研究者データベース

稲波 修(イナナミ オサム)
獣医学研究院 獣医学部門 応用獣医科学分野
教授

基本情報

所属

  • 獣医学研究院 獣医学部門 応用獣医科学分野

職名

  • 教授

論文上での記載著者名

  • Inanami Osamu

ホームページURL

J-Global ID

プロフィール

  • 学歴

    昭和53年3月 愛知県立千種高等学校卒業

    昭和53年4月 北海道大学理類入学

    昭和57年3月 北海道大学獣医学部卒業

    昭和62年3月 北海道大学大学院獣医学研究科博士後期課程卒業


    職歴

    昭和62年4月(財)東京都老人総合研究所第二生理学研究室 助手

    昭和63年9月(財)東京都老人総合研究所自律神経部門 研究員

    平成5年1月 岩手大学農学部獣医学科家畜生理学講座 助手

    平成6年10月 岩手大学農学部獣医学科家畜生理学講座 助教授

    平成7年7月 米国スクリプス医学研究所 文部省在外研究員(12ヶ月間)

    平成8年9月 北海道大学大学院獣医学研究科 環境獣医科学講座 放射線学教室 助教授

    平成19年5月 北海道大学大学院獣医学研究科 環境獣医科学講座 放射線学教室 教授

    平成29年5月 北海道大学大学院獣医学院 応用獣医科学講座 放射線学教室 教授


    研究テーマ

    昭和62年〜平成4年まで:「脳循環の自律神経調節」「脳血流と老化」

    平成5年から平成8年まで:「白血球のNADPHオキシダーゼの生化学的研究」

    平成8年〜現在まで:「放射線の生物影響」「癌の放射線・制がん剤による実験的治療」「磁気共鳴法を用いた腫瘍低酸素領域の検出」など

研究キーワード

  • NADPHオキシダーゼ   好中球   部位特異的スピンラベル法   アポトーシス   プリオン   牛   電子スピン共鳴法   ネクローシス   生体防御   細胞死   構造生物学   リンパ腫   BSE   白血病   カスパーゼ   突然変異   スーパーオキシド   株化ガン細胞   シグナル伝達   制癌剤   電子スピン共鳴(ESR)   抗酸化剤   脳   活性酸素   双極子相互作用   人獣共通感染症   リン酸化酵素   蛋白リン酸化酵素   CJD   銅イオン   放射線生物学   生理化学   Radiation Biology   physiological chemistry   

研究分野

  • ライフサイエンス / 獣医学 / 放射線学
  • ライフサイエンス / 獣医学 / 生理学・生化学
  • 環境・農学 / 環境政策、環境配慮型社会 / 放射線生物学
  • 環境・農学 / 環境影響評価 / 放射線生物学
  • ライフサイエンス / 放射線科学 / 放射線生物学

職歴

  • 2017年04月 - 現在 北海道大学大学院獣医学院 応用獣医科学講座 放射線学教室 教授
  • 2008年05月 - 2017年03月 北海道大学大学院獣医学研究科 教授
  • 1996年11月 - 2007年04月 北海道大学大学院獣医学研究科 助教授
  • 1994年10月 - 1996年10月 岩手大学農学部 助教授
  • 1995年07月 - 1996年04月 スクリプス研究所 文科省在外研究員
  • 1987年04月 - 1988年08月 財)東京都老人総合研究所 助手
  • 財)東京都老人総合研究所 研究員

学歴

  • 1982年04月 - 1994年03月   北海道大学   大学院獣医学研究科   機能形態学専攻 修士課程修了
  •         - 1987年   北海道大学
  • 1978年04月 - 1982年03月   北海道大学   獣医学部   獣医学科 卒業
  •         - 1982年   北海道大学
  •         -   北海道大学   大学院獣医学研究科   形態機能学専攻 博士課程修了

所属学協会

  • 日本酸化ストレス学会   日本基礎老化学会   日本生化学会   電子スピンサイエンス学会   日本放射線影響学会   日本獣医学会   

研究活動情報

論文

  • Tomoki Bo, Tohru Yamamori, Kumiko Yamamoto, Masaki Fujimoto, Hironobu Yasui, Osamu Inanami
    Biochemical and biophysical research communications 522 1 144 - 150 2020年01月29日 [査読有り][通常論文]
     
    Mitochondrial dynamics are crucial for cellular survival in response to various stresses. Previously, we reported that Drp1 promoted mitochondrial fission after x-irradiation and its inhibition resulted in reduced cellular radiosensitivity and mitotic catastrophe. However, the mechanisms of radiation-induced mitotic catastrophe related to mitochondrial fission remain unclear. In this study, we investigated the involvement of cellular ATP production, ROS generation, and Ca2+ levels in mitotic catastrophe in EMT6 cells. Knockdown of Drp1 and Fis1, which are mitochondrial fission regulators, resulted in elongated mitochondria and significantly attenuated cellular radiosensitivity. Reduced mitochondrial fission mainly decreased mitotic catastrophe rather than necrosis and apoptosis after irradiation. Cellular ATP contents in Drp1 and Fis1 knockdown cells were similar to those in control cells. N-acetylcysteine and 2-glucopyranoside ascorbic acid have no effect on mitotic catastrophe after irradiation. The cellular [Ca2+]i level increased after irradiation, which was completely suppressed by Drp1 and Fis1 inhibition. Furthermore, BAPTA-AM significantly reduced radiation-induced mitotic catastrophe, indicating that cellular Ca2+ is a key mediator of mitotic catastrophe induction after irradiation. These results suggest that mitochondrial fission is associated with radiation-induced mitotic catastrophe via cytosolic Ca2+ regulation.
  • Hironobu Yasui, Daisuke Iizuka, Wakako Hiraoka, Mikinori Kuwabara, Akira Matsuda, Osamu Inanami
    Nucleosides, nucleotides & nucleic acids 39 1-3 439 - 452 2020年 [査読有り][通常論文]
     
    The combination of low dose of radiation and an anticancer drug is a potent strategy for cancer therapy. Nucleoside analogs are known to have a radiosensitizing effects via the inhibition of DNA damage repair after irradiation. Certain types of nucleoside analogs have the inhibitory effects on RNA synthesis, but not DNA synthesis, with multiple functions in cell cycle modulation and apoptosis. In this review, the most up-to-date findings regarding radiosensitizing nucleoside analogs will be discussed, focusing especially on the mechanisms of action.
  • Mayuko Hashimoto, Natsuko Saito, Haru Ohta, Kumiko Yamamoto, Asuka Tashiro, Kosuke Nakazawa, Osamu Inanami, Hiroshi Kitamura
    Physiological reports 7 14 e14193  2019年07月 [査読有り][通常論文]
     
    Ubiquitin-specific protease 2 (USP2) is considered to participate in the differentiation of myoblasts to myotubes, however, its functions in myoblasts under growth conditions remain elusive. In this study, we analyzed the physiological roles of USP2 in myoblasts using Usp2 knockout (KO) C2C12 cells as well as a USP2 specific inhibitor. In addition to the disruption of differentiation, clustered regularly interspaced short palindromic repeats/Cas9-generated Usp2KO cells exhibited inhibition of proliferation compared to parental C2C12 cells. Usp2KO cells reduced the accumulation of intracellular adenosine triphosphate (ATP) content and oxygen consumption. Moreover, Usp2KO cells had fragmented mitochondria, suggesting that mitochondrial respiration was inactive. The deficiency of Usp2 did not affect the enzymatic activities of respiratory chain complexes I, III, IV, and V. However, mitochondrial membrane permeability-evaluated using calcein AM-cobalt staining-was increased in Usp2KO cells. The membrane potential of Usp2KO cells was clearly decreased. Usp2KO cells accumulated reactive oxygen species (ROS) in the mitochondria. The USP2-selective inhibitor ML364 also increased the levels of mitochondrial ROS, and modulated the membrane potential and morphology of the mitochondria. These effects were followed by a decrement in the intracellular content of ATP. Based on these findings, we speculate that USP2 may be involved in maintaining the integrity of the mitochondrial membrane. This process ensures the supply of ATP in myoblasts, presumably leading to proliferation and differentiation.
  • Yoneshiro T, Shin W, Machida K, Fukano K, Tsubota A, Chen Y, Yasui H, Inanami O, Okamatsu-Ogura Y, Kimura K
    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 33 4 5196 - 5207 2019年04月 [査読有り][通常論文]
     
    Bone marrow provides progenitors of several types of cells, including muscle and white adipocytes, ensuring peripheral tissue homeostasis. However, the role of bone marrow-derived cells (BMCs) in induction of thermogenic adipocytes is unresolved. The purpose of this study is to examine whether BMCs are involved in the emergence of thermogenic adipocytes through adrenergic activation. Irradiation of mice with 8 Gy of X-ray-depleted BMCs and peripheral blood mononucleated cells (PBMCs), which in turn impaired induction of uncoupling protein 1 (UCP1) through administration of β3 adrenergic receptor agonist, CL 316,243 (CL), in inguinal white adipose tissue (iWAT). In contrast, CL-induced UCP1 induction in brown adipose tissue was unaffected by BMC depletion. Transplantation of normal BMCs into mice depleted of BMCs recovered PBMC levels and rescued the ability of iWAT browning by CL. Furthermore, analyses of mice transplanted with green fluorescent protein (GFP)-labeled BMCs revealed that the number of GFP-positive BMCs and PBMCs were significantly decreased by CL and that GFP-positive stromal cells and GFP-positive UCP1-expressing multilocular adipocytes appeared in iWAT after CL administration, demonstrating differentiation of BMC-derived preadipocytes into UCP1-expressing thermogenic adipocytes. These results unveiled a crucial role of the BMC as a nonresident origin for a subset of thermogenic adipocytes, contributing to browning of white adipose tissue.-Yoneshiro, T., Shin, W., Machida, K., Fukano, K., Tsubota, A., Chen, Y., Yasui, H., Inanami, O., Okamatsu-Ogura, Y., Kimura, K. Differentiation of bone marrow-derived cells toward thermogenic adipocytes in white adipose tissue induced by the β3 adrenergic stimulation.
  • Hari Narayan Bhilwade, Naoto Tatewaki, Tetsuya Konishi, Miyako Nishida, Takahiro Eitsuka, Hironobu Yasui, Osamu Inanami, Osamu Handa, Yuji Naito, Nobuo Ikekawa, Hiroshi Nishida
    Nutrition and cancer 71 7 1153 - 1164 2019年 [査読有り][通常論文]
     
    Many functional foods or physiologically active ingredients derived from plants and animals are actively being investigated for their role in chronic disease prevention. Squalene (SQ) is found as active ingredient in the functional foods predominantly present in olive oil and shark liver oil. It is known that during chemotherapy anticancer drugs induce inflammation. SQ has been thought to prevent and suppress inflammation; however, there is little direct evidence available. We examined the adjuvant effect of SQ on tumor-transplanted mice along with anticancer drug doxorubicin (DOX). SQ significantly suppressed the DOX-induced increase in prostaglandin E2 (PGE2) concentration (P < 0.05) in plasma of tumor-bearing mice. SQ inhibited the numbers of writhing response (P < 0.05), formalin-induced pain and decreased COX-2 and substance P expression in the tumor tissue compared to control mice and also enhanced the antitumor efficacy of DOX in allograft mice. Thus, SQ reduces inflammation through modulation of PGE2 production indicating its potential as an adjuvant during chemotherapy in tumor-bearing mice.
  • Denis A Komarov, Yuki Ichikawa, Kumiko Yamamoto, Neil J Stewart, Shingo Matsumoto, Hironobu Yasui, Igor A Kirilyuk, Valery V Khramtsov, Osamu Inanami, Hiroshi Hirata
    Analytical chemistry 90 23 13938 - 13945 2018年12月04日 [査読有り][通常論文]
     
    An electron paramagnetic resonance (EPR)-based method for noninvasive three-dimensional extracellular pH mapping was developed using a pH-sensitive nitroxyl radical as an exogenous paramagnetic probe. Fast projection scanning with a constant magnetic field sweep enabled the acquisition of four-dimensional (3D spatial +1D spectral) EPR images within 7.5 min. Three-dimensional maps of pH were reconstructed by processing the pH-dependent spectral information on the images. To demonstrate the proposed method of pH mapping, the progress of extracellular acidosis in tumor-bearing mouse legs was studied. Furthermore, extracellular pH mapping was used to visualize the spatial distribution of acidification in different tumor xenograft mouse models of human-derived pancreatic ductal adenocarcinoma cells. The proposed EPR-based pH mapping method enabled quantitative visualization of regional changes in extracellular pH associated with altered tumor metabolism.
  • Kumiko Yamamoto, Hironobu Yasui, Tomoki Bo, Tohru Yamamori, Wakako Hiraoka, Toshihide Yamasaki, Ken-ichi Yamada, Osamu Inanami
    APPLIED MAGNETIC RESONANCE 49 8 837 - 851 2018年08月 [査読有り][通常論文]
     
    Our recent report demonstrated that genotoxic stimuli enhance mitochondrial energy metabolism in various tumor cell lines. However, the mitochondrial response against genotoxic stimuli has not been fully elucidated. In this study, to investigate mitochondrial functions in X-irradiated cells, the oxygen consumption rate (OCR) in human cervical adenocarcinoma HeLa cells was examined by electron spin resonance (ESR) spectroscopy with lithium 5,9,14,18,23,27,32,36-octa-n-butoxy-2,3-naphthalocyanine. ESR oximetry demonstrated that basal respiration, ATP-linked respiration, proton leak, maximal respiration, and reserve capacity increased in HeLa cells 24 h after X-irradiation. However, a flow cytometric analysis using MitoTracker Green showed that mitochondrial mass also increased following X-irradiation. When the OCR was standardized to the mitochondria membrane mass, the radiation-induced increases in the respiratory parameters disappeared. This finding indicated that the radiation-induced increase in cellular OCR was explained by an increase in mitochondrial mass but not by the activation of mitochondrial respiratory-related enzymes. In addition, mitochondrial semiquinone radicals at g = 2.004 were detected by low-temperature (110 K) ESR spectroscopy. The ESR signal intensity of semiquinone radicals was enhanced by X-irradiation, suggesting an increase in the electron flow in the electron transport chain. These data will be important to understand the mechanism of radio-sensitization by mitochondria-targeting reagents in tumor cells.
  • Yamamoto K, Yasui H, Bo T, Yamamori T, Hiraoka W, Yamasaki T, Yamada KI, Inanami O
    Applied Magnetic Resonance 49 8 837 - 851 2018年07月 [査読有り][招待有り]
  • Kumiko Yamamoto, Yoshinori Ikenaka, Takahiro Ichise, Tomoki Bo, Mayumi Ishizuka, Hironobu Yasui, Wakako Hiraoka, Tohru Yamamori, Osamu Inanami
    Free radical research 52 6 648 - 660 2018年06月 [査読有り][通常論文]
     
    To evaluate the metabolic responses in tumour cells exposed to ionizing radiation, oxygen consumption rate (OCR), cellular lipid peroxidation, cellular energy status (intracellular nucleotide pool and ATP production), and mitochondrial reactive oxygen species (ROS), semiquinone (SQ), and iron-sulphur (Fe-S) cluster levels were evaluated in human cervical carcinoma HeLa cells at 12 and 24 h after X-irradiation. LC/MS/MS analysis showed that levels of 8-iso PGF2α and 5-iPF2α-VI, lipid peroxidation products of membrane arachidonic acids, were not altered significantly in X-irradiated cells, although mitochondrial ROS levels and OCR significantly increased in the cells at 24 h after irradiation. LC/UV analysis revealed that intracellular AMP, ADP, and ATP levels increased significantly after X-irradiation, but adenylate energy charge (adenylate energy charge (AEC) = [ATP + 0.5 × ADP]/[ATP + ADP + AMP]) remained unchanged after X-irradiation. In low-temperature electron spin resonance (ESR) spectra of HeLa cells, the presence of mitochondrial SQ at g = 2.004 and Fe-S cluster at g = 1.941 was observed and X-irradiation enhanced the signal intensity of SQ but not of the Fe-S cluster. Furthermore, this radiation-induced increase in SQ signal intensity disappeared on treatment with rotenone, which inhibits electron transfer from Fe-S cluster to SQ in complex I. From these results, it was suggested that an increase in OCR and imbalance in SQ and Fe-S cluster levels, which play a critical role in the mitochondrial electron transport chain (ETC), occur after X-irradiation, resulting in an increase in ATP production and ROS leakage from the activated mitochondrial ETC.
  • Tatsuya Amida, Ririko Nakaoka, Denis A Komarov, Kumiko Yamamoto, Osamu Inanami, Shingo Matsumoto, Hiroshi Hirata
    IEEE transactions on bio-medical engineering 65 5 1124 - 1132 2018年05月 [査読有り][通常論文]
     
    OBJECTIVE: The purpose of this work was to develop an electronically tunable resonator operating at 750 MHz for continuous-wave electron paramagnetic resonance (CW-EPR) imaging of a mouse tumor-bearing leg. METHODS: The resonator had a multi-coil parallel-gap structure with a sample space of 16 mm in diameter and 20 mm in length. Microstrip line couplers were used in conjunction with varactor diodes to enable resonance frequency adjustment and to reduce the nonlinear effects of the varactor diodes. The resonator was modeled by the finite-element method and a microwave circuit simulation was performed to clarify its radiofrequency characteristics. RESULTS: A tunable resonator was evaluated in terms of its resonance frequency, tunable frequency band, and conversion efficiency of the RF magnetic field. The developed resonator provided a tunable frequency band of 4 MHz at a central frequency of 747 MHz and a conversion efficiency of 34 μT/W1/2. To demonstrate the application of this tunable resonator to EPR imaging, three-dimensional EPR images of a sample solution and a mouse tumor-bearing leg were obtained. CONCLUSION: The developed tunable resonator satisfied our initial requirements for in vivo EPR imaging and may be able to be further improved using the present finite-element and circuit models if any problems arise during future practical applications. SIGNIFICANCE: This work may help to promote EPR imaging of tumor-bearing mice in cancer-related studies.
  • Masaki Nagane, M. Lakshmi Kuppusamy, Jennifer An, Jesse M. Mast, Rajan Gogna, Hironobu Yasui, Tohru Yamamori, Osamu Inanami, Periannan Kuppusamy
    Radiation Research 189 5 519 - 528 2018年05月01日 [査読有り][通常論文]
     
    Endothelial nitric oxide synthase (eNOS), a constitutive enzyme expressed in vascular endothelial cells, is the main source of nitric oxide (NO), which plays key roles in diverse biological functions, including regulation of vascular tone. Exposure to radiation has been known to generate nitric oxide from eNOS however, the precise mechanism of its generation and function is not known. The goal of this study was to determine the involvement of radiation-induced DNA damage response (DDR) on eNOS transcription and its effect on cell survival after irradiation. Irradiated bovine aortic endothelial cells showed increased eNOS transcription and NO generation through upregulation of ataxia-telangiectasia mutated (ATM) kinase. Radiation exposure induced NO inhibited cell death, as well as induced cellular senescence postirradiation. This study established that radiation-induced DDR uses ATM kinase to upregulate eNOS transcription and NO generation, leading to cellular senescence, which may play a critical role in radiation-mediated cardiovascular injury.
  • Hironobu Yasui, Nobuo Kubota, Junko Nishizumi, Yuri Sakai, Tohru Yamamori, Osamu Inanami
    Oncology Letters 15 2 1993 - 1998 2018年02月01日 [査読有り][通常論文]
     
    To overcome the radioresistance of hypoxic cells in solid tumor, numerous types of radiosensitizers specifically against them have been developed. Glycididazole has a chemical structure in which two metronidazole forms are combined, and is widely used as a hypoxic radiosensitizer in China. However, a detailed investigation of its radiosensitizing properties has not been performed. The present study reported a comparative assessment of glycididazole and doranidazole, another hypoxic radiosensitizer. All experiments were performed using the murine squamous cell carcinoma cell line SCCVII. Prior to X-irradiation, the cells were treated with the test drugs at concentrations of 10 mM and 200 mg/kg in vitro and in vivo, respectively. Uptake and their intratumor chemical forms were analyzed by high performance liquid chromatography (HPLC). Both drugs enhanced the reproductive cell death induced by X-irradiation under hypoxia. However, the growth delay assay of the transplanted tumor revealed the combination of X-irradiation and glycididazole showed a similar antitumor effect to that of X-irradiation alone, whereas doranidazole significantly sensitized the cells to X-irradiation. HPLC analysis revealed that incorporated glycididazole was decomposed to metronidazole and was therefore present at a lower concentration compared with that of doranidazole. The decomposition of glycididazole to metronidazole reduced its radiosensitizing efficiency in vivo. Elucidation of the kinetics of drugs containing metabolizable chemical forms is necessary for the optimization of clinical treatment.
  • Tomoki Bo, Tohru Yamamori, Motofumi Suzuki, Yuri Sakai, Kumiko Yamamoto, Osamu Inanami
    Biochemical and biophysical research communications 495 2 1601 - 1607 2018年01月08日 [査読有り][通常論文]
     
    Mitochondrial dynamics are suggested to be indispensable for the maintenance of cellular quality and function in response to various stresses. While ionizing radiation (IR) stimulates mitochondrial fission, which is mediated by the mitochondrial fission protein, dynamin-related protein 1 (Drp1), it remains unclear how IR promotes Drp1 activation and subsequent mitochondrial fission. Therefore, we conducted this study to investigate these concerns. First, we found that X-irradiation triggered Drp1 phosphorylation at serine 616 (S616) but not at serine 637 (S637). Reconstitution analysis revealed that introduction of wild-type (WT) Drp1 recovered radiation-induced mitochondrial fission, which was absent in Drp1-deficient cells. Compared with cells transfected with WT or S637A Drp1, the change in mitochondrial shape following irradiation was mitigated in S616A Drp1-transfected cells. Furthermore, inhibition of CaMKII significantly suppressed Drp1 S616 phosphorylation and mitochondrial fission induced by IR. These results suggest that Drp1 phosphorylation at S616, but not at S637, is prerequisite for radiation-induced mitochondrial fission and that CaMKII regulates Drp1 phosphorylation at S616 following irradiation.
  • Daisuke Ueno, Hazuki Mizukawa, Osamu Inanami, Hiromitsu Nagasaka, Nozomi Tatsuta, Yukinori Narazaki, Takeshi Fujino, Izumi Watanabe, Yutaka Kameda, Kunihiko Nakai
    Landscape and Ecological Engineering 14 1 29 - 35 2018年01月01日 [査読有り][通常論文]
     
    The “Caddisfly Watch” program proposes the use of larvae of the caddisfly genus Stenopsyche (Trichoptera: Stenopsychidae) to monitor the radioactive cesium (137Cs) pollution, including that of suspended solids, in river environments, as a simple method was essential for this following the Fukushima nuclear disaster in March 2011. A variety of aquatic organisms were collected from rivers in Japan in 2012 and their levels of radioactive Cs measured. Amongst all the organisms collected, the highest concentrations of 137Cs were observed in caddisfly larvae. These larvae occur at a high density and can be collected at regular intervals in most rivers throughout Japan. It is proposed that caddisfly larvae can be used as bioindicators of radioactive Cs contamination in rivers, as their temporal and spatial changes are easily assessed.
  • Yuri Sakai, Tohru Yamamori, Yoji Yoshikawa, Tomoki Bo, Motofumi Suzuki, Kumiko Yamamoto, Tetsuro Ago, Osamu Inanami
    Free radical research 52 1 92 - 102 2018年01月 [査読有り][通常論文]
     
    Excessive DNA damage induced by ionising radiation (IR) to normal tissue cells is known to trigger cellular senescence, a process termed stress-induced premature senescence (SIPS). SIPS is often accompanied by the production of reactive oxygen species (ROS), and this is reported to be important for the initiation and maintenance of SIPS. However, the source of ROS during SIPS after IR and their significance in radiation-induced normal tissue damage remain elusive. In the present study, we tested the hypothesis that the NADPH oxidase (NOX) family of proteins mediates ROS production in SIPS-induced cells after IR and plays a role in SIPS-associated biological events. X-irradiation of primary mouse embryonic fibroblasts (MEFs) resulted in cellular senescence and the concomitant increase of intracellular ROS. Among all six murine NOX isoforms (NOX1-4 and DUOX1/2), only NOX4 was detectable under basal conditions and was upregulated following IR. In addition, radiation-induced ROS production was diminished by genetic or pharmacological inhibition of NOX4. Meanwhile, NOX4 deficiency did not affect the induction of cellular senescence after IR. Furthermore, the migration of human monocytic U937 cells to the culture medium collected from irradiated MEFs was significantly reduced by NOX4 inhibition, suggesting that NOX4 promotes the recruitment of inflammatory cells. Collectively, our findings imply that NOX4 mediates ROS production in radiation-induced senescent cells and contributes to normal tissue damage after IR via the recruitment of inflammatory cells and the exacerbation of tissue inflammation.
  • H. Shino, Y. Otsuka-Yamasaki, T. Sato, K. Ooi, O. Inanami, R. Sato, M. Yamasaki
    Journal of Veterinary Internal Medicine 32 1 165 - 171 2018年01月01日 [査読有り][通常論文]
     
