  

    Porphyromonas gingivalis is important in the development of marginal periodontitis. However, the precise role and localization of P. gingivalis in chronic periapical periodontitis remain unclear. Thus, methods that can detect P gingivalis in formalin-fixed and paraffin-embedded (FFPE) tissue samples are needed. We assessed a technique combining loop mediated isothermal amplification (LAMP) with PCR (PCR-LAMP) for detection of P gingivalis, using 110 FFPE tissue samples of chronic apical periodontitis. PCR-LAMP specifically detected P gingivalis with high sensitivity in FFPE tissue samples, and the sensitivity of the technique was higher than that of PCR or LAMP alone. The results of immunohistochemistry (IHC) confirmed the specificity of PCR-LAMP. IHC showed that P gingivalis was localized hi a granular layer of chronic apical periodontitis, a region that correlated with the localization of macrophages. This is the first study to describe the localization of P gingivalis in human periapical periodontitis. In conclusion, PCR-LAMP was an effective tool for detecting P gingivalis in periapical periodontitis. In addition, IHC results improve our understanding of the role of P gingivalis in the progression of periapical periodontitis.

    DOI: 10.2334/josnusd.15-0665

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  • Secretory leukocyte protease inhibitor inhibits expression of polymeric immunoglobulin receptor via the NF-kappa B signaling pathway 査読

    Yoshikazu Mikami, Takashi Iwase, Yusuke Komiyama, Naoyuki Matsumoto, Hidero Oki, Kazuo Komiyama

    MOLECULAR IMMUNOLOGY   67 ( 2 )   568 - 574   2015年10月

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    担当区分:筆頭著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PERGAMON-ELSEVIER SCIENCE LTD  

    Polymeric immunoglobulin receptor (pIgR) plays an important role in mucosal immune systems. Secretory immunoglobulin A, composed of secretory component of pIgR and a dimeric form of immunoglobulin A, is secreted on mucosal surfaces and serves as a biological defense factor. pIgR gene expression is reportedly induced by activation of the transcription factor nuclear factor (NF)-kappa B. On the other hand, secretory leukocyte protease inhibitor (SLPI) is a glycoprotein that functions as a serine protease inhibitor. In alveolar epithelial cells, SLPI increases the level of I kappa B beta, which indicates that it is an inhibitor of NF-kappa B at the protein level. Taken together, SLPI may regulate plgR expression; however, the specific mechanism by which this occurs is unclear. Therefore, the aim of this study was to elucidatethe influence of SLPI on pIgR expression. SLPI and pIgR localized in goblet cells and ciliated epithelial cells of the gastrointestinal tract, respectively. No cells were detected in which SLPI and pIgR were co-expressed. In addition, recombinant human SLPI stimulation of an epithelial cell line (HT-29) decreased the pIgR expression. The pIgR expression was also higher in SLPI-deficient Ca9-22 cells than in wild-type Ca9-22 cells. Furthermore, a luciferase assay using a NF-kappa B reporter plasmid and real-time RT-PCR analysis indicated that when SLPI was present, the transcriptional activity of NF-kappa B protein was suppressed, which was accompanied by anincrease in the protein, but not the mRNA,expression of I kappa B beta. These results demonstrate that SLPI down-regulates pIgR expression through the NF-kappa B signaling pathway by inhibiting degradation of I kappa B beta protein. (C) 2015 The Authors. Published by Elsevier Ltd.

    DOI: 10.1016/j.molimm.2015.07.021

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  • Whole Transcriptome Analysis Using Next-Generation Sequencing of Sterile-Cultured Eisenia andrei for Immune System Research 査読

    Yoshikazu Mikami, Atsushi Fukushima, Takao Kuwada-Kusunose, Tetsuya Sakurai, Taiichi Kitano, Yusuke Komiyama, Takashi Iwase, Kazuo Komiyama

    PLOS ONE   10 ( 2 )   e0118587   2015年2月

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    担当区分:筆頭著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PUBLIC LIBRARY SCIENCE  

    Recently, earthworms have become a useful model for research into the immune system, and it is expected that results obtained using this model will shed light on the sophisticated vertebrate immune system and the evolution of the immune response, and additionally help identify new biomolecules with therapeutic applications. However, for earthworms to be used as a genetic model of the invertebrate immune system, basic molecular and genetic resources, such as an expressed sequence tag (EST) database, must be developed for this organism. Next-generation sequencing technologies have generated EST libraries by RNA-seq in many model species. In this study, we used Illumina RNA-sequence technology to perform a comprehensive transcriptome analysis using an RNA sample pooled from sterile-cultured Eisenia andrei. All clean reads were assembled de novo into 41,423 unigenes using the Trinity program. Using this transcriptome data, we performed BLAST analysis against the GenBank non-redundant (NR) database and obtained a total of 12,285 significant BLAST hits. Furthermore, gene ontology (GO) analysis assigned 78 unigenes to 24 immune class GO terms. In addition, we detected a unigene with high similarity to beta-1,3-glucuronyltransferase 1 (GlcAT-P), which mediates a glucuronyl transfer reaction during the biosynthesis of the carbohydrate epitope HNK-1 (human natural killer-1, also known as CD57), a marker of NK cells. The identified transcripts will be used to facilitate future research into the immune system using E. andrei.

    DOI: 10.1371/journal.pone.0118587

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  • Osteocytes up-regulate the terminal differentiation of pre-osteoblasts via gap junctions 査読

    Yoichi Nishikawa, Yuko Akiyama, Kiyofumi Yamamoto, Masayuki Kobayashi, Eri Watanabe, Nobukazu Watanabe, Noriyoshi Shimizu, Yoshikazu Mikami, Kazuo Komiyama

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   456 ( 1 )   1 - 6   2015年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACADEMIC PRESS INC ELSEVIER SCIENCE  

    We examined cell-to-cell interaction between pre-osteoblasts and osteocytes using MC3T3-E1 and MLO-Y4, respectively. First, GFP expressing MC3T3-E1 (E1-GFP) cells were generated to isolate the cells from co-culture with MLO-Y4. No changes were observed in the expression of osteogenic transcription factors Runx2, Osterix, Dlx5 and Msx2, but expression of alkaline phosphatase (ALP) and bone sialoprotein (BSP) in E1-GFP co-cultured with MLO-Y4 was 300-400-fold greater than that in mono-cultured E1-GFP. In addition, mineralized nodule formation was drastically increased in co-cultured E1-GFP cells compared to mono-cultured cells. Patch clamp assay showed the presence of gap junctions between E1-GFP and MLO-Y4. Furthermore, when the gap junction inhibitor carbenoxolone (CBX) was added to the culture, increased expression of ALP and BSP in E1-GFP co-cultured with MLO-Y4 was suppressed. These results suggest that gap junction detected between pre-osteoblasts and osteocytes plays an important role on the terminal differentiation of pre-osteoblasts. (C) 2014 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.bbrc.2014.10.128

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  • Osteogenic Gene Transcription Is Regulated via Gap Junction-Mediated Cell-Cell Communication 査読

    Yoshikazu Mikami, Kiyofumi Yamamoto, Yuko Akiyama, Masayuki Kobayashi, Eri Watanabe, Nobukazu Watanabe, Masatake Asano, Noriyoshi Shimizu, Kazuo Komiyama

    STEM CELLS AND DEVELOPMENT   24 ( 2 )   214 - 227   2015年1月

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    担当区分:筆頭著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:MARY ANN LIEBERT, INC  

    An analytical study of cell-cell communications between murine osteoblast-like MLO-A5 cells and bone marrow mesenchymal stem cell (BMSC)-like C3H10T1/2 cells was performed. C3H10T1/2 cells expressing green fluorescent protein (10T-GFP cells) were generated to enable the isolation of the BMSC-like cells from co-cultures with MLO-A5 cells. The mRNA expression levels of several osteogenic transcription factors (Runx2, Osterix, Dlx5, and Msx2) did not differ between the co-cultured and mono-cultured 10T-GFP cells, but those of alkaline phosphatase (ALP) and bone sialoprotein (BSP) were 300- to 400-fold higher in the co-cultured cells. Patch clamp and biocytin transfer assays revealed gap junction-mediated communication between co-cultured 10T-GFP and MLO-A5 cells. The addition of a gap junction inhibitor suppressed the increases in the expression levels of the ALP and BSP mRNAs in co-cultured 10T-GFP cells. Furthermore, the histone acetylation levels were higher in co-cultured 10T-GFP cells than in mono-cultured 10T-GFP cells. These results suggest that osteoblasts and BMSCs associate via gap junctions, and that gap junction-mediated signaling induces histone acetylation that leads to elevated transcription of the genes encoding ALP and BSP in BMSCs.

