研究者総覧

小林 直人 (コバヤシ ナオト)

  • 大学院医学系研究科 医学専攻 教授
Last Updated :2020/10/13

研究者情報

学位

  • 博士(医学)(東京大学)

ホームページURL

J-Global ID

プロフィール

  • 愛媛大学学長特別補佐、教育・学生支援機構副機構長。学内および医学部での教育改善(アクティブラーニングなど教員を対象としたミクロ・レベルでのFD、AP/CP/DPの一貫性構築を含むミドル・レベルのFD、等)の実績をもとに、平成21年度より、愛媛大学教育企画室長として、学内外のFD関連の講習会、ワークショップの講師や企画を担当している。

研究キーワード

  • 腎糸球体   細胞生物学   解剖学教育   医学教育   高等教育   Renal glomerulus   Cell culture   Cell biology   Anatomical education   Medical education   Higher Education   

研究分野

  • ライフサイエンス / 解剖学 / 解剖学教育、医学教育

経歴

  • 2015年04月 - 現在  愛媛大学 学長特別補佐(教育企画、能力開発、学生支援)
  • 2009年04月 - 現在  愛媛大学 教育・学生支援機構Institute for Education and Student Support副機構長/教育企画室長
  • 2005年11月 - 現在  愛媛大学 医学部附属総合医学教育センター長/大学院医学系研究科医学専攻医学教育学講座Medical Education Center教授
  • 1998年07月 - 2005年10月  愛媛大学 医学部 解剖学・発生学分野School of Medicine助教授
  • 1995年 - 1998年  順天堂大学 医学部 解剖学第一講座Faculty of Medicine特任講師
  • 1991年 - 1995年  順天堂大学 医学部 解剖学第一講座Faculty of Medicine助手

学歴

  • 1988年04月 - 1991年03月   東京大学大学院   医学系研究科   第一基礎医学(解剖学・細胞生物学) 中退
  • 1982年04月 - 1988年03月   東京大学   医学部   医学科 卒業

所属学協会

  • 日本東洋医学会   日本医学教育学会   日本腎臓学会   日本解剖学会   

研究活動情報

論文

  • 「医学教育分野別評価」受審後の改善に向けた着眼点~受審20大学の比較~
    永井勅久, 小林直人
    愛媛医学 39 3 118 - 122 愛媛医学会 2020年09月 [査読有り]
     研究論文(学術雑誌) 
    (目的)医学教育の国際的質保証のため、世界医学教育連盟(WFME)の示したグローバルスタンダードに基づいた「医学教育分野別評価基準日本版」による評価を日本国内の各大学も受審しつつある。愛媛大学も2018年度に受審し認定を受けたが、実地調査では特に評価基準内の領域1「使命と学修成果」の指摘が多く、公表された評価報告書での全体の指摘事項も多かった。そこで、同時期に受審した各大学での評価報告書の指摘、評価の傾向を分析した。 (方法)2017年~2018年に「医学教育分野別評価基準日本版」Ver2.1~2.2を用いて日本医学教育評価機構(JACME)の評価を受審した20大学を対象とした。JACMEの公表した評価報告書をもとに、分野別評価基準内の領域1~9の「部分的適合」を受けた下位領域の個数、および指摘事項の個数、評価事項の個数を比較対象とした。 (結果)20大学で「部分的適合」を受けた個数は大きな違いはなかったが、指摘事項の個数は特に「基本的領域」において愛媛大学で多い結果であった。また、領域1と領域2~9の基本的領域の指摘事項の個数は高い相関を示した。評価事項に関しては、高く評価された項目は愛媛大学にはなかった。 (結論)領域1は他領域と強く相関する項目であり、領域1の達成度が全体の国際認証評価、医学部の国際基準の達成に深く関与していると考えられ、今後の改善には領域1の項目を重視する必要性が示唆された。また、評価事項に関してはより愛媛大学の良い点のアピールが必要と考えられた。
  • 医療従事に関するキャリアデザインの実態調査 愛媛大学医学部生の調査結果について
    奈須悠樹、向 平和、隅田 学、小林直人、上田敏子
    大学教育実践ジャーナル 18 9 - 20 2020年03月 [査読有り]
     研究論文(学術雑誌)
  • 医学科の進級判定と医師国家試験の合格率は相関するのか
    永井勅久, 小林直人
    愛媛医学 38 4 164 - 168 愛媛医学会 2019年12月 [査読有り]
     研究論文(学術雑誌) 
    (目的)医学科において、医師国家試験の合格率の向上のために進級判定を厳格化し、学修成果が不良な学生はより多く留年させることで、国家試験の合格率が向上する可能性があるが、国家試験の合格率と進級判定の相関を検討したデータはみられない。
    (方法)学生が多く留年し、6年間留年せず卒業した学生の率(=ストレート卒業率)が低ければ国家試験の合格率が向上するとの仮説を立て、公表されている国公立大学の国家試験合格率(新卒者)と、ストレート卒業率を用いて二変量分析を行った。
    (結果)2016年、2018年の結果においてはストレート卒業率と国家試験合格率は相関せず、2017年ではストレート卒業率が高いほど国家試験合格率が有意に高い結果となった。
    (結論)大学6年間の進級判定を厳格化し留年率を上昇させることが、必ずしも国家試験の合格率向上に寄与しないことが強く示唆された。合格率向上のためには臨床実習中の学力評価のための方策を探る必要がある。
  • Farzana Islam, Md Sakirul Islam Khan, Hiroaki Nabeka, Shouichiro Saito, Xuan Li, Tetsuya Shimokawa, Kimiko Yamamiya, Naoto Kobayashi, Seiji Matsuda
    Cell and tissue research 373 2 439 - 457 2018年08月 [査読有り]
     研究論文(学術雑誌) 
    Salivary glands produce various neurotrophins that are thought to regulate salivary function during normal and pathological conditions. Prosaposin (PSAP) is a potent neurotrophin found in several tissues and various biological fluids and may play roles in the regulation of salivary function. However, little is known about PSAP in salivary glands. As the functions of salivary glands are diverse based on age and sex, this study examines whether PSAP and its receptors, G protein-coupled receptor 37 (GPR37) and GPR37L1, are expressed in the salivary glands of rats and whether sex and aging affect their expression. Immunohistochemical analysis revealed that PSAP and its receptors were expressed in the major salivary glands of rats, although their expression varied considerably based on the type of gland, acinar cells, age and sex. In fact, PSAP, GPR37 and GPR37L1 were predominantly expressed in granular convoluted tubule cells of the submandibular gland and the intensity of their immunoreactivity was higher in young adult female rats than age-matched male rats, which was more prominent at older ages (mature adult to menopause). On the other hand, weak PSAP, GPR37 and GPR37L1 immunoreactivity was observed mainly in the basal layer of mucous cells of the sublingual gland. Triple label immunofluorescence analysis revealed that PSAP, GPR37 and GPR37L1 were co-localized in the basal layer of acinar and ductal cells in the major salivary glands. The present findings indicate that PSAP and its receptors, GPR37 and GPR37L1, are expressed in the major salivary glands of rats and their immunoreactivities differ considerably with age and sex.
  • Hiroaki Nabeka, Shouichiro Saito, Xuan Li, Tetsuya Shimokawa, Md Sakirul Islam Khan, Kimiko Yamamiya, Soichiro Kawabe, Takuya Doihara, Fumihiko Hamada, Naoto Kobayashi, Seiji Matsuda
    IBRO reports 3 17 - 32 2017年12月 [査読有り]
     研究論文(学術雑誌) 
    Prosaposin (PS) is a secretory neurotrophic factor, as well as a regulator of lysosomal enzymes. We previously reported the up-regulation of PS and the possibility of its axonal transport by GABAergic interneurons after exocitotoxicity induced by kainic acid (KA), a glutamate analog. In the present study, we performed double immunostaining with PS and three calcium binding protein markers: parvalbumin (PV), calbindin, and calretinin, for the subpopulation of GABAergic interneurons, and clarified that the increased PS around the hippocampal pyramidal neurons after KA injection existed mainly in the axons of PV positive interneurons. Electron microscopy revealed PS containing vesicles in the PV positive axon. Double immunostaining with PS and secretogranin or synapsin suggested that PS is secreted with secretogranin from synapses. Based on the results from in situ hybridization with two alternative splicing forms of PS mRNA, the increase of PS in the interneurons was due to the increase of PS + 0 (mRNA without 9-base insertion) as in the choroid plexus, but not PS + 9 (mRNA with 9-base insertion). These results were similar to those from the choroid plexus, which secretes an intact form PS + 0 to the cerebrospinal fluid. Neurons, especially PV positive GABAergic interneurons, produce and secrete the intact form of PS around hippocampal pyramidal neurons to protect them against KA neurotoxicity.
  • Haruka Watanabe, Takashi Fujiwara, Naoto Kobayashi
    Journal of general and family medicine 18 4 175 - 179 2017年08月 [査読有り]
     