    Background: In veterinary medicine, congenital methemoglobinemia associated with nicotinamide adenine dinucleotide (NADH)-cytochrome b5 reductase (b5R) deficiency is rare. It has been reported in several breeds of dogs, but little information is available about its etiology. Objectives: To analyze the NADH-cytochrome b5 reductase gene, CYB5R3, in a Pomeranian dog family with methemoglobinemia suspected to be caused by congenital b5R deficiency. Animals: Three Pomeranian dogs from a family with methemoglobinemia were analyzed. Five healthy beagles and 5 nonrelated Pomeranian dogs without methemoglobinemia were used as controls. Methods: Methemoglobin concentration, b5R activity, and reduced glutathione (GSH) concentration were measured, and a turbidity index was used to evaluate Heinz body formation. The CYB5R3 genes of the affected dog and healthy dogs were analyzed by direct sequencing. Results: Methemoglobin concentrations in erythrocytes of the affected dogs were remarkably higher than those of the control dogs. The b5R activity of the affected dogs was notably lower than that of the control dogs. DNA sequencing indicated that this Pomeranian family carried a CYB5R3 gene missense variant (ATC→CTC at codon 194) that resulted in the replacement of isoleucine (Ile) by leucine (Leu). Conclusions and Clinical Importance: This dog family had familial congenital methemoglobinemia caused by b5R deficiency, which resulted from a nonsynonymous variant in the CYB5R3 gene. This variation (c.580A> C) led to an amino acid substitution (p.Ile194Leu), and Ile194 was located in the proximal region of the NADH-binding motif. Our data suggested that this variant in the canine CYB5R3 gene would affect function of the b5R in erythrocytes.
  • Motofumi Suzuki, Tohru Yamamori, Tomoki Bo, Yuri Sakai, Osamu Inanami
    TRANSLATIONAL ONCOLOGY 10 4 491 - 500 2017年08月 [査読有り][通常論文]
     
    Checkpoint kinase 1 (Chk1) is an evolutionarily conserved serine/threonine kinase that plays an important role in G2/M checkpoint signaling. Here, we evaluate the radiosensitizing effects of a novel selective Chk1 inhibitor MK-8776, comparing its efficacy with a first-generation Chk1 inhibitor UCN-01, and attempt to elucidate the mechanism of radiosensitization. In a clonogenic survival assay, MK-8776 demonstrated a more pronounced radiosensitizing effect than UCN-01, with lower cytotoxicity. Importantly, radiosensitization by MK-8776 can be achieved at doses as low as 2.5 Gy, which is a clinically applicable irradiation dose. MK-8776, but not UCN-01, exacerbated mitotic catastrophe (MC) and centrosome abnormalities, without affecting repair kinetics of DNA double strand breaks. Furthermore, live-cell imaging revealed that MK-8776 significantly abrogated the radiationinduced G2/M checkpoint, prolonged the mitotic phase, and enhanced aberrant mitosis. This suggests that Chk1 inhibition by MK-8776 activates a spindle assembly checkpoint and increases mitotic defects in irradiated EMT6 cells. In conclusion, we have shown that, at minimally toxic concentrations, MK-8776 enhances radiation-induced cell death through the enhancement of aberrant mitosis and MC, without affecting DNA damage repair.
  • 永根 大幹, 安井 博宣, 山盛 徹, Periannan Kuppusamy, 稲波 修
    放射線生物研究 = Radiation biology research communications : 放射線生物研究会機関誌 52 2 173 - 182 放射線生物研究会 2017年06月 [査読無し][通常論文]
  • Harue Kubota, Denis A. Komarov, Hironobu Yasui, Shingo Matsumoto, Osamu Inanami, Igor A. Kirilyuk, Valery V. Khramtsov, Hiroshi Hirata
    MAGNETIC RESONANCE MATERIALS IN PHYSICS BIOLOGY AND MEDICINE 30 3 291 - 298 2017年06月 [査読有り][通常論文]
     
    Objectives The aim of this study was to demonstrate the feasibility of in vivo three-dimensional (3D) relaxation time T-2* mapping of a dicarboxy-PROXYL radical using continuous-wave electron paramagnetic resonance (CW-EPR) imaging. Materials and methods Isotopically substituted dicarboxy-PROXYL radicals, 3,4-dicarboxy-2,2,5,5-tetra(H-2(3)) methylpyrrolidin-( 3,4-H-2(2))-(1-N-15)-1-oxyl (H-2, N-15-DCP) and 3,4-dicarboxy-2,2,5,5-tetra(H-2(3)) methylpyrrolidin-(3,4-H-2(2))1- oxyl (H-2-DCP), were used in the study. A clonogenic cell survival assay was performed with the H-2-DCP radical using squamous cell carcinoma (SCC VII) cells. The time course of EPR signal intensities of intravenously injected H-2, N-15-DCP and H-2-DCP radicals were determined in tumor-bearing hind legs of mice (C3H/HeJ, male, n = 5). CW-EPR-based single-point imaging (SPI) was performed for 3D T-2* mapping. Results H-2-DCP radical did not exhibit cytotoxicity at concentrations below 10 mM. The in vivo half-life of H-2, N-15-DCP in tumor tissues was 24.7 +/- 2.9 min (mean +/- standard deviation [SD], n = 5). The in vivo time course of the EPR signal intensity of the H-2, N-15-DCP radical showed a plateau of 10.2 +/- 1.2 min (mean +/- SD) where the EPR signal intensity remained at more than 90% of the maximum intensity. During the plateau, in vivo 3D T-2* maps with H-2, N-15-DCP were obtained from tumor-bearing hind legs, with a total acquisition time of 7.5 min. Conclusion EPR signals of H-2, N-15-DCP persisted long enough after bolus intravenous injection to conduct in vivo 3D T-2* mapping with CW-EPR-based SPI.
  • Tohru Yamamori, Tomoya Sasagawa, Osamu Ichii, Mie Hiyoshi, Tomoki Bo, Hironobu Yasui, Yasuhiro Kon, Osamu Inanami
    JOURNAL OF RADIATION RESEARCH 58 3 292 - 301 2017年05月 [査読有り][通常論文]
     
    Mitochondria strongly contribute to the maintenance of cellular integrity through various mechanisms, including oxidative adenosine triphosphate production and calcium homeostasis regulation. Therefore, proper regulation of the abundance, distribution and activity of mitochondria is crucial for the maintenance of cellular homeostasis. Previous studies have shown that ionizing radiation (IR) alters mitochondrial functions, suggesting that mitochondria are likely to be an important target of IR. Though IR reportedly influences cellular mitochondrial abundance, the mechanism remains largely unknown. In this study, we examined how IR influences mitochondrial abundance in mouse fibroblasts. When mouse NIH/3T3 cells were exposed to X-rays, a time-dependent increase was observed in mitochondrial DNA (mtDNA) and mitochondrial mass, indicating radiation-induced upregulation of mitochondrial abundance. Meanwhile, not only did we not observe a significant change in autophagic activity after irradiation, but in addition, IR hardly influenced the expression of two mitochondrial proteins, cytochrome c oxidase subunit IV and cytochrome c, or the mRNA expression of Polg, a component of DNA polymerase gamma. We also observed that the expression of transcription factors involved in mitochondrial biogenesis was only marginally affected by IR. These data imply that radiation-induced upregulation of mitochondrial abundance is an event independent of macroautophagy and mitochondrial biogenesis. Furthermore, we found evidence that IR induced long-term cell cycle arrest and cellular senescence, indicating that these events are involved in regulating mitochondrial abundance. Considering the growing significance of mitochondria in cellular radioresponses, we believe the present study provides novel insights into understanding the effects of IR on mitochondria.
  • Hironobu Yasui, Kumiko Yamamoto, Motofumi Suzuki, Yuri Sakai, Tomoki Bo, Masaki Nagane, Eri Nishimura, Tohru Yamamori, Toshihide Yamasaki, Ken-ichi Yamada, Osamu Inanami
    CANCER LETTERS 390 160 - 167 2017年04月 [査読有り][通常論文]
     
    It has recently been reported that radiation enhances mitochondrial energy metabolism in various tumor cell lines. To examine how this radiation -induced alteration in mitochondrial function influences tumor cell viability, Various lipophilic triphenylphosphonium (TPP+) cation derivatives and related compounds such as 4-hydroxy-2,2,6,6-tetramethyl-l-oxy-piperidin (Tempol) with TPP+ (named."Mito-") were designed to inhibit the mitochondrial electron transport chain. Mito-(CH2)io-Tempol (M10T) and its derivatives, Mito-(CH2)(5)-Tempol (M5T), Mito-(CH2)(10)-Tempol-Methyl (M10T-Me), Mito-C10H21 (M10), and C10H21-Tempol (10T), were prepared. In HeLa human cervical adenocarcinoma cells and A549 human lung carcinoma cells, the fractional uptake of the compound into mitochondria was highest among the TTP+ analogs conjugated with Tempol (M10T, M5T, and 10T). MlOT, M10T-Me, and M10 exhibited strong cytotoxicity and enhanced X-irradiation -induced reproductive cell death, while 10T and M5T did not. Furthermore, M10T, M10T-Me, and M10 decreased basal mitochondrial membrane potential and intracellular ATP. MIOT treatment inhibited X-ray -induced increases in ATP production. These results indicate that the TPP cation and a long hydrocarbon linker are essential for radiosensitization of tumor cells. The reduction in intracellular ATP by lipophilic TP13(+) is partly responsible for the observed radiosensitization. (C) 2017 Elsevier B.V. All rights reserved.
  • Lue Sun, Takashi Moritake, Kazuya Ito, Yoshitaka Matsumoto, Hironobu Yasui, Hidehiko Nakagawa, Aki Hirayame, Osamu Inanami, Koji Tsuboi
    PLOS ONE 12 4 e0176162  2017年04月 [査読有り][通常論文]
     
    Medulloblastoma is a fatal brain tumor in children, primarily due to the presence of treatment-resistant medulloblastoma stem cells. The energy metabolic pathway is a potential target of cancer therapy because it is often different between cancer cells and normal cells. However, the metabolic properties of medulloblastoma stem cells, and whether specific metabolic pathways are essential for sustaining their stem cell-like phenotype and radioresistance, remain unclear. We have established radioresistant medulloblastoma stem-like clones (rMSLCs) by irradiation of the human medulloblastoma cell line ONS-76. Here, we assessed reactive oxygen species (ROS) production, mitochondria function, oxygen consumption rate (OCR), energy state, and metabolites of glycolysis and tricarboxylic acid cycle in rMSLCs and parental cells. rMSLCs showed higher lactate production and lower oxygen consumption rate than parental cells. Additionally, rMSLCs had low mitochondria mass, low endogenous ROS production, and existed in a low-energy state. Treatment with the metabolic modifier dichloroacetate (DCA) resulted in mitochondria dysfunction, glycolysis inhibition, elongated mitochondria morphology, and increased ROS production. DCA also increased radiosensitivity by suppression of the DNA repair capacity through nuclear oxidization and accelerated the generation of acetyl CoA to compensate for the lack of ATP. Moreover, treatment with DCA decreased cancer stem cell-like characters (e.g., CD133 positivity and sphere-forming ability) in rMSLCs. Together, our findings provide insights into the specific metabolism of rMSLCs and illuminate potential metabolic targets that might be exploited for therapeutic benefit in medulloblastoma.
  • Attempts of Radiation Dose Measurement in the Teeth of Mice Living around the Nuclear Power Plant in Fukushima Using Electron Spin Resonance Spectroscopy
    Kitaya Taichi, Maeda Teruaki, Yasui Hironobu, Mizukawa Hazuki, Inanami Osamu, Nakata Akifumi, Miura Tomisato, Hosokawa Yoichiro, Kuwabara Mikinori
    Radiation Environment and Medicine 6 1 1 - 5 弘前大学出版会 2017年02月 [査読有り][通常論文]
  • Hironobu Yasui, Tatsuya Kawai, Shingo Matsumoto, Keita Saito, Nallathamby Devasahayam, James B. Mitchell, Kevin Camphausen, Osamu Inanami, Murali C. Krishna
    FREE RADICAL RESEARCH 51 9-10 861 - 871 2017年 [査読有り][通常論文]
     
    Hypoxia is considered one of the microenvironmental factors associated with the malignant nature of glioblastoma. Thus, evaluating intratumoural distribution of hypoxia would be useful for therapeutic planning as well as assessment of its effectiveness during the therapy. Electron paramagnetic resonance imaging (EPRI) is an imaging technique which can generate quantitative maps of oxygen in vivo using the exogenous paramagnetic compound, triarylmethyl and monitoring its line broadening caused by oxygen. In this study, the feasibility of EPRI for assessment of oxygen distribution in the glioblastoma using orthotopic U87 and U251 xenograft model is examined. Heterogeneous distribution of pO(2) between 0 and 50mmHg was observed throughout the tumours except for the normal brain tissue. U251 glioblastoma was more likely to exhibit hypoxia than U87 for comparable tumour size (median pO(2); 29.7 and 18.2 mmHg, p = .028, in U87 and U251, respectively). The area with pO(2) under 10mmHg on the EPR oximetry (HF10) showed a good correlation with pimonidazole staining among tumours with evaluated size. In subcutaneous xenograft model, irradiation was relatively less effective for U251 compared with U87. In conclusion, EPRI is a feasible method to evaluate oxygen distribution in the brain tumour.
  • Maeda K, Yasui H, Yamamori T, Matsuura T, Takao S, Suzuki M, Matsuda A, Inanami O, Shirato H
    PloS one 11 11 e0166848  2016年11月 [査読有り][通常論文]
     
    The effect of 1-(3-C-ethyny1-beta-D-ribo-pentofuranosyl)cytosine (ECyd) on proton-induced cell death was evaluated in human lung carcinoma cell line A549 and Chinese hamster fibroblast cell line V79 to enhance relative biological effectiveness (RBE) within the spread-out Bragg peak (SOBP) of proton beams. Treatment with ECyd significantly enhanced the proton -induced loss of clonogenicity and increased senescence at the center, but not at the distal edge of SOBP. The p53-binding protein 1 foci formation assay showed that ECyd decelerated the rate of DNA double-strand break (DSB) repair at the center, but not the distal region of SOBP, suggesting that the ECyd-induced enhancement of proton-induced cell death is partially associated with the inhibition of DSB repair. This study demonstrated that ECyd enhances proton-induced cell killing at all positions of SOBP, except for the distal region and minimizes the site-dependent differences in RBE within SOBP. Thus, ECyd is a unique radiosensitizer for proton therapy that may be useful because it levels the biological dose within SOBP, which improves tumor control and reduces the risk of adverse effects at the distal edge of SOBP.
  • Kenichiro Maeda, Hironobu Yasui, Taeko Matsuura, Tohru Yamamori, Motofumi Suzuki, Masaki Nagane, Jin-Min Nam, Osamu Inanami, Hiroki Shirato
    JOURNAL OF RADIATION RESEARCH 57 3 307 - 311 2016年06月 [査読有り][通常論文]
     
    Variations in relative biological effectiveness (RBE) from a fixed value of 1.1 are critical in proton beam therapy. To date, studies estimating RBE at multiple positions relative to the spread-out Bragg peak (SOBP) have been predominantly performed using passive scattering methods, and limited data are available for spot-scanning beams. Thus, to investigate the RBE of spot-scanning beams, Chinese hamster fibroblast V79 cells were irradiated using the beam line at the Hokkaido University Hospital Proton Therapy Center. Cells were placed at six different depths, including the entrance of the proton beam and the proximal and distal part of the SOBP. Surviving cell fractions were analyzed using colony formation assay, and cell survival curves were obtained by the curve fitted using a linear-quadratic model. RBE10 and RBE37 were 1.15 and 1.21 at the center of the SOBP, respectively. In contrast, the distal region showed higher RBE values (1.50 for RBE10 and 1.85 for RBE37). These results are in line with those of previous studies conducted using passive scattering proton beams. Taken together, these data strongly suggest that variations in RBE should be considered during treatment planning for spot-scanning beams as well as for passive scattering proton beams.
  • Motofumi Suzuki, Tohru Yamamori, Hironobu Yasui, Osamu Inanami
    ANTICANCER RESEARCH 36 6 2783 - 2792 2016年06月 [査読有り][通常論文]
     
    Background/Aim: The monopolar spindle 1 (MPS1) is a serine/threonine kinase that plays an important role in spindle assembly checkpoint signaling. To determine the possible relationship between MPS1 inhibition and genotoxic stress responses, herein we examined whether MPS1 inhibition influences cellular susceptibility towards two genotoxic treatments, etoposide and ionizing radiation (IR). Materials and Methods: Two murine tumour cell lines, SCCVII and EMT6, were used. The effect of genotoxic treatments with or without two novel MPS1 inhibitors, NMS-P715 and AZ3146, on cellular survival, cell-cycle distribution, centrosome status and mitotic catastrophe (MC) was evaluated. Results: MPS1 inhibition sensitized murine tumour cells to etoposide but not to IR. In addition, MPS1 inhibition altered cell-cycle progression and exacerbated centrosome abnormalities, resulting in enhanced MC induced by etoposide but not by IR. Conclusion: MPS1 inhibition promotes the etoposide-induced aberrant mitosis and, consequently, the induction of tumour cell death.
  • Tohru Yamamori, Satoshi Ike, Tomoki Bo, Tomoya Sasagawa, Yuri Sakai, Motofumi Suzuki, Kumiko Yamamoto, Masaki Nagane, Hironobu Yasui, Osamu Inanami
    MOLECULAR BIOLOGY OF THE CELL 26 25 4607 - 4617 2015年12月 [査読有り][通常論文]
     
    Accumulating evidence suggests that mitochondrial dynamics is crucial for the maintenance of cellular quality control and function in response to various stresses. However, the role of mitochondrial dynamics in cellular responses to ionizing radiation (IR) is still largely unknown. In this study, we provide evidence that IR triggers mitochondrial fission mediated by the mitochondrial fission protein dynamin-related protein 1 (Drp1). We also show IR-induced mitotic catastrophe (MC), which is a type of cell death associated with defective mitosis, and aberrant centrosome amplification in mouse embryonic fibroblasts (MEFs). These are attenuated by genetic or pharmacological inhibition of Drp1. Whereas radiation-induced aberrant centrosome amplification and MC are suppressed by the inhibition of Plk1 and CDK2 in wild-type MEFs, the inhibition of these kinases is ineffective in Drp1-deficient MEFs. Furthermore, the cyclin B1 level after irradiation is significantly higher throughout the time course in Drp1-deficient MEFs than in wild-type MEFs, implying that Drp1 is involved in the regulation of cyclin B1 level. These findings strongly suggest that Drp1 plays an important role in determining the fate of cells after irradiation via the regulation of mitochondrial dynamics.
  • Sakata K, Kondo T, Mizuno N, Shoji M, Yasui H, Yamamori T, Inanami O, Yokoo H, Yoshimura N, Hattori Y
    Vascular pharmacology 70 55 - 65 2015年07月 [査読有り][通常論文]
     
    Vascular endothelial cells can absorb higher radiation doses than any other tissue in the body, and post-radiation impaired endothelial nitric oxide synthase (eNOS) function may be developed as a potential contributor to the pathogenesis of vascular injury. In this study, we investigated early alterations of eNOS signaling in human umbilical venous endothelial cells (HUVECs) exposed to X-ray radiation. We found that ionizing radiation increased eNOS phosphorylation at Ser-1177 and dephosphorylation at Thr-495 in HUVECs in a dose-dependent (<= 20 Gy) and time-dependent (6-72 h) manner. The total expression levels of eNOS were unchanged by radiation. Although a transient but significant increase in extracellular signal-regulated protein kinase 1/2 (ERK1/2) phosphorylation and a biphasic decline in Akt phosphorylation were observed after irradiation, these inhibitors were without effect on the radiation-induced changes in eNOS phosphorylation. There was an increase in protein kinase C-beta II (PKC-beta II) expression and the ablation of PKC-beta II by small interfering RNA (siRNA) negated the radiation effect on the two eNOS phosphorylation events. Furthermore, when the radiation-induced increase in reactive oxygen species (ROS) generation was prevented by the anti-oxidant N-acetyl-L-cysteine, eNOS Ser-1177 phosphorylation and Thr-495 dephosphorylation in irradiated HUVECs were significantly reduced. However, transfection of PKC-beta siRNA did not alter ROS production after irradiation, and NAC failed to block the radiation-induced increase in PKC-beta II expression. Taken together, our results suggest that ionizing radiation-induced eNOS activation in human vascular endothelial cells is attributed to both the upregulation of PKC-beta II and the increase in ROS generation which were independent of each other. (C) 2015 Elsevier Inc. All rights reserved.
  • Sakai Y, Yamamori T, Yasui H, Inanami O
    Biochemical and biophysical research communications 461 1 35 - 41 2015年05月 [査読有り][通常論文]
     
    The DNA repair enzyme apurinic/apyrimidinic endonuclease 1 (APE1) plays a central role in base excision repair and functions as a reductive activator of various transcription factors. Multiple other functionalities have been ascribed to APEI in addition to these major functions. A recent study showed that APE1 knockdown upregulated the expression of a set of genes related to extracellular matrix (ECM) production, indicating an additional novel biological role for this enzyme. Based on this finding, we have investigated the effect of APE1 downregulation on ECM-related gene expression and its biological consequences. Endogenous APEI expression was downregulated in human cervical carcinoma HeLa cells and human lung carcinoma A549 cells using siRNA. When the expression of six ECM-related genes (TGFB1, LAMC1, FN1, COL1A1, COL3A1, and COL4A1) was evaluated, we found that APE1 knockdown upregulated the expression of TGFB1 in both cell lines. APE1 downregulation promoted actin rearrangement, inducing F-actin accumulation in HeLa cells and the dissipation of stress fibers in A549 cells. We also discovered that APE1 knockdown enhanced cellular motility in A549 cells, which was suppressed by the inhibition of transforming growth factor (TGF)-fil signaling. These results suggested that APE1 controls the organization of actin cytoskeleton through the regulation of TGF-131 expression, providing novel insights into the biological significance of APE1. (C) 2015 Elsevier Inc. All rights reserved.
  • Masaki Nagane, Hironobu Yasui, Yuri Sakai, Tohru Yamamori, Koichi Niwa, Yuichi Hattori, Takashi Kondo, Osamu Inanami
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 456 1 541 - 546 2015年01月 [査読有り][通常論文]
     
    In this study, the involvement of ataxia telangiectasia mutated (ATM) kinase and heat shock protein 90 (HSP90) in endothelial nitric oxide synthase (eNOS) activation was investigated in X-irradiated bovine aortic endothelial cells. The activity of nitric oxide synthase (NOS) and the phosphorylation of serine 1179 of eNOS (eNOS-Ser1179) were significantly increased in irradiated cells. The radiation-induced increases in NOS activity and eNOS-Ser1179 phosphorylation levels were significantly reduced by treatment with either an ATM inhibitor (Ku-60019) or an HSP90 inhibitor (geldanamycin). Geldanamycin was furthermore found to suppress the radiation-induced phosphorylation of ATM-Ser1181. Our results indicate that the radiation-induced eNOS activation in bovine aortic endothelial cells is regulated by ATM and HSP90. (C) 2014 Elsevier Inc. All rights reserved.
  • Mikinori Kuwabara, Wakako Hiraoka, Osamu Inanami
    Applications of EPR in Radiation Research 9783319092164 353 - 384 2014年06月01日 [査読有り][通常論文]
     
    Ionizing radiation produces many kinds of free radicals in biologically important molecules through direct or indirect actions. In this chapter we have shown that a method that combined ESR, spin trapping and chromatographic separation made it possible to identify free radicals induced by direct ionization as well as OH-radical reactions in nucleic-acid related and protein-related compounds. In addition, combination with enzymatic digestion expanded the potential of this method to detect free radical production in molecules with high molecular weight such as polynucleotides, RNA and DNA, and polypeptides and proteins. The effects of ionizing radiation on biological molecules underlie the primary processes involved in radiation biology. ESR combined with spin trapping remains a useful method for clarifying these processes in a living organism.
  • Naoya Nishida, Hironobu Yasui, Masaki Nagane, Tohru Yamamori, Osamu Inanami
    JOURNAL OF RADIATION RESEARCH 55 3 455 - 463 2014年05月 [査読有り][通常論文]
     