    DOI: 10.1089/scd.2014.0060

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  • The P75 neurotrophin receptor regulates proliferation of the human MG63 osteoblast cell line 査読

    Yuko Akiyama, Yoshikazu Mikami, Eri Watanabe, Nobukazu Watanabe, Taku Toriumi, Tomihisa Takahashi, Kazuo Komiyama, Keitaro Isokawa, Noriyoshi Shimizu, Masaki J. Honda

    DIFFERENTIATION   87 ( 3-4 )   111 - 118   2014年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCI LTD  

    The 75 kDa transmembrane protein, p75(NTR), is a marker of mesenchymal stem cells (MSCs). Isolated MSCs are capable of differentiating into osteoblasts, but the molecular function of p75(NTR) in MSCs and osteoblasts is poorly understood. The aim of this study was to examine the function of p75(NTR) in the human MG63 osteoblast cell line compared to the murine MC3T3E-1 pre-osteoblast cell line.
    MG63 cells and MC3T3-E1 cells expressing exogenous p75(NTR) protein (denoted as p75-MG63 and p75GFP-E1, respectively) were generated to compare osteogenic differentiation and cell proliferation abilities. Overexpression of p75(NTR) induced alkaline phosphatase activity and the mRNA expression of osteoblast-related genes such as osterix and bone sialoprotein in both p75-MG63 and p75GFP-E1. Interestingly, exogenous p75(NTR) stimulated cell proliferation and cell cycle progression in p75GFP-E1, but not in p75-MG63.
    To elucidate any different effects of p75(NTR) expression on osteogenic differentiation and cell proliferation, we examined the mRNA expression of tropomyosin receptor kinase (trk) genes (trkA, trkB, trkC) and Nogo receptor (NgR), which are binding partners of p75(NTR). Although trkA, trkB, and trkC were detected in both p75-MG63 and p75GFP-E1, only NgR was detected in p75-MG63. We then used the K252a inhibitor of the trks to identify the signaling pathway for osteogenic differentiation and cell pmliferation. Inhibition of trks by K252a suppressed p75(NTR)-mediated osteogenic differentiation of p75GFP-E1, whereas deletion of the GDI domain in p75(NTR) from the p75-MG63 produced enhanced cell proliferation compared to p75-MG63. These results suggest that p75(NTR) signaling associated with Lrk receptors promotes both cell proliferation arid osteoblast differentiation, but that p75(NTR)-mediated proliferation may be suppressed by signaling from the p75(NTR)/NgR complex. (C) 2014 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

    DOI: 10.1016/j.diff.2014.01.002

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  • Up-regulation of Axin2 by dexamethasone promotes adipocyte differentiation in ROB-C26 mesenchymal progenitor cells 査読

    Masako Naito, Yoshikazu Mikami, Minoru Takagi, Tomihisa Takahashi

    CELL AND TISSUE RESEARCH   354 ( 3 )   761 - 770   2013年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SPRINGER  

    Dexamethasone (Dex) regulates osteoblastic and adipocytic differentiation in mesenchymal progenitor cells through regulation of Wnt/beta-catenin signaling. To elucidate the regulatory mechanisms underlying the effects of Dex, we examine the expression of Axin2, which is an intracellular inhibitor of Wnt/beta-catenin signaling, in ROB-C26 clonal mesenchymal progenitor cells (C26). We observed the induction of Axin2 mRNA in C26 cells in response to Dex treatment. Treatment with a glucocorticoid receptor (GR) antagonist, mifepristone, showed that Dex-induced up-regulation of Axin2 is mediated by the GR. In the absence of Dex, gene silencing by using Axin2-targeted short hairpin RNA increased the number of alkaline phosphatase (ALP)-positive and nuclear beta-catenin-positive cells and ALP activity. In the presence of Dex, Axin2 knockdown resulted in an increased number of ALP-positive and nuclear beta-catenin-positive cells. Furthermore, Axin2 knockdown in Dex-treated cells suppressed adipocyte differentiation (as determined by reduced Oil Red O staining), reduced the number of PPAR gamma-positive and aP2-positive cells and decreased the mRNA expression of PPAR gamma 2 and aP2. These results suggest that Axin2 plays a key role in adipocyte and osteoblastic differentiation by controlling beta-catenin expression.

    DOI: 10.1007/s00441-013-1696-5

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  • Phylogenic Relationship of Labridae Species Deduced from Comparative Dissection 査読

    Yoshikazu Mikami

    ANATOMICAL RECORD-ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY   296 ( 5 )   788 - 797   2013年5月

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    担当区分:筆頭著者, 最終著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-BLACKWELL  

    Labridae is a family of shallow-water coastal fish that belong to the Labroidei suborder in the Perciformes order. There has however been little comparative dissection or phylogenetic research on this family, which resulted in confusion regarding species classification. Therefore morphological characters of 12 species of Labridae containing three subfamilies and seven genera were compared for the purpose of determining the characteristics of the subfamilies and genera of Labridae and deducing their phylogenic relationship. We compiled a phylogenetic tree by using the computer program PAUP 4.0b10 and a data set composed of 17 meristic and osteological characters. In our phylogenic tree, the two species of Bodianinae, three species of Cheilininae and seven species of Corinae each formed monophyletic groups, supporting the conventional classification by lateral line shape. Furthermore, the group of three species of Cheilininae and the group of two species of Bodianinae were shown to be more closely related, because they have several common morphological characteristics, such as neuroarticular processes and anterior teeth on premaxilla. Between seven species of Corinae, not only was the group of two species of Halichoeres more closely related to Coris dorsomacula, but also, this group of three species may be closely related to the three species of Thalassoma. Suezichthys gracilis was also found to have separated from the other Corinae species at the earliest stage. (C) 2013 Wiley Periodicals, Inc.

    DOI: 10.1002/ar.22687

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  • The p75 neurotrophin receptor regulates MC3T3-E1 osteoblastic differentiation 査読

    Yoshikazu Mikami, Shinnosuke Suzuki, Yumiko Ishii, Nobukazu Watanabe, Tomihisa Takahashi, Keitaro Isokawa, Masaki J. Honda

    DIFFERENTIATION   84 ( 5 )   392 - 399   2012年12月

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    担当区分:筆頭著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCI LTD  

    While the role of p75(NTR) signaling in the regulation of nerve-related cell growth and survival has been well documented, its actions in osteoblasts are poorly understood. In this study, we examined the effects of p75(NTR) on osteoblast proliferation and differentiation using the MC3T3-E1 pre-osteoblast cell line. Proliferation and osteogenic differentiation were significantly enhanced in p75(NTR)-overexpressing MC3T3-E1 cells (p75GFP-E1). In addition, expression of osteoblast-specific osteocalcin (OCN), bone sialoprotein (BSP), and osterix mRNA, ALP activity, and mineralization capacity were dramatically enhanced in p75GFP-E1 cells, compared to wild MC3T3-E1 cells (GFP-E1). To determine the binding partner of p75(NTR) in p75GFP-E1 cells during osteogenic differentiation, we examined the expression of trkA, trkB, and trkC that are known binding partners of p75(NTR), as well as NgR. Pharmacological inhibition of trk tyrosine kinase with the K252a inhibitor resulted in marked reduction in the level of ALPase under osteogenic conditions. The deletion of the GDI binding domain in the p75(NTR)-GFP construct had no effect on mineralization. Taken together, our studies demonstrated that p75(NTR) signaling through the trk tyrosine kinase pathway affects osteoblast functions by targeting osteoblast proliferation and differentiation.

    DOI: 10.1016/j.diff.2012.07.001

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  • Inhibition of Wnt/β-catenin signaling by dexamethasone promotes adipocyte differentiation in mesenchymal progenitor cells, ROB-C26. 査読

    Naito M, Omoteyama K, Mikami Y, Takahashi T, Takagi M

    Histochem Cell Biol.   138 ( 6 )   833-845   2012年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1007/s00418-012-1007-3.