    To resolve the problem that evidence-based medicine (EBM) courses are not sufficiently taught in Japanese medical schools, we organized a year-round EBM-learning course. This study was an observational study and was designed to evaluate the participants' understanding of EBM using an original survey. The survey was given three times. In total, 18 students responded to our survey. Of those 18 students, six students answered both the first and the last surveys, and their mean score increased 1.17 of 4.00 (95% CI: 0.72-1.65). These results suggest our course improved students' ability to read clinical articles.
  • 薬学生・医学生を対象とした「バイタルサインからの臨床診断BPVS (Basic Physiology of Vital Signs) シミュレーショントレーニング」による教育効果
    秋山伸二, 山脇孝, 入江聰五郎, 高取真吾, 喜陽宗司, 伊波朋香, 難波弘行, 高田清式, 小林直人, 松岡一郎, 酒井郁也
    薬学教育 1 1 1 - 8 2017年 [査読有り]
  • 医学部との連携による薬学生4年次のバイタルサイン・フィジカルアセスメント実習
    秋山伸二, 山口巧, 山脇孝, 田中亮裕, 田中守, 難波弘行, 荒木博陽, 高田清式, 小林直人, 酒井郁也
    日本シミュレーション医療教育学会雑誌 5 1 40 - 48 2017年 [査読有り]
  • 川邊哲也, 羽野卓三, 相馬仁, 鈴木敬一郎, 赤池雅史, 小林直人, 大槻眞嗣, 鈴木利哉, 奈良信雄
    医学教育 47 2 77 - 88 2016年04月 [査読有り][招待有り]
  • Hiroaki Nabeka, Tetsuya Shimokawa, Takuya Doihara, Shouichiro Saito, Hiroyuki Wakisaka, Fumihiko Hamada, Naoto Kobayashi, Seiji Matsuda
    PLOS ONE 10 5 e0126856  2015年05月 [査読有り]
     研究論文(学術雑誌) 
    Four sphingolipid activator proteins (i.e., saposins A-D) are synthesized from a single precursor protein, prosaposin (PS), which exerts exogenous neurotrophic effects in vivo and in vitro. Kainic acid (KA) injection in rodents is a good model in which to study neurotrophic factor elevation; PS and its mRNA are increased in neurons and the choroid plexus in this animal model. An 18-mer peptide (LSELIINNATEELLIKGL; PS18) derived from the PS neurotrophic region prevents neuronal damage after ischemia, and PS18 is a potent candidate molecule for use in alleviating ischemia-induced learning disabilities and neuronal loss. KA is a glutamate analog that stimulates excitatory neurotransmitter release and induces ischemia-like neuronal degeneration; it has been used to define mechanisms involved in neurodegeneration and neuroprotection. In the present study, we demonstrate that a subcutaneous injection of 0.2 and 2.0 mg/kg PS18 significantly improved behavioral deficits of Wistar rats (n = 6 per group), and enhanced the survival of hippocampal and cortical neurons against neurotoxicity induced by 12 mg/kg KA compared with control animals. PS18 significantly protected hippocampal synapses against KA-induced destruction. To evaluate the extent of PS18- and KA-induced effects in these hippocampal regions, we performed histological evaluations using semithin sections stained with toluidine blue, as well as ordinal sections stained with hematoxylin and eosin. We revealed a distinctive feature of KA-induced brain injury, which reportedly mimics ischemia, but affects a much wider area than ischemia-induced injury: KA induced neuronal degeneration not only in the CA1 region, where neurons degenerate following ischemia, but also in the CA2, CA3, and CA4 hippocampal regions.
  • Ken Sakushima, Hiroki Mishina, Shunichi Fukuhara, Kenei Sada, Junji Koizumi, Takashi Sugioka, Naoto Kobayashi, Masaharu Nishimura, Junichiro Mori, Hirofumi Makino, Mitchell D. Feldman
    BMC MEDICAL EDUCATION 15 54  2015年03月 [査読有り]
     研究論文(学術雑誌) 
    Background: Physician-scientists play key roles in biomedical research across the globe, yet prior studies have found that it is increasingly difficult to recruit and retain physician-scientists in research careers. Access to quality research mentorship may help to ameliorate this problem in the U.S., but there is virtually no information on mentoring in academic medicine in Japan. We conducted a survey to determine the availability and quality of mentoring relationships for trainee physician-scientists in Japan. Methods: We surveyed 1700 physician-scientists in post-graduate research training programs in 6 academic medical centers in Japan about mentorship characteristics, mentee perceptions of the mentoring relationship, and attitudes about career development. Results: A total of 683 potential physician-scientist mentees completed the survey. Most reported that they had a departmental mentor (91%) with whom they met at least once a month; 48% reported that they were very satisfied with the mentoring available to them. Mentoring pairs were usually initiated by the mentor (85% of the time); respondents identified translational research skills (55%) and grant writing (50%) as unmet needs. Mentoring concerning long-term career planning was significantly associated with the intention to pursue research careers, however this was also identified by some mentees as an unmet need (35% desired assistance; 15% reported receiving it). Conclusions: More emphasis and formal training in career mentorship may help to support Japanese physician-scientist mentees to develop a sense of self-efficacy to pursue and stay in research careers.
  • Nabeka Hiroaki, Shimokawa Tetsuya, Doihara Takuya, Saito Shouichiro, Wakisaka Hiroyuki, Hamada Fumihiko, Kobayashi Naoto, Matsuda Seiji
    PloS one 10 5 e0126856  2015年 [査読有り]
     研究論文(学術雑誌) 
    Four sphingolipid activator proteins (i.e., saposins A-D) are synthesized from a single precursor protein, prosaposin (PS), which exerts exogenous neurotrophic effects in vivo and in vitro. Kainic acid (KA) injection in rodents is agood model in which to study neurotrophic factor elevation; PS and its mRNA are increased in neurons and the choroid plexus in this animal model. An 18-mer peptide (LSELIINNATEELLIKGL; PS18) derived from the PS neurotrophic region prevents neuronal damage after ischemia, and PS18 is a potent candidate molecule for use in alleviating ischemia-induced learning disabilities and neuronal loss. KA is a glutamate analog that stimulates excitatory neurotransmitter release and induces ischemia-like neuronal degeneration; it has been used to define mechanisms involved in neurodegeneration and neuroprotection. In the present study, we demonstrate that a subcutaneous injection of 0.2 and 2.0 mg/kg PS18 significantly improved behavioral deficits of Wistar rats (n = 6 per group), and enhanced the survival of hippocampal and cortical neurons against neurotoxicity induced by 12 mg/kg KA compared with control animals. PS18 significantly protected hippocampal synapses ag
  • Hiroaki Nabeka, Keigo Uematsu, Hiroko Takechi, Tetsuya Shimokawa, Kimiko Yamamiya, Cheng Li, Takuya Doihara, Shouichiro Saito, Naoto Kobayashi, Seiji Matsuda
    PLOS ONE 9 12 e110534  2014年12月 [査読有り]
     研究論文(学術雑誌) 
    Because excessive glutamate release is believed to play a pivotal role in numerous neuropathological disorders, such as ischemia or seizure, we aimed to investigate whether intrinsic prosaposin (PS), a neuroprotective factor when supplied exogenously in vivo or in vitro, is up-regulated after the excitotoxicity induced by kainic acid (KA), a glutamate analog. In the present study, PS immunoreactivity and its mRNA expression in the hippocampal and cortical neurons showed significant increases on day 3 after KA injection, and high PS levels were maintained even after 3 weeks. The increase in PS, but not saposins, detected by immunoblot analysis suggests that the increase in PS-like immunoreactivity after KA injection was not due to an increase in saposins as lysosomal enzymes after neuronal damage, but rather to an increase in PS as a neurotrophic factor to improve neuronal survival. Furthermore, several neurons with slender nuclei inside/outside of the pyramidal layer showed more intense PS mRNA expression than other pyramidal neurons. Based on the results from double immunostaining using anti-PS and anti-GABA antibodies, these neurons were shown to be GABAergic interneurons in the extraand intra-pyramidal layers. In the cerebral cortex, several large neurons in the V layer showed very intense PS mRNA expression 3 days after KA injection. The choroid plexus showed intense PS mRNA expression even in the normal rat, and the intensity increased significantly after KA injection. The present study indicates that inhibitory interneurons as well as stimulated hippocampal pyramidal and cortical neurons synthesize PS for neuronal survival, and the choroid plexus is highly activated to synthesize PS, which may prevent neurons from excitotoxic neuronal damage. To the best of our knowledge, this is the first study that demonstrates axonal transport and increased production of neurotrophic factor PS after KA injection.
  • 秋山 伸二, 酒井 郁也, 山脇 孝, 山口 巧, 高取 真吾, 田中 亮裕, 荒木 博陽, 高田 清式, 小林 直人, 難波 弘行
    社会薬学 33 Suppl. 46 - 46 日本社会薬学会 2014年09月
  • Midori Morishita, Hiroaki Nabeka, Tetsuya Shimokawa, Kyojy Miyawaki, Takuya Doihara, Shouichiro Saito, Naoto Kobayashi, Seiji Matsuda
    PLOS ONE 9 5 e95883  2014年05月 [査読有り]
     研究論文(学術雑誌) 
    Neurogenesis in the hippocampal dentate gyrus occurs constitutively throughout postnatal life. Adult neurogenesis includes a multistep process that ends with the formation of a postmitotic and functionally integrated new neuron. During adult neurogenesis, various markers are expressed, including GFAP, nestin, Pax6, polysialic acid-neural cell adhesion molecule (PSA-NCAM), neuronal nuclei (NeuN), doublecortin, TUC-4, Tuj-1, and calretinin. Prosaposin is the precursor of saposins A-D; it is found in various organs and can be excreted. Strong prosaposin expression has been demonstrated in the developing brain including the hippocampus, and its neurotrophic activity has been proposed. This study investigated changes in prosaposin in the dentate gyrus of young and adult rats using double immunohistochemistry with antibodies to prosaposin, PSA-NCAM, and NeuN. Prosaposin immunoreactivity was intense in the dentate gyrus at postnatal day 3 (P3) and P7, but decreased gradually after P14. In the dentate gyrus at P28, immature PSA-NCAM-positive neurons localized exclusively in the subgranular zone were prosaposin-negative, whereas mature Neu-N-positive neurons were positive for prosaposin. Furthermore, these prosaposin-negative immature neurons were saposin B-positive, suggesting that the neurons take up and degrade prosaposin. In situ hybridization assays showed that prosaposin in the adult dentate gyrus is dominantly the Pro+9 type, a secreted type of prosaposin. These results imply that prosaposin secreted from mature neurons stimulates proliferation and maturation of immature neurons in the dentate gyrus.
  • Saito Shouichiro, Saito Kyoko, Nabeka Hiroaki, Shimokawa Tetsuya, Kobayashi Naoto, Matsuda Seiji
    Cell and tissue research 356 1 231 - 242 2014年04月 [査読有り]
     研究論文(学術雑誌) 
    Prosaposin has two distinct profiles. One is a precursor form that is processed into saposins thuspromoting lysosomal sphingolipid hydrolase function, whereas the other is an intact form thatis not processed into saposins but is abundant in certain tissues and secretory fluids, including the cerebrospinal fluid. In rats, alternative splicing in the prosaposin gene generates mRNAs with and without a 9-base insertion (Pro+9 and Pro+0 mRNAs, respectively). Pro+9 mRNA is reported to be preferentially expressed in tissues in which the intact form of prosaposin dominates, whereas Pro+0 mRNA is preferentially expressed in tissues in which the precursor dominates. The expression patterns of Pro+9 and Pro+0 mRNAs in the rat choroid plexus are examined in the present study. The specificities of 36-meroligonucleotide probes used to detect the 9-base insertionby in situ hybridization were demonstrated by dot-blot hybridization. Next, these probes were used for in situ hybridization, which showed predominant expressionof Pro+0 mRNA and weak expression of Pro+9 mRNA in the choroid plexus. These expression patterns were confirmed by reverse transcription plus the polymerase chain reaction with Al
  • Shouichiro Saito, Kyoko Saito, Hiroaki Nabeka, Tetsuya Shimokawa, Naoto Kobayashi, Seiji Matsuda
    CELL AND TISSUE RESEARCH 356 1 231 - 242 2014年04月 [査読有り]
     研究論文(学術雑誌) 
    Prosaposin has two distinct profiles. One is a precursor form that is processed into saposins thus promoting lysosomal sphingolipid hydrolase function, whereas the other is an intact form that is not processed into saposins but is abundant in certain tissues and secretory fluids, including the cerebrospinal fluid. In rats, alternative splicing in the prosaposin gene generates mRNAs with and without a 9-base insertion (Pro+9 and Pro+0 mRNAs, respectively). Pro+9 mRNA is reported to be preferentially expressed in tissues in which the intact form of prosaposin dominates, whereas Pro+0 mRNA is preferentially expressed in tissues in which the precursor dominates. The expression patterns of Pro+9 and Pro+0 mRNAs in the rat choroid plexus are examined in the present study. The specificities of 36-mer oligonucleotide probes used to detect the 9-base insertion by in situ hybridization were demonstrated by dot-blot hybridization. Next, these probes were used for in situ hybridization, which showed predominant expression of Pro+0 mRNA and weak expression of Pro+9 mRNA in the choroid plexus. These expression patterns were confirmed by reverse transcription plus the polymerase chain reaction with AlwI restriction enzyme treatment. Expression of the intact form of prosaposin in the choroid plexus was assessed by Western blotting and immunohistochemistry. Because the choroid plexus is responsible for the generation of cerebrospinal fluid containing the intact form of prosaposin, the present study raises the possibility that Pro+0 mRNA is related to the intact form in the choroid plexus and that the alternatively spliced forms of mRNAs do not simply correspond to the precursor and intact forms of prosaposin.
  • 医学研究科大学院の若手研究者がメンターに望む指導の男女差
    三品浩基, 佐久嶋研, 佐田憲映, 小泉順二, 杉岡隆, 小林直人, 西村正治, 森淳一郎, 槇野博史, Mitchell D Feldman, 福原俊一
    医学教育 45 1 1 - 7 2014年02月 [査読有り]
  • Hiroaki Asai, Hiroshi Fujiwara, Sohei Kitazawa, Naoto Kobayashi, Toshiki Ochi, Yukihiro Miyazaki, Fumihiro Ochi, Yoshiki Akatsuka, Sachiko Okamoto, Junichi Mineno, Kiyotaka Kuzushima, Hiroaki Ikeda, Hiroshi Shiku, Masaki Yasukawa
    JOURNAL OF HEMATOLOGY & ONCOLOGY 7 3  2014年01月 [査読有り]
     研究論文(学術雑誌) 
    Because WT1 is expressed in leukemia cells, the development of cancer immunotherapy targeting WT1 has been an attractive translational research topic. However, concern of this therapy still remains, since WT1 is abundantly expressed in renal glomerular podocytes. In the present study, we clearly showed that WT1-specific cytotoxic T lymphocytes (CTLs) certainly exerted cytotoxicity against podocytes in vitro; however, they did not damage podocytes in vivo. This might be due to the anatomical localization of podocytes, being structurally separated from circulating CTLs in glomerular capillaries by an exceptionally thick basement membrane.
  • Hui-ling Gao, Cheng Li, Hiroaki Nabeka, Tetsuya Shimokawa, Naoto Kobayashi, Shouichiro Saito, Zhan-You Wang, Ya-ming Cao, Seiji Matsuda
    PLOS ONE 8 11 e80032  2013年11月 [査読有り]
     研究論文(学術雑誌) 
    Background: Duchenne muscular dystrophy caused by a mutation in the X-linked dystrophin gene induces metabolic and structural disorders in the brain. A lack of dystrophin in brain structures is involved in impaired cognitive function. Prosaposin (PS), a neurotrophic factor, is abundant in the choroid plexus and various brain regions. We investigated whether PS serves as a link between dystrophin loss and gross and/or ultrastructural brain abnormalities. Methodology/Principal Findings: The distribution of PS in the brains of juvenile and adult mdx mice was investigated by immunochemistry, Western blotting, and in situ hybridization. Immunochemistry revealed lower levels of PS in the cytoplasm of neurons of the cerebral cortex, hippocampus, cerebellum, and choroid plexus in mdx mice. Western blotting confirmed that PS levels were lower in these brain regions in both juveniles and adults. Even with low PS production in the choroids plexus, there was no significant PS decrease in cerebrospinal fluid (CSF). In situ hybridization revealed that the primary form of PS mRNA in both normal and mdx mice was Pro+ 9, a secretory-type PS, and the hybridization signals for Pro+ 9 in the above-mentioned brain regions were weaker in mdx mice than in normal mice. We also investigated mitogen-activated protein kinase signalling. Stronger activation of ERK1/2 was observed in mdx mice, ERK1/2 activity was positively correlated with PS activity, and exogenous PS18 stimulated both p-ERK1/2 and PS in SH-SY5Y cells. Conclusions/Significance: Low levels of PS and its receptors suggest the participation of PS in some pathological changes in the brains of mdx mice.
  • Cheng Li, Hui-ling Gao, Tetsuya Shimokawa, Hiroaki Nabeka, Fumihiko Hamada, Hiroaki Araki, Ya-ming Cao, Naoto Kobayashi, Seiji Matsuda
    HISTOLOGY AND HISTOPATHOLOGY 28 7 875 - 892 2013年07月 [査読有り]
     研究論文(学術雑誌) 
    The trophic factor prosaposin (PS) is strongly expressed in skeletal muscle, and reportedly, a PS-derived peptide attenuates loss of muscle mass after nerve injury in vivo and increases myoblast fusion into myotubes in vitro. However, few studies have focused on the role of PS during muscle regeneration. We examined the expression of PS in the skeletal muscles in normal, mdx, and cardiotoxin (CTX)-treated mice using immunofluorescence staining, Western blotting, and in situ hybridisation. Immunofluorescence showed intense PS immunoreactivity in the peripheral cytoplasm of uninjured myofibres of normal mice and regenerated myofibres of 8 weeks post-CTX-injection mice. In early stage CTX-treated mice (14 days and earlier), intense PS immunoreactivity was also detected in the immune cells that infiltrated damaged muscle, but it was weak for regenerating myofibres. Western blot confirmed these findings. In contrast, PS was continuously low in mdx mice in both immunofluorescence and Western blotting. In situ hybridisation confirmed the decrease of PS mRNA in regenerated myofibres and revealed the main form of PS mRNA as Pro+0 without a 9-base insertion both in normal and mdx mice. The embryonic myosin (MYH3) was clearly localized in the newly regenerated myofibres at 3, 7, and 14 days of post-CTX-injection and mdx mice, but was lower in the late stage of regenerated myofibres (28 and 56 days post-CTX injection). The inverse distribution of MYH3 and PS indicates that the PS expression is closely related to the differentiation of regenerated myofibres. Investigation of the mitogen-activated protein (MAP) kinase signal pathway showed the inversely synchronous correlation of phosphorylated ERK1/2 with myofibre PS and the synchronous correlation of phosphorylated p-38 with myofibre PS. These data suggest that PS is involved in the regulation of muscle differentiation of regenerated fibres.
  • Tetsuya Shimokawa, Hiroaki Nabeka, Kimiko Yamamiya, Hiroyuki Wakisaka, Takashi Takeuchi, Naoto Kobayashi, Seiji Matsuda
    Cell and Tissue Research 352 3 685 - 693 2013年06月 [査読有り]
     研究論文(学術雑誌) 
    Prosaposin (PSAP) is as a trophic factor and an activator protein for sphingolipid hydrolase in lysosomes. We generated a specific antibody to PSAP and examined the spatiotemporal distribution of PSAP-immunoreactive (PSAP-IR) cells in the lymphatic tissues of Wistar rats. Immunoblots of tissue homogenates separated electrophoretically showed a single band for PSAP in brain but two bands in spleen. PSAP-IR cells were distributed in both the red and white pulp of the spleen, in both the cortex and medulla of the thymus and in mesenteric lymph nodes. Many PSAP-IR cells were found in the dome portion of Peyer's patches and the number of PSAP-IR cells increased with the age of the rat. To identify the PSAP-IR cells, double- and triple-immunostainings were performed with antibodies against PSAP, CD68 and CD1d. The large number of double- and triple-positive cells suggested that antigen-presenting cells contained much PSAP in these lymphatic tissues. Intense expression of PSAP mRNA, examined by in situ hybridisation, was observed in the red pulp and corona of the spleen. In rats, the PSAP gene generates two alternative splicing forms of mRNA: Pro+9 containing a 9-base insertion and Pro+0 without the insertion. We examined the expression patterns of the alternative splicing forms of PSAP mRNA in the spleen. The presence of both types of mRNA (Pro+9 and Pro+0) indicated that the spleen contains various types of prosaposin-producing and/or secreting cells. These findings suggest diverse functions for PSAP in the immune system. © 2013 Springer-Verlag Berlin Heidelberg.
  • Terashita Takehiro, Saito Shouichiro, Nabeka Hiroaki, Hato Naohito, Wakisaka Hiroyuki, Shimokawa Tetsuya, Kobayashi Naoto, Gyo Kiyofumi, Matsuda Seiji
    Acta oto-laryngologica 133 5 462 - 468 2013年05月 [査読有り]
     研究論文(学術雑誌) 
    An 18-mer peptide derived from the neurotrophic region of prosaposin (PS-pep) preventshearing loss and cochlear damage due to transient cochlear ischaemia by activating an anti-apoptotic pathway. PS-pep is a potent candidate molecule for alleviating ischaemia-induced hearing loss.PS-pep was investigated for its protective effects against ischaemia-induced hearing loss and cochlear damage.Ischaemia was induced in both cochleae in Mongolian gerbils by pulling the ligatures around both vertebral arteries inan anterior direction using 5 g weights for 15 min. PS-pep was synthesized artificially and administered subcutaneously four times after the induction of transient cochlear ischaemia.An increase in the auditory brainstem response threshold was alleviated in animals treated with 2.0 mg/kg PS-pep. Histological examinations conducted on day 7 showed that the loss of inner hair cells (IHCs) was more prominent than that of outer hair cells. Higher doses of PS-pep significantly alleviated IHC loss. An increase in the anti-apoptotic factorbcl-2 was also noted in the IHCs treated with PS-pep.