    Considerable interest has recently been focused on the special characteristics of cancer metabolism, and several drugs designed to modulate cancer metabolism have been tested as potential anticancer agents. To date, however, very few studies have been conducted to investigate the combined effects of anticancer drugs and radiotherapy. In this study, to evaluate the role of mitochondria-derived reactive oxygen species (ROS) in the radiation-induced cell death of tumor cells, we have examined the effect of 3-methyl pyruvate (MP). MP is a membrane-permeable pyruvate derivative that is capable of activating mitochondrial energy metabolism in human lung carcinoma A549 cells and murine squamous carcinoma SCCVII cells. Pretreatment with MP significantly enhanced radiation-induced cell death in both cell lines, and also led to increases in the mitochondrial membrane potential, intracellular adenosine triphosphate content, and mitochondria-derived ROS production following the exposure of the cells to X-rays. In A549 cells, MP-induced radiosensitization was completely abolished by vitamin C. In contrast, it was partially abolished in SCCVII cells. These results therefore suggest that the treatment of the cells with MP induced radiosensitization via the production of excess mitochondria-derived ROS in tumor cells.
  • Hironobu Yasui, Ryo Takeuchi, Masaki Nagane, Shunsuke Meike, Yoshinari Nakamura, Tohru Yamamori, Yoshinori Ikenaka, Yasuhiro Kon, Hiroki Murotani, Motoi Oishi, Yukio Nagasaki, Osamu Inanami
    CANCER LETTERS 347 1 151 - 158 2014年05月 [査読有り][通常論文]
     
    High atomic number molecules, such as gold and platinum, are known to enhance the biological effect of X-irradiation. This study was aimed to determine the radiosensitizing potential of PEGylated nanogel containing gold nanoparticles (GNG) and the cellular mechanism involved. GNG pretreatment increased the levels of reproductive cell death and apoptosis induced by X-irradiation. GNG accumulated in cytoplasm and increased the expression of endoplasmic reticulum (ER) stress-related protein. GNG suppressed the repair capacity of DNA after X-irradiation by down-regulating DNA repair-related proteins. Our results suggest that GNG radiosensitized cells by enhancing apoptosis and impairing DNA repair capacity via ER stress induction. (C) 2014 Elsevier Ireland Ltd. All rights reserved.
  • Jonathan Goodwin, Katsuya Yachi, Masaki Nagane, Hironobu Yasui, Yusuke Miyake, Osamu Inanami, Andrey A. Bobko, Valery V. Khramtsov, Hiroshi Hirata
    NMR IN BIOMEDICINE 27 4 453 - 458 2014年04月 [査読有り][通常論文]
     
    The in vivo quantification of extracellular pH (pH(e)) in tumours may provide a useful biomarker for tumour cell metabolism. In this study, we assessed the viability of continuous-wave electron paramagnetic resonance (CW-EPR) spectroscopy with a pH-sensitive nitroxide for the measurement of extracellular tumour pH in a mouse model. CW-EPR spectroscopy (750 MHz) of C3H HeJ mice hind leg squamous cell tumour was performed after intravenous tail vein injection of pH-sensitive nitroxide (R-SG, 2-(4-((2-(4-amino-4-carboxybutanamido)-3-(carboxymethylamino)-3-oxoproylthio)methyl)phenyl)-4-pyrrolidino-2,5,5-triethyl-2,5-dihydro-1H-imidazol-1-oxyl) during stages of normal tumour growth and in response to a single 10-Gy dose of X-ray irradiation. An inverse relationship was observed between tumour volume and pH(e) value, whereby, during normal tumour growth, a constant reduction in pH(e) was observed. This relationship was disrupted by X-ray irradiation and, from 2-3 days post-exposure, a transitory increase in pH(e) was observed. In this study, we demonstrated the viability of CW-EPR spectroscopy using R-SG nitroxide to obtain high-sensitivity pH measurements in a mouse tumour model with an accuracy of <0.1 pH units. In addition, the measured changes in pH(e) in response to X-ray irradiation suggest that this may offer a useful method for the assessment of the physiological change in response to existing and novel cancer therapies. Copyright (c) 2014 John Wiley & Sons, Ltd.
  • Tohru Yamamori, Shunsuke Meike, Masaki Nagane, Hironobu Yasui, Osamu Inanami
    FEBS LETTERS 587 20 3348 - 3353 2013年10月 [査読有り][通常論文]
     
    In this study, we provide evidence that endoplasmic reticulum (ER) stress suppresses DNA double-strand break (DSB) repair and increases radiosensitivity of tumor cells by altering Rad51 levels. We show that the ER stress inducer tunicamycin stimulates selective degradation of Rad51 via the 26S proteasome, impairing DSB repair and enhancing radiosensitivity in human lung cancer A549 cells. We also found that glucose deprivation, which is a physiological inducer of ER stress, triggered similar events. These findings suggest that ER stress caused by the intratumoral environment influences tumor radiosensitivity, and that it has potential as a novel target to improve cancer radiotherapy. (C) 2013 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.
  • Masaki Nagane, Hironobu Yasui, Tohru Yamamori, Songji Zhao, Yuji Kuge, Nagara Tamaki, Hiromi Kameya, Hideo Nakamura, Hirotada Fujii, Osamu Inanami
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 437 3 420 - 425 2013年08月 [査読有り][通常論文]
     
    Tumor hypoxia, which occurs mainly as a result of inadequate tissue perfusion in solid tumors, is a well-known challenge for successful radiotherapy. Recent evidence suggests that ionizing radiation (IR) upregulates nitric oxide (NO) production and that IR-induced NO has the potential to increase intratumoral circulation. However, the kinetics of NO production and the responsible isoforms for NO synthase in tumors exposed to IR remain unclear. In this study, we aimed to elucidate the mechanism by which IR stimulates NO production in tumors and the effect of IR-induced NO on tumor radiosensitivity. Hoechst33342 perfusion assay and electron spin resonance oxymetry showed that IR increased tissue perfusion and pO(2) in tumor tissue. Immunohistochemical analysis using two different hypoxic probes showed that IR decreased hypoxic regions in tumors; treatment with a nitric oxide synthase (NOS) inhibitor, L-NAME, abrogated the effects of IR. Moreover, IR increased endothelial NOS (eNOS) activity without affecting its mRNA or protein expression levels in SCCVII-transplanted tumors. Tumor growth delay assay showed that L-NAME decreased the anti-tumor effect of fractionated radiation (10 Gy x 2). These results suggested that IR increased eNOS activity and subsequent tissue perfusion in tumors. Increases in intratumoral circulation simultaneously decreased tumor hypoxia. As a result, IR-induced NO increased tumor radiosensitivity. Our study provides a new insight into the NO-dependent mechanism for efficient fractionated radiotherapy. (C) 2013 Elsevier Inc. All rights reserved.
  • Hironobu Yasui, Taketoshi Asanuma, Junichi Kino, Tohru Yamamori, Shunsuke Meike, Masaki Nagane, Nobuo Kubota, Mikinori Kuwabara, Osamu Inanami
    BMC CANCER 13 106  2013年03月 [査読有り][通常論文]
     
    Background: Glioblastoma is one of the intractable cancers and is highly resistant to ionizing radiation. This radioresistance is partly due to the presence of a hypoxic region which is widely found in advanced malignant gliomas. In the present study, we evaluated the effectiveness of the hypoxic cell sensitizer doranidazole (PR-350) using the C6 rat glioblastoma model, focusing on the status of blood brain barrier (BBB). Methods: Reproductive cell death in the rat C6 glioma cell line was determined by means of clonogenic assay. An intracranial C6 glioma model was established for the in vivo experiments. To investigate the status of the BBB in C6 glioma bearing brain, we performed the Evans blue extravasation test. Autoradiography with [C-14]-doranidazole was performed to examine the distribution of doranidazole in the glioma tumor. T2-weighted MRI was employed to examine the effects of X-irradiation and/or doranidazole on tumor growth. Results: Doranidazole significantly enhanced radiation-induced reproductive cell death in vitro under hypoxia, but not under normoxia. The BBB in C6-bearing brain was completely disrupted and [C-14]-doranidazole specifically penetrated the tumor regions. Combined treatment with X-irradiation and doranidazole significantly inhibited the growth of C6 gliomas. Conclusions: Our results revealed that BBB disruption in glioma enables BBB-impermeable radiosensitizers to penetrate and distribute in the target region. This study is the first to propose that in malignant glioma the administration of hydrophilic hypoxic radiosensitizers could be a potent strategy for improving the clinical outcome of radiotherapy without side effects.
  • Masato Eitaki, Tohru Yamamori, Shunsuke Meike, Hironobu Yasui, Osamu Inanami
    BMC CANCER 12 469  2012年10月 [査読有り][通常論文]
     
    Background: Anti-cancer drugs are widely used in cancer treatment frequently combined with surgical therapy and/or radiation therapy. Although surgery and radiation have been suggested to facilitate invasion and metastasis of tumor cells in some cases, there is so far little information about the effect of anti-cancer drugs on cellular invasive ability and metastasis. In this study, using four different anti-cancer drugs (vincristine, paclitaxel, cisplatin and etoposide), we examined whether these drugs influence the invasive ability of tumor cells. Methods: Human gastric adenocarcinoma MKN45 cells were used to evaluate the effect of anti-cancer drugs. After drug treatment, cellular invasive ability was assessed using the Matrigel invasion chamber. Cytoskeletal changes after treatment were examined microscopically with F-actin staining. In addition, we monitored cellular motility in 3D matrigel environment by time-lapse microscopic analysis. The drug-induced activation of RhoA and ROCK was evaluated by pull-down assay and Western blotting using an antibody against phosphorylated myosin light chain (MLC), respectively. Where necessary, a ROCK inhibitor Y27632 and siRNA for guanine nucleotide exchange factor-H1 (GEF-H1) were applied. Results: Among all drugs tested, only vincristine stimulated the invasive ability of MKN45 cells. Microscopic analysis revealed that vincristine induced the formation of non-apoptotic membrane blebs and amoeboid-like motility. Vincristine significantly enhanced RhoA activity and MLC phosphorylation, suggesting the involvement of RhoA/ROCK pathway in the vincristine-induced cytoskeletal reorganization and cellular invasion. Furthermore, we found that Y27632 as well as the siRNA for GEF-H1, a RhoA-specific activator, attenuated MLC phosphorylation, the formation of membrane blebs and the invasive ability after vincristine treatment. Conclusions: These results indicate that vincristine activates GEF-H1/RhoA/ROCK/MLC signaling, thereby promoting amoeboid-like motility and the invasive ability of MKN45 cells.
  • Hiroko P. Indo, Osamu Inanami, Tomoko Koumura, Shigeaki Suenaga, Hsiu-Chuan Yen, Shizuko Kakinuma, Ken-Ichiro Matsumoto, Ikuo Nakanishi, William St Clair, Daret K. St Clair, Hirofumi Matsui, Richard Cornette, Oleg Gusev, Takashi Okuda, Yasuhito Nakagawa, Toshihiko Ozawa, Hideyuki J. Majima
    FREE RADICAL RESEARCH 46 8 1029 - 1043 2012年08月 [査読有り][通常論文]
     
    HLE, a human hepatocellular carcinoma cell line was transiently transfected with normal human MnSOD and MnSOD without a mitochondrial targeting signal (MTS). Mitochondrial reactive oxygen species (ROS), lipid peroxidation and apoptosis were examined as a function of time following 18.8 Gy X-ray irradiation. Our results showed that the level of mitochondrial ROS increased and reached a maximum level 2 hours after X-ray irradiation. Authentic MnSOD, but not MnSOD lacking MTS, protected against mitochondrial ROS, lipid peroxidation and apoptosis. In addition, the levels of mitochondrial ROS were consistently found to always correlate with the levels of authentic MnSOD in mitochondria. These results suggest that only when MnSOD is located in mitochondria is it efficient in protecting against cellular injuries by X-ray irradiation and that mitochondria are the critical sites of X-ray-induced cellular oxidative injuries.
  • Tohru Yamamori, Hironobu Yasui, Masayuki Yamazumi, Yusuke Wada, Yoshinari Nakamura, Hideo Nakamura, Osamu Inanami
    FREE RADICAL BIOLOGY AND MEDICINE 53 2 260 - 270 2012年07月 [査読有り][通常論文]
     
    Whereas ionizing radiation (Ir) instantaneously causes the formation of water radiolysis products that contain some reactive oxygen species (ROS), ROS are also suggested to be released from biological sources in irradiated cells. It is now becoming clear that these ROS generated secondarily after Ir have a variety of biological roles. Although mitochondria are assumed to be responsible for this Ir-induced ROS production, it remains to be elucidated how It triggers it. Therefore, we conducted this study to decipher the mechanism of Ir-induced mitochondria! ROS production. In human lung carcinoma A549 cells, Ir (10 Gy of X-rays) induced a time-dependent increase in the mitochondrial ROS level. It also increased mitochondrial membrane potential, mitochondrial respiration, and mitochondrial ATP production, suggesting upregulation of the mitochondrial electron transport chain (ETC) function after Ir. Although we found that Ir slightly enhanced mitochondrial ETC complex II activity, the complex II inhibitor 3-nitropropionic acid failed to reduce Ir-induced mitochondrial ROS production. Meanwhile, we observed that the mitochondrial mass and mitochondrial DNA level were upregulated after Ir, indicating that It increased the mitochondrial content of the cell. Because irradiated cells are known to undergo cell cycle arrest under control of the checkpoint mechanisms, we examined the relationships between cell cycle and mitochondrial content and cellular oxidative stress level. We found that the cells in the G2/M phase had a higher mitochondrial content and cellular oxidative stress level than cells in the G1 or S phase, regardless of whether the cells were irradiated. We also found that It-induced accumulation of the cells in the G2/M phase led to an increase in cells with a high mitochondrial content and cellular oxidative stress level. This suggested that Ir upregulated mitochondrial ETC function and mitochondrial content, resulting in mitochondrial ROS production, and that It-induced G2/M arrest contributed to the increase in the mitochondrial ROS level by accumulating cells in the G2/M phase. (C) 2012 Elsevier Inc. All rights reserved.
  • Kobayashi S, Abe Y, Inanami O, Oda S, Yamauchi K, Hankanga C, Yasuda J, Sato R
    Veterinary immunology and immunopathology 143 1-2 155 - 161 2011年09月 [査読有り][通常論文]
     
    Lactoferrin, a glycoprotein present in neutrophils and exocrine secretions, plays important roles in host defense. Administration of bovine lactoferrin has been reported to modulate various neutrophil functions. We found a mixed-breed male dog with novel familial neutrophil dysfunction. The disorder was caused by a decrease of beta 2-integrin expression encoding CD18 without mutation. Antibiotics therapy alone did not influence a series of neutrophil functions in the same dog. We examined the effects of oral administration of bovine lactoferrin on the neutrophil function and clinical symptoms in the same dog. Oral chronic administration of bovine lactoferrin increased neutrophilic beta 2-integrin gene expression comparable to normal dogs, followed by the upregulation of surface CD18 expression. Concurrently, the superoxide production, phagocytic activity and adherence that were beta 2-integrin-related neutrophil functions increased to normal canine levels. The chronic inflammation from bacterial upper respiratory infections and pneumonia was also alleviated in the dog. Our results indicate that oral treatment with bovine lactoferrin increases neutrophil beta 2-integrin transcript level, leading to the upregulation of neutrophil functions and improvement of clinical symptoms in the dog with familial neutrophil dysfunction. (C) 2011 Elsevier B.V. All rights reserved.
  • Shunsuke Meike, Tohru Yamamori, Hironobu Yasui, Masato Eitaki, Akira Matsuda, Osamu Inanami
    JOURNAL OF RADIATION RESEARCH 52 4 456 - 463 2011年07月 [査読有り][通常論文]
     
    The combination of a chemotherapeutic agent and radiation is widely applied to enhance cell death in solid tumor cells in cancer treatment. The purine analogue 8-aminoadenosine (8-NH(2)-Ado) is known to be a transcription inhibitor that has proved very effective in multiple myeloma cell lines and primary indolent leukemia cells. In this report, to examine whether 8-NH(2)-Ado had the ability to enhance the radiation-induced cell killing in solid tumor cells, human lung adenocarcinoma A549 cells were irradiated in the presence and absence of 8-NH(2)-Ado. 8-NH(2)-Ado significantly increased reproductive cell death and apoptosis in A549 cells exposed to X-rays. When peptide inhibitors against caspase-3, -8, and -9 were utilized to evaluate the involvement of caspases, all inhibitors suppressed the enhancement of radiation-induced apoptosis, suggesting that not only mitochondria-mediated apoptotic signal transduction pathways but also death receptor-mediated pathways were involved in this enhancement of apoptosis. In addition, in the cells exposed to the treatment combining X-irradiation and 8-NH(2)-Ado, reduction of the intracellular ATP concentration was essential for survival, and down-regulation of the expression of antiapoptotic proteins such as survivin and XIAP was observed. These results indicate that 8-NH(2)-Ado has potential not only as an anti-tumor drug for leukemia and lymphoma but also as a radiosensitizing agent for solid tumors.
  • Meike S, Yamamori T, Yasui H, Eitaki M, Matsuda A, Morimatsu M, Fukushima M, Yamasaki Y, Inanami O
    Molecular cancer 10 92  2011年07月 [査読有り][通常論文]
     
    Background: A novel anticancer drug 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine (ECyd, TAS106) has been shown to radiosensitize tumor cells and to improve the therapeutic efficiency of X-irradiation. However, the effect of TAS106 on cellular DNA repair capacity has not been elucidated. Our aim in this study was to examine whether TAS106 modified the repair capacity of DNA double-strand breaks (DSBs) in tumor cells. Methods: Various cultured cell lines treated with TAS106 were irradiated and then survival fraction was examined by the clonogenic survival assays. Repair of sublethal damage (SLD), which indicates DSBs repair capacity, was measured as an increase of surviving cells after split dose irradiation with an interval of incubation. To assess the effect of TAS106 on the DSBs repair activity, the time courses of gamma-H2AX and 53BP1 foci formation were examined by using immunocytochemistry. The expression of DNA-repair-related proteins was also examined by Western blot analysis and semi-quantitative RT-PCR analysis. Results: In clonogenic survival assays, pretreatment of TAS106 showed radiosensitizing effects in various cell lines. TAS106 inhibited SLD repair and delayed the disappearance of gamma-H2AX and 53BP1 foci, suggesting that DSB repair occurred in A549 cells. Western blot analysis demonstrated that TAS106 down-regulated the expression of BRCA2 and Rad51, which are known as keys among DNA repair proteins in the homologous recombination (HR) pathway. Although a significant radiosensitizing effect of TAS106 was observed in the parental V79 cells, pretreatment with TAS106 did not induce any radiosensitizing effects in BRCA2-deficient V-C8 cells. Conclusions: Our results indicate that TAS106 induces the down-regulation of BRCA2 and the subsequent abrogation of the HR pathway, leading to a radiosensitizing effect. Therefore, this study suggests that inhibition of the HR pathway may be useful to improve the therapeutic efficiency of radiotherapy for solid tumors.
  • Tohru Yamamori, Ayano Mizobata, Yoshiro Saito, Yasuomi Urano, Osamu Inanami, Kaikobad Irani, Noriko Noguchi
    FREE RADICAL RESEARCH 45 3 342 - 350 2011年03月 [査読有り][通常論文]
     
    6-Hydroxydopamine (6-OHDA) is a neurotoxin that has been widely used to generate Parkinson's disease (PD) models. Increased oxidative stress is suggested to play an important role in 6-OHDA-induced cell death. Given the lessened susceptibility to oxidative stress exhibited by mice lacking p66shc, this study investigated the role of p66shc in the cytotoxicity of 6-OHDA. 6-OHDA induced cell death and p66shc phosphorylation at Ser36 in SH-SY5Y cells. Pre-treatment with the protein kinase C beta (PKC beta) inhibitor hispidin suppressed 6-OHDA-induced p66shc phosphorylation. Elimination of H(2)O(2) by catalase reduced cell death and p66shc phosphorylation induced by 6-OHDA. Cells deficient in p66shc were more resistant to 6-OHDA-induced cell death than wild-type cells. Furthermore, reconstitution of wild-type p66shc, but not the S36A mutant, in p66shc-deficient cells increased susceptibility to 6-OHDA. These results indicate that H(2)O(2) derived from 6-OHDA is an important mediator of cell death and p66shc phosphorylation induced by 6-OHDA and that p66shc phosphorylation at Ser36 is indispensable for the cytotoxicity of 6-OHDA.
  • ラット脳腫瘍における間欠的低酸素の可視化と放射線感受性に与える影響
    安井博宣, 新坊弦也, 山盛徹, 永根大幹, 趙松吉, 久下祐司, 稲波修
    日本獣医学会学術集会講演要旨集 152nd 316  2011年 [査読無し][通常論文]
  • Hironobu Yasui, Nozomi Ito, Tohru Yamamori, Hideo Nakamura, Jun Okano, Taketoshi Asanuma, Takayuki Nakajima, Mikinori Kuwabara, Osamu Inanami
    FREE RADICAL RESEARCH 44 6 645 - 654 2010年06月 [査読有り][通常論文]
     
    It has previously been suggested that the spin trap agent alpha-phenyl-N-tert-butylnitrone (PBN) induces neurite outgrowth through activation of the Ras-ERK pathway in PC12 cells. However, the chemical properties of PBN contributing to its biological function and the detailed mechanism for the activation of Ras by PBN remain unknown. This study demonstrates that the hydrophobic structure of PBN is related to the activation of Ras, by comparing with hydrophilic analogues of PBN. [(14)C]-labelled PBN was found to localize in the lipid fraction and activate Ras indirectly. On the other hand, neurite outgrowth by PBN was inhibited by a nitric oxide (NO) scavenger. Moreover, the neurite outgrowth induced by PBN and the NO donor NOR4 was inhibited by the dominant negative Ras or MAPK/ERK inhibitor. Taken together, these results suggest that PBN is incorporated into the plasma membrane and induces neurite outgrowth in PC12 cells by activating the Ras-ERK pathway through NO release.
  • Yasuko Watanabe, Wakako Hiraoka, Manabu Igarashi, Kimihito Ito, Yuhei Shimoyama, Motohiro Horiuchi, Tohru Yamamoria, Hironobu Yasui, Mikinori Kuwabara, Fuyuhiko Inagaki, Osamu Inanami
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 394 3 522 - 528 2010年04月 [査読有り][通常論文]
     
    To explore Cu(II) ion coordination by His(186) in the C-terminal domain of full-length prion protein (moPrP), we utilized the magnetic dipolar interaction between a paramagnetic metal, Cu(II) ion, and a spin probe introduced in the neighborhood of the postulated binding site by the spin labeling technique (SDSL technique) Six moPrP mutants. moPrP(D143C), moPrP(Y148C), moPrP(E151C), moPrP(Y156C), moPrP(T189C). and moPrP(Y156C,H186A), were reacted with a methane thiosulfonate spin probe and a nitroxide residue (R1) was created in the binding site of each one Line broadening of the ESR spectra was induced in the presence of Cu(II) ions in moPrP(Y148R1), moPrP(Y151R1). moPrP(Y156R1), and moPrP(T189R1) but not moPrP(D143R1) This line broadening indicated the presence of electron-electron dipolar interaction between Cu(II) and the rutroxide spin probe, suggesting that each interspin distance was within 20 angstrom The interspin distance ranges between Cu(II) and the spin probes of moPrP(Y148R1), inoPrP(Y151R1), moPrP(Y156R1), and moPrP(T189R1) were estimated to be 12 1 angstrom, 18 1 angstrom, 107 angstrom, and 84 angstrom, respectively In moPrP(Y156R1,H186A), line broadening between Cu(II) and the spin probe was not observed These results suggest that a novel Cu(II) binding site is involved in His186 in the Helix2 region of the C-terminal domain of moPrP(C) (C) 2010 Elsevier Inc All rights reserved
  • Taketoshi Asanuma, Masafumi Ono, Kei Kubota, Akira Hirose, Yoshihiro Hayashi, Toshiji Saibara, Osamu Inanami, Yasuhiro Ogawa, Hideaki Enzan, Saburo Onishi, Mikinori Kuwabara, Jude A. Oben
    GUT 59 2 258 - 266 2010年02月 [査読有り][通常論文]
     