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  • Development of Collagen Fibres and Lysyl Oxidase Expression in the Presumptive Dermis of Chick Limb Bud 査読

    Y. Yamazaki, Y. Mikami, M. Yuguchi, Y. Namba, K. Isokawa

    Anatomia, Histologia, Embryologia   41 ( 1 )   68 - 74   2012年2月

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    掲載種別:研究論文(学術雑誌)   出版者・発行元:Wiley  

    DOI: 10.1111/j.1439-0264.2011.01103.x

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  • Suppression of lamin A/C by short hairpin RNAs promotes adipocyte lineage commitment in mesenchymal progenitor cell line, ROB-C26. 査読 国際誌

    Naito M, Omoteyama K, Mikami Y, Takagi M, Takahashi T

    Histochem Cell Biol.   137 ( 2 )   235-247. - 47   2012年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Lamin A/C gene encodes a nuclear membrane protein, and mutations in this gene are associated with diverse degenerative diseases that are linked to premature aging. While lamin A/C is involved in the regulation of tissue homeostasis, the distinct expression patterns are poorly understood in the mesenchymal cells differentiating into adipocytes. Here, we examined the expression of lamin A/C in a rat mesenchymal progenitor cell-line, ROB-C26 (C26). Immunocytochemical analysis showed that lamin A/C was transiently down-regulated in immature adipocytes, but its expression increased with terminal differentiation. To elucidate the role of lamin A/C expression on mesenchymal cell differentiation, lamin A/C expression was suppressed using short hairpin RNA (shRNA) molecules in C26 cells. In the absence of adipogenic stimuli, lamin A/C shRNA decreased alkaline phosphatase (ALP) activity, but induced preadipocyte factor -1 (Pref-1) mRNA expression. In the presence of adipogenic stimuli, lamin A/C knockdown promotes adipocytes differentiation, as assessed by the detection of an increase in Oil Red O staining. RT-PCR analysis showed that lamin A/C shRNA resulted in increased mRNA expression of PPARγ2 and aP2 during adipocyte differentiation. These results suggest that decreased lamin A/C expression levels not only suppress osteoblast phenotypes but also promote adipocyte differentiation in C26 cells.

    DOI: 10.1007/s00418-011-0890-3

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  • A Novel Bromomelatonin Derivative Suppresses Apoptosis with Regulating the Expression of Bcl-2 Family Genes 査読

    Yoshikazu Mikami, Masanori Somei, Eri Watanabe, Nobukazu Watanabe, Tomihisa Takahashi, Masaki J. Honda

    LETTERS IN DRUG DESIGN & DISCOVERY   8 ( 10 )   951 - 956   2011年12月

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    担当区分:責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:BENTHAM SCIENCE PUBL LTD  

    SSH-BM-I is a new synthetic compound that is synthesized from Bromomelatonin by using a newly developed synthetic method. Recently, our group demonstrated that SST-VED-I-1, which has a similar chemical structure to SSH-BM-I, inhibits starvation-induced apoptosis in osteoblasts. However, the effects of SSH-BM-I on apoptosis in osteoblasts have not yet been examined. Therefore, this study examined the effects of this compound on apoptosis in osteoblasts. In the present study, MC3T3-E1 mouse osteoblasts were used and apoptosis was induced by ultraviolet radiation (UV). We investigated the effect of SSH-BM-I on apoptosis by analyzing Caspase3/7 activity, translocation of Phosphatidylserine (PS), and mRNA expression levels of Bcl-2 and Bax. We found that SSH-BM-I suppressed UV-induced cell death in a dose-dependent manner, which was accompanied with the inhibition of Caspase activation and translocation of PS. Furthermore, SSH-BM-I up-regulated Bcl-2 expression and down-regulated Bax expression. These results suggest that SSH-BM-I has an inhibitory effect on apoptosis in osteoblasts through a Bcl-2 family-dependent signaling pathway.

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  • Bacitracin Upregulates mbrAB Transcription via mbrCD to Confer Bacitracin Resistance in Streptococcus mutans 査読

    Yoshikazu Mikami, Naoto Suzuki, Tomihisa Takahashi, Kichibee Otsuka, Hiromasa Tsuda

    JOURNAL OF PHARMACOLOGICAL SCIENCES   117 ( 3 )   204 - 207   2011年11月

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    担当区分:筆頭著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:JAPANESE PHARMACOLOGICAL SOC  

    Streptococcus mutans is a bacterial cause of dental caries that is resistant to bacitracin. The aim of this study was to elucidate the mbrABCD-related bacitracin resistance mechanism of S. mutans. Transcriptome data demonstrated that the expression levels of 33 genes were induced more than twofold by bacitracin. Fourteen genes were selected from the upregulated genes, and defective mutants of these genes were constructed for measurement of their sensitivity to bacitracin. Among the mutants, only the mbrA- or mbrB-deficient mutants exhibited 100- to 121-fold greater sensitivity to bacitracin when compared with the wild-type strain. Moreover, knockout of the mbrC and mbrD genes abolished the bacitracin-induced mbrAB upregulation. These results suggest that both mbrC and mbrD are required for mbrAB upregulation that confers the bacitracin-resistant phenotype on S. mutans. [Supplementary Table: available only at http://dx.doi.org/10.1254/jphs.11052SC]

    DOI: 10.1254/jphs.11052SC

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  • Novel approach for transient protein expression in primary cultures of human dental pulp-derived cells 査読

    Hisashi Suguro, Yoshikazu Mikami, Rieko Koshi, Bunnai Ogiso, Eri Watanabe, Nobukazu Watanabe, Masaki J. Honda, Masatake Asano, Kazuo Komiyama

    PROTEIN EXPRESSION AND PURIFICATION   78 ( 2 )   143 - 148   2011年8月

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    担当区分:責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACADEMIC PRESS INC ELSEVIER SCIENCE  

    Transfection is a powerful method for investigating variable biological functions of desired genes. However, the efficiency of transfection into primary cultures of dental pulp-derived cells (DPDC) is low. Therefore, using a recombinant vaccinia virus (vTF7-3), which contains T7 RNA polymerase, we have established a transient protein expression system in DPDCs. In this study, we used the human polymeric immunoglobulin receptor (pIgR) cDNA as a model gene. pIgR expression by the vTF7-3 expression system was confirmed by flow cytometry analysis and Western blotting. Furthermore, exogenous pIgR protein localized at the cell surface in DPDCs and formed a secretory component (SC). This suggests that exogenous pIgR protein expressed by the vTF7-3 expression system acts like endogenous pIgR protein. These results indicate the applicability of the method for cells outgrown from dental pulp tissue. In addition, as protein expression could be detected shortly after transfection (approximately 5 h), this experimental system has been used intensely for experiments examining very early steps in protein exocytosis. (C) 2011 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.pep.2011.02.005

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  • SSH-BM-I, a Tryptamine Derivative, Stimulates Mineralization in Terminal Osteoblast Differentiation but Inhibits Osteogenesis of Pre-committed Progenitor Cells 査読

    Yoshikazu Mikami, Masanori Somei, Hiromasa Tsuda

    JOURNAL OF PHARMACOLOGICAL SCIENCES   116 ( 1 )   63 - 72   2011年5月

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    担当区分:筆頭著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:JAPANESE PHARMACOLOGICAL SOC  

    SSH-BM-I was synthesized from tryptamine by using a newly developed synthetic method, and it has structural similarity to bromomelatonin. Recently, it had been reported that SSH-BM-I increases osteoblasts in scales of gold fish. However, the effect of SSH-BM-I on osteoblast differentiation in mammalian cells has not yet been examined. Therefore, this study examined the effect of SSH-BM-I on osteoblast differentiation in mesenchymal progenitor-like cells and mature osteoblast-like cells. SSH-BM-I enhanced terminal osteoblast differentiation, as indicated by mineralization, which was accompanied by upregulation of the osteogenic marker genes bone sialoprotein (BSP) and osteocalcin (OC). However, in mesenchymal progenitor ROB-C26 cultures, no mineralized nodules were observed regardless of SSH-BM-I treatment, although BMP-2 was able to induce nodule formation in these cells. Furthermore, BMP-2 induced nodule formation was suppressed by SSH-BM-I treatment in ROB-C26 cultures. We further investigated the impact of the timing and duration of SSH-BM-I treatment on osteoblast differentiation. The effect of SSH-BM-I treatment on osteoblast differentiation of ROB-C26 in the presence of BMP-2 switches from negative to positive sometime between day 6 and 9, because SSH-BM-I treatment enhanced the formation of mineralized nodules when it was started on day 9, but suppressed nodule formation when it was started at day 6 or earlier. These results suggest that the stimulatory effects of SSH-BM-I on the formation of mineralized nodules depend on the degree of cell differentiation.