  • Takehiro Terashita, Shouichiro Saito, Hiroaki Nabeka, Naohito Hato, Hiroyuki Wakisaka, Tetsuya Shimokawa, Naoto Kobayashi, Kiyofumi Gyo, Seiji Matsuda
    ACTA OTO-LARYNGOLOGICA 133 5 462 - 468 2013年05月 [査読有り]
     研究論文(学術雑誌) 
    Conclusion: An 18-mer peptide derived from the neurotrophic region of prosaposin (PS-pep) prevents hearing loss and cochlear damage due to transient cochlear ischaemia by activating an anti-apoptotic pathway. PS-pep is a potent candidate molecule for alleviating ischaemia-induced hearing loss. Objective: PS-pep was investigated for its protective effects against ischaemia-induced hearing loss and cochlear damage. Methods: Ischaemia was induced in both cochleae in Mongolian gerbils by pulling the ligatures around both vertebral arteries in an anterior direction using 5 g weights for 15 min. PS-pep was synthesized artificially and administered subcutaneously four times after the induction of transient cochlear ischaemia. Results: An increase in the auditory brainstem response threshold was alleviated in animals treated with 2.0 mg/kg PS-pep. Histological examinations conducted on day 7 showed that the loss of inner hair cells (IHCs) was more prominent than that of outer hair cells. Higher doses of PS-pep significantly alleviated IHC loss. An increase in the anti-apoptotic factor bcl-2 was also noted in the IHCs treated with PS-pep.
  • Gao Hui-Ling, Li Cheng, Nabeka Hiroaki, Shimokawa Tetsuya, Kobayashi Naoto, Saito Shouichiro, Wang Zhan-You, Cao Ya-Ming, Matsuda Seiji
    PloS one 8 11 e80032  2013年 [査読有り]
     研究論文(学術雑誌) 
    Duchenne muscular dystrophy caused by a mutation in the X-linked dystrophin gene induces metabolic and structural disorders in the brain. A lack of dystrophin in brain structures is involved in impaired cognitive function. Prosaposin (PS), a neurotrophic factor, is abundant in the choroid plexus and various brain regions. We investigated whether PS servesas a link between dystrophin loss and gross and/or ultrastructural brain abnormalities.The distribution of PS in the brains of juvenile and adult mdx mice was investigated by immunochemistry, Western blotting, and in situ hybridization. Immunochemistry revealed lower levels of PS in the cytoplasm of neurons of the cerebral cortex, hippocampus,cerebellum, and choroid plexus in mdx mice. Western blotting confirmed that PS levels were lower in these brain regions in both juveniles and adults. Even with low PS production in the choroids plexus, there was no significant PS decrease in cerebrospinal fluid (CSF). In situ hybridization revealed that the primary form of PS mRNA in both normal and mdx mice was Pro+9, a secretory-type PS, and the hybridization signals for Pro+9 in the above-mentioned brain regions were weaker in mdx mice than
  • 愛媛大学における食育実践プログラムの概要と実践
    垣原登志子, 上田博史, 藤原正幸, 小林直人, 中村慶子, 佐伯修一
    大学教育実践ジャーナル 10 81 - 87 2012年03月 [査読有り]
  • 大規模科目「こころと健康」の科目設計と標準化
    庭崎隆, 野本ひさ, 佐伯修一, 岡田克俊, 橋本巌, 山本万喜雄, 糸岡夕里, 小林直人, 上田博史, 垣原登志子
    大学教育実践ジャーナル 10 69 - 75 2012年03月 [査読有り]
  • Tetsuya Shimokawa, Takuya Doihara, Manami Makara, Kyoji Miyawaki, Hiroaki Nabeka, Hiroyuki Wakisaka, Naoto Kobayashi, Seiji Matsuda
    JOURNAL OF VETERINARY MEDICAL SCIENCE 74 2 155 - 160 2012年02月 [査読有り]
     研究論文(学術雑誌) 
    The stomach of the Pacific white-sided dolphin is divided into three parts: forestomach, proper gastric gland portion, and pyloric chamber. The histological features of the dolphin stomach are similar to those of terrestrial mammal stomachs, although the distribution of glycoconjugates in mucosal cells of the dolphin stomach is unknown. To learn about glycoconjugates in cetacean gastric mucosa, the glycoconjugate distribution in the mucous epithelium of the Pacific white-sided dolphin was studied using 21 lectins. Among the lectins tested, GSL-I and DBA specifically labelled the superficial layer of the forestomach epithelium. GSL-I, SBA, RCA-I, VVA, GSL-I I, DSL, LEL, STL, s-WGA, WGA. PNA, and Jacalin labelled the luminal surface of the chief cells in the proper gastric gland. GSL-I, SBA, RCA-I, DSL, LEL, STL, s-WGA, PNA, and LCA labelled tubular structures in the cytoplasm of parietal cells. The surface portion of the pits in the pyloric chamber strongly reacted with RCA-I, GSL-II, WGA, PNA, LCA, PHA-L, and UEA-I, whereas the neck portion reacted weakly. Although lining one tubular portion, individual secretory cells in the pyloric gland displayed a heterogeneous reaction. This is the first report on the lectin histochemistry of a cetacean stomach and reveals GSL-I and DBA as specific marker lectins for the cornified stratified squamous epithelium cells of the Pacific white-sided dolphin. The stomachs of cetaceans and terrestrial mammals have similar histological features and mucous glycoconjugate content.
  • 山田貴代, 宮崎龍彦, 寺田美穂, 小林直人, 松田正司
    形態・機能 11 1 17 - 23 2012年 [査読有り]
  • 鳥類神経系を用いた発生学研究
    松田正司, 鍋加浩明, 王敏, 下川哲哉, 土居原 拓也, 山宮公子, 脇坂浩之, 小林直人
    愛媛医学 31 1 - 5 2012年 [査読有り]
     研究論文(学術雑誌)
  • Xue Bing, Chen Jie, Gao Huiling, Saito Shouichiro, Kobayashi Naoto, Shimokawa Tetsuya, Nabeka Hiroaki, Sano Akira, Matsuda Seiji
    Neuroscience research 71 1 22  2011年09月 [査読有り]
     研究論文(学術雑誌) 
    Prosaposin is the precursor protein of four glycoproteins, saposins A, B, C, and D, which activate sphingolipid hydrolases in lysosomes. Besides this role, intact prosaposin is also known as a potent neurotrophic factor that prevents neuronalcell death and stimulates neurite outgrowth in in vivo and in vitro experiments. In the present study, we examined chronological changes in prosaposin immunoreactivity in the rat brain using immunofluorescence staining and Diaminobenzidine (DAB) immunohistochemistry. In the hippocampal regions CA1, CA3, and dentate gyrus, the strongest staining of prosaposin was observed on postnatal day 1. The prosaposin immunoreactivity then decreased gradually until postnatal day 28. But in the cerebral cortex, prosaposin staining intensity increased from postnatal day 1 to 14, then decreased until postnatal day 28. The prosaposin immunoreactivity co-localized with the lysosomal granules labeled by an anti-Cathepsin D antibody, indicating that prosaposin mainly localized inthe lysosomes of theneurons. We also examined thechronological changes in prosaposin mRNA and its two alternatively spliced variants using in situ hybridization. We found that both the mRN
  • Tetsuya Shimokawa, Takuya Doihara, Manami Makara, Kyojy Miyawaki, Hiroaki Nabeka, Hiroyuki Wakisaka, Naoto Kobayashi, Seiji Matsuda
    JOURNAL OF VETERINARY MEDICAL SCIENCE 73 9 1233 - 1236 2011年09月 [査読有り]
     研究論文(学術雑誌) 
    Sugars in the glycocalyx play an important role in the attachment of infectious agents to the respiratory mucosa. We examined the histochemistry of 23 lectins to survey the sugar expression in the glycocalyx of the respiratory mucosa of the Pacific white-sided dolphin. Lagenorhynchus obliquidens. The ciliated and basal cells were positive for all of the lectins studied. SBA, WFA, GSL-II, STL, S-WGA, and PNA staining in the cytoplasm showed different intensities between basal cells and ciliated cells. These results suggest that multiple terminal glycosylation occurs on ciliated and basal cells, such as GaINAc, GIcNAc, NeuNAc, galactose, glucose/mannose, olieosaccharide, and fucose, and that sugar residue expression changes during cell differentiation. The Pacific white-sided dolphin respiratory mucosa might express multiple sugar residues in the glycocalyx, to prevent the attachment and colonisation of infectious agents.
  • Ryusuke Tsujimura, Katsumi Mominoki, Masae Kinutani, Tetsuya Shimokawa, Takuya Doihara, Hiroaki Nabeka, Hiroyuki Wakisaka, Naoto Kobayashi, Seiji Matsuda
    NEUROSCIENCE RESEARCH 71 1 85 - 91 2011年09月 [査読有り]
     研究論文(学術雑誌) 
    Spina bifida aperta (SBA) is an open neural tube defect that occurs during the embryonic period. We created SBA chicks by incising the roof plate of the neural tube in the embryo. The area of the dorsal funiculus was smaller in the SBA chicks than in the normal controls. Additionally, the SBA group had fewer nerve fibres in the dorsal funiculus than the normal controls. The pathway of the ascending sensory nerves was revealed by tracing the degenerated nerve fibres using osmification. We cut the sciatic nerve (L5) of the control and SBA chicks at the central end of the dorsal root ganglion 1 day after hatching and fixed the tissue 3 days later. Degenerated sensory nerve fibres were observed in the ipsilateral dorsal funiculus in the control chicks. In contrast, degenerated sensory nerve fibres were observed in the ipsilateral and contralateral dorsal, ventral and lateral funiculi of the spinal cord in the SBA chicks. Consequently, fewer sensory nerve fibres ascended to the thoracic dorsal funiculus in the SBA chicks than in the normal controls. This is the first report of abnormal changes in the ascending sensory nerve fibres in SBA. (C) 2011 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
  • Bing Xue, Jie Chen, Huiling Gao, Shouichiro Saito, Naoto Kobayashi, Tetsuya Shimokawa, Hiroaki Nabeka, Akira Sano, Seiji Matsuda
    Neuroscience Research 71 1 22 - 34 2011年09月 [査読有り]
     研究論文(学術雑誌) 
    Prosaposin is the precursor protein of four glycoproteins, saposins A, B, C, and D, which activate sphingolipid hydrolases in lysosomes. Besides this role, intact prosaposin is also known as a potent neurotrophic factor that prevents neuronal cell death and stimulates neurite outgrowth in in vivo and in vitro experiments. In the present study, we examined chronological changes in prosaposin immunoreactivity in the rat brain using immunofluorescence staining and Diaminobenzidine (DAB) immunohistochemistry. In the hippocampal regions CA1, CA3, and dentate gyrus, the strongest staining of prosaposin was observed on postnatal day 1. The prosaposin immunoreactivity then decreased gradually until postnatal day 28. But in the cerebral cortex, prosaposin staining intensity increased from postnatal day 1 to 14, then decreased until postnatal day 28. The prosaposin immunoreactivity co-localized with the lysosomal granules labeled by an anti-Cathepsin D antibody, indicating that prosaposin mainly localized in the lysosomes of the neurons. We also examined the chronological changes in prosaposin mRNA and its two alternatively spliced variants using in situ hybridization. We found that both the mRNA forms, especially the one without a nine-base insertion, increased significantly from embryonic day 15 to postnatal day 7, then decreased gradually until postnatal day 28. Abundant prosaposin expression in the perinatal stages indicates a potential role of prosaposin in the early development of the rat brain. © 2011 Elsevier Ireland Ltd and the Japan Neuroscience Society.
  • Xuejiao Zhang, Cheng Li, Huiling Gao, Hiroaki Nabeka, Tetsuya Shimokawa, Hiroyuki Wakisaka, Seiji Matsuda, Naoto Kobayashi
    CELLULAR & MOLECULAR BIOLOGY LETTERS 16 2 279 - 295 2011年06月 [査読有り]
     研究論文(学術雑誌) 
    We investigated the effects of Rho-associated kinase (ROCK) on migration and cytoskeletal organization in primary human osteoblasts and Saos-2 human osteosarcoma cells. Both cell types were exposed to two different ROCK inhibitors, Y-27632 and HA-1077. In the improved motility assay used in the present study, Y-27632 and HA-1077 significantly increased the migration of both osteoblasts and osteosarcoma cells on plastic in a dose-dependent and reversible manner. Fluorescent images showed that cells of both types cultured with Y-27632 or HA-1077 exhibited a stellate appearance, with poor assembly of stress fibers and focal contacts. Western blotting showed that ROCK inhibitors reduced myosin light chain (MLC) phosphorylation within 5 min without affecting overall myosin light-chain protein levels. Inhibition of ROCK activity is thought to enhance the migration of human osteoblasts through reorganization of the actin cytoskeleton and regulation of myosin activity. ROCK inhibitors may be potentially useful as anabolic agents to enhance the biocompatibility of bone and joint prostheses.
  • Min Wang, Katsumi Mominoki, Masae Kinutani, Zhong Wang, Naoto Kobayashi, Tetsuya Shimokawa, Hiroaki Nabeka, Takashi Fujiwara, Seiji Matsuda
    JOURNAL OF VETERINARY MEDICAL SCIENCE 73 4 447 - 452 2011年04月 [査読有り]
     研究論文(学術雑誌) 
    Spina bifida aperta (SBA) is a congenital malformation of the spinal cord with complications such as spinal ataxia and bowel and bladder dysfunction. We have developed a chick model with surgery-induced SBA that shows spinal ataxia after hatching. In the present study, motor neurons in the early stages in chicks with and without SBA were observed by immunohistochemical staining with a monoclonal antibody against Islet-1, a motor neuron marker. Delay in migration and maturation of motor neurons was observed in SBA. Although the final numbers of Islet-1-positive neurons in these two groups were not different, a detect in the production and elimination of excess motor neurons in the early developmental stages in the SBA group may be involved in the pathological mechanism of the motor complications of this disease.
  • Min WANG, Katsumi MOMINOKI, Masae KINUTANI, Zhong WANG, Naoto KOBAYASHI, Tetsuya SHIMOKAWA, Hiroaki NABEKA, Takashi FUJIWARA, Seiji MATSUDA
    J Vet Med Sci 73 4 447 - 452 2011年
  • Fujiwara T, Nishimura M, Honda R, Nishiyama T, Nomoto M, Kobayashi N, Ikeda M
    Advances in medical education and practice 2 187 - 191 2011年 [査読有り]
  • Hiroyuki Machida, Shuichi Ito, Tomonori Hirose, Fumihiko Takeshita, Hisashi Oshiro, Tomoko Nakamura, Masaaki Mori, Yoshiaki Inayama, Kunimasa Yan, Naoto Kobayashi, Shumpei Yokota
    NEPHROLOGY DIALYSIS TRANSPLANTATION 25 8 2530 - 2537 2010年08月 [査読有り]
     研究論文(学術雑誌) 
    Background. Childhood-onset systemic lupus erythematosus (SLE) is frequently complicated with lupus nephritis (LN), which is characterized by the deposition of DNA-containing immune complex to the glomerulus. Toll-like receptor 9 (TLR9), capable of recognizing the microbially derived CpG oligonucleotide, plays a crucial role in the innate immunity. TLR9 is also assumed to be related to the aetiology of SLE in the recognition of anti-DNA antibody-containing immune complex, but this remains controversial. We conducted a study to elucidate the association between TLR9 and LN in childhood-onset SLE. Methods. We compared the expression and localization of TLR9 and the slit membrane-related protein in the biopsied kidney sample by immunostaining in four children with active or inactive LN. We also evaluated their laboratory findings, such as anti-DNA antibody, complement and proteinuria at biopsy, to assess the correlation to the findings of the immunostaining. Results. TLR9 is not expressed in a normal control kidney. However, TLR9 develops in podocytes only in active LN but disappears in remission. Meanwhile, the slit membrane-related proteins such as nephrin, podocin and synaptopodin in podocytes express clearly and uniformly in remission, but their expression is markedly diminished in active LN, which results in podocyte injury. When TLR9 is expressed in podocytes, all the patients simultaneously showed hypocomplementaemia, high titre of anti-double-stranded DNA (dsDNA) antibody and proteinuria. Conclusion. Injured podocytes in active LN express TLR9. This expression could be associated with proteinuria and increased anti-dsDNA antibody. This is the first report indicating that TLR9 is involved in the aetiology of LN and that it may play some role in podocyte injury.
  • Yuji Miguchi, Hiromi Takata, Takuya Doihara, Kyojy Miyawaki, Tetsuya Shimokawa, Fumihiko Hamada, Naoto Kobayashi, Seiji Matsuda
    BIOLOGICAL BULLETIN 219 1 12 - 16 2010年08月 [査読有り]
     研究論文(学術雑誌) 
    In echinoderms, the circumesophageal muscle is mesodermal in origin. Several studies of sea urchins have reported that the molecular events of myogenesis occur during the differentiation of the circumesophageal muscle in early embiyogenesis. In contrast, few detailed reports have examined the differentiation of the circumesophagus muscle in larval starfish. Here, we examined the temporal-numeric distribution and differentiation of esophagus circular muscle fibers in the starfish Patina pectinifera by using rhodamine phalloidin staining. Muscle fibers were not detected in mouth-forming larvae, but a mean of about 10 muscle fibers was observed in 48-h larvae, and about 26 bundles were observed after 60 h. During the next 12 h, the number of muscle fiber bundles increased slightly to about 31 bundles and was stable until 96 h.
  • Matsuda S, Hasegawa M, Muro H, Asano H, Hamada F, Shimokawa T, Miyawaki K, Nabeka H, Wakisaka H, Hamai M, Kobayashi N
    Kaibogaku zasshi. Journal of anatomy 84 4 103 - 109 2009年12月 [査読有り]
  • Takuya Doihara, Yuji Miguchi, Kyojy Miyawaki, Tetsuya Shimokawa, Fumihiko Hamada, Naoto Kobayashi, Seiji Matsuda
    DEVELOPMENT GENES AND EVOLUTION 219 4 199 - 206 2009年04月 研究論文(学術雑誌) 
    To examine embryogenic mechanisms in the starfish Patiria (Asterina) pectinifera, we histochemically analyzed several larval stages using Alcian Blue (AB, which stains acidic mucins), Periodic Acid Schiff (PAS, which stains neutral mucins), and 21 types of lectins. Carbohydrate distribution patterns were observed in the cytoplasm, basement membrane, and blastocoel as follows: (1) The first group of lectins showed granular signals in the mesendodermal cells, and these lectins may be useful as mesendoderm markers. (2) The second class of lectins showed diffuse signals across the entire cytoplasm from the hatched blastula until the mid gastrula. These signals became localized to the basal cytoplasm of archenteron cells at the early bipinnaria. (3) Lectin reactivity in the basement membrane peaked at the early-to-mid gastrula and was nearly gone by the early bipinnaria. These results suggest the existence of various substances in the basement membrane and imply the importance of these substances during archenteron elongation and the induction of mesenchyme differentiation. (4) Signal colors with AB-PAS double staining in the blastocoel changed from magenta (by PAS staining) into blue (by AB staining) during these stages, thus, indicating that mucin located in the blastocoel changed from neutral to acidic. The most significant part of this report is the first description regarding temporal changes in the characteristics of intra- and extracellular components with the combination of many different lectins and stains.
  • 学生の症状とホルムアルデヒドガス濃度から見た解剖実習室内の局所排気装置の効果
    松田正司, 長谷川雅則, 室 大明, 浅野 博, 濱田文彦, 下川哲哉, 宮脇恭史, 鍋加浩明, 脇坂浩之, 濱井盟子, 小林直人
    解剖学雑誌 84 103 - 109 2009年 [査読有り]
  • M. Hattori, Y. Akioka, H. Chikamoto, N. Kobayashi, K. Tsuchiya, M. Shimizu, S. Kagami, H. Tsukaguchi
    AMERICAN JOURNAL OF TRANSPLANTATION 8 7 1550 - 1556 2008年07月 研究論文(学術雑誌) 
    Recurrent focal segmental glomerulosclerosis (FSGS) is a major challenge in the field of transplantation. Integrin-linked kinase (ILK) has emerged as a key mediator of podocyte-glomerular basement membrane (GBM) interactions. To clarify the involvement of plasma factors in FSGS recurrence, we examined the effects of plasma from FSGS patients with or without posttransplant recurrence on cultured podocytes, focusing particularly on ILK activity. Podocytes from a conditionally immortalized mouse podocyte cell line were treated with plasma from 11 FSGS patients, and ILK activity was determined using an immune complex kinase assay. Treatment with plasma from three patients with recurrence induced an increase in ILK activity. In contrast, no increase in ILK activity was observed in cultured podocytes treated with plasma from the remaining three patients with recurrence and five patients without recurrence. Cultured podocytes treated with plasma that induced ILK activity showed alterations of focal contact and detachment from the laminin matrix. In conclusion, this preliminary study provides experimental evidence suggesting the possible presence of circulating toxic factors in the plasma of some patients with recurrent FSGS, which induce an increase in podocyte ILK activity that may lead to the detachment of podocytes from the GBM.
  • Jie Chen, Shouichiro Saito, Naoto Kobayashi, Kohji Sato, Takehiro Terashita, Tetsuya Shimokawa, Katsumi Mominoki, Kyojy Miyawaki, Akira Sano, Seiji Matsuda
    NEUROSCIENCE RESEARCH 60 1 82 - 94 2008年01月 [査読有り]
     研究論文(学術雑誌) 
    Prosaposin acts as a neurotrophic factor, in addition to its role as the precursor protein for saposins A, B, C, and D, which are activators for specific sphingolipid hydrolases in lysosomes. In rats, the prosaposin gene generates two alternative splicing forms of mRNA: Pro + 9 containing a 9-base insertion and Pro + 0 without. The expression of these mRNAs changes after brain injury. We examined the expression patterns of the alternative splicing forms of prosaposin mRNA in the rat facial nerve nucleus for 52 days following facial nerve transection. Pro + 0 mRNA increased within 3 days of transection, peaked after 5-10 days, and remained significantly elevated for 21 days. In contrast, the expression of Pro + 9 mRNA was constant throughout the regenerative period. Prosaposin mRNA expression increased not only in facial motoneurons, but also in microglia during facial nerve regeneration. Our findings indicate that the saposin B domain of prosaposin, which is the domain affected by alternative splicing, plays an important role in both neurons and microglia during neuroregeneration. (c) 2007 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
  • カイニン酸神経細胞障害によるプロサポシン動態
    武智浩子, 上松敬吾, 濱田文彦, 下川哲哉, 小林直人, 松田正司
    愛媛医学 27 120 - 126 2008年 [査読有り]