    Background The pathogenesis of non-alcoholic fatty liver disease (NAFLD) is incompletely understood. Kupffer cells (KCs), phagocytic liver-resident macrophages, provide a protective barrier against egress of endotoxin from the portal to the systemic circulation. It is not known if KC phagocytic function is impaired in NAFLD. Super-paramagnetic iron oxide ( SPIO) magnetic resonance imaging is a comparative technology dependent on KC phagocytic function. Objective To evaluate KC uptake function, in patients and experimental animals with NAFLD, using SPIO. Methods Abdominal CT and histological examination of liver biopsy specimens were used to estimate the degree of steatosis in patients with NAFLD and controls with chronic hepatitis C. SPIO-MRI was then performed in all patients. Normal rats fed a methionine-choline-deficient diet to induce non-alcoholic steatohepatitis (NASH), the more severe stage of NAFLD, and obese, insulin resistant, Zucker fa/fa rats with steatohepatitis, were also studied with SPIO-MRI and analysed for hepatic uptake of fluorescent microbeads. Immunohistochemical analysis evaluated the numbers of KCs in patients and rat livers. Results Relative signal enhancement (RSE), inversely proportional to KC function, was higher in patients with NAFLD than in controls and with the degree of steatosis on CT. RSE also positively correlated with the degree of steatosis on histology and was similarly higher in rats with induced severe NAFLD ( NASH). On immunohistochemistry, defective phagocytic function was the result of reduced phagocytic uptake and not due to reduced KC numbers in rats or patients with NAFLD. Conclusions KC uptake function is significantly impaired in patients with NAFLD and experimental animals with NASH, worsens with the degree of steatosis and is not due to a reduction of KC numbers.
  • Saori Kobayashi, Reeko Sato, Yuya Abe, Osamu Inanami, Hironobu Yasui, Katsuhiko Omoe, Jun Yasuda, Careen Hankanga, Shinichi Oda, Juso Sasaki
    VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 130 3-4 187 - 196 2009年08月 [査読有り][通常論文]
     
    Canine leukocyte adhesion deficiency (CLAD) in Irish setters is caused by genetic defects of leukocyte integrin CD18 leading to recurrent bacterial infections. We report clinical features and analysis of neutrophil function from two mixed-breed canine littermates (one female and one male dog) similar to CLAD. The symptoms of pyogenic infection were first recognized at 3 months of age and since then the patients suffered from recurrent bacterial infections. These clinical findings were strongly suggestive of genetic phagocyte dysfunction. Neutrophil function tests revealed a marked reduction of serum-opsonized zymosan-mediated superoxide production in the two littermates. Neutrophils of the male dog revealed impaired integrin-mediated adherence and phagocytic activity, whereas ability of serum opsonization was normal. There was also a profound decrease of surface expression of CD11b/CD18 and beta 2-integrin transcript level, detected by real-time RT-PCR without missense mutations unlike CLAD. Immunoblot analysis indicated that protein expression of cytochrome 15558 component gp91(phox), the cytosolic components p47(phox) and P67(phox) of NADPH oxidase components increased profoundly in the male. Our study suggests that decreased transcriptional levels of beta 2-integrin without mutations, lead to downregulation of surface expression, resulting in multiple defects in adhesion-related neutrophil functions and consequently, recurrent bacterial infections from puppyhood. (C) 2009 Elsevier B.V. All rights reserved.
  • Aki Ogura, Shigeru Oowada, Yasuhiro Kon, Aki Hirayama, Hironobu Yasui, Shunsuke Meike, Saori Kobayashi, Mikinori Kuwabara, Osamu Inanami
    CANCER LETTERS 277 1 64 - 71 2009年05月 [査読有り][通常論文]
     
    Mitochondria in mammalian cells are well-known to play an important role in the intrinsic pathway of genotoxic-agent-induced apoptosis by releasing cytochrome c into cytosol and to be a major source of reactive oxygen species (ROS). The aim of this study was to examine whether mitochondrial ROS are involved in radiation-induced apoptotic signaling in A549 cells. Post-irradiation treatment with N-acetyl-L-cystein (NAC) inhibited cytochrome c release from mitochondria but did not affect expression levels of Bcl-2, Bcl-X-L and Bax, suggesting that late production of ROS triggered cytochrome c release. Experiments using DCFDA (a classical ROS fluorescence probe) and MitoAR (a novel mitochondrial ROS probe) demonstrated that intracellular and mitochondrial ROS were enhanced 6 h after X irradiation. Furthermore, the O-2(-center dot) production ability of mitochondria isolated from A549 cells was evaluated by ESR spectroscopy combined with a spin-trapping reagent (CYPMPO). When isolated mitochondria were incubated with NADH, succinate and CYPMPO, an ESR spectrum due to CYPMPO-OOH was detected. This NADH/succinate-dependent O-2(-center dot) production from mitochondria of irradiated cells was significantly increased in comparison with that of unirradiated cells. These results indicate that ionizing radiation enhances O-2(-center dot), production from mitochondria to trigger cytochrome c release in A549 cells. (C) 2008 Elsevier Ireland Ltd. All rights reserved.
  • Yun-Wei Chiang, Yuki Otoshima, Yasuko Watanabe, Osamu Inanami, Yuhei Shimoyama
    JOURNAL OF BIOMOLECULAR STRUCTURE & DYNAMICS 26 3 355 - 365 2008年12月 [査読有り][通常論文]
     
    Valine 160 on beta-sheet-2 (S2) of mouse prion (moPrP(C)) has been previously identified as the most highly pH-sensitive site on moPrP(C) by ESR spectroscopy using site-directed spin labeling (SDSL) technique. However, no further theoretical analysis to reveal the molecular dynamics reported on the experimental ESR spectra is available. The X-band ESR spectra of R1 nitroxide spin label at V160 and four other sites are carefully analyzed over large pH and temperature ranges using a spectral simulation method based upon stochastic Liouville equation (SLE). The results clearly reveal the dynamics and ordering of the local environment of V160R1 showring that (i) molecular mobility of V160r1 on S2 gradually increases with a decrease of pH from 7.5 to 4.5: (ii) two distinctly different spectral components are simulateneously present in all spectra of V160R1 studied. The spectral components are, respectively, denoted as immobile (lm), characterized by lower molecular mobility and higher ordering, and mobile (Mb) component of high mobility and low ordering. The population ratio (lm/Mb) increases with increasing pH. while lm remains dominant in all V160R1 spectra. It suggests a more mobile and disordered dynamic molecular structure for mouse PrP(c), which is very likely correlated with increased beta-sheet content at low pH, as the environment changes from neutral to acidic pH. Together with the results of the SLE-based analyses on the spectra of other sites that appear pH-insensitive. we suggest that the simultaneous presence of the spectral components for V160R1 is strongly correlated with the coexistence of multiple protein conformations in local structure of PrP(C) over the varied pH range. It demonstrates that the combined approach of the SDSL technique and the SLE-based analysis leads to a powerful method for unraveling the complexity of protein dynamics.
  • H. Yasui, A. Ogura, T. Asanuma, A. Matsuda, I. Kashiwakura, M. Kuwabara, O. Inanami
    BRITISH JOURNAL OF CANCER 99 9 1442 - 1452 2008年10月 [査読有り][通常論文]
     
    In a previous study, we showed that a novel anticancer drug, 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl) cytosine (TAS106, ECyd) increased the antitumour efficacy of X-irradiation. However, its effects on hypoxic cells in tumours remain unclarified. Here, we show that TAS106 enhances the induction of apoptosis in X-irradiated human gastric adenocarcinoma MKN45 and MKN28 cells under hypoxia in vitro. At the same time, the accumulation of HIF-1 alpha observed under hypoxia was shown to be decreased to the level of normoxia in the presence of 0.1 mu M TAS106. To study the function of HIF-1 alpha protein in apoptosis of hypoxic cells, we employed an HIF-1 alpha reductive approach using its specific antisense oligodeoxynucleotide. The reduction of HIF-1 alpha gene expression dramatically enhanced X-ray-induced apoptosis in hypoxic cells. In in vivo experiments in which MKN45 cells were transplanted into severe combined immunodeficient (SCID) mice, TAS106 (0.5 mg kg(-1)) suppressed HIF-1 alpha expression and subsequently reduced the area of the hypoxic region in the tumour and enhanced the induction of apoptosis in the hypoxic region when combined with 2Gy of X-irradiation. These results suggest the possibility that TAS106 acts as a potent radiosensitiser through the inhibition of HIF-1 alpha expression and can be a useful agent against radiotherapy-resistant hypoxic cells in solid tumours.
  • Taketoshi Asanuma, Sabrina Doblas, Yasvir A. Tesiram, Debra Saunders, Rebecca Cranford, Hironobu Yasui, Osamu Inanami, Nataliya Smith, Robert A. Floyd, Yashige Kotake, Rheal A. Towner
    JOURNAL OF MAGNETIC RESONANCE IMAGING 28 3 574 - 587 2008年09月 [査読有り][通常論文]
     
    Purpose: To apply fiber tractography to assess the effect of a possible antiglioma drug, phenyl N-tert-butyl nitrone (PBN), on glioma-affected neuronal fibers. The fiber tractography method was able to differentiate between different tumor types. such as the C6 and F98 rat glioma models. Materials and Methods: C6 or F98 cells were intracranially injected into the cortex of male Fischer 344 rats. PBN treatment was initiated before or after cell implantation. Tumor growth was monitored with diffusion tensor imaging (DTI) and fiber tractography using diffusion-weighting gradients in 30 noncolinear directions. neuronal fiber tractography. we were able to evaluate the protective effect of PBN against invasive glioma growth in rat brains. PBN provided protection of the neuronal fibers against tumor-induced ischemia and tumor invasion. Results: Although proton density-weighted (PDw) and T2-weighted (T2w) images did not show any difference between C6 and F98 gliomas without edema, the apparent diffusion coefficient (ADC) and fractional anisotropy (FA) maps were able to discriminate between these two tumor models. Fiber tractography was used to) visualize C6 glioma-induced ischemia of tumor-surrounding tissues, whereas F98 glioma was found to infiltrate and penetrate into the corpus callosum (CC). During glioma growth, neuronal fibers were found to disappear at the border regions between the tumor and surrounding tissues. PBN treatment was shown to inhibit glioma growth with accompanying changes in the surrounding tissue. Conclusion: By noninvasively monitoring the degree of neuronal fiber integrity and connectivity with the use of
  • Mikinori Kuwabara, Taketoshi Asanuma, Koichi Niwa, Osamu Inanami
    JOURNAL OF CLINICAL BIOCHEMISTRY AND NUTRITION 43 2 51 - 57 2008年09月 [査読有り][通常論文]
     
    Oxidative stress stimulates two opposite signaling pathways leading to cell death and cell survival. Preferential selection of survival signals leads to the protection of cells against damage induced by reactive oxygen species, whereas preferential acceleration of death signals can be used to advantage in tumor therapy with oxidizing agents such as ionizing radiation and anticancer drugs. In vitro and in vivo experiments using cultured mammalian cells and experimental animals showed that ERK was included in survival signals and SAPK and p38 MAPK in death signals in oxidative stress. The activation of SAPK/JNK and subsequent expression of death receptor Fas on the cell surface caused the induction of cell death. The results mean that the acceleration of the activation of SAPK/JNK might lead to the enhancement of cell death by oxidizing agents like ionizing radiation and anticancer drugs. In fact, when cultured mammalian cells were exposed to ionizing radiation with 2-nitroimidazole derivatives having electrophilicity, the lethal effect of ionizing radiation was found to be enhanced together with the activation of SAPK/JNK and the enhancement of Fas expression. The activation of both survival and death signals was suppressed by the antioxidants N-acetylcystein and Trolox, suggesting that both signaling pathways are redox-regulated.
  • Taketoshi Asanuma, Hironobu Yasui, Masayasu Sato, Osamu Inanami, Mikinori Kuwabara
    JAPANESE JOURNAL OF VETERINARY RESEARCH 56 2 99 - 107 2008年08月 [査読有り][通常論文]
     
    This study was performed to examine whether the brain activities induced by noxious algesic chemical substances in anesthetized animals could be detected by blood oxygen-level-dependent functional magnetic resonance imaging (BOLD-fMRI). Multislice gradient echo images of the primary somatosensory cortex were obtained using a 7.05 T superconducting system and a one-turned surface coil centered over the primary somatosensory cortex of the 1.0%-isoflurane-anesthetized rat. The Z-score t-map of BOLD signals and its time-course analysis revealed that subcutaneous injection of formalin into the left forepaw immediately induced an early response in the contralateral primary sensory cortex lasting for a few minutes, followed by a late response until 20 min after stimulation. In contrast, injection of capsaicin into the left forepaw evoked only the early response. Furthermore, pretreatment with morphine completely abolished these responses induced by the chemical algesic substances. Thus BOLD-fMRI is a useful method to analyze the brain activities of painful stimulation in anesthetized animals.
  • Daisuke Iizuka, Aki Ogura, Mikinori Kuwabara, Osamu Inanami
    ANTI-CANCER DRUGS 19 6 565 - 572 2008年07月 [査読有り][通常論文]
     
    To clarify the mechanisms of purvalanol A in the induction of apoptosis, we investigated whether purvalanol A influenced the RNA synthesis and expression of RNA polymerase II and signal transducer and activator of transcription 3 (STAT3). When MKN45 cells were treated with 30 mu mol/l purvalanol A, mitochondrial dysfunction occurred before the induction of the apoptosis and the expression of antiapoptotic proteins survivin, Bcl-X(L), and Bcl-2 was reduced. The treatment with parvalanol A was also shown to reduce not only mRNA for these proteins but also global RNA synthesis. The phosphorylation of the carboxy-terminal domain of RNA polymerase II, which was involved in transcriptional regulation, was strongly inhibited by purvalanol A, followed by the partial inhibition of the expression of RNA polymerase II. Furthermore, the phosphorylation at Tyr705 of STAT3, which is known to be a phosphorylation site for Janus kinase 2 (JAK2), was completely inhibited by purvalanol A early (3 h) after drug treatment, although the phosphorylation of STAT3 at Ser727, which is a phosphorylation site for Ras/Raf/MEK and extracellular signal-regulated protein kinase 1/2, was still detectable until late (12 h) after treatment. In addition, the tyrosine phosphorylation of JAK2 was efficiently inhibited by purvalanol A. These results suggest that the inhibition of JAK2/STAT3 and RNA polymerase II is crucial in the downregulation of antiapoptotic proteins leading to the apoptotic cell death induced by parvalanol A.
  • Akihiro Kamikawa, Tatsuya Ishii, Kohei Shimada, Kennedy Makondo, Osamu Inanami, Naoki Sakane, Toshihide Yoshida, Masayuki Saito, Kazuhiro Kimura
    DIABETES-METABOLISM RESEARCH AND REVIEWS 24 4 331 - 338 2008年05月 [査読有り][通常論文]
     
    Background Proinsulin C-peptide shows ameliorative effects on diabetic complications, possibly through the production of nitric oxide (NO). On the contrary, increased local availability of NO and expression of endothelial NO synthase (eNOS) in the renal endothelium are shown to be involved in the progression of diabetic nephropathy. The aim of this study was to elucidate the effect of C-peptide and insulin as a reference on the eNOS expression in the early phase of type 1 diabetic rat kidney. Methods Type 1 diabetes in rats was produced by streptozotocin injection and some of the rats were treated with either C-peptide or insulin by the aid of an osmotic pump for 1 week. Conventional biochemical and histological analyses were performed on tissue samples. Results The diabetic rats showed hyperglycemia with over 90% reduction of endogenous insulin and C-peptide. Replacement with C-peptide or insulin resulted in recovery of weight lost, but only insulin infusion lowered plasma-glucose concentration. The eNOS protein was localized in glomeruli and endothelial cells of arterioles, and its amounts in the kidneys, but not in the lungs, of diabetic rats was increased. Replacement with C-peptide or insulin-abrogated diabetes-induced increase of renal eNOS protein. Conclusion The results indicate that C-peptide suppresses diabetes-induced abnormal renal eNOS expression, by which C-peptide may exert beneficial effects on diabetic nephropathy. Copyright (C) 2007 John Wiley & Sons, Ltd.
  • Saori Kobayashi, Reeko Sato, Takako Aoki, Katsuhiko Omoe, Osamu Inanami, Careen Hankanga, Yuichi Yamada, Nobuyuki Tomizawa, Jun Yasuda, Juso Sasaki
    JOURNAL OF VETERINARY MEDICAL SCIENCE 70 5 429 - 435 2008年05月 [査読有り][通常論文]
     
    Feline immunodeficiency virus (FIV) infection is characterized by chronic overactivation of immune and inflammatory system, resulting in anergic state and dysfunction of immune cells. Lactoferrin (LF), a glycoprotein present in exocrine secretions and neutrophils, plays an important role in host defense system. Our previous study showed that oral administration of bovine LF (bLF) suppressed oral inflammation, improved the clinical symptoms and decreased serum gamma-globulin as a marker of inflammation in FIV-infected cats with intractable stomatitis. The anti-inflammatory effect was partly involved in regulation of neutrophil function by bLF. In this study, to clarify the relationship between anti-inflammatory effects of bLF and peripheral blood mononuclear cells (PBMC), we examined the effect of bLF on proliferation, cell cycle progression and cytokine expression in mitogen-activated PBMC. MTT [3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide] assay showed that bLF inhibited the concanavalin A (ConA)-induced cell proliferation in FIV-infected cats with the asymptomatic carrier and AIDS-related complex (ARC) phase. Bovine LF restored ConA-induced cell cycle progression and resulted in suppression of the induced apoptosis in feline PBMC. Real-time RT-PCR showed that bLF suppressed ConA-induced expression of interferon-gamma and interleukin-2 in cells of the ARC group regardless of the time of its addition to the medium. These results suggest the hypothesis that therapy with bLF may have the potential to improve and protect functions of overactivated lymphocytes by modulating the cell proliferation, cell cycle and cytokines expression in cats in terminal stage of FIV infection.
  • Yuichi Yamada, Reeko Sato, Saori Kobayashi, Careen Hankanga, Osamu Inanami, Mikinori Kuwabara, Yutaka Momota, Nobuyuki Tomizawa, Jun Yasuda
    JOURNAL OF VETERINARY MEDICAL SCIENCE 70 5 443 - 448 2008年05月 [査読有り][通常論文]
     
    Lactoferrin has several biological activities, including antitumor activities in some human and animal tumor cells. Clinical trials have been carried out in human medicine based on these effects. However, the antitumor effects of lactoferrin in veterinary medicine remain unknown. In this in vitro study, we demonstrated that co-incubation of canine mammary gland tumor cells (CIPp and CHMp) and bovine lactoferrin induced growth arrest of tumor cells. This growth arrest was associated with induction of G1 arrest. Furthermore, this effect was stronger in tumor cells than in normal cells. These findings demonstrate that bovine lactoferrin has anti-tumor activity in canine mammary tumors and has the potential for use in tumor-bearing dogs.
  • Tatsuro Kosugi, Koichi Kawahara, Motoki Tanaka, Yasuko Watanabe, Osamu Inanami
    NEUROCHEMISTRY INTERNATIONAL 52 4-5 887 - 896 2008年03月 [査読有り][通常論文]
     
    "Ca2+ paradox" is the phenomenon whereby the intracellular concentration of Ca2+ paradoxically increases during reperfusion with normal Ca2+-containing media after brief exposure to a Ca2+-free solution. The present study aims to characterize the Ca2+ paradox induced cell injury in neuron/astrocyte co-cultures. Prior exposure of the co-cultures to a low Ca2+ solution for 60 min significantly injured only neurons after reperfusion with a normal Ca2+ medium for 24 h, but astrocytes remained intact. An analysis of the Ca2+ paradox-induced changes in the intracellular concentration of Na+ revealed that the concentration in astrocytes increased significantly during the reperfusion episode, resulting in a reversal of the operation of the astrocytic Na+-dependent glutamate transporter GLT-1. These results suggested that Ca2+ paradox-induced accumulation of Na+ in astrocytes was crucially involved in the excitotoxic neuronal injury resulting from the reversed astrocytic GLT-1 during the reperfusion episode. Previous studies have suggested that Ca2+ paradox-induced injury in the brain occurs first in astroglial cells and only later in neurons resulting from the prior damage of astrocytes. Here we show that if "Ca2+ paradox" occurs in the brain, neurons would be the primary target of Ca2+ paradox-induced cell injury in the central nervous system. (C) 2007 Elsevier Ltd. All rights reserved.
  • Asami Oriya, Kenji Takahashi, Osamu Inanami, Toshiaki Miura, Yoshinao Abe, Mikinori Kuwabara, Ikuo Kashiwakura
    JOURNAL OF RADIATION RESEARCH 49 2 113 - 121 2008年03月 [査読有り][通常論文]
     
    The aim of this study is to evaluate the individual differences in radiosensitivity of lineage-committed myeloid hematopoietic progenitors, colony-forming cells (CFC), detected in steady-state human peripheral blood (PB). Mononuclear cells were prepared from the buffy-coat of 30 individuals PB, and were assayed for CFC by semi-solid culture supplemented with cytokines. X irradiation was performed in the range of 0.5 - 4 Gy at a dose rate of about 80 cGy/min. The mean number of hematopoietic progenitor cells is 5866 +/- 3408 in 1 ml of buffy-coat, suggesting that the erythroid progenitor cells are the major population. The total CFC radiosensitivity parameter D-0 and n value are 1.18 +/- 0.24 and 1.89 +/- 0.98, respectively. Using a linear regression analysis, a statistically significant correlation is observed between the D-0 value and the surviving fraction at 4 Gy (r = 0.611 p < 0.001). Furthermore, we evaluate the relationship between individual radio sensitivity and the level of antioxidants, plasma uric acid, plasma bilirubin, and intracellular glutathione. No statistically significant correlations are observed, however, between the D-0 parameter and the level of antioxidants, plasma uric acid, plasma bilirubin, and intracellular glutathione. The present study demonstrates that there are large individual differences in the radiosensitivity of hematopoietic progenitor cells as detected in steady-state human PB. These differences demonstrate almost no correlation with plasma or intracellular antioxidants. The prediction of individual differences in radiosensitivity of CFC can only be measured by 4 Gy irradiation.
  • Momoko Takahashi, Hironobu Yasui, Aki Ogura, Taketoshi Asanuma, Nobuo Kubota, Michihiko Tsujitan, Mikinori Kuwabara, Osamu Inanami
    JOURNAL OF RADIATION RESEARCH 49 2 153 - 161 2008年03月 [査読有り][通常論文]
     
    Our previous study showed that X irradiation induced the expression of death receptor DR5 on the cell surface in tumor cell lines under not only normoxia but also hypoxia. X irradiation combined with TNF alpha-related apoptosis-inducing ligand (TRAIL), which is the ligand of DR5, induced apoptosis in vitro (Takahashi et al., (2007) Journal of Radiation Research, 48: 461-468). In this report, we examined the in vivo antitumor efficacy of X irradiation combined with TRAIL treatment in tumor xenograft models derived from human gastric adenocarcinoma MKN45 and MKN28 cells in SCID mice. X irradiation combined with TRAIL synergistically suppressed the tumor growth rates in the xenograft models derived from MKN45 and MKN28 cells, which have wild type Tp53 and mutated Tp53, respectively, indicating that the antitumor effects occurred in a Tp53-independent manner. Histological analysis showed that the combination of X irradiation and TRAIL induced caspase-3-dependent apoptotic cell death. Moreover, the immunohistochemical detection of hypoxic regions using the hypoxic marker pimonidazole revealed that caspase-3-dependent apoptosis occurred in the hypoxic regions in the tumors. These results indicated that X irradiation combined with TRAIL may be a useful treatment to reduce tumor growth in not only normoxic but also hypoxic regions.
  • Yasuko Watanabe, Wakako Hiraoka, Yuhei Shimoyama, Motohiro Horiuchi, Mikinori Kuwabara, Osamu Inanami
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 366 1 244 - 249 2008年02月 [査読有り][通常論文]
     
    We examined the influence of D177N (D178N in humans) mutation on the conformational stability of the S2 region of moPrP(C) with varying pHs by using the SDSL-ESR technique. The ESR spectrum of D177N at pH 7.5 was narrower than that of Y161R1, referred to as WT*. The ESR spectrum of D177N did not change when pH in the solution decreased to pH 4.0. Our results suggested that the disappearance of a salt bridge (D177-R163) induced the increase in the instability of S2 region. Moreover, the line shape of the ESR spectrum obtained from H176S neighboring the salt bridge linked to the S2 region was similar to D177N. These results indicate that the protonation of H176 is strongly associated with the stability of S2 region. These findings are important for understanding the mechanism by which the disruption of the salt bridge in the S2 region forms the pathogenic PrPSc structure in hereditary prion disease. (c) 2007 Elsevier Inc. All rights reserved.
  • Aki Ogura, Yasuko Watanabe, Daisuke Iizuka, Hironobu Yasui, Makoto Amitani, Saorl Kobayashi, Mikinori Kuwabara, Osamu Inanami
    CANCER LETTERS 259 1 71 - 81 2008年01月 [査読有り][通常論文]
     