    DOI: 10.1254/jphs.10329FP

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  • CD271/p75(NTR) Inhibits the Differentiation of Mesenchymal Stem Cells into Osteogenic, Adipogenic, Chondrogenic, and Myogenic Lineages 査読

    Yoshikazu Mikami, Yumiko Ishii, Nobukazu Watanabe, Tetsuo Shirakawa, Shinnosuke Suzuki, Seiko Irie, Keitaro Isokawa, Masaki J. Honda

    STEM CELLS AND DEVELOPMENT   20 ( 5 )   901 - 913   2011年5月

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    担当区分:筆頭著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:MARY ANN LIEBERT INC  

    We describe a novel role for CD271 in the differentiation of mesenchymal stem cells (MSCs), including deciduous dental pulp stem cells (DDPSCs) and murine multipotent MSCs (C3H10T1/2 cells). The CD271(+) sub-population of deciduous dental pulp cells (CD271(+)/DDPSCs) and the forced expression of CD271 in C3H10T1/2 (10T271) were analyzed by fluorescence-activated cell sorting. CD271 expression was detected in DDPSCs that expressed both CD44 and CD90, simultaneously, and the clonogenic capacity of the CD271(+)/DDPSCs was higher than that of the CD271(-)/DDPSCs that expressed both CD44 and CD90. Further, the differentiation of CD271(+)/DDPSCs into osteoblasts and adipocytes was inhibited although CD271(-)/DDPSCs were capable of differentiating into osteoblasts and adipocytes. CD271 was overexpressed in C3H10T1/2 cells, which have the potential to differentiate into osteoblasts, adipocytes, chondrocytes, and myocytes. CD271 inhibited the differentiation of C3H10T1/2 cells into any of these lineages. These results indicate a role for CD271 in inhibiting the differentiation of MSCs.

    DOI: 10.1089/scd.2010.0299

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  • Dexamethasone Modulates Osteogenesis and Adipogenesis With Regulation of Osterix Expression in Rat Calvaria-Derived Cells 査読

    Yoshikazu Mikami, Mio Lee, Seiko Irie, Masaki J. Honda

    JOURNAL OF CELLULAR PHYSIOLOGY   226 ( 3 )   739 - 748   2011年3月

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    担当区分:筆頭著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-BLACKWELL PUBLISHING, INC  

    Osteoblasts and adipocytes originate from common mesenchymal progenitor cells and although a number of compounds can induce osteoblastic and adipogenic differentiation from progenitor cells, the underlying mechanisms have not been elucidated. The present study examined the synergistic effects of dexamethasone (Dex) and bone morphogenetic protein (BMP)-2 on the differentiation of clonal mesenchymal progenitor cells isolated from rat calvaria into osteoblasts and adipocytes, as well as the effects of the timing of treatment. Cells were cultured for various periods of time in the presence of Dex and/or BMP-2. When cells were treated with Dex+BMP-2 during the early phase of differentiation, they differentiated into adipocytes. However, when cells were treated with Dex+BMP-2 during the late phase of differentiation, a synergistic effect on in vitro matrix mineralization was observed. To examine differences between the early and late phases of differentiation, ALP activity was measured in the presence of BMP-2. ALP activity increased markedly on Day 9, corresponding to the onset of the synergistic effect of Dex. Dex treatment inhibited osterix (OSX) expression in cells committed to adipogenic differentiation, but not in cells committed to osteogenic differentiation following BMP-2 treatment. The isoform2 OSX promoter region was found to be involved in the effects of Dex on cells during the early phase of differentiation. Furthermore, cells stably expressing OSX (isoform2) formed mineralized nodules even though they had been treated with Dex+BMP-2 during the early phase of differentiation. It appears that Dex modulates osteogenesis and adipogenesis in mesenchymal stem cells by regulating OSX expression. J. Cell. Physiol. 226: 739-748, 2011. (C) 2010 Wiley-Liss, Inc.

    DOI: 10.1002/jcp.22392

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  • Inhibitory Effects of a Tryptamine Derivative on Ultraviolet Radiation-Induced Apoptosis in MC3T3-E1 Mouse Osteoblasts 査読

    Yoshikazu Mikami, Motoki Senoo, Mio Lee, Kiyoshi Yamada, Kuniyasu Ochiai, Masaki J. Honda, Eri Watanabe, Nobukazu Watanabe, Masanori Somei, Minoru Takagi

    JOURNAL OF PHARMACOLOGICAL SCIENCES   115 ( 2 )   214 - 220   2011年2月

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    担当区分:筆頭著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:JAPANESE PHARMACOLOGICAL SOC  

    MS-IPA1 is a new synthetic compound that is synthesized from tryptamine. Recently, our group demonstrated that SST-VED-I-1, which has a similar chemical structure to MS-IPA1, inhibits starvation-induced apoptosis in osteoblasts. However, the effects of MS-IPA I on apoptosis in osteoblasts have not yet been examined. Therefore, this study examined the effects of this compound on apoptosis in osteoblasts. In this study, MC3T3-E1 mouse osteoblasts were used and apoptosis was induced by ultraviolet radiation (UV). We investigated the effect of MS-IPA1 on apoptosis by analyzing caspase3/7 activity, translocation of phosphatidylserine (PS), and mRNA expression levels of Bcl-2 and Bax. In addition, it was investigated whether MS-IPA1 affects cell proliferation and cell cycle progression. We found that MS-IPA I had no effect on cell proliferation or cell cycle progression. However, MS-IPA I suppressed UV-induced cell death in a dose-dependent manner, which was accompanied with the inhibition of caspase activation and translocation of PS. Furthermore, after UV exposure, Bcl-2 expression was increased in the MS-IPA1 treated cells as compared to that in the vehicle-treated cells. In contrast, Bax expression was decreased in the MS-IPA1 treated cell as compared to that in the vehicle-treated cells. These results suggest that MS-IPA1 has an inhibitory effect on apoptosis in osteoblasts through a Bcl-2 family-dependent signaling pathway.

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  • Bone Morphogenetic Protein 2 and Dexamethasone Synergistically Increase Alkaline Phosphatase Levels Through JAK/STAT Signaling in C3H10T1/2 Cells 査読

    Yoshikazu Mikami, Masatake Asano, Masaki J. Honda, Minoru Takagi

    JOURNAL OF CELLULAR PHYSIOLOGY   223 ( 1 )   123 - 133   2010年4月

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    担当区分:筆頭著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-LISS  

    Alkaline phosphatase (ALP) is generally believed to be a faithful marker of osteoblast differentiation, and its expression is induced by bone morphogenetic protein-2 (BMP-2) and dexamethasone (Dex). However, the effects of combined administration of BMP-2 and Dex on ALP transcription have not been extensively examined. In this study, we found that BMP-2 and Dex synergistically increase ALP levels in mouse C3H10T1/2 pluripotent stem cells. However, switching from one inducer to the other, by adding BMP-2 or Dex to cell cultures at different times, was no more effective than continuous treatment with either inducer alone. A significant induction of ALP mRNA expression was observed only in cells continuously treated with both inducers. This result suggests that both BMP-2 and Dex may act in the same pathway or at the same stage of differentiation. A luciferase assay using ALP promoter deletion constructs showed that a region of the promoter containing a putative signal transducer and activator of transcription 3 (STAT3) response element (SRE) responds to treatment with a combination of BMP-2 and Dex. Furthermore, a ChIP assay indicated that STAT3 bound to the SRE. In addition, a STAT3 siRNA suppressed the synergistic effect of BMP-2 and Dex on ALP levels. These results indicate that STAT3 may play an important role in regulating ALP expression. To our knowledge, this is the first time that STAT3 has been implicated in the regulation of ALP expression by BMP-2 and Dex. These findings raise the possibility of developing new strategies for the enhancement of bone formation using a combination of BMPs and Dex. J. Cell. Physiol. 223: 123-133, 2010. (C) 2009 Wiley-Liss, Inc.