  • 本学院における人体解剖実習についての学生アンケートの解析-人体解剖実習および実習前後のセミナーによる学生の意識の変化について-
    山田貴代, 信埼良子, 藤原雅弘, 澤田昌宏, 松田正司, 小林直人
    理学療法学 35 2 70 - 79 2008年 [査読有り]
  • アンケート解析に基づく、人体解剖実習前後のセミナー授業の効果の考察
    山田貴代, 信埼良子, 澤田昌宏, 藤原雅弘, 松田正司, 小林直人
    リハビリテーション教育研究 13 141 - 144 2008年 [査読有り]
  • 山田貴代, 信崎良子, 藤原雅弘, 澤田昌宏, 松田正司, 小林直人
    形態・機能 6 2 99 - 109 2008年 [査読有り]
  • Myasthenia Gravis and Related Disorders.
    Annals of the New York Academy of Sciences 1132 93 - 98 2008年
  • Jie Chen, Shouichiro Saito, Naoto Kobayashi, Kohji Sato, Takehiro Terashita, Tetsuya Shimokawa, Katsumi Mominoki, Kyojy Miyawaki, Akira Sano, Seiji Matsuda
    Neuroscience Research 60 1 82 - 94 2008年01月 研究論文(学術雑誌) 
    Prosaposin acts as a neurotrophic factor, in addition to its role as the precursor protein for saposins A, B, C, and D, which are activators for specific sphingolipid hydrolases in lysosomes. In rats, the prosaposin gene generates two alternative splicing forms of mRNA: Pro + 9 containing a 9-base insertion and Pro + 0 without. The expression of these mRNAs changes after brain injury. We examined the expression patterns of the alternative splicing forms of prosaposin mRNA in the rat facial nerve nucleus for 52 days following facial nerve transection. Pro + 0 mRNA increased within 3 days of transection, peaked after 5-10 days, and remained significantly elevated for 21 days. In contrast, the expression of Pro + 9 mRNA was constant throughout the regenerative period. Prosaposin mRNA expression increased not only in facial motoneurons, but also in microglia during facial nerve regeneration. Our findings indicate that the saposin B domain of prosaposin, which is the domain affected by alternative splicing, plays an important role in both neurons and microglia during neuroregeneration. © 2007 Elsevier Ireland Ltd and the Japan Neuroscience Society.
  • Kazuhiro Shigemoto, Sachiho Kubo, Chen Jie, Naohito Hato, Yasuhito Abe, Norifumi Ueda, Naoto Kobayashi, Kenji Kameda, Katsumi Mominoki, Atsuo Miyazawa, Akihito Ishigami, Seiji Matsuda, Naoki Maruyama
    MYASTHENIA GRAVIS AND RELATED DISORDERS: 11TH INTERNATIONAL CONFERENCE 1132 93 - 98 2008年 [査読有り]
     研究論文(学術雑誌) 
    Here we present the first evidence that muscle-specific kinase (MUSK) antigen can cause myasthenia in animals. MUSK is expressed at the postsynaptic membranes of neuromuscular junctions (NMJ) and forms complexes with acetylcholine receptors (AChR) and rapsyn. MUSK is activated by agrin, which is released from motoneurons, and induces AChR clustering and subsequent formation of NMJ in embryos. Notably, autoantibodies against MUSK were found in a proportion of patients with generalized myasthenia gravis (MG) but without the characteristic AChR autoantibodies. However, MUSK autoantibodies had no known pathogenic potential, and animals immunized with purified MUSK proteins did not develop MG in former studies. In contrast, we have now injected rabbits with MUSK ectodomain protein in vivo and evoked a MG-like muscle weakness with a reduction of AChR clustering at the NMJ. Our results showed that MUSK is required for maintenance of synapses and that interference with that function by MUSK antibodies causes myasthenic weakness. In vitro, AChR clustering in myotubes is induced by agrin and agrin-independent inducers, which do not activate MUSK. Neither the receptor nor the activation mechanisms of AChR clustering induced by agrin-independent inducers has been identified with certainty, but MUSK autoantibodies in myasthenic animals inhibited both agrin and agrin-independent AChR clustering. MUSK plays multiple roles in pre-patterning of the postsynaptic membrane before innervation and formation of NMJ in embryos. Some of these mechanisms may also participate in the maintenance of mature NMJ. This model system would provide new knowledge about the molecular pathogenesis of MG and MUSK functions in mature NMJ.
  • Hosoda Yoshiki, Miyawaki Kyojy, Saito Shouichiro, Chen Jie, Bing Xue, Terashita Takehiro, Kobayashi Naoto, Araki Nobukazu, Shimokawa Tetsuya, Hamada Fumihiko, Sano Akira, Tanabe Hirotaka, Matsuda Seiji
    Cell and tissue research 330 2 197  2007年11月 [査読有り]
     研究論文(学術雑誌) 
    Prosaposin is the precursor of four sphingolipid activator proteins (saposins A, B, C, and D) for lysosomal hydrolases and is abundant in the nervous system and muscle. In addition to its role as a precursor of saposins in lysosomes, intact prosaposin has neurotrophic effects in vivo or in vitro when supplied exogenously. We examined the distribution of prosaposin in the central and peripheral nervous systems and its intracellular distribution. Using a monospecific antisaposin D antibody that crossreacts with prosaposin but not with saposins A, B, or C, immunoblot experiments showed that both the central and peripheral nervous systems express unprocessed prosaposin and little saposin D. Using the antisaposin D antibodies, we demonstrated that prosaposin is abundant in almost all neurons of both the central and peripheral nervous systems, including autonomic nerves, as wellas motor and sensory nerves. Immunoelectron microscopy using double staining with antisaposin D and anticathepsin D antibodies showed strong prosaposin immunoreactivity mainly in the lysosomal granules in the neurons in both the central and peripheral nervous systems. The expression of prosaposin mRNA, examined us
  • Yoshiki Hosoda, Kyojy Miyawaki, Shouichiro Saito, Jie Chen, Xue Bing, Takehiro Terashita, Naoto Kobayashi, Nobukazu Araki, Tetsuya Shimokawa, Fumihiko Hamada, Akira Sano, Hirotaka Tanabe, Seiji Matsuda
    Cell and Tissue Research 330 2 197 - 207 2007年11月 研究論文(学術雑誌) 
    Prosaposin is the precursor of four sphingolipid activator proteins (saposins A, B, C, and D) for lysosomal hydrolases and is abundant in the nervous system and muscle. In addition to its role as a precursor of saposins in lysosomes, intact prosaposin has neurotrophic effects in vivo or in vitro when supplied exogenously. We examined the distribution of prosaposin in the central and peripheral nervous systems and its intracellular distribution. Using a monospecific antisaposin D antibody that crossreacts with prosaposin but not with saposins A, B, or C, immunoblot experiments showed that both the central and peripheral nervous systems express unprocessed prosaposin and little saposin D. Using the antisaposin D antibodies, we demonstrated that prosaposin is abundant in almost all neurons of both the central and peripheral nervous systems, including autonomic nerves, as well as motor and sensory nerves. Immunoelectron microscopy using double staining with antisaposin D and anticathepsin D antibodies showed strong prosaposin immunoreactivity mainly in the lysosomal granules in the neurons in both the central and peripheral nervous systems. The expression of prosaposin mRNA, examined using in situ hybridization, was observed in these same neurons. Our results suggest that prosaposin is synthesized ubiquitously in neurons of both the central and peripheral nervous systems. © 2007 Springer-Verlag.
  • Shuang-yan Gao, Chun-yu Li, Tetsuya Shimokawa, Takehiro Terashita, Seiji Matsuda, Eishin Yaoita, Naoto Kobayashi
    CELL AND TISSUE RESEARCH 328 2 391 - 400 2007年05月 [査読有り]
     研究論文(学術雑誌) 
    Intercellular adhesions between renal glomerular epithelial cells (also called podocytes) are necessary for the proper function of the glomerular filtration barrier. Although our knowledge of the molecular composition of podocyte cell-cell contact sites has greatly progressed, the underlying molecular mechanism regulating the formation of these cell-cell contacts remains largely unknown. We have used forskolin, an activator of adenylyl cyclase that elevates the level of intracellular cAMP, to investigate the effect of cAMP and three Rho-family small GTPases (RhoA, Cdc42, and Rac1) on the regulation of cell-cell contact formation in a murine podocyte cell line. Transmission electron microscopy and the immunostaining of cell adhesion molecules and actin-associated proteins have revealed a structural change at the site of cell-cell contact following forskolin treatment. The activity of the Rho-family small GTPases before and after forskolin treatment has been evaluated with a glutathione-S-transferase pull-down assay. Forskolin reinforces the integrity of cell-cell contacts, resulting in the closure of an intercellular adhesion zipper, accompanied by a redistribution of cell adhesion molecules and actin-associated proteins in a continuous linear pattern at cell-cell contacts. The Rho-family small GTPases Rac1 and Cdc42 are activated during closure of the adhesion zipper, whereas RhoA is suppressed. Thus, cAMP promotes the assembly of cell-cell contacts between podocytes via a mechanism that probably involves Rho-family small GTPases.
  • Takehiro Terashita, Shouichiro Saito, Kyojy Miyawaki, Masamitsu Hyodo, Naoto Kobayashi, Tetsuya Shimokawa, Kyoko Saito, Seiji Matsuda, Kiyofumi Gyo
    NEUROSCIENCE RESEARCH 57 3 372 - 378 2007年03月 [査読有り]
     研究論文(学術雑誌) 
    Prosaposin, the precursor of the sphingolipid hydrolase activator proteins called saposins A, B, C, and D, is abundant in the nervous system and muscles. Besides its role as the precursor of saposins, prosaposin is reported to function as a neurotrophic factor, initiating neural differentiation and preventing neuronal cell death in vivo and in vitro. In this study, we examined the localization and synthesis of prosaposin in the rat cochlea. Intense prosaposin immunoreactivity was observed in the organ of Corti, stria vascularis, and spiral ganglion. In an immuno-electron microscopic study, prosaposin immunoreactivity was found mainly in lysosomal granules of the cells in these regions. In the lysosome, prosaposin does not always colocalize with cathepsin D, but was localized mainly in the dark area of the lysosome. Prosaposin mRNA was observed in these same regions. Our results suggest that prosaposin plays a role in homeostasis in the peripheral auditory system. (c) 2006 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
  • Takehiro Terashita, Shouichiro Saito, Kyojy Miyawaki, Masamitsu Hyodo, Naoto Kobayashi, Tetsuya Shimokawa, Kyoko Saito, Seiji Matsuda, Kiyofumi Gyo
    NEUROSCIENCE RESEARCH 57 3 372 - 378 2007年03月 研究論文(学術雑誌) 
    Prosaposin, the precursor of the sphingolipid hydrolase activator proteins called saposins A, B, C, and D, is abundant in the nervous system and muscles. Besides its role as the precursor of saposins, prosaposin is reported to function as a neurotrophic factor, initiating neural differentiation and preventing neuronal cell death in vivo and in vitro. In this study, we examined the localization and synthesis of prosaposin in the rat cochlea. Intense prosaposin immunoreactivity was observed in the organ of Corti, stria vascularis, and spiral ganglion. In an immuno-electron microscopic study, prosaposin immunoreactivity was found mainly in lysosomal granules of the cells in these regions. In the lysosome, prosaposin does not always colocalize with cathepsin D, but was localized mainly in the dark area of the lysosome. Prosaposin mRNA was observed in these same regions. Our results suggest that prosaposin plays a role in homeostasis in the peripheral auditory system. (c) 2006 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
  • カイニン酸投与後ラット海馬におけるプロサポシン免疫反応の変化
    上松敬吾, 武智浩子, 下川哲哉, 宮脇恭史, 小林直人, 松田正司
    愛媛医学 26 313 - 318 2007年 [査読有り]
  • Shouichiro Saito, Naoto Kobayashi, Yasuro Atoji
    Anatomy and embryology 211 5 395 - 402 2006年10月 研究論文(学術雑誌) 
    Lectin binding patterns in the olfactory bulb of the Japanese common toad, Bufo japonicus, were examined using 21 types of lectin. Ten out of 21 lectins, WGA, s-WGA, LEL, STL, DBA, VVA, SJA, RCA-I, PNA, and PHA-L, stained the olfactory nerve, the glomeruli in the main olfactory bulb (MOB), the vomeronasal nerve, and the glomeruli in the accessory olfactory bulb (AOB). The binding patterns of LEL, STL, DBA, and PHA-L subdivided AOB glomeruli into rostral and caudal regions, where LEL, STL, and DBA stained the rostral region more intensely than the caudal region, and PHA-L had the opposite effect. Another lectin, BSL-I, stained both AOB glomeruli and the vomeronasal nerve, but not MOB glomeruli or the olfactory nerve. This is the first report of histological subdivision in the AOB of an amphibian, which suggests that the AOB development in Bufo may be unique.
  • CY Li, SY Gao, T Terashita, T Shimokawa, H Kawahara, S Matsuda, N Kobayashi
    CELL AND TISSUE RESEARCH 324 3 369 - 375 2006年06月 [査読有り]
     研究論文(学術雑誌) 
    The first event occurring at the boundary between a metal implant and living tissue is the attachment of cells onto the metal surface of the implant. The attachment characteristics of the metal in this situation are critical in determining its biocompatibility and usefulness as artificial bone and tooth implants. Using the human osteosarcoma cell line Saos-2, we attempted to establish simple and reliable methods for evaluating the attachment of cultured osteoblastic cells onto titanium samples that had been subjected to various surface treatments. Fluorescence actin imaging showed that cells cultured on titanium with hydrofluoric acid etching (HF-Ti) exhibited delayed spreading of their cytoplasm, as compared to cells cultured for the same length of time on nitrided titanium or physically polished titanium. The HF-Ti-cultured cells also exhibited poor assembly of focal contacts, as visualized by vinculin immunofluorescence. Furthermore, in motility assays based on an in vitro wound model, cells cultured on HF-Ti migrated more slowly than cells cultured on other titanium surfaces. These data suggest that Saos-2 cells attach less effectively to the HF-Ti surface. The methods described in this study should be useful for assessing the initial interactions of cultured cells with various materials, including metals.
  • N Kobayashi, T Takebayashi, S Saito, T Terashita, T Shimokawa, S Matsuda
    CLINICAL ANATOMY 19 4 354 - 357 2006年05月 [査読有り]
     研究論文(学術雑誌) 
    An anomalous branch of the right coronary artery was found in a 71-year-old male cadaver with a right-sided aortic arch. The anomalous artery arose from the proximal portion of the right coronary artery and ran in a retroaortic course, before reaching the posterior wall of the heart. It was recognized as the right-sided variation of the circumflex coronary artery. The aortic arch had as branches the left common carotid, right common carotid, right subclavian, and left subclavian arteries, in that order, and the descending aorta was located in the right thorax. The left subclavian artery arose from a Kommerell's diverticulum and ran behind the esophagus, and the left-sided ligamentum arteriosum was also connected at the diverticulum. Therefore, the right aortic arch was classified as type N according to Adachi-Willimis-Nakagawa and type III-B1 in accordance with Stewart-Edwards. The Kornmerell's diverticulum in this case seemed to press on the posterior wall of the esophagus.
  • K Shigemoto, S Kubo, N Maruyama, N Hato, H Yamada, C Jie, N Kobayashi, K Mominoki, Y Abe, N Ueda, S Matsuda
    JOURNAL OF CLINICAL INVESTIGATION 116 4 1016 - 1024 2006年04月 研究論文(学術雑誌) 
    Muscle-specific kinase (MuSK) is critical for the synaptic clustering of nicotinic acetylcholine receptors (AChRs) and plays multiple roles in the organization and maintenance of neuromuscular junctions (NMJs). MuSK is activated by agrin, which is released from motoneurons, and induces AChR clustering at the postsynaptic membrane. Although autoantibodies against the ectodomain of MuSK have been found in a proportion of patients with generalized myasthenia gravis (MG), it is unclear whether MuSK autoantibodies are the causative agent of generalized MG. In the present study, rabbits immunized with MuSK ectodomain protein manifested MG-like muscle weakness with a reduction of AChR clustering at the NMJs. The autoantibodies activated MuSK and blocked AChR clustering induced by agrin or by mediators that do not activate MuSK. Thus MuSK autoantibodies rigorously inhibit AChR clustering mediated by multiple pathways, an outcome that broadens our general comprehension of the pathogenesis of MG.
  • Naoto Kobayashi, T. Takebayashi, S. Saito, T. Terashita, T. Shimokawa, S. Matsuda
    Clinical Anatomy 19 4 354 - 357 2006年 [査読有り]
     研究論文(学術雑誌) 
    An anomalous branch of the right coronary artery was found in a 71-year-old male cadaver with a right-sided aortic arch. The anomalous artery arose from the proximal portion of the right coronary artery and ran in a retroaortic course, before reaching the posterior wall of the heart. It was recognized as the right-sided variation of the circumflex coronary artery. The aortic arch had as branches the left common carotid, right common carotid, right subclavian, and left subclavian arteries, in that order, and the descending aorta was located in the right thorax. The left subclavian artery arose from a Kommerell's diverticulum and ran behind the esophagus, and the left-sided ligamentum arteriosum was also connected at the diverticulum. Therefore, the right aortic arch was classified as type N according to Adachi-Williams-Nakagawa and type III-B1 in accordance with Stewart-Edwards. The Kommerell's diverticulum in this case seemed to press on the posterior wall of the esophagus. © 2005 Wiley-Liss, Inc.
  • ラット蝸牛におけるプロサポシンの局在と mRNA 発現部位
    寺下健洋, 兵藤政光, 下川哲哉, 齋藤正一郎, 小林直人, 暁 清文
    愛媛医学 25 178 - 184 2006年 [査読有り]
  • K Mominoki, M Kinutani, H Wakisaka, S Saito, N Kobayashi, T Fujiwara, S Matsuda
    EXPERIMENTAL NEUROLOGY 197 1 133 - 142 2006年01月 研究論文(学術雑誌) 
    We created chicks with spina bifida aperta (SBA) by incising the roof plate of the neural tube of embryos at Hamburger Hamilton stage 18 or 19. Incision over the length of three somites caused spina bifida occulta (SBO)-like malformation in 47% of the hatchlings. Incision over the length of five and seven somites caused SBA-like malformation in 100% of the hatchlings. The SBO chicks exhibited no symptoms, whereas the SBA chicks exhibited paralysis of a leg muscle and imbalance between all agonist and all antagonist leg muscles. Lesions in these SBA chicks were located in the spinal segments that give rise to motor neurons that innervated the dysfunctional muscles. Histological analysis revealed that there were fewer small spinal neurons (interneurons) at the site of the lesion in SBA chicks than in the normal chicks and that there was no such difference in the number of the large spinal neurons (motor neurons). Leg dysfunctions in this model of SBA may be attributable to the smaller number of interneurons in the spinal segments that contain motor neurons that innervate the dysfunctional muscle. This model may facilitate studies of the pathological mechanisms that lead to leg dysfunctions in SBA chicks. (c) 2005 Elsevier Inc. All rights reserved.
  • S Matsuda, N Kobayashi, T Terashita, T Shimokawa, K Shigemoto, K Mominoki, H Wakisaka, S Saito, K Miyawaki, K Saito, F Kushihata, J Chen, SY Gao, CY Li, M Wang, T Fujiwara
    JOURNAL OF COMPARATIVE NEUROLOGY 491 3 234 - 245 2005年10月 [査読有り]
     研究論文(学術雑誌) 
    Cajal's initial glomeruli (IG) and Dogiel's pericellular nests (PCNs) were first described from methylene blue preparations of healthy animal tissues around the beginning of the last century. Since that time, although many reports have been published concerning these structures, few have focused on their development and phylogeny in healthy animals. The aim of this study was to examine the phylogenetic development of the sensory neurons in Cajal's IG (also called axonal glomeruli) and Dogiel's PCNs in the dorsal root ganglion (DRG) of the healthy adult frog, chick, rat, and rabbit. The three-dimensional architecture of the neurons was observed in ganglia by scanning electron microscopy after removal of the connective tissue. The neurons in the DRG of fish are known to be bipolar, but DRG neurons in the species examined here were found to be pseudounipolar, with single stem processes. The proportion of neurons having IG or PCNs increased with increasing phylogenetic complexity in the species examined here. Cajal's initial glomeruli, the convolution of the stem process near the parent cell body: In frogs, the ganglia were small and the neuronal stem processes were very short and straight. In chicks, the stem processes were longer; sometimes very long, tortuous processes were observed. However, no neurons with typical IG were observed in either species. Typical IG were observed in rats and rabbits; their occurrence was much more frequent in rabbits. Pseudounipolarization, i.e., the transition from bipolar to pseudounipolar neurons, is thought to save space, limit the length of neuronal processes, and reduce conduction time. However, an explanation of the evolutionary advantage of the IG, which is formed by the excessive prolongation of the stem process, remains elusive. The cytological and electrophysiological importance of IG has been discussed. Dogiel's pericellular nests (PCNs), which resemble balls of yarn made of thin unmyelinated nerve fibers around DRG neurons, have been observed in the DRG of rats and rabbits, but not in frogs or chicks. This interesting structure shows not only ontogenetic development in healthy animals but also phylogenetic development among species. The nerve fibers in the PCNs were less than 1.2 mu m in diameter and had some varicosities. An immunohistochemical study using anti-tyrosine hydroxylase (TH) antibody revealed that some PCNs contain TH-positive nerve fibers and varicosities. Such TH-positive PCNs disappear after sympathectomy. These results suggest that the PCNs are made up of autonomic nerve fibers.