    To investigate the mechanism of radioresistance of solid tumor cells, we created two expression vectors encoding Survivin mutants, T34A and D53A. When T34A and D53A were overexpressed in NIH3T3, A549 and HeLa cells, radiation-induced apoptosis was significantly enhanced. Furthermore, we examined the binding capability of Survivin with Smac/DIABLO in the cells that overexpressed these mutants. Coimmunoprecipitation analysis revealed that mutant form of Survivin, D53A and T34A could bind to Smac/DIABLO, but with much less affinity compared to the authentic form. These results suggest that radiation-induced apoptosis of tumor cells is increased by inhibition of the interaction between Survivin and Smac/DIABLO through overexpression of T34A and D53A. (c) 2007 Elsevier Ireland Ltd. All rights reserved.
  • 飯塚 大輔, 稲波 修, 安井 博宣
    放射線生物研究 42 4 444 - 455 放射線生物研究会 2007年12月 [査読無し][通常論文]
  • Momoko Takahashi, Osamu Inanami, Nobuo Kubota, Michihiko Tsujitan, Hironobu Yasui, Aki Ogura, Mikinori Kuwabara
    JOURNAL OF RADIATION RESEARCH 48 6 461 - 468 2007年11月 [査読有り][通常論文]
     
    Our previous study showed that ionizing radiation induced the expression of death receptor DR5 on the cell surface in tumor cell lines and that the death receptor of the TNF alpha-related apoptosis-inducing ligand TRAIL enhanced the apoptotic pathway (Hamasu et al., (2005) Journal of Radiation Research, 46:103-110). The present experiments were performed to examine whether treatment with TRAIL enhanced the cell killing in tumor cells exposed to ionizing radiation under hypoxia, since the presence of radioresistant cells in hypoxic regions of solid tumors is a serious problem in radiation therapy for tumors. When human lung carcinoma A549 cells were irradiated under normoxia and hypoxia, respectively, radiation-induced enhancement of expression of DR5 was observed under both conditions. Incubation in the presence of TRAIL enhanced the caspase-dependent and chymotrypsin-like-protease-dependent apoptotic cell death in A549 cells exposed to X rays. Furthermore, it was shown that treatment with TRAIL enhanced apoptotic cell death and loss of clonogenic ability in A549 cells exposed to X rays not only under normoxia but also under hypoxia, suggesting that combination treatment with TRAIL and X irradiation is effective for hypoxic tumor cells.
  • Hajime Nagahata, Hidetoshi Higuchi, Osamu Inanami, Mikinori Kuwabara
    JOURNAL OF VETERINARY MEDICAL SCIENCE 69 10 993 - 997 2007年10月 [査読有り][通常論文]
     
    The present study evaluated the costimulatory effects of complement receptor type 3 (CR3) and Fc receptor for IgG (Fc gamma R) on superoxide production and intracellular signal transduction in bovine neutrophils. Stimulation with opsonized zymosan (OPZ) and heat-aggregated bovine IgG (Agg-IgG) resulted in much greater superoxide production and chemiluminescent (CL) responses in normal neutrophils compared with those stimulated with OPZ or Agg-IgG only. Superoxide production and CL response were closely associated with the stimulant-induced rise of the intracellular calcium ([Ca2+](i)) concentration, amount of tyrosine phosphorylated 100 kDa protein, and activation of p38 mitogen-activated protein kinase (p38 MAPK). No costimulatory effect was found for these receptors on superoxide production in CR3-deficient neutrophils. Costimulation of CR3 and Fc gamma R on bovine neutrophils leads to enhancement of superoxide production and their signaling pathways and appears to be associated with enhancement of neutrophil functions. KEY WORDS: bovine neutrophils, costimulation, CR3, Fc gamma R, signaling pathway.
  • Hironobu Yasui, Osamu Inanami, Taketoshi Asanuma, Daisuke Iizuka, Takayuki Nakajima, Yasuhiro Kon, Akira Matsuda, Mikinori Kumwabara
    INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS 68 1 218 - 228 2007年05月 [査読有り][通常論文]
     
    Purpose: To examine the in vivo antitumor efficacy of X-irradiation combined with administration of a ribonucleoside anticancer drug, 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine (TAS106, ECyd), to tumor cell-transplanted mice. Methods and Materials: Colon26 murine rectum adenocarcinoma cells and MKN45 human gastric adenocarcinoma cells were inoculated into the footpad in BALB/c mice and severe combined immunodeficient mice, respectively. They were treated with a relatively low dose of X-irradiation (2 Gy) and low amounts of TAS106 (0.1 mg/kg and 0.5 mg/kg). The tumor growth was monitored by measuring the tumor volume from Day 5 to Day 16 for Colon26 and from Day 7 to Day 20 for MKN45. Histologic analyses for proliferative and apoptotic cells in the tumors were performed using Ki-67 immunohistochemical and terminal deoxynucleotidyl transferase-mediated nick end labeling staining. The expression of survivin, a key molecule related to tumor survival, was assessed by quantitative polymerase chain reaction and immunohistochemical analysis. Results: When X-irradiation and TAS106 treatment were combined, significant inhibition of tumor growth was observed in both types of tumors compared with mice treated with X-irradiation or TAS106 alone. Marked inhibition of tumor growth was observed in half of the mice that received the combined treatment three times at 2-day intervals. Parallel to these phenomena, the suppression of survivin expression and appearance of Ki-67-negative and apoptotic cells were observed. Conclusions: X-irradiation and TAS106 effectively suppress tumor growth in mice. The inhibition of survivin expression by TAS106 is thought to mainly contribute to the suppression of the tumor growth. (c) 2007 Elsevier Inc.
  • Daisuke Iizuka, Osamu Inanami, Ikuo Kashiwakura, Mikinori Kuwabara
    RADIATION RESEARCH 167 5 563 - 571 2007年05月 [査読有り][通常論文]
     
    To clarify the relationship between CDC2 kinase activity and radiation-induced apoptosis, we examined whether the cyclin-dependent kinase (CDK) inhibitor purvalanol A enhanced radiation-induced apoptosis in gastric tumor cells. MKN45 cells exposed to 20 Gy of X rays increased the CDC2 kinase activity and the expression of regulatory proteins (phospho-CDC2 and cyclin B1) of the G(2)/M phase, followed by activation of the G(2)/M checkpoint, whereas the treatment of X-irradiated MKN45 cells with 20 mu M purvalanol A suppressed the increase in the CDC2 kinase activity and expression of the G(2)/M-phase regulatory proteins and reduced the fraction of the cells in the G,/M phase in the cell cycle. Furthermore, this treatment resulted in not only a significant increase in radiation-induced apoptosis but also the loss of clonogenicity in both MKN45 (p53-wild) and MKN28 (p53-mutated) cells. The expression of anti-apoptosis proteins, inhibitor of apoptosis protein (IAP) family members (survivin and XIAP) and BCL2 family members (Bcl-X(L) and Bcl-2), in purvalanol A-treated cells with and without X rays was significantly lower than for cells exposed to X rays alone. These results suggest that the inhibition of radiation-induced CDC2 kinase activity by purvalanol A induces apoptosis through the enhancement of active fragments of caspase 3. (c) 2007 by Radiation Research Society.
  • Mohamed Ahmed, Kazuhiro Kimura, Mohamed Soliman, Daisuke Yamaji, Yuko Okamatsu-Ogura, Kennedy Makondo, Osamu Inanami, Masayuki Saito
    JOURNAL OF VETERINARY MEDICAL SCIENCE 69 2 125 - 131 2007年02月 [査読有り][通常論文]
     
    Leptin, a pleiotropic hormone regulating food intake and energy expenditure, has been shown to directly modulate human polymorphonuclear neutrophil (PMN) functions or indirectly through the action of tumor necrosis factor-alpha (TNF-alpha). Bovine PMN have considerable different characteristics from human PMN. For example, it does not respond to N-formyl-Methionyl-Leucyl-phenylalanine, a well known human PMN activator. In the present study, we tested the effects of leptin and TNF-alpha on superoxide production and degranulation of bovine peripheral PMN, in which both long isoform of leptin receptor (Ob-Rb) and TNF receptor I were expressed. Human leptin, human TNF-a, phorbol myristate acetate (PMA) and opsonized zymosan particles (OZP) did not stimulate degranulation responses, while zymosan-activated serum (ZAS) did. Neither leptin nor TNF-alpha enhanced the ZAS-induced degranulation responses. TNF-alpha, PMA, OZP and ZAS increased superoxide production in different magnitudes, whereas leptin did not. TNF-alpha, but not leptin, enhanced OZP- and ZAS-induced superoxide production, possibly, in part due to facilitating translocation of p47(phox), a component of NADPH oxidase. These results indicate that, unlike in human PMN, leptin does not have any direct effect on degranulation and superoxide production in bovine PMN, although TNF-alpha. influences superoxide production.
  • Hironobu Yasui, Taketoshi Asanuma, Yasuko Watanabe, Kenji Waki, Osamu Inanami, Mikinori Kuwabara
    BIOFACTORS 29 2-3 113 - 121 2007年 [査読有り][通常論文]
     
    Oxidative damage due to ischemia/reperfusion has been implicated as one of the leading causes for delayed neuronal cell death in a number of neurodegenerative diseases, including stroke. The purpose of this research was to investigate whether oral administration of a fermented grain food mixture (AOB((R))) might offer protective effects against ischemia/reperfusion-induced neuronal damage in Mongolian gerbils, a model known for delayed neuronal death in the hippocampal CA1 region. Histological analysis revealed that AOB administration ad libitum for 3 weeks (preoperative administration) and I week (postoperative administration) dose-dependently suppressed the induction of transient ischemia/reperfusion-induced neuronal cell death. TUNEL assay also revealed that AOB suppressed it by inhibiting the induction of apoptosis. A significant increase of superoxide dismutase-like (SOD-like) activity was observed in the hippocampal CA1 region of the AOB-treated gerbil. Furthermore, immunoblot analysis showed that AOB administration down-regulated the expression of heat shock proteins HSP27 and HSP70 in the same region. These results indicated that oral administration of AOB protected against ischemia/reperfusion-induced brain injury by minimizing oxidative damage via its SOD-like activity and inhibiting apoptosis.
  • Taketoshi Asanuma, Hironobu Yasui, Osamu Inanami, Kenji Waki, Momoko Takahashi, Daisuke Iizuka, Taketo Uemura, Gregory Durand, Ange Polidori, Yasuhiro Kon, Bernard Pucci, Mikinori Kuwabara
    CHEMISTRY & BIODIVERSITY 4 9 2253 - 2267 2007年 [査読有り][通常論文]
     
    An amphiphilic alpha-phenyl-N-(tert-butyl) nitrone (PBN) derivative, N-{[4-(lactobionamido)methyl]-benzylidene}-1,1-dimethyl-2-(octylsulfanyl)ethylamine N-oxide (LPBNSH), newly synthesized from its original form PBN in hopes of clinical use, was intraperitoneally administered to Long-Evans Cinnamon (LEC) rats every 2 days at the concentrations of 0.1, 0.5, 1.0, and 2.0 mg/kg. We found that LPBNSH protected against copper-induced hepatitis with jaundice in LEC rats at concentrations of 0.1 and 0.5 mg/ kg, which were extremely low compared with that of PBN. It also effectively prevented the loss of body weight, reduced the death rate, and suppressed the increase in serum aspartate aminotransferase and alanine aminotransferase values arising from fulminant hepatitis with jaundice at the same concentrations. Similar results were observed when PBN was administered at the concentration of 150 mg/kg. immunohistochemical analysis of 8-hydroxy-2'-deoxyguanosine and measurement of thiobarbituric acid-reactive substances in the liver showed that LPBNSH largely suppressed the formation of these oxidative products at same concentrations. No difference in the abnormal accumulation of copper in the liver between the LPBNSH administered and control groups was observed. From these results, it was concluded that LPBNSH exhibited liver-protective effects against fulminant hepatitis with jaundice at ca. 1/1000, 500 the molar concentration of PBN and, therefore, was clinically promising.
  • Eriko Takahashi, Osamu Inanami, Toshio Ohta, Akira Matsuda, Mikinori Kuwabara
    LEUKEMIA RESEARCH 30 12 1555 - 1561 2006年12月 [査読有り][通常論文]
     
    To clarify the role of lipid rafts in 2-chloro-2'-deoxyadenosine (2CdA; Cladribine)-induced apoptosis, the effects of disruption of lipid rafts by methyl-beta-cyclodextrin (M beta CD) and filipin on 2CdA-induced apoptosis were investigated in four human acute lymphoblastic leukemia (ALL) cell lines comprised of T cells (MOLT-4, Jurkat) and B cells (NALM, BALL-1). The disruption of lipid rafts significantly inhibited 2CdA-induced apoptosis, indicating the crucial role of lipid rafts in the induction of apoptosis in leukemia cells. These reagents significantly inhibited 2CdA-induced elevation of the intracellular calcium concentration ([Ca2+](i)) in MOLT-4 cells, and 2CdA-induced apoptosis was partly inhibited by the Ca2+ chelators BAPTA-AM and EGTA, and the L-type Ca2+ channel blocker nifedipine. On the other hand, they had no effects on the cellular uptake of 2CdA. These results indicated that lipid rafts partly contributed to 2CdA-induced apoptosis by regulating Ca2+ influx via the plasma membrane. (c) 2006 Elsevier Ltd. All rights reserved.
  • Yasuko Watanabe, Osamu Inanami, Motohiro Horiuchi, Wakako Hiraoka, Yuhei Shimoyama, Fuyuhiko Inagaki, Mikinori Kuwabara
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 350 3 549 - 556 2006年11月 [査読有り][通常論文]
     
    We analyzed the pH-induced mobility changes in moPrP(C) alpha-helix and beta-sheets by cysteine-scanning site-directed spin labeling (SDSL) with ESR. Nine amino acid residues of alpha-helix1 (HI, codon 143-151), four amino acid residues of beta-sheet1 (SI, codon 127-130), and four amino acid residues of beta-sheet2 (S2, codon 160-163) were substituted for by cysteine residues. These recombinant mouse PrPC (moPrP(C)) mutants were reacted with a methane thiosulfonate sulfhydryl-specific spin labeling reagent (MTSSL). The 1/delta H of the central (N-14 hyperfine) component (M-1 = 0) in the ESR spectrum of spin-labeled moPrPC was measured as a mobility parameter of nitroxide residues (R1). The mobilities of E145R1 and Y149R1 at pH 7.4, which was identified as a tertiary contact site by a previous NMR study of moPrP, were lower than those of D143R1, R147R1, and R150R1 reported on the helix surface. Thus, the mobility in the HI region in the neutral solution was observed with the periodicity associated with a helical structure. On the other hand, the values in the S2 region, known to be located in the buried side, were lower than those in the SI region located in the surface side. These results indicated that the mobility parameter of the nitroxide label was well correlated with the 3D structure of moPrP. Furthermore, the present study clearly demonstrated three pH-sensitive sites in moPrP, i.e., (1) the N-terminal tertiary contact site of H1, (2) the C-terminal end of H1, and (3) the S2 region. In particular, among these pH-sensitive sites, the N-terminal tertiary contact region of HI was found to be the most pH-sensitive one and was easily converted to a flexible structure by a slight decrease of pH in the solution. These data provided molecular evidence to explain the cellular mechanism for conversion from PrPC to PrPSc in acidic organelles such as the endosome. (c) 2006 Elsevier Inc. All rights reserved.
  • Masato Kamibayashi, Shigeru Oowada, Hiroaki Kameda, Taiichi Okada, Osamu Inanami, Shunsaku Ohta, Toshihiko Ozawa, Keisuke Makino, Yashige Kotake
    FREE RADICAL RESEARCH 40 11 1166 - 1172 2006年11月 [査読有り][通常論文]
     
    5-(2,2-Dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1-pyrroline N-oxide (CYPMPO), a new cyclic DEPMPO-type nitrone was evaluated for spin-trapping capabilities toward hydroxyl and superoxide radicals. CYPMPO is colorless crystalline and freely soluble in water. Both the solid and diluted aqueous solution did not develop electron spin resonance (ESR) signal for at least 1 month at ambient conditions. CYPMPO can spin-trap superoxide and hydroxyl radicals in both chemical and biological systems, and the ESR spectra are readily assignable. Half life for the superoxide adduct of CYPMPO produced in UV-illuminated hydrogen peroxide solution was approximately 15 min, and in biological systems such as hypoxanthine (HX)/xanthine oxidase (XOD) the half-life of the superoxide adduct was approximately 50 min. In UV-illuminated hydrogen peroxide solution, there was no conversion from the superoxide adduct to the hydroxyl adduct. Although overall spin-trapping capabilities of CYPMPO are similar to DEPMPO, its high melting point, low hygroscopic property, and the long shelf-life would be highly advantageous for the practical use.
  • Ikuo Kashiwakura, Osamu Inanami, Yoshinao Abe, Kei Satoh, Tsuneo A. Takahashi, Mikinori Kuwabara
    RADIATION RESEARCH 166 2 345 - 351 2006年08月 [査読有り][通常論文]
     
    In the present study, we investigated whether X-irradiated hematopoietic stem cells can be induced to undergo megakaryocytopoiesis and thrombopoiesis in vitro using cytokine combinations that have been demonstrated to be effective for conferring increased survival on irradiated human CD34(+) megakaryocytic progenitor cells (colony-forming unit megakaryocytes; CFU-Meg), such as thrombopoietin (TPO), interleukin 3 (IL3), stem cell factor and FLT3 ligand. Culture of nonirradiated CD34(+) cells in serum-free medium supplemented with multiple cytokine combinations led to an approximately 200- to 600-fold increase in the total cell numbers by day 14 of culture. In contrast, the growth of X-irradiated cells was observed to be one-sixth to one-tenth that of the nonirradiated cultures. Similarly, total megakaryocytes were increased by 50- to 130-fold, while culture of X-irradiated cells yielded one-fourth to one-eighth of the control numbers. At this time, CD41(+) particles, which appeared to be platelets, were produced in the medium harvested from nonirradiated and irradiated cultures. Although radiation suppressed cell growth and megakaryocytopoiesis, there were no significant differences in thrombopoiesis between the two types of culture. These results suggest that X-irradiated CD34(+) cells can be induced to undergo nearly normal terminal maturation through megakaryocytopoiesis and thrombopoiesis by stimulation with appropriate cytokine combinations. (c) 2006 by Radiation Research Society
  • Mika Hori, Taketoshi Asanuma, Osamu Inanami, Mikinori Kuwabara, Hideyoshi Harashima, Hiroyuki Kamiya
    BIOLOGICAL & PHARMACEUTICAL BULLETIN 29 6 1087 - 1091 2006年06月 [査読有り][通常論文]
     
    The Escherichia coli Orf17 (NtpA, NudB) protein, a Muff-type enzyme, hydrolyzes oxidized deoxyribonucleotides, including 8-hydroxy-2'-deoxyadenosine 5'-triphosphate and 8-hydroxy-2'-deoxyguanosine 5'-triphosphate, in vitro. To examine its in vivo role(s) in bacteria, plasmid DNAs containing the orf17 gene in the sense and antisense orientations were introduced. When the Orf17 protein was overexpressed in mutT cells, the rpoB mutant frequency was decreased. On the other hand, similar effects were not observed when Orf17 was overexpressed in wild type and orf135 cells. Expression of the antisense RNA of the orf17 gene did not produce an obvious phenotype, such as increased mutant frequency and resistance to ionizing radiation. These results suggest that the role of the Orf17 protein is to back up the MutT function, and to assist in the elimination of 8-hydroxy-2'-deoxyguanosine nucleotides.
  • Satoru Monzen, Takao Mori, Kenji Takahashi, Yoshinao Abe, Osamu Inanami, Mikinori Kuwabara, Ikuo Kashiwakura
    JOURNAL OF RADIATION RESEARCH 47 2 213 - 220 2006年06月 [査読有り][通常論文]
     
    Epigallocatechin-3-gallate (EGCg) has been widely recognized as a powerful antioxidant and free radical scavenger. The effects of EGCg on the proliferation and differentiation of X-irradiated megakaryocytic progenitor cells (colony-forming unit-megakaryocyte, CFU-Meg) using CD34(+) cells prepared from human placental and umbilical cord blood have been shown. In the absence of exogenous thrombopoietin (TPO), no colonies are observed in cultures containing or lacking EGCg (1 nM-100 mu M). In the presence of TPO, in contrast, EGCg significantly promotes CFU-Meg-derived colony formations within the 10-100 nM range. A 1.5-fold increase in the total number of CFU-Meg has been counted compared with the control. These favorable effects of EGCg are also observed in the culture of CD34(+) cells before and after X irradiation with 2 Gy. Moreover, in order to investigate the function of EGCg promoting megakaryocytopoiesis and thrombopoiesis in ex vivo cultures, both non-irradiated and X-irradiated CD34(+) cells are grown in liquid cultures supplemented with TPO. In both cultures, EGCg increases the total number of cells and megakaryocytes. It has been suggested that the favorable effects of EGCg reduce the risk factor from radiation damage in megakaryocytopoiesis.
  • 桑原 幹典, 稲波 修, 安井 博宣, 飯塚 大輔, 松田 彰
    癌の臨床 52 1 31 - 36 (株)篠原出版新社 2006年04月 [査読無し][通常論文]
     
    ヒトおよびマウスの腫瘍細胞を用い,X線致死効率がこのエチニルシチジン(ECyd)との併用によりそれぞれ単独処理の和に比べて上昇するか否かを培養細胞および移植固形腫瘍で検討した.全ての細胞でX線照射のみではほとんどアポトーシスは誘導されなかった.X線とECydを併用した場合,それぞれ単独の和に比べて大きくアポトーシスが誘導された.ECydによるアポトーシス増感がそのまま線量効果曲線での増感に繋がることが判明した.シチジン添加によりECydのアポトーシス増感が消失することから,ウリジン/シチジン2によるECydのリン酸化がこの化合物の活性化の必要条件であった.マウスの固形移植腫瘍においても腫瘍組織内の増殖性細胞数の低下とアポトーシス細胞数の増加を観察した
  • K Waki, O Inanami, T Yamamori, H Nagahata, M Kuwabara
    FREE RADICAL RESEARCH 40 4 359 - 367 2006年04月 [査読有り][通常論文]
     
    This experiment was performed to clarify the role of protein kinase C (PKC) delta in NADPH oxidase-dependent O-2(-) production and actin polymerization followed by phagocytosis in neutrophils. Bovine neutrophils and human neutrophil-like differentiated HL-60 (dHL-60) cells were stimulated with serum-opsonized zymosan (OZ) and fMet-Leu-Phe (fMLP), respectively. Rottlerin, a specific inhibitor of PKC delta, attenuated the production of from NADPH oxidase in both neutrophils and dHL-60 cells. However, it did not inhibit the translocation of p47(phox) from the cytosol to the membrane in either type of cell or the phosphorylation of p47(phox) in dHL-60 cells. GF109203X (GFX), an inhibitor of cPKC, attenuated not only the production of O-2(-) but also the translocation of p47(phox) in both cells. Furthermore, rottlerin significantly attenuated the ingestion of opsonized particles and the formation of F-actin in OZ-stimulated neutrophils, whereas, GFX did not affect those phagocytic processes. These results suggest that both PKC delta and cPKC regulate NADPH oxidase through different pathways, but only PKC delta regulates the phagocytic function in neutrophils.
  • T Mizutani, S Fukushi, D Iizuka, O Inanami, M Kuwabara, H Takashima, H Yanagawa, M Saijo, Kurane, I, S Morikawa
    FEMS IMMUNOLOGY AND MEDICAL MICROBIOLOGY 46 2 236 - 243 2006年03月 [査読有り][通常論文]
     