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  • Oral bacterial extracts facilitate alkaline phosphatase activity in human dental pulp-derived cells 査読

    Abe S, Imaizumi M, Mikami Y, Wada Y, Tsuchiya S, Suzuki S, Irie S, Satomura K, Ishihara K, Honda J M

    Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology and Endodontology   109, 149-154   2010年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1016/j.tripleo.2009.08.028

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  • A Tryptamine Derivative, SST-VEDI-1, Inhibits Apoptosis and Stimulates Mineralization in Osteoblasts 査読

    Yoshikazu Mikami, Masanori Somei, Minoru Takagi

    ENDOCRINE JOURNAL   56 ( 5 )   665 - 678   2009年8月

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    担当区分:筆頭著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:JAPAN ENDOCRINE SOC  

    SST-VEDI-1(VEDI-1) is a new synthetic compound that is synthesized from tryptamine, and has structural similarity to the SSH-BM family of compounds. However, the biological effects of VEDI-1 have yet to be well characterized. A recent report has demonstrated that SSH-BM-type compounds can stimulate osteoblast activity in cultured scales of goldfish. In this study, we examined the effects of VEDI-1 on osteoblastic differentiation as well as its effects on apoptosis, which is known to be closely related to osteoblastic differentiation. We found that VEDI-1 enhanced the formation of mineralized nodules in rat osteoblast cell lines, including ROS17/2.8 cells, and in mouse pre-osteoblast cell lines, including MC3T3-E1 cells, in a dose dependent manner, which was accompanied by increased expression of late osteoblast markers, bone sialoprotein (BSP) and osteocalcin (OC). Furthermore, VEDI-1 inhibited apoptotic cell death and regulated the expression of proteins in the Bcl-2 family. These results suggest that VEDI-1 may facilitate late differentiation of osteoblasts and may have an inhibitory effect on apoptosis.

    DOI: 10.1507/endocrj.K08E-340

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  • Poly I:C-induced expression of intercellular adhesion molecule-1 in intestinal epithelial cells 査読

    D. Omagari, Y. Mikami, H. Suguro, K. Sunagawa, M. Asano, E. Sanuki, I. Moro, K. Komiyama

    CLINICAL AND EXPERIMENTAL IMMUNOLOGY   156 ( 2 )   294 - 302   2009年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-BLACKWELL PUBLISHING, INC  

    Intercellular adhesion molecul-1 (ICAM-1) is a transmembrane glycoprotein belonging to the immunoglobulin superfamily of adhesion molecules and plays perdominant roles in recruitment and trafficking of leucocytes to sites of inflammation. ICAM-1 expression in intestinal epithelial cells (IECs) is enhanced by several stimuli, such as proinflammatory cytokines, bacterial infections or pathogen-associated molecular patterns. One of these stimuli, double-stranded RNA (dsRNA), is a by-product of viral replication and can be recognized by its cognate receptor Toll-like receptor 3 (TLR-3). In spite of expression of both TLR-3 and ICAM-1 in IECs, correlation between TLR-3-signalling and ICAM-1 expression has never been examined in IECs. In the present study, we investigated whether poly I:C, an analogue of dsRNA, can stimulate the expression of ICAM-1 in IEC line, HT-29. Poly I:C-stimulation up-regulated the expression of ICAM-1 mRNA by real-time polymerase chain reaction. Enhanced expression of ICAM-1 was confirmed in protein level by immunofluoresense cell staining and enzyme-linked immunosorbent assay by measuring the released soluble ICAM-1 in culture supernatant. As the stimulation effect was reduced by pre-treatment of the cells with anti-TLR-3 antibody, poly I:C-binding signal was thought to be sensed by TLR-3 on the surface of HT-29. The results of luciferase assay and nuclear factor kappa-b (NF-kB) inhibitor treatment experiments indicated that the downstream signal was mainly transduced by transcription factor, NF-kB. All these results demonstrated the connection between TLR-3 signalling and ICAM-1 expression in HT-29 cells and indicated the importance of coordinated function of both innate and adaptive immunity against viral infections.

    DOI: 10.1111/j.1365-2249.2009.03892.x

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  • A New Synthetic Compound, SST-VEDI-1, Inhibits Osteoblast Differentiation with a Down-Regulation of the Osterix Expression 査読

    Yoshikazu Mikami, Masanori Somei, Minoru Takagi

    JOURNAL OF BIOCHEMISTRY   145 ( 2 )   239 - 247   2009年2月

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    担当区分:筆頭著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:OXFORD UNIV PRESS  

    SST-VEDI-1(VEDI-1) is a new synthetic compound which is synthesized from tryptamine. However, the effect of VEDI-1 on various bio-phenomena in cells has not yet been examined. Tryptamine is one of the known trace amines. Trace amines are present in the central nervous system at very low concentrations and they are generally considered to have potent sympathomimetic actions. On the other hand, SSH-BM-I and SSH-BM-II-type compounds have been demonstrated to stimulate osteoblast activity in the cultured scales of goldfish. These compounds are also synthesized from tryptamine. VEDI-1 has a similar chemical structure to that of SSH-BM-I and SSH-BM-II-type compounds. Therefore, this study examined the effect of VEDI-1 on osteoblastic differentiation. VEDI-1 inhibited the osteoblast differentiation identified by mineralization, which was accompanied by the down-regulation of the expression of an osteogenic transcription factor, Osterix (OSX). Furthermore, as well as VEDI-1-treatment, the suppression of the OSX expression by stable-transfection with OSX/shRNA decreased the formation of mineralized nodules. These results suggest a possibility that VEDI-1 inhibits the osteoblast differentiation by suppressing the OSX expression.

    DOI: 10.1093/jb/mvn164

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  • Dexamethasone promotes DMP1 mRNA expression by inhibiting negative regulation of Runx2 in multipotential mesenchymal progenitor, ROB-C26 査読

    Yoshikazu Mikami, Tomihisa Takahashi, Shigeyuki Kato, Minoru Takagi

    CELL BIOLOGY INTERNATIONAL   32 ( 2 )   239 - 246   2008年2月

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    担当区分:筆頭著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD  

    Dentin matrix protein 1 (DMP1) is an acidic phosphorylated extracellular protein and essential for mineralization of dentin and bone; however, the precise mechanism regulating DMP1 expression is not fully understood. A synthetic glucocorticoid (GC), dexamethasone (Dex), promotes an early osteoblast differentiation of a mesenchymal progenitor, ROB-C26 (C26), in parallel with inductive expression of an osteoblast-specific transcription factor, Runx2, and other extracellular matrix proteins such as osteocalcin and bone sialoprotein (BSP). We have examined the effect of Dex on DMP1 expression via induction of Runx2 in C26 cells. Real time RT-PCR showed that Dex increases DMP1 mRNA expression levels at time- and dose-dependent manners and a GC antagonist, RU486, drastically inhibited DMP1 mRNA expression levels. Furthermore, Dex increased the luciferase activity of six-repeated osteoblast-specific cis-acting element 2 (6 x OSE2), which is the binding sequence of Runx2, suggesting that Dex stimulates DMP1 expression via activation of Runx2. However, unexpected results showed that overexpression of exogenous Runx2 depressed DMP1 mRNA expression level, even after cells had been treated with Dex, while downregulated expression of endogenous Runx2 enhanced Dex-induced DMP1 mRNA expression level. These results imply that large amounts of exogenous Runx2 inhibit DMP1 expression, whereas small amounts are more effective for Dex-induced DMP1 expression in C26 cells. Therefore, Dex may activate some factors that inhibit negative action of Runx2 on DMP1 expression. Since mitogen-activating protein kinase (MAPK) phosphatase-1 (MKP-1) has been reported to affect the Dex-induced osteoblast differentiation via decrease of Runx2-phosphorylation, we focus on the relationship between MKP-1 and DMP1 expression. Dex increases MKP-1 expression, and overexpression of exogenous MKP-1 showed significant increase of luciferase activity of 6 x OSE up to the level detected in Dex-treated C26 cells. However, no inductive DMP1 mRNA expression level was found in C26 cells unlike BSP and OPN. These results suggest that although MKP-1 increases DNA-binding activity of Runx2, DMP1 expression may require the collaboration of MKP-1 and additive factors to stimulate Runx2-mediated DMP1 expression in the post-transcriptional event of Dex-treated C26 cells. (C) 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

    DOI: 10.1016/j.cellbi.2007.08.033

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  • Inductive effects of dexamethasone on the mineralization and the osteoblastic gene expressions in mature osteoblast-like ROS17/2.8 cells 査読

    Yoshikazu Mikami, Kazuki Omoteyama, Shigeyuki Kato, Minoru Takagi

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   362 ( 2 )   368 - 373   2007年10月

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    担当区分:筆頭著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACADEMIC PRESS INC ELSEVIER SCIENCE  