  • Kana Unuma, Jie Chen, Shouichiro Saito, Naoto Kobayashi, Kohji Sato, Kyoko Saito, Hiroyuki Wakisaka, Katsumi Mominoki, Akira Sano, Seiji Matsuda
    Neuroscience Research 52 3 220 - 227 2005年07月 研究論文(学術雑誌) 
    Prosaposin is the precursor of saposins A, B, C and D, which are activators of sphingolipid hydrolases. In addition, unprocessed prosaposin functions as a neurotrophic factor in the central and peripheral nervous systems by acting to prevent neuronal apoptosis, to elongate neurites and to facilitate myelination. In this study, the expression pattern of prosaposin in the facial nerve nucleus after facial nerve transection was examined by immunohistochemistry and in situ hybridization. Prosaposin immunoreactivity in the neurons on the operated side facial nerve nucleus showed a biphasic pattern: it was significantly increased on day 3 after transection, decreased dramatically on day 7, started to increase gradually on day 14 and reached another peak on day 21 after transection. Significant increases in the levels of prosaposin mRNA were identified in the neurons on the operated side, suggesting that prosaposin was synthesized vigorously by the neurons themselves in the case of facial nerve transection. The diverse changes in prosaposin immunoreactivity during the process of facial nerve regeneration may reflect the diverse neurotrophic activities of prosaposin in facial motoneurons. © 2005 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
  • M Hino, M Nagase, S Kaname, S Shibata, T Nagase, S Oba, M Funaki, N Kobayashi, H Kawachi, P Mundel, T Fujita
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 330 1 178 - 185 2005年04月 [査読有り]
     研究論文(学術雑誌) 
    Adrenomedullin (AM) is postulated to exert organ-protective effects. It is expressed in the renal glomeruli, but its roles in the glomerular podocytes have been poorly elucidated. In the present study, we investigated the expression and regulation of AM in recently established conditionally immortalized mouse podocyte cell line in vitro and podocyte injury model in vivo. The cultured differentiated podocytes expressed AM mRNA and secreted measurable amount of AM. AM secretion from the podocytes was increased by H2O2, hypoxia, puromycin aminonucleoside (PAN), albumin overload, and TNF-alpha. Real-time RT-PCR analysis revealed that AM mRNA expression in the podocytes was enhanced by PAN and TNF-alpha, both of which were suppressed by mitochondrial antioxidants. Furthermore, AM expression was upregulated in the glomerular podocytes of PAN nephrosis rats. These results indicated that AM expression in the podocytes was upregulated by stimuli or condition relevant to podocyte injury, suggesting its potential role in podocyte pathophysiology. (c) 2005 Elsevier Inc. All rights reserved.
  • 当学院における人体解剖実習― 導入の経緯と今後の課題 ―
    澤田昌宏, 藤原雅弘, 三澤一登, 山田貴代, 内田勝之, 信崎良子, 福田 靖, 寒竹千夏, 荒川慶子, 小林直人, 松田正司
    愛媛十全医療学院紀要 3 21 - 24 2005年
  • ラット神経系内でのプロサポシンの分布
    細田能希, 齋藤正一郎, 小林直人, 寺下健洋, 松田正司, 田邊敬貴
    愛媛医学 24 1 29 - 34 2005年 [査読有り]
  • 愛媛大学医学部医学科における肉眼解剖学実習の改善への試み-学部教育改革への対応とマンパワー不足の克服に向けて-
    小林直人, 齋藤正一郎, 寺下健洋, 下川哲哉, 松田正司
    大学教育実践ジャーナル 3 65 - 73 2005年
  • K Saito, S Saito, K Taniguchi, N Kobayashi, T Terashita, T Shimokawa, K Mominoki, K Miyawaki, J Chen, SY Gao, CY Li, S Matsuda
    NEUROSCIENCE RESEARCH 50 2 219 - 225 2004年10月 研究論文(学術雑誌) 
    In the developing central nervous system, apoptosis plays an important role in the normal organization of the neuronal circuit. The timing of neurogenesis, proliferation, and migration of the neurons in the developing olfactory bulb (OB) is well studied; however, the involvement of apoptosis in this process is not fully understood. In this study, we examined the changes in the distribution and the number of apoptotic cells in the rat OB during embryonic and postnatal periods, by using terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end-labeling (TUNEL) staining. Although the number of TUNEL-positive cells was relatively small during the embryonic period, it gradually increased after birth, and peaked on postnatal day 20 with statistical significance, especially in the granule cell layer of the main OB. This transient increase of TUNEL-positive cells on postnatal day 20 may be involved in a critical event during maturation of the OB. (C) 2004 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
  • J Watanabe, F Kushihata, K Honda, A Sugita, N Tateishi, K Mominoki, S Matsuda, N Kobayashi
    SURGERY 135 6 604 - 612 2004年06月 [査読有り]
     研究論文(学術雑誌) 
    Background. Proliferation and apoptosis of liver cancer cells are closely related phenomena. We investigated the correlation between overexpression of Bcl-xL, an anti-apoptosis-related protein of the Bcl-2 family, and the clinical course of hepatocellular carcinoma (HCC). Methods. Specimens from 7 HCC patients were used for Western blotting and immunoelectron microscopy tests. Samples from 33 HCC patients who had undergone hepatectomies were used for immunohistochemical staining. The degrees of expression of Bcl-xL and Ki-67, as an index of HCC mitosis severity, were each classified into 2 groups. Results. With the use of Western blot analysis, enhanced immunoreactivity of Bcl-xL was found in cancerous specimens. Bcl-xL overexpression was found in cancer specimens in 21 of 33 patients (63.6%). The overall survival (P = .019) and disease-free survival (P = .030) rates of the group overexpressing Bcl-xL were definitely poorer. The Ki-67 higher labeling index LI > 10) group had a poorer survival rate (P = .016). There were significant correlations between Bcl-xL and overall survival and disease-free survival. Multivariate analyses revealed that. Bcl-xL, tumor size, histologic portal invasion, and histologic metastatic foci were independent prognostic factors for overall survival and disease-free survival. Conclusions. These results showed Bcl-xL in HCC specimens, suggesting that Bcl-xL was a significant Prognostic factor for disease progression in human HCC.
  • Naoto Kobayashi, Shuang-Yan Gao, Jie Chen, Kyoko Saito, Kyojy Miyawaki, Chun-Yu Li, Lei Pan, Shouichiro Saito, Takehiro Terashita, Seiji Matsuda
    Anatomical Science International 79 1 1 - 10 2004年03月 [招待有り]
     研究論文(学術雑誌) 
    The renal glomerular podocyte exhibits a highly arborized morphology. In comparison with the neuron, which is the best studied process-bearing cell, the podocyte major processes share many cell biological characteristics with neuronal dendrites. Both podocytes and neurons develop microtubule-based thick processes with branching morphology and both have thin actin-based projections (i.e. podocyte foot processes and dendritic spines). Formation of podocyte processes and neuronal dendrites depends on the assembly of microtubules. Because the assembly of microtubules is regulated by phosphorylation of microtubule-associated proteins, inhibition of protein phosphatases abolishes and inhibition of protein kinases promotes process formation. Podocytes and dendrites also share the machinery of intracellular traffic of membranous vesicles, as well as cytoskeletal elements, which is indispensable for the elongation of these processes. Furthermore, these two cell types share expression of various molecules working for signal transduction, transmembranous transport and intercellular contacts. Such common gene expression implies a similar transcriptional regulation in these cells. Concerning the formation of podocyte foot processes and dendritic branches, actin filaments are thought to play a central role in orchestrating the function of various molecules and the regulation of actin assembly is necessary to establish and maintain such sophisticated cellular architecture. The molecular mechanism of foot process formation seems to include Rho family small GTP-binding proteins, which are known to be responsible for the establishment of dendritic branching morphology.
  • Gao SY, Li CY, Chen J, Pan L, Saito S, Terashita T, Saito K, Miyawaki K, Shigemoto K, Mominoki K, Matsuda S, Kobayashi N
    Nephron. Experimental nephrology 97 2 e49 - 61 2004年 [査読有り]
  • 冠状動脈の破格に右側大動脈弓を併せ持つ1解剖例.
    竹林孝晃, 齋藤正一郎, 寺下健洋, 下川哲哉, 松田正司, 小林直人
    愛媛医学 23 342 - 344 2004年 [査読有り]
     研究論文(大学,研究機関等紀要)
  • ラット顔面神経切断後の顔面神経核内におけるプロサポシン mRNA の発現変化
    陳 潔, 齋藤正一郎, 齋藤恭子, 寺下健洋, 小林直人, 松田正司
    愛媛医学 23 152 - 157 2004年 [査読有り]
  • H Yamazaki, A Saito, H Ooi, N Kobayashi, P Mundel, F Gejyo
    NEPHRON EXPERIMENTAL NEPHROLOGY 96 2 E52 - E58 2004年 研究論文(学術雑誌) 
    Background/Aims: Megalin is a multiligand endocytic receptor expressed in a number of epithelia. In the Lewis rat kidney, podocytes, as well as proximal tubule cells, express megalin that acts as a pathogenic antigen for Heymann nephritis (HN), an experimental model of membranous nephropathy. To obtain a tool to investigate the molecular mechanisms of megalin-mediated endocytosis and the pathogenesis of HN, we examined whether a differentiation-inducible mouse podocyte cell line expressed endocytically active megalin. Methods: Immunofluorescence and immunoprecipitation analyses with an anti-rat megalin antibody were carried out to investigate whether megalin was expressed in the differentiated and undifferentiated podocytes. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was performed to elucidate whether the cells synthesize megalin mRNA. I-125-labeled receptor-associated protein (RAP), an endocytic ligand for megalin, was used for cellular internalization and degradation assays. Results: Immunofluorescence, immunoprecipitation and RT-PCR analyses revealed that megalin was synthesized in both differentiated and undifferentiated cells and localized to the cell surfaces. Effective endocytosis of RAP via megalin was shown under the differentiated condition. Conclusion: Endocytically active megalin is expressed in differentiation-induced cultured podocytes. This cell line could be a useful tool for studies on megalin-mediated endocytosis and the pathogenesis of HN. Copyright (C) 2004 S. Karger AG, Basel.
  • SY Gao, CY Li, J Chen, L Pan, S Saito, T Terashita, K Saito, K Miyawaki, K Shigemoto, K Mominoki, S Matsuda, N Kobayashi
    NEPHRON EXPERIMENTAL NEPHROLOGY 97 2 E49 - E61 2004年 研究論文(学術雑誌) 
    Background: Podocytes, renal glomerular visceral epithelial cells, have two kinds of processes, namely major processes containing microtubules (MTs) and foot processes with actin filaments (AFs). The present study investigated how MTs are organized by the Rho-ROCK signal transduction pathway during process formation of podocytes. Method: After induction of differentiation, podocytes of the conditionally immortalized mouse cell line were treated with Y-27632, a specific inhibitor of ROCK, and exoenzyme C3, an inhibitor of RhoA, as well as with forskolin whose effects include inhibition of RhoA, in order to inhibit the Rho-ROCK pathway. Results: Inhibition of ROCK significantly enhanced the formation of thick processes containing MT bundles. Y27632 promoted process formation even in the presence of latrunculin A which disrupts AFs, strongly suggesting that ROCK directly regulates MT assembly. Treatment with Y-27632 increased MT stability, and stabilized MTs preferentially localized in podocyte processes. Moreover, when treated with a combination of Y-27632 and forskolin, and with Y-27632 and C3 as well, podocytes developed not only MT-based thick processes but also AF-based thin projections. Conclusions: These data indicate a contribution of ROCK in MT organization to promote podocyte process formation, although it was originally thought to regulate AF assembly. AF-based thin projections seem to be induced mainly by inhibition of RhoA and ROCK. The present study reveals a significant role of the Rho-ROCK signal pathway in the reorganization of both MTs and AFs during process formation of podocytes. Copyright (C) 2004 S. Karger AG, Basel.
  • GD Han, H Koike, T Nakatsue, K Suzuki, H Yoneyama, S Narumi, N Kobayashi, P Mundel, F Shimizu, H Kawachi
    JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY 14 12 3111 - 3126 2003年12月 [査読有り]
     研究論文(学術雑誌) 
    IFN-inducible protein-10 (IP-10/CXCL10) is a potent chemoattractant for activated T lymphocytes and was recently reported to have several additional biologic activities. In this study, the expression and the function in normal glomeruli and in Thy1.1 glomerulonephritis (GN) were investigated. The expression of IP-10 was detected in normal rat glomeruli mainly in the podocyte. The expression of IP-10 was also detected on the cultured podocyte. The IP-10 expression was elevated at the early phase of Thyl.1 GN. The double staining immunofluorescence study clearly demonstrated that the elevated expression of IP-10 was mostly detected in the podocyte and very partly in mesangial area. A receptor for IP-10, CXCR3, showed similar expression patterns to that of IP-10. Expressions of neither of IP-10 nor of CXCR3 were detected on the inflammatory cells. For elucidating the role of IP-10, the blocking study was carried out with monoclonal anti-IP-10 antibody. The monoclonal anti-IP-10 antibody treatment decreased the expression of IP-10 and podocyte associated proteins such as nephrin and podocin that are reported to be essential for maintaining the podocyte function (IP-10, 53.0% to control; nephrin, 43.5%; podocin, 60.4%). The findings indicated that the anti-IP-10 treatment disturbed the podocyte function. The anti-IP-10 treatment given to the rats with Thy1.1 nephritis exacerbated proteinuria, mesangiolysis, and matrix expansion. Collectively, the findings indicated that IP-10 plays a role in maintaining the podocyte function. Also, the findings suggested that anti-IP-10 treatment exacerbated the glomerular alterations in Thyl.1 GN by disturbing the podocyte function.
  • H Shinomiya, K Nagai, H Hirata, N Kobayashi, H Hasegawa, FZ Liu, K Sumita, Y Asano
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 67 6 1368 - 1375 2003年06月 [査読有り]
     研究論文(学術雑誌) 
    We previously identified a 65-kDa protein (p65) that was phosphorylated in activated macrophages. It has turned out to be a murine homologue of human L-plastin, which was identified as a novel protein in human cancer cells. p65/L-plastin is characterized by a series of Ca2+-, calmodulin-, and actin-binding domains, and is thought to play a crucial role in leukocytes and cancer cells. We have expressed a recombinant (r) p65/L-plastin in Escherichia coli that binds to beta-actin and prepared high-titer antibodies using large amounts of the protein as immunogen. Anti-rp65/L-plastin antibodies recognize native p65/L-plastin as well as rp65/L-plastin and have enabled us to detect the fine structures of intracellular p65/L-plastin, and it was found that its localization was extensively changed by stimulation with bacterial components. We further developed an enzyme-linked immunosorbent assay system and a flow cytometry method using these reagents, which made it possible to measure antibodies, including autoantibodies, against p65/L-plastin and to evaluate the maturation-dependent expression of the protein in leukocytes.
  • S Saito, T Matsui, N Kobayashi, H Wakisaka, K Mominoki, S Matsuda, K Taniguchi
    ANATOMY AND EMBRYOLOGY 206 5 349 - 356 2003年04月 [査読有り]
     研究論文(学術雑誌) 
    Expression patterns of glycoconjugates were examined by lectin histochemistry in the nasal cavity of the Japanese red-bellied newt, Cynops pyrrhogaster. Its nasal cavity consisted of two components, a flattened chamber, which was the main nasal chamber (MNC), and a lateral diverticulum called the lateral nasal sinus (LNS), which communicated medially with the MNC. The MNC was lined with the olfactory epithelium (OE), while the diverticulum constituting the LNS was lined with the vomeronasal epithelium (VNE). Nasal glands were observed beneath the OE but not beneath the VNE. In addition, a secretory epithelium was revealed on the dorsal boundary between the MNC and the LNS, which we refer to as the boundary secretory epithelium (BSE) in this study. The BSE seemed to play an important role in the construction of the mucous composition of the VNE. Among 21 lectins used in this study, DBA, SBA and Jacalin showed different staining patterns between the OE and the VNE. DBA staining showed remarkable differences between the OE and the VNE; there was intense staining, in the free border and the supporting cells of the VNE, whereas there was no staining or weak staining in the cells of the OE. SBA and Jacalin showed different stainings in the receptor neurons for the OE and the VNE. Furthermore, UEA-I and Con A showed different stainings for the nasal glands. UEA-I showed intense staining in the BSE and in the nasal glands located in the ventral wall of the MNC (VNG), whereas Con A showed intense staining in the BSE and in the nasal glands located in the dorsal and medial wall of the MNC (DMNG). The DMNG were observed to send their excretory ducts into the OE, whereas no excretory ducts were observed from the VNG to the OE or the VNE. These results suggested that the secretion by the supporting cells as well as the BSE and the DMNG establishes that there are heterogeneous mucous environments in the OE and the VNE, although both epithelia are situated in the same nasal cavity.
  • K Miyawaki, M Yamamoto, K Saito, S Saito, N Kobayashi, S Matsuda
    DEVELOPMENT GROWTH & DIFFERENTIATION 45 2 121 - 128 2003年04月 [査読有り]
     研究論文(学術雑誌) 
    Recently, beta-catenin has been reported to control the expression of morphogenetic genes through the Wnt signaling pathway in invertebrate embryogenesis. In this study, the distribution pattern of beta-catenin during starfish embryogenesis was investigated using immunohistochemistry. In 16-cell stage embryos, beta-catenin began to accumulate in some nuclei at the vegetal pole. During the early cleavage stage, the cells expressing nuclear beta-catenin increased in number in the vegetal pole region of the embryos, and the beta-catenin signal increased in intensity in each nucleus. At the blastula stage, signal for beta-catenin was also found in the cytoplasm of the cells with nuclear beta-catenin. At the vegetal plate stage, almost all vegetal plate cells expressed beta-catenin in both the nucleus and cytoplasm. When the embryos developed to early gastrulae, cells with nuclear beta-catenin were restricted to the archenteron tip, and the signal gradually faded in later stages. The localization and temporal change of beta-catenin expression suggests that beta-catenin has a pivotal role in archenteron formation in starfish embryos.
  • 鋤鼻器:両生類から人間へ
    齋藤正一郎, 小林直人, 寺下健洋, 宮脇恭史, 齋藤恭子, 松田正司
    愛媛医学 22 277 - 284 2003年 [査読有り]
     研究論文(大学,研究機関等紀要)
  • 両生類鋤鼻器の系統発生
    齋藤正一郎, 松井利康, 小林直人, 脇坂浩之, 樅木勝巳, 宮脇恭史, 齋藤恭子, 松田正司, 谷口和之
    アニテックス 15 9 - 14 2003年 研究論文(学術雑誌)
  • 愛媛大学における解剖実習についての学生アンケートの解析
    小林直人, 齋藤正一郎, 脇坂浩之, 松田正司
    大学教育実践ジャーナル 1 1 17 - 28 2003年
  • Lectin-histochemical studies on the oltactory organ of the newt, Cynops pyrrhogaster. 'jointly authored'
    Anatomy and Embryology 206 349 - 356 2003年
  • Clinical Anatomy 16 40 - 43 2003年
  • H Wakisaka, N Hato, N Honda, H Takahashi, H Kisaki, S Murakami, K Gyo, K Mominoki, N Kobayashi, S Matsuda
    EXPERIMENTAL NEUROLOGY 178 1 68 - 79 2002年11月 [査読有り]
     研究論文(学術雑誌) 
    In 1995, we developed an animal model of transient homolateral facial nerve paralysis by inoculating Herpes simplex virus type 1 (HSV-1) into the auricle of mice. This study examined the mechanism of facial nerve paralysis in this model histopathologically. Using the immunofluorescence technique with anti-HSV-1 antibody, the time course of viral spread and the site of viral replication were investigated over the entire course of the facial nerve. Furthermore, viral replication and nerve degeneration at the site of viral replication were observed by electron microscopy. On the 7th day after inoculation, facial paralysis was observed in more than 60% of mice. Immunofluorescence study revealed HSV-1 in the geniculate ganglion, the descending root, and the facial nucleus at this stage. On the 9th day, the descending root in the sections stained with osmium. looked pale, because prominent demyelination had occurred in this region; electron micrographs showed many degenerated oligodendrocytes and large naked axons. In contrast, the facial nucleus neurons showed no remarkable degeneration, despite HSV-1 particles in their cytoplasm. From these findings, we concluded that facial nerve paralysis in this model is caused mainly by facial nerve demyelination in the descending root. (C) 2002 Elsevier Science (USA).
  • J Watanabe, F Kushihata, K Honda, K Mominoki, S Matsuda, N Kobayashi
    INTERNATIONAL JOURNAL OF ONCOLOGY 21 3 515 - 519 2002年09月 [査読有り]
     研究論文(学術雑誌) 
    It is known that proliferation and apoptosis are closely related phenomena in liver cancer cells. In this study, using surgical specimens from 42 patients with hepatocellular carcinoma (HCC), we investigated the expression and localization of Bcl-xL, an antiapoptosis-related protein of the Bcl-2 family. Using Western blotting, Bcl-xL expression was detected in both cancerous and non-cancerous specimens from all of the HCC patients, and elevated Bcl-xL levels were found in cancerous specimens from two thirds of the patients. In normal human liver specimens, Bcl-xL was found mainly in the cytoplasm of hepatocytes, although it was also found in the cytoplasm of bile duct cells in Glisson's capsule by immunohistochemical staining. To the best of our knowledge, this is the first report of Bcl-xL overexpression and localization in HCC specimens. Bcl-xL was found not only in the cytoplasm of HCC cells, but also in the nuclei of some HCC cells, suggesting that Bcl-xL is involved in the progression of HCC cells in vivo.
  • ラット顔面神経切断後の顔面神経核内プロサポシン免疫反応の変化
    鵜沼 香奈, 細田 能希, 佐野 輝, 脇坂 浩之, 斎藤 正一郎, 小林 直人
    愛媛医学 21 3 296 - 300 愛媛医学会 2002年09月 [査読有り]
     