    Severe acute respiratory syndrome (SARS) is caused by SARS-coronavirus (SARS-CoV). Infection of Vero E6 cells with SARS-CoV inhibits cell proliferation. Our previous study indicated that Akt, which is poorly phosphorylated in confluent cultures of Vero E6 cells, is phosphorylated and then dephosphorylated upon infection by SARS-CoV. In the present study, we showed that a serine residue of Akt was phosphorylated in Vero E6 cells in subconfluent culture and that Akt was dephosphorylated rapidly after SARS-CoV infection without up-regulation of its phosphorylation. Phosphorylation of glycogen synthase kinase-3 beta, which is one of the downstream targets of Akt, was prevented in SARS-CoV-infected cells. However, treatment with glycogen synthase kinase-3 beta small interfering RNA indicated that the glycogen synthase kinase-3 beta signaling pathway was not related to inhibition of cell proliferation. Treatment of Vero E6 cells with the phosphatidylinositol 3'-kinase/Akt inhibitor, LY294002, which induces dephosphorylation of Akt, inhibited cell proliferation. As shown in our previous studies, apoptosis occurred in virus-infected cells within 18 h postinfection. Cellular mRNA transcription, which was reported to be up-regulated in SARS-CoV-infected Caco-2 cells, was not up-regulated in virus-infected Vero E6 cells, partially as a result of apoptosis. These results suggested that inhibition of cell proliferation is regulated by both the phosphatidylinositol 3'-kinase/Akt signaling pathway and by apoptosis in SARS-CoV-infected Vero E6 cells. This is the first study to analyze SARS-CoV-induced cell growth inhibition.
  • E Takahashi, O Inanami, T Asanuma, M Kuwabara
    JOURNAL OF RADIATION RESEARCH 47 1 19 - 25 2006年03月 [査読有り][通常論文]
     
    In the present study, using inhibitors of ceramide synthase (fumonisin B-1), ketosphinganine synthetase (L-cycloserine), acid sphingomyelinase (D609 and desipramine) and neutral sphingomyelinase (GW4869), the role of ceramide in X-ray-induced apoptosis was investigated in MOLT-4 cells. The diacylglycerol kinase (DGK) assay showed that the intracellular concentration of ceramide increased timedependently after X irradiation of cells, and this radiation-induced accumulation of ceramide did not occur prior to the appearance of apoptotic cells. Treatment with D609 significantly inhibited radiation-induced apoptosis, but did not inhibit the increase of intracellular ceramide. Treatment with desipramine or GW4869 prevented neither radiation-induced apoptosis nor the induced increase of ceramide. On the other hand, fumonisin B, and L-cycloserine had no effect on the radiation-induced induction of apoptosis, in spite of significant inhibition of the radiation-induced ceramide. From these results, it was suggested that the increase of the intracellular concentration of ceramide was not essential for radiation-induced apoptosis in MOLT-4 cells.
  • T Nakajima, T Wakasa, Y Okuma, O Inanami, Y Nomura, M Kuwahara, K Kawahara
    JOURNAL OF NEUROSCIENCE RESEARCH 83 3 459 - 468 2006年02月 [査読有り][通常論文]
     
    In the present study, we examined how the cell survival signaling via cyclic AMP-responsive element binding protein (CREB) and Akt, and the cell death signaling via cystein proteases, calpain and caspase-3, are involved in oxygen-glucose deprivation (OGD) followed by reoxygenation (OGD/reoxygenation)-induced cell death in nerve growth factor (NGF)-differentiated PC12 cells. OGD/reoxygenation-induced cell death was evaluated by LDH release into the culture medium. The level of LDH release was low (9.0% +/- 4.1%) immediately after 4 hr of OGD (0 hr of reoxygenation), was significantly increased to 28.6% +/- 6.6% at 3 hr of reoxygenation, and remained at similar levels at 6 and 20 hr of reoxygenation, suggesting that reoxygenation at least for 3 hr resulted in the loss of cell membrane integrity. After 4 hr of OGD followed by 3 hr of reoxygenation, dephosphorylation of phosphorylated CREB (pCREB), but not phosphorylated Akt (pAkt), was induced. Under these conditions, calpain- but not caspase-3-mediated alpha-spectrin breakdown product was increased, indicating that OGD/reoxygenation also induced an increase in calpain activity. The restoration of pCREB by protein phosphatase (PP)-1/2A inhibitors or the inhibition of excessive activation of calpain by calpain inhibitor did not reduce OGD/reoxygenation-induced LDH release. Cotreatment with PP-1/2A and calpain inhibitors reduced OGD/reoxygenation-induced LDH release. The present study suggests that a balance in the phosphorylation and proteolytic signaling is involved in the survival of NGF-differentiated PC 12 cells. (c) 2005 Wiley-Liss, Inc.
  • K Niwa, J Sakai, T Karino, H Aonuma, T Watanabe, T Ohyama, O Inanami, M Kuwabara
    FREE RADICAL RESEARCH 40 2 167 - 174 2006年02月 [査読有り][通常論文]
     
    To elucidate the role of shear stress in fluid-phase endocytosis of vascular endothelial cells (EC), we used a rotating-disk shearing apparatus to investigate the effects of shear stress on the uptake of lucifer yellow (LY) by cultured bovine aortic endothelial cells ( BAEC). Exposure of EC to shear stress (area-mean value of 10 dynes/cm(2)) caused an increase in LY uptake that was abrogated by the antioxidant, N-acetyl-L-cysteine (NAC), the NADPH oxidase inhibitor, acetovanillone, and two inhibitors of protein kinase C (PKC), calphostin C and GF109203X. These results suggest that fluid-phase endocytosis is regulated by both reactive oxygen species (ROS) and PKC. Shear stress increased both ROS production and PKC activity in EC, and the increase in ROS was unaffected by calphostin C or GF109203X, whereas the activation of PKC was reduced by NAC and acetovanillone. We conclude that shear stress-induced increase in fluid-phase endocytosis is mediated via ROS generation followed by PKC activation in EC.
  • T Asanuma, T Kawahara, O Inanami, M Nakao, K Nakaya, A Ito, M Takiguchi, A Hashimoto, M Kuwabara
    JOURNAL OF VETERINARY MEDICAL SCIENCE 68 1 15 - 20 2006年01月 [査読有り][通常論文]
     
    Pulmonary alveolar echinococcosis (AE) caused by the metacestode of Echinococcus multilocularis is a lethal zoonosis and 14 is a lesion secondarily induced by hematogenous dissemination from hepatic AE lesions. In the present study, a hematogenous pulmonary AE model was experimentally induced in rats by the injection of echinococcal larval tissue homogenate to the tail vein, and then the pathological and diagnostic aspects of pulmonary AE were examined by magnetic resonance imaging (MRI). Histological primary, mature and degenerated AE lesions were observed 5, 18 and 50 weeks after injection, respectively. These lesions were discriminated as signal-void, hypointense and hyperintense regions in T1-weighted MRI (T1WI), respectively. The change in signal intensity in T1WI 1 might reflect the content of proteinaceous fluid as a result of AE cyst degeneration. Western blot analysis of sera with antibodies of two epitopes (Em18 and Em16) of E. multilocularis provided evidence for AE infection in the early stage. T1WI in combination with Western blot analysis could possibility become definitive and early signs of hematogenous pulmonary A-E infection.
  • O Inanami, S Hashida, D Iizuka, M Horiuchi, W Hiraoka, Y Shimoyama, H Nakamura, F Inagaki, M Kuwabara
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 335 3 785 - 792 2005年09月 [査読有り][通常論文]
     
    The structure of the mouse prion (moPrP) was studied using site-directed spin-labeling electron spin resonance (SDSL-ESR). Since a previous NMR study by Hornemanna et al., [Hornemanna, Korthb, Oeschb, Rieka, Widera, Wuthricha, Glockshubera, Recombinant full-length murine prion protein, mPrP (23-231): purification and spectroscopic characterization, FEBS Lett. 413 (1997) 277-281] has indicated that N96, D143, and T189 in moPrP are localized in a Cu2+ binding region, Helix1 and Helix2, respectively, three recombinant moPrP mutations (N96C, D143C, and T189Q were expressed in an Escherichia coli system, and then refolded by dialysis under low pH and purified by reverse-phase HPLC. By using the preparation, we succeeded in preserving a target cystein residue without alteration of the alpha-helix structure of moPrP and were able to apply SDSL-ESR with a methane thiosulfonate spin label to the full-length prion protein. The rotational correlation times (tau) of 1.1, 3.3, and 4.8 ns were evaluated from the X-band ESR spectra at pH 7.4 and 20 degrees C for N96R1, D143R1, and T189R1, respectively. c reflects the fact that the Cu2+ binding region is more flexible than Helix1 or Helix2. ESR spectra recorded at various temperatures revealed two phases together with a transition point at around 20 degrees C in D143R1 and T189R1, but not in N96R1 With the variation of pH from 4.0 to 7.8, ESR spectra of T189R1 at 20 degrees C showed a gradual increase of tau from 2.9 to 4.8 ns. On the other hand, the pH-dependent conformational changes in N96R1 and D143R1 were negligible. These results indicated that T189 located in Helix2 possessed a structure sensitive to physiological pH changes; simultaneously, N96 in the Cu2+ binding region and D143 in Helix1 were conserved. (C) 2005 Elsevier Inc. All rights reserved.
  • Kashiwakura, I, O Inanami, Y Abe, TA Takahashi, M Kuwabara
    RADIATION RESEARCH 164 1 10 - 16 2005年07月 [査読有り][通常論文]
     
    CD34 antigen is a novel marker for human hematopoietic stem/progenitor cells. In the present study, two cell fractions, CD34(low) and CD34(high), were prepared from steady-state human peripheral blood on the basis of CD34 antigen expression. The colony-forming unit megakaryocytes (CFU-Meg) contained in each cell fraction were compared for X-radiation sensitivity and cytokine action. The content of CD34(+)CD45(+) cells in the CD34(low) and CD34(high) cell fractions was 74.8% and 88.8%, respectively, and the frequency of thrombopoietin (TPO)-supported CFU-Meg in the CD34(low) cell fraction was 1.9 times higher than that in CD34(high). The CFU-Meg in CD34(high) were more radiosensitive than those in CD34(low), indicating that steady-state human peripheral blood contains different types of CFU-Meg. However, no significant differences were observed between cell fractions in the radiation survival curves of CFU-Meg stimulated by TPO plus cytokines except granulocyte colony-stimulating factor (G-CSF). TPO plus interleukin 3 was the optimal combination for survival of both types of CFU-Meg after X irradiation. The present study also demonstrated that TPO plus G-CSF is able to increase the survival of irradiated CD34(low) CFU-Meg. These results suggest that two megakaryocytic progenitor populations with different radiosensitivity and cytokine responses are found in steady-state human peripheral blood. (c) 2005 by Radiation Research Society.
  • D Iizuka, O Inanami, A Matsuda, Kashiwakura, I, T Asanuma, M Kuwabara
    RADIATION RESEARCH 164 1 36 - 44 2005年07月 [査読有り][通常論文]
     
    The clonogenic ability (reproductive cell death) of Chinese hamster V79 cells was measured after treatment with X radiation and a newly developed anti-cancer drug, 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine (ECyd, TAS106). Amplification in the loss of clonogenicity was observed compared to that obtained for cells exposed to X rays alone. Addition of benzyloxycarbonyl-val-ala-asp-fluoromethylketone (Z-VADFMK), a broad-spectrum caspase inhibitor, attenuated the increased lethality, but the dose-response curve obtained was found to merely revert to that obtained for cells exposed to X rays alone. Flow cytometric analysis showed that the number of cells arrested at the G(2)/M phase by X irradiation was decreased by co-treatment with TAS106, and instead the number of cells in the sub-G, phase increased. Western blot analysis proved that TAS106 treatment down-regulated the expression of the G(2)/M arrest-related proteins cyclin B1, phospho-CDC2 and WEE1. From these results, it was concluded that (1) no apoptosis was included in the dose-response curve obtained from cells exposed to X rays alone, (2) X radiation induced a potentially apoptotic (proapoptotic) state in cells independent of the loss of their clonogenic ability, and (3) TAS106 enhanced the loss of their clonogenic ability by converting the proapoptotic cells to apoptotic cells through the abrogation of arrest at the G2/M phase. (c) 2005 by Radiation Research Society.
  • S Kobayashi, R Sato, O Inanami, T Yamamori, O Yamato, Y Maede, J Sato, M Kuwabara, Y Naito
    VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 105 1-2 75 - 84 2005年05月 [査読有り][通常論文]
     
    Lactofenin (LF), a glycoprotein present in milk, mucosal secretions and neutrophils, contributes to host defense and immunomodulation. In the present study, we investigated the effect of bovine LF (bLF) on cytokine messenger RNA (mRNA) expression in concanavalin A (ConA)-stimulated feline peripheral blood mononuclear cells (PBMC). Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and real-time PCR showed a ConA-induced increase of interferon-gamma (IFN-gamma) mRNA expression but not of interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha) and IL-12 p40 mRNA in feline PBMC. This ConA-induced increase of IFN-gamma mRNA expression was inhibited by addition of bLF not only 30 min before ConA stimulation but also 10, 20 and 40 min after ConA stimulation. Western blotting showed that protein tyrosine kinase (PTK) and extracellular signal-regulated kinase (ERK) in feline PBMC were activated within 10 min after the ConA stimulation and that the activation of both kinases had almost disappeared by 40 min after stimulation. Moreover, the ConA-induced IFN-gamma mRNA expression was partly prevented by genistein, a global PTK inhibitor, and PD-98059, an ERK inhibitor, respectively. These results suggest that bLF is able to inhibit the ConA-induced IFN-gamma mRNA expression by abrogation of intracellular signaling activated after interaction between ConA and its receptor. (c) 2004 Elsevier B.V. All rights reserved.
  • T Hamasu, O Inanami, M Tsujitani, K Yokoyama, E Takahashi, Kashiwakura, I, M Kuwabara
    APOPTOSIS 10 3 557 - 567 2005年05月 [査読有り][通常論文]
     
    To elucidate radiobiological effects of hypoxia on X-ray-induced apoptosis, MOLT-4 cells were treated under four set of conditions: (1) both X irradiation and incubation under normoxia, (2) X irradiation under hypoxia and subsequent incubation under normoxia, (3) X irradiation under normoxia and subsequent incubation under hypoxia, and (4) both X irradiation and incubation under hypoxia, and the induction of apoptosis was examined by fluorescence microscopy. About 28-33% apoptosis was observed in cells treated under conditions 1 and 2, but this value was significantly reduced to around 18-20% in cells treated under conditions 3 and 4, suggesting that post-irradiation hypoxic incubation rather than hypoxic irradiation mainly caused the reduction of apoptosis. The activation and expression of apoptosis signal-related molecules SAPK/JNK, Fas and caspase-3 were also suppressed by hypoxic incubation. Effects of hypoxic incubation were canceled when cells were treated under conditions 3 and 4 with an oxygen-mimicking hypoxic cell radiosensitizer, whereas the addition of N-acetyl-L-cysteine again reduced the induction of apoptosis. From these results it was concluded that hypoxia reduced the induction of apoptosis by changing the intracellular redox state, followed by the regulation of apoptotic signals in X-irradiated MOLT-4 cells.
  • 丹羽 光一, 稲波 修
    放射線生物研究 40 1 1 - 13 放射線生物研究会 2005年03月 [査読無し][通常論文]
  • T Hamasu, O Inanami, T Asanuma, M Kuwabara
    JOURNAL OF RADIATION RESEARCH 46 1 103 - 110 2005年03月 [査読有り][通常論文]
     
    The death receptors Fas and DR5 are known to be expressed not only in immune cells but also in various tumor cells. The aim of the present study was to determine whether X irradiation enhanced induction of apoptosis in Tp53 wild type and Tp53-mutated tumor cell lines treated with agonists against these death receptors. We showed that 5 Gy of X irradiation significantly up-regulated the expression of death receptors Fas and DR5 on the plasma membrane in gastric cancer cell lines MKN45 and MKN28, lung cancer cell line A549, and prostate cancer cell line DU145, and that subsequent treatments with agonistic molecules for these death receptors, Fas antibody CH11 and TRAIL, increased the formation of active fragment p20 of caspase 3 followed by the induction of apoptosis. This death -receptor- mediated apoptosis was independent of Tp53 status since MKN28 and DU145 were Tp53-mutated. The post-irradiation treatment of the cells with N-acetyl-L-cysteine (NAC) abolished the up-regulation of the expression of Fas and DR5 on the plasma membrane. NAC also attenuated the increase in the formation of p20 and the induction of apoptosis by agonistic molecules. These results suggested that the increase in the induction of apoptosis by combined treatment with X irradiation and CH11 or TRAIL occurred through a change of the intracellular redox state independent of Tp53 status in human carcinoma cell lines.
  • O Inanami, D Iizuka, A Iwahara, T Yamamori, Y Kon, T Asanuma, A Matsuda, Kashiwakura, I, K Kitazato, M Kuwabara
    RADIATION RESEARCH 162 6 635 - 645 2004年12月 [査読有り][通常論文]
     
    1-(3-C-Ethynyl-beta-(D)-ribo-pentofuranosyl)cytosine (ECyd, TAS106) is a newly developed anti-tumor agent that targets RNA synthesis. We report here that a low dose of ECyd induces radiosensitization of caspase-dependent apoptosis and reproductive cell death in cells of the gastric tumor cell lines MKN45 and MKN28 and murine rectum adenocarcinoma Colon26. Flow cytometry demonstrated that TAS106 induced the abrogation of the X-ray-induced G2/M checkpoint. Western blot analysis showed that X rays increased the expression of cyclin B1, phospho-Cdc2 and Weel, whereas co-treatment with X rays and TAS106 decreased the expression of these cell cycle proteins associated with the G2/M checkpoint. Furthermore, TAS106 was shown to decrease the radiation-induced expression of survivin but not Bcl2 and BcIX(1) regardless of TP53 status and cell type. Overexpression of wild-type survivin in MKN45 cells inhibited the induction of apoptosis induced by co-treatment with X rays and TASI06. These results suggest that TAS106 enhances X-ray-induced cell death through down-regulation of survivin and abrogation of the cell cycle machinery. (C) 2004 by Radiation Research Society.
  • SJ Hosseinimehr, O Inanami, T Hamasu, M Takahashi, Kashiwakura, I, T Asanuma, M Kuwabara
    JOURNAL OF RADIATION RESEARCH 45 4 557 - 561 2004年12月 [査読有り][通常論文]
     
    We investigated the effect of SCF, a c-kit ligand, on the radiosensitivity of HL60 cells. X-ray-induced apoptosis in HL60 cells was significantly lower in the presence of SCF than in the absence of SCF. This attenuation of X-ray-induced apoptosis by SCF was abolished by PD98059 (an ERK inhibitor), but not by wortmannin (a P13-K inhibitor) or GF109203X (a PKC inhibitor). The expression of phospho-ERK1/2 (active form) and the ERK1/2-regulated expression of survivin were found to increase in cells treated with X irradiation and SCF. However, X irradiation alone induced down-regulation of the expression of phospho-ERK1/2. Our findings suggest that activation of c-kit by SCF confers radioresistance through up-regulation of ERK-dependent survivin expression in HL60 cells.
  • 中島 崇行, 岩淵 禎弘, 宮崎 浩之, 大熊 康修, 稲波 修, 桑原 幹典, 野村 靖幸, 河原 剛一
    日本病態生理学会雑誌 13 2 46 - 46 日本病態生理学会 2004年12月 [査読有り][通常論文]
  • ZG Cui, T Kondo, LB Feril, K Waki, O Inanami, M Kuwabara
    APOPTOSIS 9 6 757 - 763 2004年11月 [査読有り][通常論文]
     
    Hydroxyl radicals (circleOH) and superoxide anion radicals (O-2(circle-)) are known to play cardinal roles in cell killing and various types of cell damage. In order to elucidate the mechanism of the involvement of both free radicals on apoptosis, the correlation between anti-apoptotic effects and free radical scavenging abilities of anti-oxidants was studied. As an indicator of anti-apoptotic effects, C1/2 (antioxidant concentration to inhibit DNA fragmentation by 50%) was evaluated in human lymphoma cell line U937 cells 6 hr after X-ray (10 Gy) or hyperthermia (44degreesC, 30 min) treatment. Rate constants of the reactions between antioxidants and circleOH or O-2(circle-) were calculated as the scavenging ability of the antioxidants with graded concentration estimated by EPR spectroscopy. No apparent correlation between C1/2 obtained in apoptosis induced by X-rays or hyperthermia and the rate constants of antioxidants for circleOH or O-2(circle-) was observed. On the other hand, the partition coefficients in 1-octanol/water of the antioxidants, an indicator of hydrophobicity, revealed a correlation with the C1/2 of the agents with hyperthermia, but not with X-ray irradiation. These results indicate that the prevention of apoptosis by an antioxidant is not simply associated with its scavenging ability for circleOH or O-2(circle-.) The hydrophobicity of the antioxidant, among other possible factors, is involved in the inhibition of hyperthermia-induced apoptosis.
  • T Yamamori, O Inanami, H Nagahata, M Kuwabara
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 316 3 720 - 730 2004年04月 [査読有り][通常論文]
     
    Superoxide production by NADPH oxidase is essential for the bactericidal properties of phagocytes. Phosphorylation of p47(phox), one of the cytosolic components of NADPH oxidase, is a crucial step of the oxidase activation. Some evidences suggest that phosphoinositide 3-kinase (PI3K) is involved in p47(phox) phosphorylation, but it has not been fully understood how PI3K regulates it. The aim of this study was to examine the mechanism underlying the PI3K regulation of p47(phox) phosphorylation. Pharmacological inhibition of PI3K attenuated both fMLP-stimulated p47(phox) phosphorylation and NADPH oxidase activity in HL-60 cells differentiated to a neutrophil-like phenotype. Although fMLP elicited Akt activation in a PI3K-dependent manner, an Akt inhibitor had no effect on the oxidase activity triggered by fMLP. In vitro kinase assay revealed that Akt was unable to catalyze p47(phox) phosphorylation. Interestingly, the activation of cPKC and PKCdelta after fMLP stimulation was dependent on PI3K. Furthermore, PI3K inhibitors reduced the activation of phospholipase Cgamma2 without affecting tyrosine phosphorylation on it. These results suggest that PI3K regulates the phosphorylation of NADPH oxidase component p47(phox) by controlling diacylglycerol-dependent PKCs but not Akt. (C) 2004 Elsevier Inc. All rights reserved.
  • T Asanuma, O Inanami, K Tabu, K Waki, Y Kon, M Kuwabara
    NEUROSCIENCE LETTERS 359 1-2 57 - 60 2004年04月 [査読有り][通常論文]
     
    The present experiments were carried out to provide direct in vivo evidence for the involvement of c-Jun N-terminal kinase (JNK) in the induction of ischemic brain injury. Malonate, which produces lesions similar to those of focal ischemia-reperfusion by a reversible inhibition of succinate dehydrogenase in mitochondria, was injected into the left striatum in the rat brain without or with the simultaneous injection of a cell permeable peptidic JNK inhibitor, (L)-HIV-TAT(48-57)-PP-JP-JBD(20). Two regions of malonate-induced brain injury were visualized as a hyperintense region with surrounding hypointense regions by apparent diffusion coefficient mapping magnetic resonance imaging. The INK inhibitor significantly counteracted both hyper- and hypointense regions at the early stage of brain injury. Histological examination clarified that the inhibitor suppressed the induction of coagulation necrosis and spongy degeneration at early and late stages. (C) 2004 Elsevier Ireland Ltd. All rights reserved.
  • H Nagahata, H Higuchi, H Teraoka, K Takahashi, O Inanami, M Kuwabara
    IMMUNOLOGY AND CELL BIOLOGY 82 1 32 - 37 2004年02月 [査読有り][通常論文]
     
    Stimulant-induced viability of neutrophils, nuclear-fragmentation, increase in intracellular calcium ([Ca2+](i)), expression of annexin V on neutrophils and proteolysis of a fluorogenic peptide substrate Ac-DEVD-MCA (acetyl Asp-Glu-Val-Asp alpha-[4-methyl-coumaryl-7-amide]) by neutrophil lysates from five normal calves and three calves with leucocyte adhesion deficiency were determined to evaluate the apoptosis of normal and CD18-deficient neutrophils. Viability was markedly decreased in control neutrophils stimulated with opsonized zymosan (OPZ), compared to CD18-deficient neutrophils at 37degreesC after incubation periods of 6 and 24 hours. The rate of apoptosis of control neutrophils stimulated with OPZ increased significantly depending on the incubation time, whereas no apparent increase in apoptosis was found in CD18-deficient neutrophils under the same conditions. Aggregated bovine (Agg) IgG-induced apoptosis of control neutrophils was not significantly different from that of CD18-deficient neutrophils. The expression of annexin V on OPZ-stimulated control neutrophils was greater than that of unstimulated ones 6 h after stimulation. No apparent increase in annexin V expression on CD18-deficient neutrophils was found with OPZ stimulation. A delay in apoptosis was demonstrated in CD18-deficient bovine neutrophils and this appeared to be closely associated with lowered signalling via [Ca2+](i), diminished annexin V expression on the cell surface, and decreased caspase 3 activity in lysates.
  • M Kuwabara, T Asanuma, O Inanami
    SFRR: PROCEEDINGS OF THE XII BIENNIAL MEETING OF THE SOCIETY FOR FREE RADICAL RESEARCH INTERNATIONAL 257 - 260 2004年 [査読有り][通常論文]
     