    We examined the effects of dexamethasone (Dex), a synthetic glucocorticoid, on the formation of mineralized bone nodules and the gene expressions of the late ostcoblastic markers, bone sialoprotein (BSP), osteocalcin (OC), and osteopontin (OPN) in mature osteoblast ROS17/2.8 cells. Treatment of ROS17/2.8 cells with Dex resulted in the induction of mineralization accompanied with increasing BSP and OC expressions. Previous reports have demonstrated that BSP and OC expressions are regulated by Runx2. Then, we hypothesized that Dex might promote osteoblastic differentiation and mineralization on ROS 17/2.8 by Runx2. In this study, no effect was observed in mRNA and protein expression of Runx2. However, the transcriptional activity of Runx2 was enhanced by Dex treatment. Furthermore, the Dex-induced BSP and OC expressions decreased after the transfection of Runx2 small-interfering RNAs (siRNAs). These results suggested that the enhancement of Runx2 transcriptional activity by Dex treatment may be followed by the activation of ostcoblast marker genes, such as BSP and OC to thereby produce a bone-specific matrix that subsequently becomes mineralized. (C) 2007 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.bbrc.2007.07.192

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  • Foxc2 induces expression of MyoD and differentiation of the mouse myoblast cell line C2C12 査読

    Kazuki Omoteyama, Yoshikazu Mikami, Minoru Takagi

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   358 ( 3 )   885 - 889   2007年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACADEMIC PRESS INC ELSEVIER SCIENCE  

    The Fox family of transcription factors is expressed in various organs and tissues during development, and is involved in a variety of developmental and cellular differentiation processes. Foxc2 mRNA is strongly expressed in mesoderm-derived tissues in the embryo, but the molecular mechanism of Foxc2-induced cellular differentiation and the physiological role of Foxc2 are unclear.
    In mouse myoblast C2C12 cells, over-expression of Foxc2 increased the expression of desmin, the muscle-specific member of the intermediate filament family of proteins, and induced the synthesis of myotubes. Transient expression of Foxc2 increased MyoD mRNA and protein levels, as assessed by real-time PCR and Western blot, respectively. Chromatin immunoprecipitation (ChIP) analysis showed that Foxc2 does not bind to the promoter region of the MyoD gene, which indicated that Foxc2 does not directly activate MyoD. In contrast to reports that Foxc2 regulates the production of basement membrane components in endothelial cells, we found no evidence of Foxc2-mediated regulation of Collagen type IV alpha 1 (Col4a1) or Col4a2 in myoblast cells. Taken together, these results indicate that Foxc2 plays an important role in the development of the mesenchyme through the regulation of MyoD gene expression. (C) 2007 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.bbrc.2007.05.009

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  • The functional region of CENP-H interacts with the Nuf2 complex that localizes to centromere during mitosis 査読

    Y Mikami, T Hori, H Kimura, T Fukagawa

    MOLECULAR AND CELLULAR BIOLOGY   25 ( 5 )   1958 - 1970   2005年3月

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    担当区分:筆頭著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC MICROBIOLOGY  

    CENP-H is a constitutive centromere component that localizes to the centromere throughout the cell cycle. Because CENP-H is colocalized with CENP-A and CENP-C, it is thought to be an inner centromere protein. We previously generated a conditional loss-of-function mutant of CENP-H and showed that CENP-H is required for targeting of CENP-C to the centromere in chicken DT40 cells. In the present study, we used this mutant to identify the functional region of chicken CENP-H necessary for centromere targeting and cell viability. This region was found by yeast two-hybrid analysis to interact with Hec1, which is a member of the Nuf2 complex that transiently localizes to the centromere during mitosis. Coimmunoprecipitation experiments revealed that CENP-H interacts with the Nuf2 complex in chicken DT40 cells. Photobleaching experiments showed that both Hec1 and CENP-H form stable associations with the centromeres during mitosis, suggesting that Hec1 acts as a structural component of centromeres during mitosis. On the basis of these results and previously published data, we propose that the Nuf2 complex functions as a connector between the inner and outer kinetochores.

    DOI: 10.1128/MCB.25.5.1958-1970.2005

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  • CENP-H, a constitutive centromere component, is required for centromere targeting of CENP-C in vertebrate cells 査読

    T Fukagawa, Y Mikami, A Nishihashi, Regnier, V, T Haraguchi, Y Hiraoka, N Sugata, K Todokoro, W Brown, T Ikemura

    EMBO JOURNAL   20 ( 16 )   4603 - 4617   2001年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:OXFORD UNIV PRESS  

    CENP-H has recently been discovered as a constitutive component of the centromere that co-localizes with CENP-A and CENP-C throughout the cell cycle. The precise function, however, remains poorly understood. We examined the role of CENT-H in centromere function and assembly by generating a conditional loss-of-function mutant in the chicken DT40 cell line. In the absence of CENP-H, cell cycle arrest at metaphase, consistent with loss of centromere function, was observed. Immunocytochemical analysis of the CENP-H-deficient cells demonstrated that CENP-H is necessary for CENP-C, but not CENP-A, localization to the centromere. These findings indicate that centromere assembly in vertebrate cells proceeds in a hierarchical manner in which localization of the centromere-specific histone CENP-A is an early event that occurs independently of CENP-C and CENP-H.

    DOI: 10.1093/emboj/20.16.4603

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  • External and Internal Morpholigy and Nucleotide Sequence of Mitochondrial Cytochrome b Gene in Three Thalassoma Species (Perciformes:Labridae)

    Mikami Y, Machida Y

    Mem.Fac.Sci.Kochi Univ.   20   35-46   1999年

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    担当区分:筆頭著者   記述言語:日本語   掲載種別:研究論文(大学,研究機関等紀要)  

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書籍等出版物

  • Synergistic effects of BMP-2 and other osteogenic inducers. In: Nohe A, editor. Bone Morphogenetic Proteins: New Research

    Mikami Y( 範囲: pp175-186)

    Nova Science Publishers, Inc.  2011年 

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    記述言語:英語 著書種別:学術書

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  • Autophagy in periodontal disease. In: Gorbunov NV, editor. Autophagy: Principles, Regulation and Roles in Disease

    Tsuda H, Mikami Y( 担当: 共著 ,  範囲: pp85-96)

    Nova Science Publishers, Inc. Hauppauge  2011年 

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    記述言語:英語 著書種別:学術書

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MISC

  • コメペプチドとそのアミノ酸置換体はPorphyromonas gingivalis,Fusobacterium nucleatumのバイオフィルム形成を阻害する

    松岸葵, 松岸葵, 野中由香莉, 竹内麻衣, 原実生, 原実生, 早津学, 三上剛和, 牛木辰男, 土門久哲, 山崎和久, 多部田康一

    日本歯周病学会会誌(Web)   62   2020年

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  • Current status of drug therapies for osteoporosis and the search for stem cells adapted for bone regenerative medicine 査読

    Yoshikazu Mikami, Taro Matsumoto, Koichiro Kano, Taku Toriumi, Masanori Somei, Masaki J. Honda, Kazuo Komiyama

    ANATOMICAL SCIENCE INTERNATIONAL   89 ( 1 )   1 - 10   2014年1月

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    担当区分:筆頭著者, 責任著者   記述言語:英語   掲載種別:記事・総説・解説・論説等(学術雑誌)   出版者・発行元:SPRINGER  

    A number of factors can lead to bone disorders such as osteoporosis, in which the balance of bone resorption vs. bone formation is upset (i.e., more bone is resorbed than is formed). The result is a loss of bone mass, with a concomitant decrease in bone density. Drugs for osteoporosis can be broadly classified as "bone resorption inhibitors", which impede bone resorption by osteoclasts, and "bone formation accelerators", which augment bone formation by osteoblasts. Here, we describe representative drugs in each class, i.e., the bisphosphonates and the parathyroid hormone. In addition, we introduce two novel bone formation accelerators, SST-VEDI and SSH-BMI, which are currently under investigation by our research group. On the other hand, regenerative therapy, characterized by (ideally) the use of a patient's own cells to regenerate lost tissue, is now a matter of global interest. At present, candidate cell sources for regenerative therapy include embryonic stem cells (created from embryos based on the fertilization of oocytes), induced pluripotent stem cells (created artificially by using somatic cells as the starting material), and somatic stem cells (found in the tissues of the adult body). This review summarizes the identifying features and the therapeutic potential of each of these stem cell types for bone regenerative medicine. Although a number of different kinds of somatic stem cells have been reported, we turn our attention toward two that are of particular interest for prospective applications in bone repair: the dedifferentiated fat cell, and the deciduous dental pulp-derived stem cell.