    ヒト顔面神経麻痺の再生過程を知ることを目的に,ラットのモデルを用い,顔面神経切断後の顔面神経細胞内のプロサポシンの増減を免疫組織化学的手法により検討した.ラットの右側顔面神経の切断を行い,正常側の免疫反応と比較した.その結果,顔面神経核内のプロサポシン免疫反応は術後3日に一過性に増加し,7日後に著しく低下し,14日後に回復傾向を示し,21日後に再増加のピークを示した.術後3日の免疫反応は,障害された神経細胞がプロサポシンを産出し,自らを保護する働きによると判断した.術後21日は神経軸索の再形成期と一致しており,増加したプロサポシンが髄鞘再形成を刺激すると推測した.7日後,14日後は,軸索膜損傷後の神経細胞内タンパク合成能の低下によりプロサポシン産生が減少したため,反応が低下したと判断した
  • N Kobayashi, W Kriz
    MICROSCOPY RESEARCH AND TECHNIQUE 57 4 187 - 188 2002年05月 [査読有り]
  • N Kobayashi
    MICROSCOPY RESEARCH AND TECHNIQUE 57 4 217 - 223 2002年05月 研究論文(学術雑誌) 
    In this review article we discuss the common mechanism for cellular process formation. Besides the podocyte, the mechanism of process formation, including cytoskeletal organization and signal transduction, etc., has been studied using neurons and glias as model systems. There has been an accumulation of data showing common cell biological features of the podocyte and the neuron: 1) Both cells possess long and short cell processes equipped with highly organized cytoskeletal systems; 2) Both show cytoskeletal segregation; microtubules (MTs) and intermediate filaments (IFs) in podocyte primary processes and in neurites, while actin filaments (AFs) are abundant in podocyte foot processes in neuronal synaptic regions; 3) In both cells, process formation is mechanically dependent on MTs, whose assembly is regulated by various microtubule-associated proteins (MAPS); 4) In both cells, process formation is positively regulated by PP2A, a Ser/Thr protein phosphatase; 5) In both cells, process formation is accelerated by laminin, an extracellular matrix protein. In addition, recent data from our and other laboratories have shown that podocyte processes share many features with neuronal dendrites: 1) Podocyte processes and neuronal dendrites possess MTs with mixed polarity, namely, plus-end-distal and minus-end-distal MTs coexist in these processes; 2) To establish the mixed polarity of MTs, both express CHO1/MKLP1, a kinesin-related motor protein, and when its expression is inhibited formation of both podocyte processes and neuronal dendrites is abolished; 3) The elongation of both podocyte processes and neuronal dendrites is supported by rab8-regulated basolateral-type membrane transport; 4) Both podocyte processes and neuronal dendrites express synaptopodin, an actin-associated protein, in a development-dependent manner; interestingly, in both cells, synaptopodin is localized not in the main shaft of processes but in thin short projections from the main shaft. We propose that the podocyte process and the neuronal dendrite share many features, while the neuronal axon should be thought of as an exceptionally differentiated cellular process. (C) 2002 Wiley-Liss, Inc.
  • 両生類の嗅球におけるサブセット構造
    齋藤正一郎, 脇坂浩之, 小林直人, 松田正司, 谷口和之
    Aroma Research 3 16 - 25 2002年 研究論文(学術雑誌)
  • ラット嗅球の発生におけるTUNEL陽性細胞の出現様式について(共著)
    齋藤恭子, 齋藤正一郎, 脇坂浩之, 小林直人, 松田正司
    愛媛医学 21 156 - 161 2002年 [査読有り]
  • H Wakisaka, N Kobayashi, K Mominoki, S Saito, N Honda, N Hato, K Gyo, S Matsuda
    JOURNAL OF NEUROCYTOLOGY 30 8 685 - 693 2001年08月 [査読有り]
     研究論文(学術雑誌) 
    This study presents the first direct evidence for herpes simplex virus type 1 (HSV-1) infection in the neurons of the vestibular ganglion. Although many investigators have reported electron microscopic evidence of HSV-1 infection in sensory ganglia, HSV-1 infection in the vestibular ganglion has not been described. Vestibular ganglion neurons have a unique structure, with a loose myelin sheath instead of the satellite cell sheath that is seen in other ganglia. This loose myelin is slightly different from compact myelin which is known as too tight for HSV-1 to penetrate. The role of loose myelin in terms of HSV-1 infection is completely unknown. Therefore, in an attempt to evaluate the role of loose myelin in HSV-1 infection, we looked for HSV-1 particles, or any effects mediated by HSV-1, in the vestibular ganglion as compared with the geniculate ganglion. At the light microscopic level, some neurons with vacuolar changes were observed, mainly in the distal portion of the vestibular ganglion where the communicating branch from the geniculate ganglion enters. At the electron microscopic level, vacuoles, dilated rough endoplasmic reticulum and Golgi vesicles occupied by virus were observed in both ganglia neurons. In contrast, viral infections in Schwann and satellite cells were observed only in the geniculate ganglion, but not in the vestibular ganglion. These results suggest that loose myelin is an important barrier to HSV-1 infection, and it must play an important role in the prevention of viral spread from infected neurons to other cells.
  • N Kobayashi, J Reiser, K Schwarz, T Sakai, W Kriz, P Mundel
    HISTOCHEMISTRY AND CELL BIOLOGY 115 3 255 - 266 2001年03月 研究論文(学術雑誌) 
    Podocytes possess major processes containing microtubules (MTs) and intermediate filaments and foot processes containing actin filaments (AFs) as core cytoskeletal elements. Although the importance of these cytoskeletal elements for maintaining podocyte processes was previously shown, so far no data are available concerning the developmental regulation of podocyte process formation. A conditionally immortalized mouse podocyte cell line, which can be induced to develop processes similar to those found in vivo, was treated with various reagents to disrupt cytoskeletal elements or to inhibit protein phosphatases, MTs colocalized with vimentin intermediate filaments but not with AFs. After AF disassembly, major processes were maintained, whereas after depolymerization of MTs, podocytes lost their processes, rounded up, and maintained only actin-based pe ripheral projections. Suppression of MT elongation by nanomolar vinblastine or inhibition of serine/threonine phosphatase PP2A with okadaic acid abolished process formation. PP2A was expressed in undifferentiated but not in differentiated podocytes. One- and two-dimensional western blot analyses revealed a dose-dependent increase in serine/threonine phosphorylation after okadaic acid treatment. Hence, morphogenetic activity of MTs induces podocyte process formation via serine/threonine protein dephosphorylation by PP2A. These results may open new avenues for understanding the signaling mechanism underlying podocyte cytoskeleton alterations during development and in glomerular diseases.
  • ラット後腎組織培養系と阻害ペプチドを用いた、後腎の形態形成におけるラミニンの機能についての研究
    石原美佐, 野水基義, 長田道夫, 樅木勝巳, 脇坂浩之, 齋藤正一郎, 小林直人
    発達腎研究会誌 9 28 - 32 2001年 [査読有り]
  • イトマキヒトデ胚発生に対するRNA合成阻害の影響(共著)
    宮脇恭史, 山本雅道, 脇坂浩之, 齋藤正一郎, 小林直人, 松田正司
    愛媛医学 20 138 - 143 2001年 [査読有り]
  • N Kobayashi, K Mominoki, H Wakisaka, Y Shimazaki, S Matsuda
    ADVANCES IN MICROANATOMY OF CELLS AND TISSUES, BIOPHYSICAL AND BIOCHEMICAL CORRELATES 7 Suppl.1 423 - 430 2001年 研究論文(国際会議プロシーディングス) 
    Morphogenetic effects of various extracellular matrix proteins on the renal podocyte were investigated using the conditionally immortalized podocyte cell line. Podocytes were plated on glass coverslips and coated with the following matrix proteins: laminin-10/11, laminin-1, fibronectin, collagen type IV, collagen type L Three hours after plating, podocytes on laminins developed prominent processes, while those on other matrix proteins started to elongate processes after two days. Vinculin-immunolabeling showed that podocytes plated on laminins possessed thin rod-shaped focal contacts, whereas those on fibronectin showed large dot-shaped focal contacts. Inhibition of serine/threonine protein kinases induced podocyte process formation in an extracellular matrix-independent manner. The present study reveals the significance of laminin on podocyte morphogenesis in vitro, and shows that different extracellular matrix proteins trigger different intracellular signals governing podocyte morphogenesis. Taken together with our previous studies, podocyte process formation is thought to be regulated by protein Ser/Thr phosphorylation.
  • ラット後腎組織培養系を用いた,ラミニンの形態形成誘導能の研究
    石原 美佐, 小林 直人, 野水 基義, 長田 道夫, 山宮 公子, 樅木 勝巳, 脇坂 浩之, 島崎 由美子, 松田 正司
    解剖学雑誌 75 5 478 - 478 (一社)日本解剖学会 2000年10月
  • 越智俊元, 小林直人, 松田正司, 重本和宏
    解剖学雑誌 75 1 116  2000年02月
  • ラット後腎の形態形成におけるラミニンの機能について 組織培養系を用いた研究
    石原 美佐, 小林 直人, 野水 基義, 長田 道夫, 山宮 公子, 樅木 勝巳, 脇坂 浩之, 島崎 由美子, 松田 正司
    解剖学雑誌 75 1 61 - 61 (一社)日本解剖学会 2000年02月
  • N Kobayashi, HW Heid, T Sakai, W Kriz, G Huber, P Mundel
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 268 2 306 - 309 2000年02月 研究論文(学術雑誌) 
    The identity of two microtubule-associated proteins, MAP3 and MAP4, was verified both immunologically and biochemically. MAP3 was enriched from the heat-stable fraction of rat brain extracts by reverse-phase HPLC and preparative two-dimensional gel electrophoresis, Both MAP3 and MAP4 antibodies reacted with the corresponding spots on two-dimensional Western blots. Amino acid sequences of internal peptides derived from rat MAP3 matched with corresponding sequence stretches of mouse MAP4. In the kidney cortex, the MAP3 antibody stained not only glomerular podocytes but also interstitial cells. This distribution pattern of MAP3 is identical to that of MAP4 reported previously. These results indicate that MAP3 and MAP4 are identical. (C) 2000 Academic Press.
  • Matsuda S, Kobayashi N, Wakisaka H, Saito S, Saito K, Miyawaki K, Mominoki K, Shigemoto K, Murakami S, Fujiwara T
    Biomedical Reviews 11 39 - 52 2000年 [査読有り]
     研究論文(学術雑誌) 
    The development of sensory ganglion neurons and satellite cells examined by scanning electron microscopy after removal of the connective tissue is reviewed. Sensory neurons are bipolar at early stages of development and later became pseudounipolar. This maturation event starts earlier but proceeds more slowly in chick than in rat embryos. These may due to the difference in the extent and intimacy of satellite cell investments between these two animal species. The neuronal perikaryal projections are observed by scanning electron microscopy after removal of the connective tissue and satellite cells. The morphometric analysis reveals that perikaryal projections are mom numerous on the surface of mature pseudounipolar neurons than on that of premature bipolar neurons; they increase in number as the neuronal cell bodies grow larger. This may support the hypothesis that perikaiγal projections are structural devices for increasing the neuron-satellite interface and for improving the efficiency of metabolic exchange between these two cell types. The important role of satellite cells in neuronal maturation is discussed. © The Bulgarian-American Center.
  • 小林直人, 樅木勝巳, 脇坂浩之, 松田正司, 坂井建雄
    解剖学雑誌 74 4 436 - 439 1999年 [査読有り]
     研究論文(学術雑誌) 
    Microtubular cytoskeletons play a crucial role in the morphogenesis of process-bearing cells, such as the neuron and the renal glomerular podocyte. Microtubules are bundled and stabilized by various microtubule-associated proteins, providing a mechanical basis to maintain the deviated morphology of cell processes. To support the process morphology, microtubules are also associated with other cytoskeletal elements such as actin and intermediate filaments. The microtubular polarity is uniformly plus-end-distal in neuronal axons, whereas in dendrites as well as in podocytes, the polarity is revealed to be non-uniform (i.e., both plus-end-distal and minus-end-distal microtubules are present in cell processes). Recently, this non-uniformity is reported to be established by a microtubule-dependent motor protein. Motor proteins are capable to drive the intracellular transport of cytoskeletal elements in addition to that of membrane vesicles. It is still an open question whether cytoskeletal elements are transported along cell processes as subunits or as polymers.
  • Moiphological transformation of sensory ganglion neurons and satelite cells(jointly authored)
    Biomedical Review 10 603 - 613 1999年
  • Morphological change of sensory neurons and perikaryal projections analysed by SEM.
    Microscopy and Analysis 1999年
  • Naoto Kobayashi, Jochen Reiser, Wilhelm Kriz, Ryoko Kuriyama, Peter Mundel
    Journal of Cell Biology 143 7 1961 - 1970 1998年12月 [査読有り]
     研究論文(学術雑誌) 
    Podocytes are unique cells that are decisively involved in glomerular filtration. They are equipped with a complex process system consisting of major processes and foot processes whose function is insufficiently understood (Mundel, P., and W. Kriz. 1995. Anat. Embryol. 192:385-397). The major processes of podocytes contain a microtubular cytoskeleton. Taking advantage of a recently established cell culture system for podocytes with preserved ability to form processes (Mundel, P., J. Reiser, A. Zuniga Mejia Borja, H. Pavenstadt, G.R. Davidson, W. Kriz, and R. Zeller. 1997b. Exp. Cell Res. 36:248-258), we studied the functional significance of the microtubular system in major processes. The following data were obtained: (a) Microtubules (MTs) in podocytes show a nonuniform polarity as revealed by hook-decoration. (b) CHO1/ MKLP1, a kinesin-like motor protein, is associated with MTs in podocytes. (c) Treatment of differentiating podocytes with CHO1/MKLP1 antisense oligonucleotides abolished the formation of processes and the nonuniform polarity of MTs. (d) During the recovery from taxol treatment, taxol-stabilized (nocodazoleresistant) MT fragments were distributed in the cell periphery along newly assembled nocodazole-sensitive MTs. A similar distribution pattern of CHO1/MKLP1 was found under these circumstances, indicating its association with MTs. (e) In the recovery phase after complete depolymerization, MTs reassembled exclusively at centrosomes. Taken together, these findings lead to the conclusion that the nonuniform MT polarity in podocytes established by CHO1/MKLP1 is necessary for process formation.
  • Seiji Matsuda, Naoto Kobayashi, Katsumi Mominoki, Hiroyuki Wakisaka, Masahiko Mori, Shingo Murakami
    Acta Anatomica Nipponica 73 6 612 - 613 1998年 [査読有り]
     研究論文(学術雑誌) 
    Sensory ganglion neurons in higher vertebrates are unique in that they are pseudounipolar with a single stem process that divides at some distance from the cell body into central and peripheral processes. In the early stages of development, these neurons are bipolar but later they became pseudounipolar. This developmental process of sensory ganglion neurons with satellite cells was examined by scanning electron microscopy following removal of connective tissue. This pseudo-unipolarization began earlier but proceeded at a slower rate in chick than in rat embryos. This difference may due to the difference found in the extent and intimacy of satellite cell investments in these two animals, which was due to the fact that sensory neurons undergo pseudo-unipolarization only in the presence of satellite cells in vitro. The neuronal perikaryal projections were observed by scanning electron microscopy after removal of connective tissue and satellite cells. Morphometric analysis revealed that perikaryal projections were more numerous on the surface of mature pseudounipolar neurons than on the surface of premature bipolar neurons, and that the number of projections increased as the neuronal cell bodies grew larger. This may support the hypothesis that perikaryal projections are structural devices for increasing the neuron-satellite interface and for improving the efficiency of metabolic exchange between these two cell types. These results suggest that satellite cells play an important role in neuronal maturation.
  • 知覚神経節細胞の形態変化と衛星細胞の働き
    松田正司, 小林直人, 樅木勝巳, 脇坂浩之, 森正彦, 村上信五
    解剖学雑誌 73 6 603 - 613 1998年 [査読有り]
  • Clinical and Experimental Nephrology 2 2 85 - 99 1998年
  • N Kobayashi, T Sakai
    CELL AND TISSUE RESEARCH 289 3 487 - 497 1997年09月 [査読有り]
     研究論文(学術雑誌) 
    The distribution of aortic intimal smooth muscle cells in the normal rat during postnatal development was studied by electron microscopy and by staining with fluorescence-labeled phalloidin. The phenotypes of intimal and medial smooth muscle cells were almost identical at first; however, during development, the former remained synthetic, whereas the latter became contractile. Confocal laser scanning microscopy was utilized to observe intimal and medial cells separately. Intimal smooth muscle cells were rarely observed in neonatal rats, but appeared by 10 days of age and increased during postnatal development. A combination of confocal and conventional fluorescent microscopy clearly demonstrated that the intimal smooth muscle cells were preferentially distributed in: (1) the right-lateral and dorsal wall of the upper thoracic aorta, (2) the left-lateral and ventral wall of distal two-thirds of the descending aorta, and (3) the downstream side of branch orifices. Intimal smooth muscle cells in group (1) were oriented randomly, whereas most in group (2) ran longitudinally. Intimal smooth muscle cells at branches in group (3) ran obliquely from the edges at the downstream side in an upstream direction. They tended to accumulate in regions of the aortic wall considered to be under high tensile stress.
  • S Inokuchi, Shirato, I, N Kobayashi, H Koide, Y Tomino, T Sakai
    KIDNEY INTERNATIONAL 50 4 1278 - 1287 1996年10月 [査読有り]
     研究論文(学術雑誌) 
    The sequence of morphological changes during foot process effacement in acute puromycin aminonucleoside (PAN) nephrosis was examined by means of NaOH maceration and freeze cracking for scanning electron microscopy (SEM). The micrographs of SEM and those of transmission electron microscopy (TEM) were quantitatively analyzed by computerized morphometry, and were correlated with renal function. On day 2 after PAN injection, the slit length was moderately decreased by both shortening and degradation of the foot processes. On day 4, membrane-bounded vesicles were scattered in the lamina rara externa. During foot process effacement, the basal surface of podocytes developed palm-like domains that represented the cytoplasmic areas between interdigitation. The decrease in the length of podocyte cell borders paralleled the decrease of 2 C-hour creatinine clearance. The developement of the palm-like domains on the basal aspects of podocytes estimated by distance class analysis closely correlated with the sudden onset of proteinuria. We conclude that foot process effacement in PAN nephrosis caused by the retraction and degradation of foot processes leads to the development of palm-like domains, which is correlated with podocyte detachment as well as massive proteinuria.
  • K InkyoHayasaka, T Sakai, N Kobayashi, Shirato, I, Y Tomino
    KIDNEY INTERNATIONAL 50 2 672 - 683 1996年08月 研究論文(学術雑誌) 
    The three-dimensional structure of the mesangium was analyzed by means of reconstruction from serial semithin and ultrathin sections of the rat glomerulus. The mesangial domains traced on light micrographs of semithin sections were transferred to styrene models, which were stacked up to reconstruct the whole mesangium. The reconstructed mesangium was tree-like in shape nd was divided into three lobes that were connected to the vascular pole by a slender neck. The glomerulus contained no islets of mesangium which were not connected to the vascular pole. The mesangium contained mesangial loops that were penetrated by capillaries. Reliability of the findings on the mesangial loops was ascertained by various methods including reconstruction of part of the mesangium from ultrathin sections. Electron microscopic observations revealed that the arms of the mesangial loops were frequently very slender and consisted of mesangial cell processes containing prominent bundles of actin filaments. The mesangial loops were distributed evenly within the mesangium. Considering previous reports showing about 400 capillary the rat glomerulus as well as the present findings, we the mesangial loops may change the distribution of intraglomerular blood flow bq dynamic contraction of the mesangial cells, in or serve a's an additional safety device to prevent the expansion of glomerular capillaries.
  • H Watanabe, T Sakai, N Kobayashi, Y Fukuda, K Yabuta
    PEDIATRIC NEPHROLOGY 10 4 461 - 466 1996年08月 研究論文(学術雑誌) 
    Distribution of glomerular basement membrane (GEM) outpockets and dimensional growth of glomeruli were studied in the maturing stage of rat glomeruli after completion of nephrogenesis. We observed the postnatal rat glomeruli from 5 to 60 days of age by transmission electron microscopy and estimated the structural development of glomeruli by computerized morphometry. On day 5, the GEM was double in structure, possessing an epithelial and endothelial lamina densa. After day 10, the lamina densa of the GEM was single and sent branches toward the epithelial side making outpockets. There is no change in the distributional pattern of the outpockets, at least from day 10 to day 60, although they decreased considerably in number between days 20 and 40. They were found almost exclusively on the peripheral surface of the glomerulus. The rat glomeruli increased in volume constantly in this period, and the capillary volume increased more significantly than the mesangial volume. The GEM surface area increased in parallel with the glomerular tuft volume. The growing mode of capillaries was different before and after day 40; namely before day 40 elongation was predominant, whereas after day 40 widening was more pronounced. These results indicate that if the outpockets are the other site of GEM assembly after fusion of double basement membranes, the GEM must be redistributed from the peripheral to the paramesangial site to enable elongation and branch formation of capillaries during the growth of glomeruli.
  • H NONOGUCHI, A OWADA, N KOBAYASHI, M TAKAYAMA, Y TERADA, J KOIKE, K UJIIE, F MARUMO, T SAKAI, K TOMITA
    JOURNAL OF CLINICAL INVESTIGATION 96 4 1768 - 1778 1995年10月 [査読有り]
     研究論文(学術雑誌) 
    We investigated immunohistochemical localization of V2 vasopressin receptor along the nephron using a specific polyclonal antibody. Staining was observed in some of thick ascending limbs and all of principal and inner medullary collecting duct (IMCD) cells. Not only basolateral but also luminal membrane was stained in collecting ducts, especially in terminal IMCD (tIMCD). To learn the functional role of luminal V2 receptor in tIMCD, we studied the luminal effects of arginine vasopressin (AVP) on osmotic water permeability (Pf), urea permeability (Pu), and cAMP accumulation using isolated perfused rat tIMCD. In the absence of bath AVP, luminal AVP caused a small increase in cAMP accumulation, Pf and Pu, confirming the presence of V2 receptor in the lumen of tIMCD. In contrast, luminal AVP inhibited Pf and Pu by 30-65% in the presence of bath AVP by decreasing cAMP accumulation via V1a or oxytocin receptors and by an unknown mechanism via V2 receptors in the luminal membrane of tIMCD. These data show that V2 receptors are localized not only in the basolateral membrane but also in the luminal membrane of the distal nephron, Luminal AVP acts as a negative feedback system upon the basolateral action of AVP in tIMCD.
  • SI SASAKI, N KOBAYASHI, T DAMBARA, S KIRA, T SAKAI
    ANATOMY AND EMBRYOLOGY 191 6 477 - 489 1995年06月 研究論文(学術雑誌) 
    The structure of the normal pulmonary arteries in the rat was studied with light and electron microscopy after use of a newly devised technique of perfusion fixation and tissue preparation. We distinguished two main types of artery in the rat lung on the basis of the structure of the media, an elastic artery and a muscular artery. The elastic artery was characterized by an abundance of extracellular matrix in the media and by an oblique arrangement of smooth muscle cells to connect neighboring elastic laminae. It was subdivided into two segments, a classical elastic and a transitional elastic segment. The muscular artery was distinguished by a paucity of extracellular matrix in the media and by a circumferential arrangement of smooth muscle cells (or pericytes) enclosing the lumina, and was subdivided into four segments, a thick muscular, an ordinary muscular, a partially muscular and a nonmuscular segment. The smooth muscle cells in the muscular artery contained well-developed microfilament bundles compared with those in the elastic artery. Structural differences in smooth muscle cells and in extracellular matrix in the media between the elastic and muscular arteries may re fleet the functional heterogeneity of pulmonary arteries in response to hypoxic pulmonary vasoconstriction and to vasoactive substances such as endothelium-derived relaxing and hyperpolarizing factors, and endothelin.
  • Kenjiro Kimura, Ryozo Nagai, Tatsuo Sakai, Masanori Aikawa, Makoto Kuro-o, Naoto Kobayashi, Isao Shirato, Tadashi Inagami, Masaya Oshi, Naoe Suzuki, Shigeyoshi Oba, Naobumi Mise, Akihiro Tojo, Yasunobu Hirata, Atsuo Goto, Yoshio Yazaki, Masao Omata
    Kidney International 48 2 372 - 382 1995年 [査読有り]
     研究論文(学術雑誌) 
    The contractility and distensibility of renal arterioles are important in the regulation of glomerular filtration. However, little is known regarding the characteristics of contractile proteins in these arterioles. Recently it was demonstrated that vascular smooth muscles contain two types of myosin heavy chain (MHC) isoforms, SM1 and SM2, which are unique molecular markers of smooth muscle cell phenotypes. SM1 is constitutively expressed in all types of smooth muscles, whereas SM2 exists only in mature smooth muscles. We characterized the expression of MHC isoforms as well as the ultrastructural myofilament assembly of renal arteriolar smooth muscles in human, rat and rabbit by immunohistochemical techniques. SM1 and α-smooth muscle actin were localized in both the preglomerular vessels (including the afferent arterioles) and efferent arterioles, whereas SM2 was present only in the preglomerular vessels. Renin-producing cells in the afferent arterioles (juxtaglomerular granular cells, JG cells) were positive for α-smooth muscle actin but negative for SM2. When renin synthesis was stimulated, the more proximal afferent arteriolar smooth muscles turned renin-positive and SM2 disappeared. Glomerular mesangial cells did not show immunoreactivities for SM1, SM2 or α-smooth muscle actin. The difference in MHC isoform expression in these arterioles was also reflected by ultrastructures the afferent arteriolar smooth muscles contained abundant myofilaments including thick filaments, whereas the efferent arteriolar smooth muscles had a few myofilaments composed only of thin microfilaments. The JG cells displayed a myofilament assembly similar to that in the efferent arteriolar smooth muscles. We conclude from these observations that smooth muscles in pre- and postglomerular arterioles, the glomerular mesangial cells and JG cells differ in phenotypes, suggesting that they may have different contractile properties which may be critically involved in the regulation of glomerular filtration.
  • 内皮細胞におけるアクチン線維の局在の時間的・空間的多様性
    小林直人
    東京大学学位論文(医学博士) 1995年 [査読有り]
  • N KOBAYASHI, T SAKAI
    CELL AND TISSUE RESEARCH 278 3 471 - 482 1994年12月 [査読有り]
     研究論文(学術雑誌) 
    Postnatal change in the distribution of actin filaments in endothelial cells was studied in the rat aorta by use of rhodamine-phalloidin staining and confocal laser scanning microscopy. Endothelial cells of the rat aorta possessed two populations of actin filament bundles, namely, peripheral bands at the cell border and stress fibers running longitudinally in the cytoplasm. Aortic endothelial cells of the neonatal rat contained only stress fibers, whereas those of the 10-day-old rat developed both peripheral bands and stress fibers. After 20 days of age, aortic endothelial cells had predominantly peripheral bands with occasional stress fibers around the branch orifices. During postnatal development the length density of stress fibers in aortic endothelial cells decreased, whereas individual stress fibers in endothelial cells were shortened. Electron-microscopic observation revealed that the high intercellular boundaries of aortic endothelial cells at birth decreased in height and developed cytoplasmic interdigitations after 20 days of age. The occurrence of peripheral bands at the cell border is thought to be closely related to formation of cytoplasmic interdigitation which strengthens the mechanical connection between endothelial cells against increasing transmural pressure. Expression of stress fibers in aortic endothelial cells of the neonatal rat is supposed to be affected by longitudinal elongation of the developing aorta, whereas their postnatal decrease is thought to be correlated with the change of fluid shear stress loaded on the aortic endothelium.
  • N KOBAYASHI, T SAKAI
    EXTRACELLULAR MATRIX IN THE KIDNEY 107 10 - 20 1994年 [査読有り]
     研究論文(国際会議プロシーディングス)
  • N KOBAYASHI, T SAKAI
    EUROPEAN JOURNAL OF CELL BIOLOGY 60 1 57 - 66 1993年02月 [査読有り]
     研究論文(学術雑誌) 
    The distribution of actin filaments (AFs) in arterial and arteriolar endothelial cells (ECs) was examined in situ by staining with rhodamine-phalloidin in the rat kidney after perfusion-fixation under physiological pressure. ECs possessed two populations of AF bundles, namely, peripheral bands (PBs) at the cell border and stress fibers (SFs) in the cytoplasm. The distribution of AFs was heterogeneous and exhibited three patterns in the arterial tree. In large arteries (> 120 mum in luminal diameter) ECs contained predominantly PBs in addition to occasional SFs mainly in the upstream side of cells (PB-pattern). In arteries with intermediate diameters (30-120 mum), both PBs and SFs were conspicuous (PB-SF-pattern). In small arteries and arterioles (<45 mum), SFs were prominent and PBs were rare (SF-pattern). In electron micrographs, we found that PBs were attached not to the lateral but to the basal plasma membrane via electron-dense materials and that SFs included two subpopulations, namely, thick basal and thin apical SFs. Morphometric analysis revealed that the length density of PBs was higher in segments with the PB-pattern than in those with the PB-SF-pattern. The length density of SFs was significantly higher in the SF-pattern than in the PB-SF-pattern, whereas there was little difference in this parameter among vessel segments with the SF-pattern. We suggest that PBs and SFs are fundamentally structures that anchor ECs on the substratum, and are differentiated according to their different mechanical burdens.
  • カニクイザル虫様筋の機能構成の定量分析
    榎謙一郎, 山内裕雄, 坂井建雄, 小林直人
    日本手の外科学会雑誌 9 868 - 872 1993年 [査読有り]
  • T SAKAI, N KOBAYASHI
    ANATOMY AND EMBRYOLOGY 186 5 467 - 476 1992年10月 研究論文(学術雑誌) 
    The structure of the intracellular actin filaments and the extracellular matrices was studied in the distal interlobular arteries in the rat kidney, employing three different morphological techniques, including rhodamin-phalloidin staining of cryosections, resorcin-fuchsin staining of paraffin sections, and a cold dehydration procedure for electron microscopy. The endothelial cells possess longitudinally running stress fibers. The inner elastic layer is composed of meshworks of elastic fibers encompassing numerous pores. The smooth muscle cells containing abundant actin filaments are arranged circumferentially around the vascular axis. The endothelial stress fibers are found mainly in the basal half of the endothelial cells, and anchor onto the basal cell membranes. The elastic meshworks send off longitudinal branch fibers to contact the endothelial cell membranes at the anchoring sites of stress fibers. In addition circumferential branches run toward the smooth muscle cells. The functional significance of the intracellular contractile apparatus and the extracellular tensile component in small arteries was discussed.
  • 解剖実習における遺体の処置および管理について
    坂井建雄, 本間敏彦, 村田一彰, 小泉憲司, 小林直人, 井田勝弘, 五十嵐弘一, 大根田辰子, 齋藤紘昭, 淺見一羊
    解剖学雑誌 67 2  1992年 [査読有り]
  • N KOBAYASHI, N HIROKAWA
    CELL MOTILITY AND THE CYTOSKELETON 11 3 167 - 177 1988年 研究論文(学術雑誌)