    In the present study, as a model of oxidative stress, ischemic brain injury was induced by injecting malonate into the left striatum in the brain of male Sprague-Dawley rat. Then, a cell permeable peptidic JNK inhibitor, (L)-HIV-TAT(48-57)-PP-JBD(20), was simultaneously injected into it to confirm whether the activation of c-jun by JNK is involved in the ischemia-induced brain injury. The brain was examined with the MRI/ADC mapping method after injection. The malonate injection induced two regions of brain injury visualized as a hyperintense region with surrounding hypointense one. The peptide injection was found to significantly counteract both hyper- and hypointense regions at the early stage of ischemic injury, suggesting that the JNK pathway is involved in malonate-induced ischemic brain injury.
  • K Niwa, O Inanami, T Yamamori, T Ohta, T Hamasu, M Kuwabara
    ANTIOXIDANTS & REDOX SIGNALING 5 6 713 - 722 2003年12月 [査読有り][通常論文]
     
    To clarify the apoptotic and survival signal transduction pathways in activated vascular endothelial cells exposed to oxidative stress, the effects of inhibitors of signal transduction on hydrogen peroxide (H2O2)-induced apoptosis in bovine aortic vascular endothelial cells (BAEC) were examined. Treatment of BAEC with 1 mM H2O2 caused increases of DNA fragmentation, p53 expression, Bax/Bcl-2 ratio, and the activities of caspases 3 and 9. The increases of DNA fragmentation, Bax/Bcl-2 ratio, and caspase activities were abrogated by BAPTA-AM (an intracellular Ca2+ chelator) and N-acetyl-L-cysteine (an antioxidant), and augmented by wortmannin [a phosphatidylinositol 3-kinase (PI3K) inhibitor]. The increase of the intracellular Ca2+ concentration ([Ca2+](i)) observed in H2O2-stimulated cells was unaffected by wortmannin, suggesting that the potentiating effect of wortmannin on the apoptosis was not due to an alteration of [Ca2+](i).H2O2 increased the levels of PI3K activity and Akt phosphorylation. Both were attenuated by wortmannin and, to a lesser extent, by genistein (a tyrosine kinase inhibitor) and suramin (a growth factor receptor inhibitor), but not affected by BAPTA-AM. These results suggest that H2O2 induces Ca2+-dependent apoptosis and Ca2+-independent survival signals such as redox-regulated activation of PI3K/Akt, which is partly mediated by the activation of growth factor receptors in BAEC.
  • T Asanuma, Y Matsumoto, M Takiguchi, O Inanami, M Nakao, K Nakaya, A Ito, A Hashimoto, M Kuwabara
    COMPARATIVE MEDICINE 53 6 649 - 656 2003年12月 [査読有り][通常論文]
     
    The early stage of experimentally induced secondary cerebral alveolar echinococcosis (AE) in rats was investigated by use of magnetic resonance imaging (MRI) and immunoblot (western blot) analyses. Thirty-six female Wistar rats (6 to 8 weeks old) were injected intracranially with a 10% homogenate of echinococcal larval tissues in which the concentrations of microvesicles and protoscolices were estimated to be 3.8 and 1.5 x 10(4)/ml, respectively. To observe the fine structure of the rat brain, MRI was performed under a high magnetic field of 7.05 T. Histologic examination also was performed. The T2-weighted MR images revealed a hyperintense region in the cerebral cortex at two weeks after injection of the homogenate. At three weeks after injection, this region was found to have cysts on the basis of results of histologic examination. Signal-void regions corresponding to hyperplasia and the subsequent calcification of the cuticle layer at six and 13 weeks after injection, respectively, were observed in T2-weighted and proton density MR images. On the other hand, at nine weeks after injection, AE was discernible by use of western blot analysis of sera with antibodies of two epitopes (Em18 and Em16) of E. multilocularis. Using this secondary cerebral AE animal model, it was concluded that the MRI method was suitable for early detection of secondary cerebral AE.
  • M Ogata, O Inanami, M Nakajima, T Nakajima, W Hiraoka, M Kuwabara
    PHOTOCHEMISTRY AND PHOTOBIOLOGY 78 3 241 - 247 2003年09月 [査読有り][通常論文]
     
    To clarify the role of the Golgi apparatus in photodynamic therapy-induced apoptosis, its signaling pathway was studied after photodynamic treatment of human cervix carcinoma cell line HeLa, in which a photosensitizer, 2,4,5,7-tetrabromorhod-amine 123 bromide (TBR), was incorporated into the Golgi apparatus. Laser scanning microscopic analysis of TBR-loaded HeLa cells confirmed that TBR was exclusively located in the Golgi apparatus. HeLa cells incubated with TBR for 1 h were then exposed to visible light using an Xe lamp. Light of wavelength below 670 nm was eliminated with a filter. Morphological observation of nuclei stained with Hoechst 33342 revealed that apoptosis of cells was induced by exposure to light. Electron spin resonance spectrometry showed that light-exposed TBR produced both singlet oxygen (102) and superoxide anion (O-2(-)). Apoptosis induction by TBR was inhibited by pyrrolidine dithiocarbamate, an O-2(-) scavenger, but not by NaN3, a quencher of O-1(2). Furthermore, TBR-induced apoptosis was inhibited by aurintricarboxylic acid and ZnCl2, which are known as inhibitors of deoxyribonuclease (DNase) gamma, and (acetoxymethyl)-1,2-bis(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, a chelator of Ca2+, but not by acetyl Asp-Glu-Val-Asp-aldehyde, an inhibitor of caspase-3. These results suggested that O-2(-) was responsible for TBR-induced apoptosis, and Ca2+-dependent and caspase-3-independent nuclease such as DNase gamma played an important role in apoptotic signaling triggered by Golgi dysfunction.
  • M Kuwabara, K Takahashi, O Inanami
    JOURNAL OF RADIATION RESEARCH 44 3 203 - 209 2003年09月 [査読有り][通常論文]
     
    A post-irradiation treatment of the human leukemia cell line MOLT-4 with the anitoxidant Trolox attenuated capase-3 dependent apoptosis. The increase in the p53 expression and SAPK/JNK activation after X irradiation was also inhibited by a Trolox treatment, but the expression of BCL-2 and BAX, which would occur downstream from p53, was not changed. Studies on the effects of the intracellular calcium chelator BAPTA-AM on the induction of apoptosis and the activation of SAPK/JNK and capase-3 proved that the chelation of calcium merely delayed the onset of radiation-induced apoptosis and the activation of SAPK/JNK and caspase-3. When the effects of the protein synthesis inhibitor cycloheximde on the apoptotic signaling pathways, including the activation of caspase family proteins and SAPK/JNK, were investigated, the expression of death receptor Fas through SAPK/JNK activation was found to be required for radiation-induced apoptosis. Finally, the relationship between the amounts of DNA dsb and induction of apoptosis was examined by irradiating BrdU-incorporated cells. An increase in DNA dsb caused by BrdU was found, but the induction of apoptosis was not enhanced. From these data, we could get no positive evidence for DNA as a target of X-rays and p53 as an indispensable factor to induced apoptosis in X-irradiated MOLT-4 cells.
  • Kashiwakura, I, O Inanami, K Takahashi, TA Takahashi, M Kuwabara, Y Takagi
    RADIATION RESEARCH 160 2 210 - 216 2003年08月 [査読有り][通常論文]
     
    In previous studies we characterized the radiosensitivity of CFU-megakaryocytes from human placental and umbilical cord blood and the effects of various early-acting cytokines. We found that the maximal clonal growth of CFU-megakaryocytes in vitro and maximal protection against X-ray damage were supported by a combination of thrombopoietin and stem cell factor. However, the mechanism by which the two cytokines exert a synergistic effect remained unclear, so we extended these studies to investigate the radioprotective action of synergistic thrombopoietin and stem cell factor on the survival of X-irradiated CD34(+) CFU-megakaryocytes. A combination of thrombopoietin and stem cell factor led to activation of mitogen-activated protein kinase and extracellular signal-regulated protein kinase and to suppression of caspase 3 in X-irradiated CD34+ cells. When PD98059 and various synthetic substrates-specific inhibitors of these proteins-were used, the combination had less effect on the clonal growth of X-irradiated CD34+ CFU-megakaryocytes. However, the addition of wortmannin, a specific inhibitor of the phosphatidylinositol-3 kinase pathway, did not alter the synergistic action of thrombopoietin plus stem cell factor. We suggest that part of this synergistic effect can be explained by activation of mitogen-activated protein kinase and extracellular signal-regulated protein kinase and by suppression of the caspase cascade. (C) 2003 by Radiation Research Society.
  • K Waki, O Inanami, T Yamamori, M Kuwabara
    FREE RADICAL RESEARCH 37 6 665 - 671 2003年06月 [査読有り][通常論文]
     
    This experiment was performed to clarify the role of extracellular signal-regulated kinase, ERK1/2, in NADPH oxidase-dependent O-2(-) production in rat peritoneal neutrophils. When neutrophils were exposed to N-formylmethionyl-leucyl-phenylalanine (fMLP) to stimulate an N-formyl peptide receptor, not only the production of O-2(-) but also the activation of ERK1/2 was observed. The translocation of an NADPH oxidase component, p47(phox), from cytosol to membrane also occurred in neutrophils stimulated with fMLP. U0126, an ERK1/2 kinase inhibitor, inhibited both the production of O-2(-) and the translocation of p47(phox) elicited by fMLP. On the other hand, when complement receptor 3 of neutrophils was stimulated with opsonized zymosan (OZ), weaker activation of ERK1/2 than that by fMLP was observed. In this case, U0126 showed no inhibition against the production of O-2(-) and slight inhibition against the translocation of p47(phox). Large inhibition against the OZ-induced production of O-2(-) was only observed in neutrophils treated with GF109203X, a PKC inhibitor. The present study indicates that receptor dependence exists in the ERK1/2 signaling pathway leading to the activation of NADPH oxidase.
  • R Sato, O Inanami, B Syuto, J Sato, M Kuwabara, Y Naito
    JOURNAL OF VETERINARY MEDICAL SCIENCE 65 4 465 - 469 2003年04月 [査読有り][通常論文]
     
    To clarify the relationship between plasma antioxidant activity and diseases in dogs, plasma samples were collected from 6 healthy dogs and 16 diseased dogs (6 dogs with cancer, 5 dogs with hepatic disease, and 5 dogs with inflammation), and measured superoxide anion scavenging activities. Antioxidant activities of canine plasma were evaluated by measuring their superoxide anion (O-2(-)) scavenging activities with electron spin response spectroscopy combined with spin trapping reagent, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Total O-2(-) scavenging activities in the presence of plasma of diseased dogs tended to be higher than those in healthy controls, especially significant higher activities in the presence of canine plasma of hepatic disease and inflammation were observed. In diseased dogs, KCN-insensitive activities, suggesting the activity of manganese-containing superoxide dismutase (Mn-SOD), were significantly higher than those in healthy controls. Therefore, it seems that there is a possibility of utilizing of plasma O-2(-). scavenging activity as one of clinical indicators for oxidative-related diseases such as cancer, hepatic disease and inflammation in dogs.
  • T Mizutani, M Kobayashi, Y Eshita, O Inanami, T Yamamori, A Goto, Y Ako, H Miyoshi, H Miyamoto, H Kariwa, M Kuwabara, Takashima, I
    INSECT MOLECULAR BIOLOGY 12 1 61 - 66 2003年02月 [査読有り][通常論文]
     
    When Western blot analysis of heat-killed bacteria- and lipopolysaccharide (LPS)-treated Aedes albopictus mosquito cell line C6/36 was performed using antiphospholyrated c-Jun amino-terminal kinase (JNK) antibodies, approximately 46 kDa protein was clearly detected with a peak around 30 min. After the C6/36 cells were incubated at 45 degreesC in order to induce apoptosis, the 46 kDa protein continued to be detected for at least 3 h. The internalization of fluorescein-labelled bacteria was inhibited by a JNK-specific inhibitor SP600125, suggesting that phagocytosis involves the JNK signalling pathway in mosquito cells. Based on these results, we found one candidate for the nucleotide sequence of JNK (Ae-JNK) from the C6/36 cells. This study is the first report regarding the mitogen-activated protein kinase (MAPK) of mosquito.
  • K Niwa, O Inanami, T Yamamori, T Ohta, T Hamasu, T Karino, M Kuwabara
    FREE RADICAL RESEARCH 36 11 1147 - 1153 2002年11月 [査読有り][通常論文]
     
    To clarify the signaling pathways of oxidative stress-induced apoptosis in bovine aortic endothelial cells (BAEC), we treated cells with 1 mM H2O2 and investigated the roles of protein kinase C delta (PKCdelta) and Ca2+ in the accumulation of p53 associated with apoptosis. The treatment of cells with H2O2 caused the accumulation of p53, which was inhibited by rottlerin (a PKCdelta inhibitor) but not by BAPTA-AM (an intracellular Ca2+ chelator). PKCdelta itself was activated through the phosphorylation at tyrosine residues. H2O2 induced the release of cytochrome c and the activation of caspases 3 and 9, and these apoptotic signals were inhibited by rottlerin and BAPTA-AM. These results suggest that PKCdelta contributes to the accumulation of p53 and that Ca2+ plays a role in downstream signals of p53 leading to apoptosis in H2O2-treated BAEC.
  • Takayuki Nakajima, Sadahiro Iwabuchi, Hiroyuki Miyazaki, Yasunobu Okuma, Osamu Inanami, Mikinori Kuwabara, Yasuyuki Nomura, Koichi Kawahara
    Neuroscience letters 331 1 13 - 6 2002年10月04日 [査読有り][通常論文]
     
    Application of a brief period of ischemia, i.e. preconditioning treatment of the middle cerebral artery territory, has been known to produce ischemic tolerance, reducing cerebral infarction volume in the penumbra region after lethal ischemia. However, little is known about the molecular mechanisms responsible for preconditioning-induced ischemic tolerance. In the present study, we examined the difference in the phosphorylation pattern of cyclic AMP responsive element binding protein (CREB) after 1 h of focal cerebral ischemia between preconditioned and non-preconditioned rats by immunohistochemistry and Western blotting. The phosphorylation of CREB in the penumbra region was more rapidly enhanced in the preconditioned rats than in the non-preconditioned rats after 1 h of ischemia. The result suggested that the immediate enhancement in the phosphorylation of CREB in the penumbra region prevented the spread of infarction in the preconditioned rats.
  • T Asanuma, H Ishibashi, A Konno, Y Kon, O Inanami, M Kuwabara
    NEUROSCIENCE LETTERS 329 3 281 - 284 2002年09月 [査読有り][通常論文]
     
    Ischemic brain injury induced by injection of 3-mumol malonate into the left striatum of male Sprague-Dawley rats was examined by apparent water diffusion coefficient (ADC) mapping of magnetic resonance imaging. The region surrounding the injection core was imaged as a hypointense area in ADC mapping and ADC values in the regions were significantly decreased 3 and 6 h after ischemia. Significant reduction of the hypointense area and the recovery of ADC values were observed in rats to which alpha-phenyl-N-tert-butylnitrone (PBN) was intraperitoneally administered 1 h before ischemia. Since ADC mapping has been reported to be a suitable method for evaluating the extent and the degree of cytotoxic edema in the early period after the onset of ischemia, the present results prove that PBN is able to prevent early ischemic insults such as cytotoxic edema. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.
  • Kashiwakura, I, O Inanami, M Murakami, TA Takahashi, M Kuwabara, Y Takagi
    RADIATION RESEARCH 158 2 202 - 209 2002年08月 [査読有り][通常論文]
     
    Combinations of thrombopoietin and cytokines that act on megakaryocyte development (stem cell factor, IL3, IL6, IL11, W) ligand (now known as FLT3LG), erythropoietin, GM-CSF and G-CSF were evaluated for their ability to enhance clonal growth in vitro of X-irradiated CD34(+) megakaryocytic progenitor cells (CFU-megakarvocytes) purified from normal human peripheral blood. These data were compared with corresponding results described previously for CD341 CFU-megakaryocytes from human placental/umbilical cord blood (I. Kashiwakura, Radiat. Res. 153, 144-152, 2000). All cytokines, except IL3, promoted thrombopoietin-induced colony formation, but they resulted in exponential radiation survival curves. No significant differences in the D-0 (46-61 cGy) and extrapolation number n (1.00-1.04) were observed between thrombopoietin alone and in combination with these cytokines. IL3 did not promote colony formation, but marked shoulders were observed on the survival curves (D-0 = 91 cGy, n = 2.83). Flow cytometric analysis of cells harvested from cultures of X-irradiated cells stimulated with thrombopoietin plus IL3 showed no significant differences in the expression of surface antigens and DNA ploidy distribution of megakaryocytes from the control. These findings suggest that IL3 plays a key role in promoting the survival of Virradiated CD34(+) CFU-megakaryocytes from peripheral blood as well as those from cord blood, though the former are more radiosensitive. (C) 2002 by Radiation Research Society.
  • T Yamamori, O Inanami, H Sumimoto, T Akasaki, H Nagahata, M Kuwabara
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 293 5 1571 - 1578 2002年05月 [査読有り][通常論文]
     
    Superoxide production by NADPH oxidase is essential for bactericidal properties of neutrophils. However, molecular mechanisms underlying the activation of this enzyme remain largely unknown. Here, using bovine neutrophils we examined the role of p38 mitogen-activated protein kinase (p38 MAPK) in the signaling pathways of the NADPH oxidase activation. Superoxide production was induced by stimulation with serum-opsonized zymosan (OZ) and attenuated by p38 MAPK inhibitor, SB203580. OZ stimulation induced the translocation of P47(phox) and Rac to the plasma membrane and SB203580 completely blocked the translocation of Rac, but only partially blocked that of p47(Phox). Furthermore, SB203580 abolished the OZ-elicited activation of Rac, which was assessed by detecting the GTP-bound form of this protein. Phosphatidylinositol 3-kinase (PI3K) inhibitors, wortmannin and LY294002, blocked not only p38 MAPK activation but also Rac activation. However, SB203580 showed no effect on the PI3K activity. These results suggested that PI3K/p38 MAPK/Rac pathway was present in the activation of NADPH oxidase in bovine neutrophils. (C) 2002 Elsevier Science (USA). All rights reserved.
  • O Inanami, K Sugihara, T Okui, M Hayashi, M Tsujitani, M Kuwabara
    INTERNATIONAL JOURNAL OF RADIATION BIOLOGY 78 4 267 - 274 2002年04月 [査読有り][通常論文]
     
    Purpose: To examine how hypoxia influences ionizing irradition-induced apoptosis in cultured mammalian cells and how a hypoxic cell radiosensitizer sensitizes apoptosis under hypoxic conditions. Materials and methods: Two cell lines derived from human lymphocytes, HL60 and MOLT-4, were exposed to 15 Gy X-rays under aerobic and hypoxic conditions. Etanidazole was used as a hypoxic cell radiosensitizer. The apoptotic morphological changes of nuclei and the induction of ladder-like DNA fragmentation were assessed by fluorescence microscopy and agarose gel electrophoresis, respectively. Results: In HL60 cells, apoptotic cell death and the activation of caspases 8, 9 and 3 were less induced under the hypoxic conditions than under the aerobic ones. Treatment of hypoxic cells with etanidazole enhanced X-ray-induced apoptosis and caspase activation. However, in MOLT-4 cells, neither hypoxia nor etanidazole influenced X-ray-induced apoptosis and caspase activation. In both cell lines, the frequency of X-ray-induced DNA double-stand breaks (DSB) under hypoxia was significantly smaller than that in aerobic conditions. Treatment of hypoxic cells with etanidazole enhanced them. Conclusion: These results suggested that X-ray-induced apoptosis in HL60 cells was initiated by DNA DSB and the treatment of hypoxic cells with etanidazole sensitized them through the enhancement of DSB induction, whereas X-ray-induced apoptosis in MOLT-4 cells occurred through damage other than to DNA.
  • M Kuwabara, Y Iida, O Inanami, S Sawamura, K Yokoyama, M Tsujitani
    JOURNAL OF RADIATION RESEARCH 43 1 77 - 88 2002年03月 [査読有り][通常論文]
     
    This study was performed to confirm the radiation-chemical properties of the 2-nitroimidazole derivative doranidazole, (+/-)-(2RS,3SR)-3-[(2-nitroimdazol-1-yl)-methoxy]butane-1,2,4-triol [CAS 137339-64-1], PR-350, which was synthesized as a hypoxic cell radiosensitizer with low toxicity. Radiation-chemical experiments using doranidazole showed that (1) unlike 02, it had high reactivity toward not only hydrated electrons (e(aq)(-)), but also hydroxyl radicals ( . OH), (2) the reduced intermediates of doranidasole had no ability to induce immediate strand breaks of colE1 plasmid DNA, (3) doranidazole enhanced radiation-induced DNA strand breaks of colE1 plasmid DNA in the aqueous state, whereas it did not enhance the base alteration, such as 8-oxo-deoxyguanosine, (4) it enhanced the radiation-induced formation of strand breaks with 3'-phosophate and 3'-phosphoglycolate termini, and (5) it was bound to DNA after irradiation. These facts revealed that the majority of radiation-chemical properties of doranidazole, except for the high reactivity toward (OH)-O-., were similar to those of oxygen.
  • K Takahashi, O Inanami, M Hayashi, M Kuwabara
    INTERNATIONAL JOURNAL OF RADIATION BIOLOGY 78 2 115 - 124 2002年02月 [査読有り][通常論文]
     
    Purpose: To demonstrate whether protein synthesis is required for ionizing radiation-induced apoptosis through activation of caspases in human leukaemia cell line MOLT-4, the effects of a protein synthesis inhibitor, cycloheximide, on the apoptotic signalling pathway including the activation of caspase family and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and the expression of Fas/CD95/APO-1 (Fas) were examined in X-irradiated MOLT-4 cells. Materials and methods: MOLT-4 cells pretreated with 0.5 mug/ml cycloheximide for 1 h were exposed to 7.5 Gy of X-rays. The appearance of apoptosis, expression of Fas, activation of caspases-3, -8, -9, SAPK/JNK and AP-1, the release of mitochondrial cytochrome-C and the formation of death-induced signalling complex (DISC) between Fas and the Fas-associated death domain (FADD) were measured by fluorescence microscopy, Western blotting, flow cytometry, gel shift assay and immunoprecipitation, respectively. Results: Nuclear fragmentation and chromatin condensation were observed at 6 h after X-irradiation and gradually increased up to 12 h. These phenomena were significantly attenuated by cycloheximide. Cycloheximide also inhibited the activation of caspases and AP-1, the expression of Fas, the formation of DISC and the release of cytochrome-C, but not the activation of SAPK/JNK in X-irradiated MOLT-4 cells. Conclusion: These results indicate that apoptosis of X-ray-induced MOLT-4 cells is dependent on the activation of caspases regulated by de novo protein synthesis through SAPK/JNK activation.
  • Kashiwakura, I, M Murakami, O Inanami, Y Hayase, TA Takahashi, M Kuwabara, Y Takagai
    EUROPEAN JOURNAL OF PHARMACOLOGY 437 1-2 19 - 25 2002年02月 [査読有り][通常論文]
     
    This study investigated the effects of amifostine, a clinically usable radioprotector or chemoprotector, on the proliferation and differentiation of normal and X-irradiated cluster of differentiation 34 positive (CD34(+)) megakaryocytic progenitor cells (colony-forming unit in megakaryocytes, CFU-Meg) from human placental and umbilical cord blood (CB) in vitro. Amifostine significantly accelerated megakaryocyte colony formation in a plasma clot culture supplemented with recombinant human thrombopoietin because of an increase in immature CFU-Meg-derived large megakaryocyte colony formation. An analysis of the cells that were harvested from the culture showed that amifostine induced a 70- and an 83-fold increase in the total cell and CFU-Meg numbers, respectively, and produced hyperploid megakaryocytes of more than 8 N ploidy. The radioprotective effect of amifostine on the clonal growth of X-irradiated CD34(+) CFU-Meg was observed by treatment before or after irradiation. These findings suggest that the action of amifostine extends from immature CFU-Meg to the terminal differentiation of megakaryopoiesis, and its radioprotective effect is shown in megakaryopoiesis and thrombopoiesis, 2002 Elsevier Science B.V. All rights reserved.
  • K Niwa, O Inanami, T Ohta, S Ito, T Karino, M Kuwabara
    XI BIENNIAL MEETING OF THE SOCIETY FOR FREE RADICAL RESEARCH INTERNATIONAL 389 - 393 2002年 [査読有り][通常論文]
     