    DOI: 10.1007/s12565-013-0208-8

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  • Mesenchymal Dental Stem Cells for Tissue Regeneration 査読

    Masaki J. Honda, Eri Watanabe, Yoshikazu Mikami, Yoko Saito, Taku Toriumi, Tetsuo Shirakawa, Noriyoshi Shimizu, Nobukazu Watanabe, Keitaro Isokawa

    The International Journal of Oral & Maxillofacial Implants   28 ( 6 )   e451 - e460   2013年

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    記述言語:英語   掲載種別:記事・総説・解説・論説等(学術雑誌)   出版者・発行元:Quintessence Publishing  

    DOI: 10.11607/jomi.te25

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  • Advances in Tooth Regeneration Techniques 査読

    Yoshikazu Mikami

    Dentistry   02 ( 03 )   2012年

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    担当区分:筆頭著者, 最終著者, 責任著者   記述言語:英語   掲載種別:記事・総説・解説・論説等(学術雑誌)   出版者・発行元:OMICS Publishing Group  

    DOI: 10.4172/2161-1122.1000e105

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講演・口頭発表等

  • 骨芽細胞は Gap junction を介して間葉系幹細胞分化を制御する

    三上剛和

    第33回日本骨代謝学会学術集会  2015年 

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    記述言語:日本語   会議種別:口頭発表(一般)  

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  • Regulation of osteogenic gene transcription via gap junction-mediated cell-cell communication 国際会議

    Mikami Y, Kitano T, Asano M, Komiyama K

    96th Annual Meeting, Scientific Sessions & Exhibition in conjunction with the Japanese Society and Korean Association of Oral and Maxilllofacial Surgeons  2014年 

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    記述言語:英語   会議種別:ポスター発表  

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  • 熟骨芽細胞MLO-A5はGap junctionを介して未分化間葉系幹細胞C3H10T1/2の分化を制御する

    三上剛和

    第55回歯科基礎医学会学術大会・総会サテライトシンポジウム  2013年 

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    記述言語:日本語   会議種別:シンポジウム・ワークショップ パネル(公募)  

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  • 骨芽細胞分化におけるBMP-2とDexamethasoneの影響

    三上剛和, 内藤昌子, 高橋富久

    第117回日本解剖学会総会・全国学術集会  2012年 

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    記述言語:日本語   会議種別:口頭発表(一般)  

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  • Bone morphogenetic protein 2とDexamethasoneはJAK/STATシグナルを介して相乗的にAlkaline Phosphataseを活性化させる

    三上剛和, 高橋富久

    第53回歯科基礎医学会学術大会・総会  2011年 

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    記述言語:日本語  

    開催地:岐阜  

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  • トリプタミン派生化合物、SST-VEDI-1の骨芽細胞分化に対する影響

    三上剛和, 染井正徳, 高城稔

    第114回日本解剖学会総会・全国学術集会  2009年 

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    記述言語:日本語   会議種別:ポスター発表  

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  • ROS17/2.8細胞における石灰化と骨芽細胞分化関連因子の発現に対するデキサメサゾンの影響

    三上剛和, 表山和樹, 高城稔

    日本解剖学会  2008年 

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    記述言語:日本語   会議種別:口頭発表(一般)  

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  • Functional region of CENP-H interacts with the Nuf2 complex, which functions as a connector between the inner and outer kinetochores

    三上剛和, 堀哲也, 深川竜郎

    日本分子生物学会  2004年 

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    記述言語:日本語   会議種別:口頭発表(一般)  

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  • Functional region of CENP-H interacts with the Nuf2 complex, which functions as a connector between the inner and outer kinetochores

    Yoshikazu Mikami, Tetsuya Hori, Tatsuro Fukagawa

    2004年 

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    記述言語:英語   会議種別:口頭発表(一般)  

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  • セントロメアタンパク質CENP-Hの機能解析

    Yoshikazu Mikami, Ai Nishihashi, Tatsuro Fukagawa

    日本分子生物学会  2003年 

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    記述言語:日本語   会議種別:口頭発表(一般)  

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  • 新規セントロメアタンパク質CENP-Hの機能解析

    三上剛和, 西橋愛, 深川竜郎

    日本分子生物学会  2000年 

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    記述言語:日本語   会議種別:ポスター発表  

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産業財産権

  • アポトーシス抑制剤

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    出願番号:特願2008-212044 

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受賞

  • JBMM論文賞(入賞)

    2019年   日本骨代謝学会   Alkaline phosphatase determines polyphosphate-induced mineralization in a cell-type independent manner

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  • 奨励賞

    2013年   日本解剖学会   骨芽細胞の分化機構の解明と骨再生に関する研究

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    受賞区分:国内学会・会議・シンポジウム等の賞 

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共同研究・競争的資金等の研究

  • 骨芽細胞のⅠ型コラーゲンと基質小胞の分泌経路におけるRabタンパク質の機能解明

    研究課題/領域番号:23K09117

    2023年4月 - 2026年3月

    制度名:科学研究費助成事業

    研究種目:基盤研究(C)

    提供機関:日本学術振興会

    柿原 嘉人, 加来 賢, 三上 剛和

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    配分額:4680000円 ( 直接経費:3600000円 、 間接経費:1080000円 )

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  • ヒト歯肉オルガノイドを用いた歯肉炎症および自己免疫疾患誘導メカニズムの探索

    研究課題/領域番号:20K09913

    2020年4月 - 2023年3月

    制度名:科学研究費助成事業

    研究種目:基盤研究(C)

    提供機関:日本学術振興会

    津田 啓方, 三上 剛和, 鳥海 拓, 篠塚 啓二

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    配分額:4290000円 ( 直接経費:3300000円 、 間接経費:990000円 )

    今年度は患者由来の初代ヒト歯肉上皮細胞および線維芽細胞の入手が難しかったため、本研究において最もベースとなる初代歯肉上皮細胞および線維芽細胞を用いた歯肉三次元培養系を作成することができなかった。そのため、本研究の次のステップに必要なヒト単球性白血病THP-1細胞株に緑色蛍光タンパク質EGFPの安定発現株を作成した。次いで、昨年までに構築していた歯肉株化細胞を用いた三次元培養系を作成し、そこに歯周病原細菌代謝産物である酪酸等を作用させたときに歯肉上皮が示す反応について調べた。酪酸を24時間以降72時間まで作用させ培養上清中に放出された2本鎖DNAをSYTOX-green色素を用いて測定したところ、コントロールと比較して酪酸を作用させた三次元歯肉培養系からは有意に多くのDNAの放出が確認された。この結果は二次元歯肉上皮株化細胞単独における同様の実験とほぼ同じ結果であった。このDNA放出はネクローシス様の細胞死によるものであるので、次にDamage-associated molecular patterns (DAMPs)の放出についても調べたところ、酪酸濃度依存的にDAMPsであるSin3A associated protein 130 (SAP130)やHigh mobility group box 1 (HMGB1)等の放出が確認された。これらの結果から、株化細胞を用いた三次元培養系では酪酸誘導の歯肉上皮細胞の細胞死及びそれに伴うDAMPs放出誘導が起こる事がわかった。

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  • 細胞運動の3次元ダイナミクス解析への新しいアプローチ:SICMと機械学習の融合

    研究課題/領域番号:20K07217

    2020年4月 - 2023年3月

    制度名:科学研究費助成事業

    研究種目:基盤研究(C)

    提供機関:日本学術振興会

    水谷 祐輔, 三上 剛和

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    配分額:4030000円 ( 直接経費:3100000円 、 間接経費:930000円 )