書籍

  • 大学教員のための授業方法とデザイン
    玉川大学出版部 2010年
  • 医学書院 医学大辞典 第2版
    医学書院 2009年
  • 人体の構造と機能および疾病の成り立ち/人体の構造と生理機能
    医歯薬出版株式会社 2007年
  • 愛媛大学FDハンドブック Vol.1「もっと!!授業を良くする -シラバス作成から成績評価まで-」第2版
    愛媛大学 教育・学生支援機構 教育企画室 2007年
  • 看護師国家試験のための看護学 Core Note
    医学芸術社 2005年
  • 愛媛大学FDハンドブック Vol.2「もっと!!授業を良くする -成功するスタディスキルの教え方-」
    愛媛大学教育開発センター 2005年
  • 医学書院 医学大辞典
    医学書院 2004年
  • からだの百科事典
    朝倉書店 2004年
  • "Cell Biology of the Podocyte", Microscopy Research and Technique, volume 57 issue 4, May 15, 2002
    Wiley-Liss 2002年
  • "Cell Biology of the Podocyte", Microscopy Research and Technique, volume 57 issue 4, May 15, 2002
    Wiley-Liss 2002年

講演・口頭発表等

  • 学生が「学ばんと欲する」クラスを実現するヒントとしての「問いかけ」@ワークショップ2「アクティブ・ラーニング模擬授業に参加してみよう」  [招待講演]
    小林 直人
    第51回日本医学教育学会大会 2019年07月 シンポジウム・ワークショップパネル(指名)
  • 今改めて医学教育におけるアクティブラーニングとは? @シンポジウム6「病理学の教育はこう変わる!」  [招待講演]
    小林 直人
    第108回日本病理学会総会 2019年05月 シンポジウム・ワークショップパネル(公募)
  • 解剖学教育を活性化する~医学教育全般を広く見据えた上で~ @ワークショップ「肉眼解剖学周辺の解剖学教育のあり方~特に、組織学・細胞生物学・発生学・神経解剖学教育の視点から~」  [招待講演]
    小林 直人
    第123回日本解剖学会総会・全国学術集会 2018年03月 シンポジウム・ワークショップパネル(指名)
  • 大学医学部を中心としたオール愛媛態勢での医療専門職学生の解剖学教育 @ワークショップ「医療専門職人体解剖学実習のこれから」  [招待講演]
    小林 直人
    日本解剖学会第72回中国・四国支部学術集会 2017年10月 シンポジウム・ワークショップパネル(指名)
  • アクティブ・ラーニングと教育観・学習観のパラダイムシフト @シンポジウム「アクティブ・ラーニングの実践例の紹介」  [招待講演]
    小林 直人
    第49回日本医学教育学会大会 2017年08月 シンポジウム・ワークショップパネル(指名)
  • アクティブ・ラーニングとは? @ワークショップ「明日からできるアクティブ・ラーニング~さまざまなアクティブ・ラーニングモデルを共有する~」  [招待講演]
    小林 直人
    第64回医学教育セミナーとワークショップ 2017年04月 シンポジウム・ワークショップパネル(指名)
  • 原点に還る 医学教育者に求められること  [招待講演]
    小林 直人
    医学教育 2016年07月 (一社)日本医学教育学会
  • 愛媛県の地域医療についての住民アンケート調査 特に医学生に望むこと  [通常講演]
    池田 祐一, 高橋 敏明, 高田 清式, 小林 直人
    医学教育 2016年07月 (一社)日本医学教育学会
  • 愛媛大学における初年次教育~プロフェッショナリズム、スタディ・スキル、リメディアル教育~ @シンポジウム「初年次教育を考える」  [招待講演]
    小林 直人
    日本医学教育学会第47回大会 2015年07月 シンポジウム・ワークショップパネル(指名)
  • 招聘講演、基礎医学教育:学生の“深い学び”を導くために  [招待講演]
    小林 直人
    第120回日本解剖学会全国学術集会(第92回日本生理学会大会と合同開催) 2015年03月 口頭発表(招待・特別)

MISC

  • 愛媛大学における新任教員の研修制度、特集「新任教員の研修制度」
    仲道雅輝、小林直人 IDE現代の高等教育 記事・総説・解説・論説等(学術雑誌) (619) 40 -44 2020年04月 [招待有り]
  • 泉 美貴, 小林 直人 薬学教育 記事・総説・解説・論説等(学術雑誌) 3 2019年09月 [査読有り][招待有り]
     
    薬学を含む本邦の医療系教育は,長く一方向性で受け身の講義が主体であった.しかし,昨今人口に膾炙しているアクティブ・ラーニングは,学修効果の点でも,近年における医療情報の激増により授業中にすべてを教え込むことが不可能になってきたという社会情勢からもその有用性が増している.本来,アクティブ・ラーニングにおける「アクティブ」とは身体的なアクティビティーの高さではなく,主体的で協同的な深い学びを指している.学生が深く思考するためには,教育者は良い「問い」を発することが重要で,どの手法を用いるかはさほど重要ではない.アクティブ・ラーニングは大教室でも充分に実施が可能で,「シンク・ライト・ペア・シェア」やミニッツ・ペーパーなどは比較的簡単に導入できる.アクティブ・ラーニングを成功させるための重要な要素として,①雰囲気作り,②発問による刺激,③話させる・書かせること,④学生相互での学び,⑤経験や事例を通じた学びがあげられる.
  • 四国地区大学教職員能力開発ネットワーク(SPOD)~10年間の活動と課題~
    小林 直人 IDE現代の高等教育 記事・総説・解説・論説等(大学・研究所紀要) 605 51 -55 2018年12月 [招待有り]
  • 愛媛大学における特色ある取り組み① 大学が一体となって教育改革を進める ~入試改革を例として~
    小林 直人 文部科学教育通信 記事・総説・解説・論説等(商業誌、新聞、ウェブメディア) 447 26 -27 2018年11月 [招待有り]
  • 解剖学の意味を考える どう理解し、どう教えるか 体幹の機能解剖を例にして
    小林 直人 理学療法えひめ 記事・総説・解説・論説等(学術雑誌) 31 1 -3 2018年01月 [招待有り]
  • 次世代の医師をどうやって育てるか
    小林 直人 愛媛医学 記事・総説・解説・論説等(学術雑誌) 36 (4) 215 -217 2017年12月 [招待有り]
  • 卒前・卒後の地域医療教育
    小林 直人 愛媛医学 記事・総説・解説・論説等(学術雑誌) 36 (2) 82 -84 2017年06月 [招待有り]
  • 医学教育のトピックス アクティブ・ラーニングのすすめ
    小林 直人, 永井 勅久, 山脇 孝 愛媛医学 記事・総説・解説・論説等(学術雑誌) 36 (1) 9 -16 2017年03月 [招待有り]
  • 【グラフィック・シラバスとカリキュラム・マップ 授業とカリキュラムを「見える化」しよう!】 カリキュラム・マップの作成とその効果
    小林 直人 看護教育 記事・総説・解説・論説等(学術雑誌) 56 (12) 1170 -1175 2015年12月 [招待有り]
  • 教養教育における内部質保証
    小林 直人 大学評価研究 記事・総説・解説・論説等(学術雑誌) 11 21 -34 2012年 [招待有り]
  • R. Tsujimura, H. Nabeka, T. Terashita, T. Shimokawa, S. Matsuda, N. Kobayashi ANNALS OF ANATOMY-ANATOMISCHER ANZEIGER 189 (2) 197 -201 2007年 
    During our dissection of a Japanese elderly female cadaver, a double aortic arch with a deformed trachea was found in the cadaver. The ascending aorta was bifurcated to form the left (anterior) and right (posterior) aortic arches. Encircling and compressing the trachea and esophagus, they confluenced into the descending aorta. We concluded that it was a case of the double aortic arch forming a vascular ring. In the vascular ring the trachea was deformed. In addition, the left innominate vein coursed under the aortic arches (subaortic Left innominate vein, SLIV) and crossed the mediastinum posterior to the ascending aorta and anterior to the trachea. (c) 2006 Elsevier GmbH. All rights reserved.
  • Seiji Matsuda, Takehiro Terashita, Tetsuya Shimokawa, Kyoujy Miyawaki, Yuji Miguchi, Takuya Doihara, Jie Chen, Shuang-yan Gao, Chun-yu Li, Min Wang, Zhong Wang, Bing Xue, Naoto Kobayashi, Kazuhiro Shigemoto NEUROSCIENCE RESEARCH 研究発表ペーパー・要旨(国際会議) 55 S157 -S157 2006年
  • Takuya Doihara, Yuji Miguchi, Hiroyuki Kaneko, Kyoji Miyawaki, Hiroaki Komori, Naoto Kobayashi, Jie Chen, Shuang-yan Gao, Chun-yu Li, Min Wang, Zhong Wang, Bing Xue, Takehiro Terashita, Tetsuya Shimokawa, Seiji Matsuda, Masato Nose ZOOLOGICAL SCIENCE 研究発表ペーパー・要旨(国際会議) 22 (12) 1471 -1471 2005年12月
  • Yuji Miguchi, Takuya Doihara, Hiromi Takata, Kyoji Miyawaki, Hiroaki Komori, Naoto Kobayashi, Jie Chen, Shuang-yan Gao, Chun-yu Li, Min Wang, Zhong Wang, Bing Xue, Takehiro Terashita, Tetsuya Shimokawa, Seiji Matsuda, Masato Nose ZOOLOGICAL SCIENCE 研究発表ペーパー・要旨(国際会議) 22 (12) 1471 -1471 2005年12月
  • Kyojy Miyawaki, Takuya Doihara, Yuji Miguchi, Hiroaki Komori, Kazuhiro Shigemoto, Katsurm Mominoki, Masahito Ogasawara, Chun-yu Li, Jie Chen, Shuang-yan Gao, Kyoko Saito, Takehiro Terashita, Tetsuya Shimokawa, Shouichiri Saito, Naoto Kobayashi, Seiji Matsuda, Masato Nose ZOOLOGICAL SCIENCE 研究発表ペーパー・要旨(国際会議) 21 (12) 1294 -1294 2004年12月
  • 小笠原正人, 斎藤正一郎, 山内広平, 小林直人, 松田正司, 前山一隆 日本薬理学雑誌 123 (3) 69P 2004年03月
  • UKリポート、イギリスの大学における教育改革-平成14年度 学長裁量経費 イギリス視察班 報告書-
    折本 素, 小林直人, 矢野博子 大学教育実践ジャーナル 記事・総説・解説・論説等(大学・研究所紀要) 2 1 -18 2004年
  • M Hattori, Y Akioka, H Chikamoto, K Tsuchiya, SY Gao, N Kobayashi, S Kagami, P Mundel, K Ito JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY 研究発表ペーパー・要旨(国際会議) 14 375A -375A 2003年11月
  • J Chen, L Pan, S Saito, T Terashita, K Saito, K Miyawaki, K Shigemoto, K Mominoki, S Matsuda, N Kobayashi, SG Gao JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY 研究発表ペーパー・要旨(国際会議) 14 358A -358A 2003年11月
  • 嗅上皮および鋤鼻器における神経栄養因子プロサポシンの発現性および発現部位についての検討
    齋藤 正一郎, 小林 直人, 脇坂 浩之, 樅木 勝巳, 宮脇 恭史, 齋藤 恭子, 陳 潔, 高 双燕, 佐野 輝, 松田 正司 解剖学雑誌 78 (Suppl.) 202 -202 2003年04月
  • 小林直人, 齋藤正一郎, 脇坂浩之, 樅木勝巳, 宮脇恭史, 齋藤恭子, 松田正司 生体の科学 記事・総説・解説・論説等(学術雑誌) 54 (2) 76 -81 2003年 [招待有り]
  • 足細胞(糸球体上皮細胞)の形態と病理
    小林直人, 齋藤正一郎, 脇坂浩之, 宮脇恭史, 齋藤恭子, 松田正司 腎と透析 記事・総説・解説・論説等(学術雑誌) 54 (2) 195 -200 2003年 [招待有り]
  • M Hattori, Y Akioka, N Iwamoto, H Chikamoto, K Tsuchiya, N Kobayashi, K Ito JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY 研究発表ペーパー・要旨(国際会議) 13 123A -123A 2002年09月
  • N Kobayashi, T Iwayama, T Takebayashi, S Saito, H Wakisaka, KJ Miyawaki, K Saito, S Matsuda, P Mundel JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY 研究発表ペーパー・要旨(国際会議) 13 298A -298A 2002年09月
  • ラット一次嗅覚系における神経栄養因子プロサポシンのmRNAの局在について
    齋藤 正一郎, 佐藤 康二, 齋藤 恭子, 樅木 勝巳, 留守 ゆう子, 佐野 輝, 脇坂 浩之, 宮脇 恭史, 小林 直人, 松田 正司 日本獣医学会学術集会講演要旨集 134回 170 -170 2002年08月
  • ラット嗅球の生後発生における神経栄養因子プロサポシンの組織化学的局在
    齋藤 恭子, 齋藤 正一郎, 脇坂 浩之, 宮脇 恭史, 樅木 勝巳, 佐野 輝, 小林 直人, 松田 正司 日本獣医学会学術集会講演要旨集 134回 171 -171 2002年08月
  • ラット神経系におけるプロサポシンの免疫組織化学的検討
    細田 能由, 山宮 公子, 小林 直人, 脇坂 浩之, 斉藤 正一郎, 佐野 輝, 松田 正司, 田辺 敬貴 解剖学雑誌 76 (5) 496 -496 2001年10月
  • ラットの一次嗅覚系における神経栄養因子プロサポシンの局在について
    齋藤 正一郎, 樅木 勝巳, 齋藤 恭子, 小林 直人, 脇坂 浩之, 宮脇 恭史, 佐野 輝, 松田 正司 日本獣医学会学術集会講演要旨集 132回 103 -103 2001年09月
  • ラット顔面神経切断後の顔面神経核内プロサポシンの増加
    鵜沼 香奈, 脇坂 浩之, 小林 直人, 齋藤 正一郎, 樅木 勝巳, 佐野 輝, 松田 正司 解剖学雑誌 76 (1) 92 -92 2001年02月
  • ラットの一次嗅覚系における神経栄養因子プロサポシンの局在についての組織化学的研究
    齋藤 正一郎, 脇坂 浩之, 宮脇 恭史, 齋藤 恭子, 佐野 輝, 小林 直人, 松田 正司 解剖学雑誌 76 (1) 145 -145 2001年02月
  • 神経系内でのプロサポシンの分布
    細田 能由, 島崎 由美子, 小林 直人, 脇坂 浩之, 齋藤 正一郎, 山宮 公子, 松田 正司, 佐野 輝, 田辺 敬貴, 荒木 伸一 解剖学雑誌 76 (1) 142 -142 2001年02月
  • 培養糸球体の上皮細胞からの情報とその限界
    小林直人 医学のあゆみ 記事・総説・解説・論説等(学術雑誌) (198) 697 -700 2001年 [招待有り]
  • 不死化細胞を用いた、細胞の形態形成へのアプローチ-腎糸球体足細胞を例にして-
    小林直人, 石原美佐, 山宮公子, 樅木勝巳, 脇坂浩之, 島崎由美子 愛媛医学 記事・総説・解説・論説等(学術雑誌) 17 (1) 1 -4 2000年 [招待有り]
  • 糸球体足細胞における微小管の構築と機能-培養細胞株を用いたアプローチ-
    小林直人, 樅木勝巳, 脇坂浩之, 松田正司, 坂井建雄 腎と透析 記事・総説・解説・論説等(学術雑誌) 47 (6) 849 -853 1999年 [招待有り]
  • 足細胞(糸球体上皮細胞)における細胞骨格の解析-培養細胞株を用いて-
    小林直人 医学のあゆみ 記事・総説・解説・論説等(学術雑誌) 190 (1) 31 -34 1999年 [招待有り]
  • N Kobayashi, P Mundel CELL AND TISSUE RESEARCH 書評論文,書評,文献紹介等 291 (2) 163 -174 1998年02月 
    This review discusses the role of microtubules in the formation of processes from neuronal and non-neuronal cells. In elongating axons of the neuron, tubulin molecules are transported toward the end of pre-existing microtubules, which may be nucleated at the centrosome, via a mechanism called slow axonal flow. Two different hypotheses are presented to explain this mechanism; the transport of soluble monomers and/or oligomers versus the transport of polymerized microtubules. The majority of tubulin seems to be transported as small oligomers as shown by the data presented so far. Alternatively, an active transport of polymerized microtubules driven by microtubule-based motor proteins is postulated as being responsible for the non-uniform polarity of microtubule bundles in dendrites of the neuron. Microtubule-associated proteins (MAPs) play a crucial role in stabilizing the microtubular arrays, whereas the non-uniform polarity of microtubules may be established with the aid of microtubule-based motor proteins. The signals activating centrosomal proteins and MAPs, resulting in process formation, include phosphorylation and dephosphorylation of these proteins. Not only neuronal cells, but also renal glomerular podocytes develop prominent cell processes equipped with well-organized microtubular cytoskeletons, and intermediate and actin filaments. A novel cell culture system for podocytes, in which process formation can be induced, should provide further evidence that microtubules play a pivotal role in process formation of non-neuronal cells.
  • N Kobayashi, J Reiser, W Kriz, P Mundel JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY 研究発表ペーパー・要旨(国際会議) 8 A1669 -A1669 1997年09月
  • 内皮細胞におけるアクチン線維の局在パターン
    小林直人, 坂井建雄 医学のあゆみ 記事・総説・解説・論説等(学術雑誌) 180 (7) 444 -445 1997年 [招待有り]
  • K HAYASAKA, T SAKAI, N KOBAYASHI, SHIRATO, I, Y TOMINO JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY 研究発表ペーパー・要旨(国際会議) 6 (3) 867 -867 1995年09月
  • 血管壁の微細構造を決めるのは何か
    小林直人 日経サイエンス 記事・総説・解説・論説等(その他) 25 (6) 14 -15 1995年 [招待有り]
  • H WATANABE, T SAKAI, N KOBAYASHI JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY 研究発表ペーパー・要旨(国際会議) 5 (3) 640 -640 1994年09月
  • 糸球体の力学的構築と濾過障壁の生後発達
    小林直人, 坂井建雄 発達腎研究会誌 記事・総説・解説・論説等(学術雑誌) 2 (1) 3 -10 1994年 [招待有り]
  • 血管系の微細構造とその調節機構
    小林直人, 坂井建雄 腎と透析 記事・総説・解説・論説等(学術雑誌) 37 (増刊) 274 -279 1994年 [招待有り]
  • 腎血管系の特性-微細形態からの考察
    小林直人, 坂井建雄 腎と透析 記事・総説・解説・論説等(学術雑誌) 35 (臨時増刊号) 274 -279 1993年 [招待有り]
  • 腎血管構築の特異性
    坂井建雄, 小林直人 循環制御 記事・総説・解説・論説等(学術雑誌) 13 (4) 567 -573 1992年 [招待有り]

受賞

  • 2017年01月 愛媛大学教育・学生支援機構 愛媛大学共通教育貢献賞
     JPN others 
    受賞者: 小林直人他;共同受賞
  • 2000年03月 日本解剖学会 日本解剖学会奨励賞
     JPN japan_society 
    受賞者: 小林直人

共同研究・競争的資金等の研究課題

  • 文部科学省:科学研究費補助金(基盤研究(C))
    研究期間 : 2012年 -2014年 
    代表者 : 小林 直人
     
    プロサポシン(Prosaposin)はサポシンA~Dの前駆体蛋白質であり、細胞質のリソソーム内で蛋白質水解性に切り出されて各サポシンを生ずる。一方、プロサポシンは単なるサポシンの前駆体蛋白質であるだけではなく、神経細胞においてプロサポシン自身が神経栄養因子として作用すると報告されている。しかしながらその作用機序の詳細は未解明である。当研究室はこれまでにラットプロサポシン遺伝子を培養細胞株にトランスフェクションし、ラットプロサポシン蛋白質を過剰発現させる事に成功している。新たにヒトプロサポシン遺伝子をヒト神経培養細胞株SH-SY5Yにトランスフェクションし、ヒトプロサポシン蛋白質を過剰発現させ、細胞の形態変化を観察した。本実験ではヒトプロサポシンcDNA全長を発現するベクターの他、赤色蛍光蛋白質DsRedとの融合蛋白質を発現するベクターも作成した。DsRed融合蛋白質は蛍光顕微鏡で検出可能であるため、培養しながら発現細胞のタイムラプス撮影が可能である。従来は化学的に遺伝子導入をおこなってきたが、今回購入した電気的導入装置のお陰で、実験効率は格段に上がった。プロサポシンの神経保護作用の仕組みは未解明であるが、本研究を通してプロサポシンの機能解明が進む事が期待される。
  • 文部科学省:科学研究費補助金(基盤研究(C))
    研究期間 : 2008年 -2010年 
    代表者 : 小林 直人, 濱田 文彦
     
    平面内細胞極性形成シグナルは、細胞膜上に存在するFrizzled-1受容体を中心に構成されるが、この受容体と結合するリガンド分子は不明である。本研究では未知のリガンド分子の同定を目的とし、培養細胞を用いて受容体とリガンド分子との結合の評価系を確立した。種々の膜タンパク質をコードする遺伝子を細胞株で発現させるコンストラクトも構築中であり、これらの膜タンパク質と同受容体との結合について検討を進める。
  • 文部科学省:科学研究費補助金(萌芽研究)
    研究期間 : 2007年 -2008年 
    代表者 : 松田 正司, 小林 直人
     
    二分脊椎は下肢の麻痺変形を原因とする歩行障害等が出生後に顕在化する重要な疾患である。二分脊椎症の病態は十分に解明されているとは言い難く、治寮法は確立していない。その一因として運動障害を示す適切な動物モデルはほぼ無であったことが考えられる。申請者の研究室では脊椎再開裂手術により歩行異常を示すモデルを世界で始めて開発しその病態解明を続けている。一方、プロサポシンに関しては共同研究者の佐野輝による世界に先駆けた研究に端を発し、虚血海馬、神経切断後脊髄運動神経の障害に対する治療効果を報告してきた。本研究は二分脊椎の病態を明らかにするとともにプロサポシン分子由来合成ペプチド(PS12)がニワトリの二分脊椎が治癒するか否かを明らかにすることが目的で有る。本研究の結果、歩行障害を起こす二分脊椎モデル動物において、二分脊椎様状態の脊髄で運動神経細胞の発生プロセスに異常が起こっている可能性をIslet-1疫染織により示した。正常ではIslet-1陽性ニューロンは初期に増加し5日にピークに達し、その後減少する。一方、二分脊椎ではこのようなピークは認められず、緩やかな減少を続けていく。発生初期と8日を見ると両群にはほとんど違いが無いように見えるが、5日前後における正常脊椎のIslet-1陽性ニューロンの急激な増減は重要な所見である。また、二分脊椎においては運動神経細胞とその突起以外にも異常が認められることを示した。さらに、PS12はニワトリ培養神経細胞に対し生存刺激作用を有し、PS12投与により二分脊椎の病態が一部軽減することを認めている。
  • 文部科学省:科学研究費補助金(基盤研究(C))
    研究期間 : 2006年 -2007年 
    代表者 : 小林 直人
     
    本研究では、ヒト骨芽細胞の培養系を用いて、金属表面などでの細胞の動的な挙動を客観的に解析して医療材料の生体親和性に関する新しい評価方法を確立することを目的とし、以下の項目に関して成果をあげた。1)ヒト骨芽細胞の培養株を用いて、金属材料(チタン)の表面での培養細胞の挙動を、細胞接着と細胞運動の双方から解析し、この結果を英文論文として国際誌に発表した(Li C, et. al., 2006)。さらに、細胞の移動速度を制御するシグナル伝達系の解析のため、種々のシグナル伝達系に対する阻害剤や賦活剤をヒト骨芽細胞の培養系に負荷し、その影響を解析した。その結果、骨芽細胞の移動には、アクチン系を中心とした細胞骨格の動的な制御が必要であること、さらに移動の方向を制御する機構にはIP3キナーゼが関与すること、が示唆された。2)ヒト骨芽細胞の一次培養系(市販のもの)は株化された骨芽細胞とは異なる挙動を示すことが明らかとなっていた。そこで細胞運動の評価方法をいくつか試し、一次培養系の細胞に適したものを見いだした。実際には、細胞をまきこむ際にプラスティックのディッシュの中央にガラス製のスライドグラスをおき、それを鉛の重りで押さえておくと、スライドグラスを載せた部分では細胞がディッシュに接着せず、後にスライドグラスを取り除くことで、細胞が巻き込まれていない四角形の領域を作ることができた。この領域に侵入する細胞の移動速度を計測することにより、一次培養系の細胞でも再現性良く細胞の移動度を解析することが可能になった。
  • 文部科学省:科学研究費補助金(基盤研究(B))
    研究期間 : 2002年 -2005年 
    代表者 : 松田 正司, 樅木 勝巳, 佐野 輝, 小林 直人, 齋藤 正一郎
     
    本研究テーマは、手術部位と歩行異常との関係、運動神経の異常、脊髄の神経細胞におけるホメオドメイン転写調節因子発現の変化、感覚神経細胞の異常、感覚回路の異常等より成る。(1)本研究では、ニワトリ胚神経管の蓋板切開により二分脊椎胚を作製し、これを孵化させることに成功した。孵卵開始後3日目のニワトリ胚の背側要仙部で神経管を再開裂させ、二分脊椎モデルを作製した。(2)二分脊椎ヒヨコはヒト二分脊椎症患者の併発症、すなわち後肢運動機能障害に似た症状を示した。(3)ニワトリ胚二分脊椎モデルの脊髄前角運動ニューロンの発生をLIMホメオドメイン型転写因子の1つであるIslet-1を用いて免疫組織化学的に検討した。孵卵開始後3日目に神経管を再開裂させ、その後、発生を継続させ、孵卵開始後4日から12日の各発生段階において採材した。採取した胚は固定後、定法に従い、第3腰髄節横断面での凍結切片を作製した。一次抗体としてマウス抗Islet-1モノクローナル抗体を用いDABで染色し、陽性細胞数をカウントした。正常発生群ではIslet-1陽性細胞数は孵化開始後4.5日においてピークを迎え、その後徐徐に減少し、孵化開始後12日以降では、陽性細胞数の大きな変化は観察されなかった。一方、二分脊椎群においては、Islet-1陽性細胞数の増加が正常発生群に比べ遅延し、孵化開始後5.5日において陽性細胞数が最多となった。正常発生群においては脊髄前角のlateral motor column(LMC)の内側の細胞群(LMCm)にのみIslct-1に対する免疫染色性が観察されたが、二分脊椎群では、LMC全域に染色性が観察された。(4)感覚線維の走行について検討した。正常と二分脊椎において後根神経を切断してその変成線維染色により走行を追跡した。感覚線維の走行は両者で大きく異なり、このことが二分脊椎の病態の一つであると考えられる。
  • 医学教育
    研究期間 : 2005年
  • Medical Education
    研究期間 : 2005年
  • 文部科学省:科学研究費補助金(基盤研究(C))
    研究期間 : 2002年 -2004年 
    代表者 : 小林 直人, 齋藤 正一郎
     
    腎臓で尿が生成される過程の第一段階は糸球体濾過障壁を介した選択的濾過であり、この濾過機能が破綻することが難治性慢性腎疾患の一因である。このような腎疾患の病態生理の解明と新しい治療法の確立のためには、信頼できる疾患モデルの樹立が不可欠である。本研究では、新規の疾患モデルの構築のための「糸球体濾過障壁のin vitroにおける再現」を試みた。糸球体濾過障壁の主要な構成要素は足細胞(糸球体上皮細胞)と糸球体基底膜である。我々はこれまでに、本研究の海外共同研究者らから供与を受けた足細胞の条件的不死化細胞株を用いて、足細胞の形態形成(突起形成、細胞間接着形成)を調節する細胞内シグナル伝達系について研究し、RHOファミリー低分子量G蛋白の関与を明らかにした(Kobayashi N,2002、Kobayashi et al.,2004、Gao et al.,2004、Gao et al.,投稿準備中)。糸球体濾過障壁の一部であるスリット膜は、足細胞の間に形成される非常に特殊な細胞間接着装置であり、スリット膜の異常は蛋白尿と密接に関係している。我々は、薬理学的にRhoAを阻害することでRHOファミリー低分子量G蛋白(RhoA, Rac1,Cdc42)の間の活性のバランスが変化し、その結果として足細胞間の細胞接着装置がより強固になることを明らかにした。これは、必ずしも複合培養系を用いなくても糸球体濾過障壁を培養下で再現できる可能性を示すものである。また、足細胞独自の形態である非常に発達した細胞突起の形成についても、RhoAやその下流で働くキナーゼであるROCKを阻害することにより、培養下でin vivoの形態に類似した突起を誘導することができた。なお、足細胞の形態形成に関する研究に重点を置いたため、計画当初予定していた糸球体内皮細胞の細胞株の樹立は現在も進行中である。
  • 文部科学省:科学研究費補助金(奨励研究(A))
    研究期間 : 2000年 -2001年 
    代表者 : 小林 直人
     
    血管の機能や微細構造はその部位によって多様であり、その内面を覆う内皮細胞も、臓器・組織ごとにそれぞれ異なった性質を有しているはずである。そこで本研究では、「臓器・組織特異的な内皮細胞」として、腎糸球体毛細血管内皮細胞の培養株を樹立し、その細胞種特異的な性質を精密に解析することを目的とする。本補助金の支援を受けて昨年度より、2系統のトランスジェニックマウス(分与および購入)を掛け合わせてF1の仔を得、そこから腎糸球体を単離して培養している。現在、糸球体由来の細胞の中から内皮細胞だけを選択的に選び出してクローン化・株化する作業を進めている。現時点ではまだ培養株の樹立には至っていない。今後、早期に培養株を確立し、それを用いて微細形態や細胞内情報伝達系の特性に関して解析する予定である。なお、マウスの繁殖と掛け合わせは、愛媛大学医学部附属実験動物施設のトランスジェニックマウス専用飼育室にて、本学の「実験動物取り扱い指針」に沿って行っている。本研究に関連して、糸球体足細胞の培養株を用いた細胞機能の解析方法について総説を発表した(小林,200;Kobayashi,2002)(足細胞は、内皮細胞とともに糸球体濾過障壁を構成している。この培養株は「条件的不死化遺伝子」を持つトランスジェニックマウスから樹立されたもので、本研究のプロトタイプとなっている)。さらに、足細胞の培養系を用いてその形態形成の仕組みを分子レベルで解明し、これらの成果を国際誌に発表した(Kobayashi et al.,2001a,2001b)。
  • 文部科学省:科学研究費補助金(基盤研究(C))
    研究期間 : 1998年 -2000年 
    代表者 : 坂井 建雄, 小林 直人, 工藤 宏幸, 小泉 憲司, 栗原 秀剛
     
    1)足細胞の形態形成とスリット膜の分子構築:足細胞の細胞骨格の要素の一つとして、あらたに中間径線維に付属するタンパク質を認識するモノクローナル抗体P-31を見いだし、そのタンパク質であるp250を同定した。また微小管付属タンパク質の一種で足細胞を含め多くの細胞で発現するMAP4が、ニューロンに特異的なMAP3と同一のものであることを証明した。スリット膜の構成タンパク質であるnephrinは、もともとフィンランド型の先天性ネフローゼ症候群の原因遺伝子として見いだされたものであるが、そのラット型のものが、かつて新潟大の腎研究施設で作られた5-1-6抗体が認識するタンパク質そのものであることを証明した。スリット膜の分子構築全般についても報告した。2)糸球体の支持構造としての間質の特性:メサンギウム細胞の収縮力や細胞外基質産成に対するアンジオテンシンIIの影響を知るために、その受容体遺伝子をノックアウトしたマウスの糸球体構造を解析した。また腎臓の間質全般の構成細胞ならびに間質と上皮との界面をなす基底膜についても調べている。さらに腎臓の間質の特異性を知る一助として、肝臓のグリソン鞘の間質を微細形態学的に解析し、そこに含まれるコラーゲン線維に2つのポピュレーションがあることを見いだしている。3)腎血管系の進化・発生:腎臓は血管系と密接な関係を有する臓器であり、その発生・進化の過程は、密接に相関する。足細胞の構造的な多様性は、腎血管系の発生進化という文脈の中で理解すべきものである。本研究では、血管系の発生過程を知るモデルとして、胎盤の幹絨毛内の血管を電子顕微鏡を用いて観察し、そこに含まれる動脈と静脈を初めて同定することに成功するとともに、その微細構造的な特徴を報告した。また腎血管系の特性について、発生について、進化についても報告した。
  • 文部科学省:科学研究費補助金(奨励研究(A))
    研究期間 : 1998年 -1999年 
    代表者 : 小林 直人
     
    腎糸球体足細胞は、形態学的にもっとも分化した細胞の一つである。本研究では、足細胞の培養株を用いて、この細胞の持つ複雑な突起の形態がどのようにして形成されてゆくかを明らかにしようとしている。本年度には特に、微小管の機能を制御する因子について研究した。細胞質内で伝達される情報としては、蛋白質のSer/Thr残基におけるリン酸化が足細胞における微小管機能調節の鍵を握ることが明らかになった。すなわち、蛋白質キナーゼの阻害剤(H-7,K-252a)は突起形成を促進するのに対し、脱リン酸化酵素の阻害剤(okadaic acid)はこれを抑制した(kobayashi et al.,投稿中)。また、細胞外からの情報としては、さまざまな細胞外マトリックス蛋白がそれぞれ異なった影響を与えることが明らかとなった。すなわち、ラミニン(正常な糸球体基底膜の構成分子の一つである)は突起形成を促進したが、フィブロネクチン(通常は糸球体基底質に含まれない)はこれを抑制した(kobayashi et al.,投稿中)。これに関連して、細胞外マトリックスに関する総説を発表した(小林&坂井,2000)。微小管の機能、特にその重合を直接調節しているのは、微小管関連蛋白MAPsと呼ばれる一群の蛋白である。足細胞に発現している微小管関連蛋白としてこれまでに、MAP3とMAP4が別々に報告されていた。我々は分子レベルの解析により、その二つの蛋白が同一であることを示して論文として報告した(kobayashi et al.,2000)。また、足細胞における微小管の機能についての総説(小林ら,1999,1999)や、足細胞全般に関して考察した総説を発表した(小林&坂井,1999)。さらに本研究に関連して、突起形成に共通する機構を考察した総説を発表した(小林ら,1999;Matsuda et al.,1999,1999)。
  • 肉眼解剖学と解剖学教育
    研究期間 : 1998年
  • Macroscopic Anatomy and Abatomical Education
    研究期間 : 1998年
  • 腎糸球体足細胞の微細形態に関する研究
    研究期間 : 1996年
  • Study on the Ultrastructure of Podocytes in the renal Glomerulus
    研究期間 : 1996年
  • 文部科学省:科学研究費補助金(奨励研究(A))
    研究期間 : 1995年 -1995年 
    代表者 : 小林 直人
     
    蛍光標識したファロイジンにより大動脈内膜のアクチン線維を染色し、得られた蛍光顕微鏡像をモンタージュとして再構成して、ラット大動脈における内膜平滑筋の分布を可視化した。この結果、以下の結果を得た。1.正常ラット大動脈では、内膜平滑筋は出生時にはほとんど認められず、生後10日ごろから出現が確認される。2.内膜平滑筋細胞の出現する部位はほぼ一定しており、生後発達が進んで内膜において平滑筋細胞の占める面積が増加しても、分布パターンは維持される。分布が顕著に認められる領域は、(1)大動脈弓内側面から胸大動脈前半部の背側面にかけて、(2)胸大動脈後半部から腹大動脈前半部にかけての腹側面、(3)各動脈分岐部の遠位側の縁、であった。さらに、細胞の走行の方向についても、各領域ごとに一定の規則性があった。逆に、動脈分岐部の上流側では、内膜平滑筋細胞の極めて少ない領域が認められた。また、電子顕微鏡による観察から、以下の所見を得た。3.正常ラット大動脈において、内膜平滑筋細胞と中膜平滑筋細胞は、当初はほぼ同様の表現型phenotypeを示す。4.生後発達が進むにつれて、前者は合成型の表現型を、後者は収縮型の表現型を示すように分化してゆく。内膜平滑筋細胞は、動脈硬化の発症と密接に関係していると考えられるている。しかし、今回の結果が示す正常ラット大動脈における内膜平滑筋細胞の分布パターンは、当初の予想に反して、よく知られているヒト動脈硬化病変の好発部位とは明らかに異なっていた。内膜平滑筋細胞の分布は、壁の張力が局所的に大きくなる部位と一致すると思われる。内膜平滑筋は、その収縮により、不均一な張力による局所的な血管壁のゆがみを修正するように機能する可能性がある。(以上の研究結果は、英文の論文にまとめて投稿中である。)
  • 文部科学省:科学研究費補助金(一般研究(C))
    研究期間 : 1994年 -1995年 
    代表者 : 坂井 建雄, 小泉 憲司, 小林 直人
     
    弾性動脈である大動脈の内皮細胞について、アクチンフィラメントの配列を調べたところ、筋性動脈である腎臓内動脈の内皮細胞と基本的な差はなく、むしろ筋性動脈領域内での部域差の方が顕著であることがわかった。それに対し、大動脈において、内皮細胞のアクチンフィラメントが、生後発達期にstress fiberパターンからperipheral bandパターンへと、配列を変えることが明らかになった。また腎臓内動脈について、内皮下の基底膜の構造を調べたところ、直径に対応して、微細構造上の変化があることが見つかった。弾性動脈と筋性動脈の中膜については、一つの臓器内で両者の移行部の観察できる肺動脈を対象に、微細構造を観察した。その結果、肺動脈に関しては、弾性動脈から筋性動脈への移行が、急激に起こり、基本的に両者の間に移行形が存在しないことが明らかになった。また腎臓内の動脈について、中膜平滑筋の重鎖インフォームを調べたところ、輸入細動脈と輸出細動脈で、異なる型を示すことが明らかになった。すなわち、輸入細動脈は、成体型の大きな収縮力をもつと考えられるSM2を発現し、これに対し輸入細動脈は、胎児型で小さな収縮力を持つと考えられるSMembを発現していた。糸球体については、特に足細胞の細胞骨格を、実験的なモデルを使って解析した。単離潅流腎のモデルで、糸球体濾過障壁が伸展性を有することが明らかになったが、足細胞の細胞骨格には、その伸展性を調節する役割があると考えられる。また馬杉腎炎のモデルで、足細胞の足突起の消失に伴って、アクチンフィラメントが再配分され、細胞が基底膜から剥がれるのに抵抗する力を発生する役割を持つことが明らかになった。糸球体の成長に伴い、おもに毛細血管の総延長が延びることにより糸球体の体積が増大すること、またその際、糸球体基底膜の表面積の拡大が並行して起こることがわかった。そして基底膜の拡大装置と考えられるoutpockets構造が、糸球体の辺縁部にのみ分布するという所見を得たが、これは糸球体基底膜が糸球体の内部で再配分されることを示唆している。
  • 文部科学省:科学研究費補助金(奨励研究(A))
    研究期間 : 1994年 -1994年 
    代表者 : 小林 直人
     
    1.出生直後から60日令までのラット大動脈を用い、アクチン線維(AF)を特異的に染色するphalloidinで蛍光染色した。観察は、光学的断層像の得られる共焦点レーザー走査顕微鏡を用いて行なった。また、低温脱水法で処理した胸部大動脈の標本を、透過型電子顕微鏡で観察した。さらに内皮細胞の光顕・電顕写真上で、種々の形態計測を行ない、発達過程を追って統計学的な比較を行なった。2.出生直後の大動脈内皮細胞では、成体の場合と異なり、大動脈の全域で長いstressfiber(SF)が局在していた。10日令では、SFとperipheralband(PB)が共在していた。20日令以降では、PBが主体でSFが散在する成体のパターンになり、動脈の分岐の周辺では短いSFが発達していた。また、AFの局在パターンの変化と平行して、内皮細胞間の接着面の超微細構造が、平坦な面から複雑にかみあった形へと変化した。形態計測の結果、個々のSFの長さや線密度(単位面積当たりの総延長)は、20日令以降の内皮に比べて、10日令以前の方が有意に長いことが示された。また、生後発達の過程で、電顕切片像上での内皮細胞間の接触部分が長くなるのに対して、内皮細胞の厚さは減少し、接着面の形状が次第に複雑になってゆく過程が定量的にも示された。細胞周辺部のAFは、主に細胞間接着装置の周辺や細胞質の突起の中に局在していた。3.大動脈内皮細胞は、生後発達の過程での血管壁の張力の増大に対抗して、細胞周囲に配置したPBのサポートによって、隣り合う細胞間の細胞質のかみあいを形成して細胞間の接着を強化する、と考えられる。また、出生直後の内皮細胞に長いSFが発達している理由については、ズリ応力に対する反応の感度が高い、細胞分裂時にはSFの方が都合が良い、出生前後の大動脈の伸長に関係している、等の可能性が考えられた。
  • 文部科学省:科学研究費補助金(一般研究(B))
    研究期間 : 1991年 -1993年 
    代表者 : 坂井 建雄, 小林 直人, 小泉 憲治
     
    血管を構成する細胞および細胞外基質の力学的な役割について、研究の進んでいる糸球体、および動脈を研究対象として、形態学的に解析した。本研究ではまず、(1)糸球体の力学構造の要であるメサンギウム細胞の役割を、さまざまな実験モデルを用いて解析した。メサンギウム細胞では、収縮装置としての細胞骨格が、糸球体基底膜を内向きに牽引している。この収縮能が障害された単離潅流腎のモデルでは、毛細血管が拡張するという構造変化が見られた。(2)糸球体濾過障壁の力学的な主体は基底膜であるが、この基底膜の微細構造を、糸球体ならびに腎臓の他の部分で観察した。基底膜は、その力学的な環境によって構造が多様であることを明らかにした。(3)腎臓内の動脈において、細胞骨格と細胞外基質の構造を観察したところ、内皮細胞のアクチンフィラメントの配列および内弾性板の形状が、部位によって多様性を示すことを明らかにした。とくに小葉間動脈遠位部では、内皮細胞のアクチンフィラメントが応力線維の形をとり、内弾性板の網目状構造との間に機械的な結合を作ることが明らかになった。これにより、腎臓内の動脈で、内皮細胞の収縮装置が、動脈の構造保持の役割をもち、さらに能動的に血管の収縮状態を調節する可能性が示唆された。

委員歴

  • 2008年 - 現在   日本医学教育学会   評議員   日本医学教育学会
  • 2000年 - 現在   日本解剖学会   学術評議員   日本解剖学会

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