    本研究は,走査型イオンコンダクタンス顕微鏡(SICM)を用いて取得できる生きた細胞の頂上膜面の細胞質突起の画像からダイナミクスを解析し,そのダイナミクスと細胞機能の関係性を解明することを目的としている。そのダイナミクスの解析には,本研究を通して構築する機械学習を用いたコンピュータ支援構造判別評価システムを使用し,困難さを多く含む画像判別を自動化することでより効率的におこなう事を並行して実施するものである。
    取り組んでいる課題を以下に示した。
    1. 画像データセットの収集と機械学習用データを用いたシステムの構築: 申請者は現在までに各種実験を通して撮像したSICM像を所有し,継続的に画像データセットの収集および本研究に学習データとして用いるデータセットを選別しタグ付け作業をおこなった。また,システムの構築は,フリーウエアで構築を進め,テスト画像データ等を処理させるなど適したものの選定をおこない,機械学習の作業環境整備を進め,識別アルゴリズムの作成に着手した。
    2. 各細胞の細胞質突起の構造観察: SICMを用いた画像取得は,探針であるガラスピペットの形状や細胞の状態に依って適した画像取得に関するパラメータは異なる。本研究では,細胞質突起の観察における各種条件を精査することで,現状において適した画像取得パラメータを見出した。
    3. Tet受容体を恒常的に発現する細胞の樹立: 遺伝子導入により細胞質突起構造の遷移状態をコントロール可能なCa9-22細胞の作製を試みた。当初はTetの発現を確認したが,発現していても数日で発現が消失することが明らかとなった。そのため,非メチル化プロモーターに組み換えた発現ベクターを作製し,細胞にトランスフェクションおよびセレクションを行うことでTet受容体恒常発現細胞(TetR-Ca9-22 cells) の樹立に成功した。

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  • タンパク質分解酵素阻害因子(SLPI)によるがん病態の悪性化誘導機構の検討

    研究課題/領域番号:18K07226

    2018年4月 - 2021年3月

    制度名:科学研究費助成事業

    研究種目:基盤研究(C)

    提供機関:日本学術振興会

    三上 剛和, 上原 範久, 早津 学, 水谷 祐輔, 津田 啓方, 福島 敦史

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    配分額:4420000円 ( 直接経費:3400000円 、 間接経費:1020000円 )

    Secretory leukocyte protease inhibitor (SLPI) は,白血球が生産する酵素から,正常な皮膚や粘膜を保護するタンパク質である。その一方で,SLPIを発現するがん細胞は,「悪性がん細胞」であることが示唆されている。そこで,がん細胞の悪性化に対するSLPIの役割と作用機序を解明するために,SLPIを発現するがん細胞に対してSLPI遺伝子を欠損させて、その影響を解析した。その結果,SLPIはゲノムDNAを修飾することによって,細胞接着因子の発現を抑制し,がん細胞の移動能と浸潤能を亢進することを明らかにした。

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  • 神経伝達物質を起点とした癌根治療法の創出

    研究課題/領域番号:17K19748

    2017年6月 - 2019年3月

    制度名:科学研究費助成事業

    研究種目:挑戦的研究(萌芽)

    提供機関:日本学術振興会

    照沼 美穂, 山崎 学, 三上 剛和, 天谷 吉宏, 原田 史子

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    配分額:6370000円 ( 直接経費:4900000円 、 間接経費:1470000円 )

    がん抑制効果が期待されるAMPKと抑制性神経伝達を担うGABAB受容体のクロストークによる新しい抗がん剤の開発を目指すために研究を行った。その結果、口腔がん細胞に発現するGABAB受容体は脳細胞に発現するものとは異なり、その活性化による抗癌作用は確認できなかった。しかし、AMPKの活性化は単独で強力に抗癌作用を発揮した。今後その下流シグナルを標的とした、より特異的な分子の同定が必要である。

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  • 三次元培養系を用いた歯周病由来関節リウマチ予防法の開発

    研究課題/領域番号:17K11686

    2017年4月 - 2020年3月

    制度名:科学研究費助成事業

    研究種目:基盤研究(C)

    提供機関:日本学術振興会

    津田 啓方, 三上 剛和, 好士 亮介

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    配分額:4550000円 ( 直接経費:3500000円 、 間接経費:1050000円 )

    関節リウマチ(RA)と歯周病との関連がいわれている。これまで、歯周病原細菌の産生する酪酸の作用が歯肉上皮の細胞死を引き起こし、それに伴い、炎症促進およびRA発症に重要である因子の放出が認められている。本研究では、その細胞死誘導メカニズムを探る事を目的としている。酪酸、プロピオン酸、イソ酪酸、イソ吉草酸がヒト歯肉細胞の細胞死を誘導し、その誘導にはオートファジー、活性酸素種の産生、およびヒストンアセチル化酵素の活性化が必要であることが解った。また、ヒト歯肉の三次元培養系を作成し、株化細胞を用いた二次元培養系より生体に近いと考えられている実験系を用いて、これまでの結果を確認できる状況作成に成功した。

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  • 骨芽細胞の脱分化・多能性再獲得機構の解明

    2014年4月 - 2017年3月

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    資金種別:競争的資金

    配分額:4940000円

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  • ヤマトヒメミミズ再生幹細胞に発現する遺伝子grimpのタンパク質機能解析

    研究課題/領域番号:25440116

    2013年4月 - 2017年3月

    制度名:科学研究費助成事業

    研究種目:基盤研究(C)

    提供機関:日本学術振興会

    野呂 知加子, 栃内 新, 三上 剛和

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    配分額:5200000円 ( 直接経費:4000000円 、 間接経費:1200000円 )

    本研究は、砕片分離と再生による無性生殖を行うヤマトヒメミミズEnchytraeus japonensisにおいて、再生初期に再生芽先端付近および中胚葉系列幹細胞に発現する新規遺伝子grimpに着目し、そのタンパク質の性質と再生における機能を明らかにすることを目的とする。grimpに対する抗体を作成し、組織蛍光抗体染色により、grimpタンパク質が再生芽およびその周辺の中胚葉系細胞に局在することを示した。また、RNAレベルでは再生6-12時間で発現していたが、タンパク質レベルでは再生24時間でも発現が見られた。ウェスタンブロッティングにより、予想分子量付近に抗体と反応するバンドが認められた。

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  • 新規合成トリプトファン誘導体の効果的骨芽細胞促進条件の同定と作用機構の解明

    2012年12月 - 2014年3月

    制度名:研究成果展開事業 研究成果最適展開支援プログラム(A-STEP)フージビリスタディ・ステージ 探索タイプ

    提供機関:科学技術振興機構(JST)

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    資金種別:競争的資金

    配分額:1690000円

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  • トリプトファン誘導体およびDexを用いた骨芽細胞分化促進法の確立と作用機構の解明

    2010年4月 - 2013年3月

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    資金種別:競争的資金

    配分額:3900000円

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  • 骨芽細胞分化誘導因子の効果的作用条件の同定とその分子機構の解明

    2009年4月 - 2010年3月

    制度名:中冨健康科学振興財団 研究助成金

    提供機関:中冨健康科学振興財団

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    資金種別:競争的資金

    配分額:1000000円

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  • 生後の歯胚細胞による歯周組織を備えた歯(歯根)の再生法

    研究課題/領域番号:21390528

    2009年 - 2011年

    制度名:科学研究費助成事業

    研究種目:基盤研究(B)

    提供機関:日本学術振興会

    本田 雅規, 磯川 桂太郎, 湯口 眞紀, 三上 剛和, 渡辺 信和, 山崎 洋介

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    配分額:18070000円 ( 直接経費:13900000円 、 間接経費:4170000円 )

    3年間の研究期間において,以下の事を明らかとした。初年度においては,ブタ/ヒト歯小嚢細胞,ヒト歯根膜間葉系幹細胞,ヒト歯髄間葉系幹細胞の単離と効率的な培養方法の開発。次年度においては,歯髄細胞から象牙質塊を作製するための適した担体の模索。最終年度はブタの歯胚から採取したエナメル上皮細胞,歯髄細胞および培養歯小嚢細胞による疑似歯胚を作製し,人工的に作製した疑似歯槽陥凹モデルに疑似歯胚を移植することで歯周組織様構造を持つ歯根の再生技術の確立を試みた。

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  • 骨芽細胞分化誘導因子の効果的作用条件の解析

    2008年4月 - 2009年3月

    制度名:総合歯学研究所研究費(一般研究A)

    提供機関:日本大学 歯学部 総合歯学研究所

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    資金種別:競争的資金

    配分額:2000000円

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  • 副甲状腺ホルモン(PTH)の骨芽細胞分化に対する作用機序の解明

    2008年3月 - 2009年4月

    制度名:佐藤研究費

    提供機関:日本大学 歯学部

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    資金種別:競争的資金

    配分額:1000000円

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  • デキサメサゾンの骨芽細胞分化に対する作用機構の解明

    2007年4月 - 2010年3月

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    資金種別:競争的資金

    配分額:3860000円

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▶ 全件表示

 

担当経験のある授業科目

  • 人体の構造と機能Ⅰ(組織学総論)

    2020年
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    現在
    機関名:新潟大学

  • 人体の構造と機能Ⅱ(組織学各論)

    2020年
    -
    現在
    機関名:新潟大学