    We examined the roles of p38 MAPK, ERK and intracellular Ca2+ in H2O2-induced increase of permeability of endothelial cells (EC). The permeation rate of FITC-dextran 40 through monolayers of bovine pulmonary arterial endothelial cells was measured. H2O2 (1. mM) caused an increase of EC permeability, which was abrogated by SB203580 (a p38 MAPK inhibitor) and BAPTA-AM (a chelator of intracellular Ca2+), but not by PD90859 (an ERK inhibitor). H2O2 caused phosphorylation of both p38 MAPK and ERK, which was unaffected by BAPTA-AM. The increase of intracellular Ca2+ concentration ([Ca2+](i)) induced by H2O2 was not affected by SB203580 and PD98059. These results suggest that the activation of p38 MAPK and the increase of [Ca2+](i) contribute separately to the H2O2-induced increase of EC permeability.
  • M Tsuji, O Inanami, M Kuwabara
    JOURNAL OF BIOLOGICAL CHEMISTRY 276 35 32779 - 32785 2001年08月 [査読有り][通常論文]
     
    The spin trap alpha -phenyl-N-tert-butylnitron (PBN) is widely used for studies of the biological effects of free radicals. We previously reported the protective effects of PBN against ischemia-reperfusion injury in gerbil hippocampus by its activation of extracellular signal-regulated kinase (ERK) and suppression of both stress-activated protein kinase and p38 mitogen-activated protein kinase. In the present study, we found that PBN induced neurite outgrowth accompanied by ERK activation in PC12 cells in a dose-dependent manner. The induction of neurite outgrowth was inhibited significantly not only by transient transfection of PC12 cells with dominant negative Ras, but also by treatment with mitogen-activated protein kinase/ERK kinase inhibitor PD98059. The activation of receptor tyrosine kinase TrkA was not involved in PBN-induced neurite outgrowth. A protein kinase C (PKC) inhibitor, GF109203X, was found to inhibit neurite outgrowth. The activation of PKC epsilon was observed after PBN stimulation. PBN-induced neurite outgrowth and ERK activation were counteracted by the thiol-based antioxidant N-acetylcysteine. From these results, it was concluded that PBN induced neurite outgrowth in PC12 cells through activation of the Ras-ERK pathway and PKC.
  • YD Cui, O Inanami, T Yamamori, M Kuwabara
    PROGRESS IN BIOCHEMISTRY AND BIOPHYSICS 28 3 377 - 380 2001年06月 [査読有り][通常論文]
     
    The chemotactic peptide fMLP was known to induce adhesion, migration and phagocytosis of neutrophil. To clear the mechanism of the chemotaxis, the effects of PU-K, p38 and ERK on actin polymerization were studied with the inhibitors of these kinases in neutrophil-like, differentiated HL-60 cells stimulated with fMLP. 0.1 mu mol/L Wortmannin (PI3-kinase inhibitor) inhibited the fMLP-induced polymerization of actin. 50 mu mol/L, SB203580 (p38 inhibitor) and 50 mu mol/L PD98059 (ERK inhibitor) did not inhibited it, though p38 and ERK were regulated by PI3-K. These results suggest the signaling pathway of actin polymerization mediated by PI3-K was different from that of p38 and ERK activation mediated by PI3-K.
  • K Iwasaki, M Morimatsu, O Inanami, E Uchida, B Syuto, M Kuwabara, M Niiyama
    JOURNAL OF BIOLOGICAL CHEMISTRY 276 12 9400 - 9405 2001年03月 [査読有り][通常論文]
     
    Acute-phase serum proteins were induced by administrating a chicken with turpentine oil. One of these proteins was a new protein that appeared in front of albumin in polyacrylamide disc gel electrophoresis using a 4.5-16% gel. To purify this protein, turpentine-administrated chicken serum was fractionated by ammonium sulfate precipitation at 50% saturation, and the supernatant fraction was chromatographed on a DEAE-Toyopearl 650S column. The purified protein is a mannose-glycoprotein, and its N-terminal sequence, determined by the Edoman method, is not homologous from that of other reported acute-phase proteins. An analysis of physiological function with two different test systems, chemiluminescence measurement and electron spin resonance spectroscopy, showed that the purified protein has antioxidant, activity and inhibits superoxide (O-2(radical anion)) mediated by activation of the receptor. In support of these results, the complete amino acid sequence of 18-B is homologous to the scavenger receptor cysteine-rich (SRCR) family of proteins that participate in the regulation of leukocyte function. 18-B is composed of four SRCR domains, which is different from the previously characterized SRCR family of proteins such as Sp alpha, CD6, and CD163. These findings indicate that turpentine-induced 18-B, a new member of scavenger receptor cysteine-rich family, may be implicated in regulation of cell function in a manner of inhibition of the overproduction of the reactive oxygen species.
  • O Inanami, T Yamamori, TA Takahashi, H Nagahata, M Kuwabara
    FREE RADICAL RESEARCH 34 1 81 - 92 2001年 [査読有り][通常論文]
     
    We applied a spin trap, 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO), to detect O-2(.-) generation during phagocytosis in human polymorphonuclear leukocytes (PMNs). PMNs were activated with serum-opsonized zymosan (sOZ) in the presence of DEPMPO. The ESR spectra mainly consisted of Cu,Zn-SOD-sensitive DEPMPO-OOH spin adducts. To clarify where these spin-adducts were present, cells after stimulation were separated from extracellular fluid by brief centrifugation and resuspended in Hanks' balanced salt solution. ESR examination showed that DEPMPO-OOH adducts were present in both fractions. When cells were stimulated by phorbol myristate acetate (PMA), the DEPMPO-OOH was detected in extracellular fluid but not in the cell fraction. Furthermore, DEPMPO-OOH adducts were quickly converted into ESR-silent compounds by addition of cell lysate of PMNs. These results indicate that DEPMPO is useful to detect O-2(.-) of extracellular space including the intraphagosome but not that of intracellular space in sOZ-stimulated phagocytes.
  • H Abe, K Ikebuchi, K Niwa, O Inanami, M Kuwabara, M Fujihara, J Hirayama, H Ikeda
    ARTIFICIAL CELLS BLOOD SUBSTITUTES AND IMMOBILIZATION BIOTECHNOLOGY 29 4 275 - 283 2001年 [査読有り][通常論文]
     
    We investigated the interactions between liposome-encapsulated hemoglobin (Neo Red Cells: NRC) and human polymorphonuclear leukocytes as assessed by superoxide generation. NRC triggered superoxide generation from neutrophils in a dose-dependent manner. Empty liposomes also induced superoxide production of neutrophils. Superoxide generation of neutrophils induced by phorbol myristate acetate (PMA) was delayed but intensified both by NRC and empty liposomes. The intensity of superoxide generation induced by NRC was smaller than that by the empty liposomes. As NRC contained superoxide dismutase (SOD) that was copurified with hemoglobin from red blood cells and its activity remained, SOD contained in NRC. may partially eliminate superoxide.
  • K Niwa, O Inanami, T Ohta, S Ito, T Karino, M Kuwabara
    FREE RADICAL RESEARCH 35 5 519 - 527 2001年 [査読有り][通常論文]
     
    To examine the involvement of p38 mitogen-activated protein kinase (p38 MAPK) and extracellular signal-regulated kinase (ERK) in the oxidative stress-induced increase of permeability in endothelial cells, the effects of a p38 MAPK inhibitor (SB203580) and ERK inhibitor (PD90859) on the H2O2-induced increase of permeability in bovine pulmonary artery endothelial cells (BPAEC) were investigated using a two-compartment system partitioned by a semi-permeable filter. H2O2 at 1 mM caused an increase of the permeation rate of fluorescein isothiocyanate (FITC)-labeled dextran 40 through BPAEC monolayers. SB203580 inhibited the H2O2-induced increase of permeability but PD98059 did not, though activation (phosphorylation) of both p38 MAPK and ERK was observed in H2O2-treated cells in Western blot analysis. An H2O2-induced increase of the intracellular Ca2+ concentration ([Ca2+](i)) was also observed and an intracellular Ca2+ chelator (BAPTA-AM) significantly inhibited the H2O2-induced increase of permeability. However, it showed no inhibitory effects on the H2O2-induced phosphorylation of p38 MAPK and ERK. The H2O2-induced increase of [Ca2+](i) was not influenced by SB203580 and PD98059. These results indicate that the activation of p38 MAPK and the increase of [Ca2+](i) are essential for the H2O2-induced increase of endothelial permeability and that ERK is not.
  • O Inanami, T Yamamori, H Shionoya, M Kuwabara
    BIOLOGY OF ASCIDIANS 457 - 462 2001年 [査読有り][通常論文]
     
    Ascidian is known to have antioxidant compounds such as quinone derivatives. Recently, we developed the freeze-dried powder of the ascidians as an addition to improve properties of the foods. In this experiment, to evaluate whether the property of the ascidians was preserved during freeze-drying process, we tried to identify quinone derivatives and measure its antioxidant activity. For this purpose, electron spin resonance (ESR) technique was employed. ESR spectrum of the ethyl acetate-soluble fraction of freeze-dried powder of ascidians showed the existence of semiquinone radicals derived from prenylated hydroquinone. This fraction also showed to have significant scavenging activity for superoxide produced by xanthine oxidase/hypoxanthine system in vitro. These results suggested that several quinone derivatives in the ascidians were preserved during freeze-drying process and had the superoxide scavenging activity. The freeze-dried powder of the ascidians may be useful for an addition of antioxidantive property to foods.
  • YD Cui, O Inanami, T Yamamori, K Niwa, H Nagahata, M Kuwabara
    INFLAMMATION RESEARCH 49 12 684 - 691 2000年12月 [査読有り][通常論文]
     
    Objective and Design: To further understand the mechanisms of signal transduction pathways for the formation of F-actin (polymerization of actin) and the activation of NADPH oxidase in phagocytic cells, the effects of various inhibitors on them were studied. Materials and Methods: Differentiated HL60 cells were studied to examine their N-formyl-methionyl-leucyl-phenyl-alanine (fMLP)-stimulated formation of F-actin and activation of NADPH oxidase following treatment with various inhibitors. These included a protein kinase C (PKC) inhibitor (GF 109203X), a phosphatidylinositide 3 kinase (PI3-K) inhibitor (wortmannin), an extracellular response kinase (ERK) inhibitor (PD 98059), a p38 mitogen-activated protein kinase (MAPK) inhibitor (SB 203580) and an intracellular Ca2+-chelator (BAPTA-AM). Results: The treatment with wortmannin suppressed the formation of F-actin, with less suppression of the activation of NADPH oxidase. BAPTA-AM and GF 109203X did not attenuate the formation of F-actin but completely inhibited the activation of NADPH oxidase. PD 98059 and SE 203580 partially inhibited the activation of NADPH oxidase without influence on the formation of F-actin. Furthermore, wortmannin but not BAPTA-AM and GF 109203X inhibited the fMLP-induced activation of Akt, which is known to regulate NADPH oxidase. Conclusions: These results suggest that the formation of F-actin is dependent on PI3-K and independent of PKC, ERK and p38 MAPK as well as the increase in intracellular Ca2+, whereas the activation of NADPH oxidase is partly dependent on ERK, p38 MAPK, Akt regulated by PI3-K, and strongly dependent on the activation of PKC and the increase in intracellular Ca2+.
  • Y Iida, O Inanami, H Inoue, E Ohtsuka, M Kuwabara
    RADIATION RESEARCH 154 5 582 - 589 2000年11月 [査読有り][通常論文]
     
    Two kinds of double-stranded oligonucleotides containing a single 8-oxo-7,8-dihydroguanine were labeled with P-32 at their 5' ends and exposed to gamma rays in the frozen aqueous state at 77 K, where both direct and quasi-direct effects of ionizing radiation predominate. Analysis of the oligonucleotides with 20% denaturing polyacrylamide gel electrophoresis revealed no difference in the immediate induction of strand breaks between oligonucleotides containing 8-oxo-7,8-dihydroguanine and their corresponding oligonucleotides with normal guanine, but piperidine-sensitive damage was induced more frequently in the former than in the latter. Sequence analysis of irradiated oligonucleotides showed that not only 8-oxo-7,8-dihydroguanine but also its neighboring bases and the cytosine residue that is paired to it became piperidine-sensitive in both oligonucleotides, These results suggest that 8-oxo-7,8-dihydroguanine, its neighboring bases and the opposite cytosine are candidates for radiation damage hot spots. (C) 2000 by Radiation Research Society.
  • M Kuwabara, TA Takahashi, H Nagahata, O Inanami
    JAPANESE JOURNAL OF VETERINARY RESEARCH 48 1 3 - 13 2000年05月 [査読有り][通常論文]
     
    To clarify where oxygen radicals are generated in polymorphonuclear leukocytes (PMNs) during phagocytosis, superoxides (O-2(-)) from opsonized zymosan (OZ) stimulated human PMNs were detected by the ESR and spin-trapping methods. PMNs were preactivated with OZ for the indicated periods of time at 37 degrees C. Then a spin-trapping agent, 5, 5-dimethyl- 1 -pyrroline N-oxide (DMPO), was added to them, and they were further incubated for 30sec for ESR observations. The ESR spectra consisted of two components due to the DMPO-OOH and DMPO-OH spin adducts. To clarify where these spin-adducts were present, cells were separated from extracellular fluid by brief centrifugation and resuspended in Hanks' balanced salt solution. ESR examination of two fractions showed that the DMPO-OOH adducts was present in the cell fraction, whereas the DMPO-OH adducts were present in the extracellular fluid. When DMSO was used as a scavenger of hydroxyl radicals ((OH)-O-.), DMPO-CH adducts were observed in the fluid fraction but not in the cell fraction. Both spin adducts were completely abolished by Cu, Zn-SOD but not catalase. These results indicated that O-2(-) were produced inside phagosomes of OZ-stimulated PMNs and (OH)-O-. were produced outside them by spontaneous decomposition of the DMPO-OOH adducts.
  • M Tsuji, O Inanami, M Kuwabara
    NEUROSCIENCE LETTERS 282 1-2 41 - 44 2000年03月 [査読有り][通常論文]
     
    alpha-Phenyl-N-tert-butylnitrone (PBN), a spin trap, is known as a protective agent against delayed-neuronal death after ischemia-reperfusion. To investigate this neuroprotective effect of PBN, we examined the effect of PBN on the mitogen-activated protein kinase (MAPK) signaling pathway and the expression of heat shock proteins (HSPs) in the gerbil hippocampus following transient (5 min) ischemia. Immunoblot analysis revealed that intraperitoneal (i.p.) injection of PBN (200 mg/kg) enhanced the activation of extracellular-response kinase (ERK) and suppressed the activation of stress-activated protein kinase/c-Jun N-terminal protein kinase (SAPK/JNK) and p38 mitogen-activated protein kinase (p38) at 6 h after ischemia. Elevated levels of HSP27 and HSP70 were seen at the same period. These data suggest that PEN protects against delayed-neuronal death not only by its inherent radical-trapping activity but also by regulating the MAPK pathway and up-regulating HSPs. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.
  • 稲波 修, 高橋 賢次, 飯田 義晴
    細胞 32 2 47 - 51 ニュー・サイエンス社 2000年02月 [査読無し][通常論文]
  • Kashiwakura, I, M Kuwabara, O Inanami, M Murakami, Y Hayase, TA Takahashi, Y Takagi
    RADIATION RESEARCH 153 2 144 - 152 2000年02月 [査読有り][通常論文]
     
    The in vitro radiation sensitivity of CPU-Meg isolated from human placental and umbilical cord blood was evaluated in plasma clot cultures stimulated by recombinant human cytokines, including thrombopoietin, the FLT3 ligand (FLT3LG), interleukin-3, interleukin-11 and stem cell factor. The CD34(+) cells were irradiated with X rays at a dose rate of 73 cGy/min, The megakaryocyte colonies were identified by using an FITC-conjugated antibody to glycoprotein IIbIIIa and were classified into two groups based on colony size: large colonies (immature CPU-Meg) and small colonies (mature CPU-Meg), Treatment with thrombopoietin alone or in combination with FLT3LG and/or interleukin-11 gave exponential radiation survival curves (D-0 for immature CFU-Meg = 56-77 cGy, D-0 for mature CPU-Meg = 86 cGy-1.12 Gy), while marked shoulders were observed on the survival curves for colonies supported by the combination of thrombopoietin, interleukin-3 and stem cell factor (D-0 for immature CFU-Meg = 89-98 cGy; D-0 for mature CFU-Meg = 1.25-1.31 Gy). Our results showed that the immature CPU-Meg were more radiosensitive than the mature CPU-Meg and that the combination of cytokines, including thrombopoietin, interleukin-3 and stem cell factor, affected the radiation sensitivity of CFU-Meg to the same extent as with thrombopoietin alone or in combination with FLT3LG and/or interleukin-11. (C) 2000 by Radiation Research Society.
  • Y Nomura, O Inanami, K Takahashi, A Matsuda, M Kuwabara
    LEUKEMIA 14 2 299 - 306 2000年02月 [査読有り][通常論文]
     
    The mechanism of apoptosis induced by 2-chloro-2'-deoxyadenosine (2CdA) in human leukemia cell line MOLT-4 was investigated. 2CdA induced increases of 3'-OH ends of genomic DNA, ladder-like DNA fragmentation and phosphatidylserine translocation to the outer membrane, which are apoptotic characteristics. These apoptotic phenomena induced by 2CdA were inhibited by cycloheximide (CHX; a protein synthesis inhibitor), deoxycytidine (dC; a substrate of deoxycytidine kinase), acetyl Ile-Glu-Thr-Asp aldehyde (Ac-IETD-CHO; a caspase-8 inhibitor) and acetyl Asp-Glu-Val-Asp aldehyde (Ac-DEVD-CHO; a caspase-3 inhibitor). The protein synthesis-dependent expression of Fas and Fas ligand (Fas-L) was detected by treatment with 2CdA. The proteolytic processing of procaspases-8 and -3 to produce active fragments, caspases-8 (p18) and -3 (p17), respectively, was observed after treatment with 2CdA, and suppressed by cycloheximide. Increases in the activities of caspases-8 and -3 were observed after 2CdA treatment. Their activation was also dependent on protein synthesis. These results indicated that 2CdA-induced apoptosis was triggered by phosphorylation of 2CdA followed by the protein synthesis-dependent expression of Fas and Fas-L and activation of caspases-8 and -3.
  • T Yamamori, O Inanami, H Nagahata, YD Cui, M Kuwabara
    FEBS LETTERS 467 2-3 253 - 258 2000年02月 [査読有り][通常論文]
     
    Stimulation of bovine polymorphonuclear leukocytes (PMN) with serum-opsonized zymosan (sOZ) induced the activation of p38 mitogen-activated protein kinase (MAPK), protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3-K) and sOZ-induced O-2(-) production was significantly attenuated by their inhibitors (SB203580 for p38 MAPK, GF109203X for PBC and wortmannin for PD-K). They caused significant attenuation of sOZ-induced phosphorylation of p47phox as well, Flow cytometric analysis, however, revealed that SB203580 and wortmannin attenuated phagocytosis, but GF109203X facilitated it. The results suggest that p38 MAPK and PI3-K participated in both signaling pathways of NADPH oxidase activation (O-2(-) production) and phagocytosis, and PKC participated in the signaling pathway of NADPH oxidase activation alone, (C) 2000 Federation of European Biochemical Societies.
  • O Inanami, A Yoshito, K Takahashi, W Hiraoka, M Kuwabara
    PHOTOCHEMISTRY AND PHOTOBIOLOGY 70 4 650 - 655 1999年10月 [査読有り][通常論文]
     
    The mechanism of caspase-3-dependent apoptosis induced by photodynamic therapy (PDT) of cultured Chinese hamster V79 cells with pheophorbide a (PPa) was investigated. The PPa-PDT induced rapid apoptosis within 30 min after irradiation of cells. This apoptosis was inhibited by the O-1(2) quencher N-3(-) and caspase-3 inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde, suggesting that O-1(2) activated caspase-3 and then caused apoptosis. The intra-cellular calcium [Ca2+](i) chelator (acetoxymethyl)-1,2-bis(o-aminophenoxy)ethane N,N,N',N'-tetraacetic acid (BAPTA- AM) and the cyclic adenosine monophosphate (cAMP)-increasing agent forskolin also inhibited not only the PPa-PDT-induced activation of caspase-3 but also apoptosis in V79 cells. Furthermore, PPa-PDT-induced cytochrome c release from mitochondria was found to be inhibited by the treatment with BAPTA-AM but not forskolin. These results indicated that [Ca2+](i) and cAMP independently serve as regulators for PPa-PDT-induced apoptosis in the upstream of caspase-3.
  • K Takahashi, O Inanami, M Kuwabara
    INTERNATIONAL JOURNAL OF RADIATION BIOLOGY 75 9 1099 - 1105 1999年09月 [査読有り][通常論文]
     
    Purpose: To examine the roles of intracellular calcium in radiation-induced apoptosis of MOLT-4 cells, the effects of intracellular calcium chelator BAPTA-AM on the induction of apoptosis and the activation of apoptosis-relating enzymes SAPK/JNK and caspase-3 were studied. Materials and methods: MOLT-4 cells pretreated with 5 mu M BAPTA-AM were exposed to X-rays. DNA fragmentation, the expression of phosphorylated SAPK/JNK and the activation of caspase-3 and calcium concentration were measured by agarose gel electrophoresis, Western blotting and spectrofluorometry. Results: Time-dependent ladder-like DNA fragmentation was observed at 4 h, 5 h and 6 h after exposure to 15 Gy of X-rays. This fragmentation was significantly attenuated by pretreatment with BAPTA-AM up to 5 h after irradiation, but the attenuation due to BAPTA-AM was no longer detectable at 6 h. Activation of SAPK/JNK and caspase-3 was observed at 1 and 4 h after X-irradiation, respectively, and BAPTA-AM retarded the activation for 2 h. The pretreatment with BAPTA-AM was found to suppress the increase of calcium concentration for 6 h after irradiation. Conclusion: These results revealed that chelation of calcium merely delayed the onset of the radiation-induced apoptosis regulated by the activation of SAPK/JNK and caspase-3, and calcium was not essential for the induction of apoptosis in X-irradiated MOLT-4 cells.
  • O Inanami, A Shiga, KJ Okada, R Sato, Y Miyake, M Kuwabara
    AMERICAN JOURNAL OF VETERINARY RESEARCH 60 4 452 - 457 1999年04月 [査読有り][通常論文]
     
    Objective-To examine the association between oxidative stress and antioxidants in neonatal calves after birth. Sample Population-Sera from 6 healthy Holstein-Friesian cows and 7 of their newborn calves were obtained at various intervals after birth. Procedure-Lipid peroxides in serum of cows and their newborn calves were estimated by measuring concentrations of thiobarbituric acid-reactive substances (TBARS). The antioxidative activities of neonatal sera were evaluated by measuring their superoxide-scavenging activities, ferroxidase activities, and the concentration of bilirubin-associated albumin. Results-Concentration of TEARS in neonatal sera within 1 day after birth was significantly higher than concentrations greater than or equal to 2 days after birth and concentrations in serum of the dams. In contrast, antioxidative activities of neonatal sera, evaluated on the basis of super oxide-scavenging activities, ferroxidase activities, and concentration of bilirubin-associated albumin 3 hours after birth, were significantly lower than antioxidative activities in sera of the dams. Conclusions-Susceptibility of calves to oxidative stress during the neonatal period may be explained by the immature defense system against superoxide radicals.
  • F Okada, K Nakai, T Kobayashi, T Shibata, S Tagami, Y Kawakami, T Kitazawa, R Kominami, S Yoshimura, K Suzuki, N Taniguchi, O Inanami, M Kuwabara, H Kishida, D Nakae, Y Konishi, T Moriuchi, M Hosokawa
    BRITISH JOURNAL OF CANCER 79 3-4 377 - 385 1999年02月 [査読有り][通常論文]
     
    We isolated six clones of weakly tumorigenic fibrosarcoma (QR) from the tumorigenic clone BMT-11 cl-9. The QR clones were unable to grow in normal C57BL/6 mice when injected s.c. (1 x: 10(5) cells). However, they formed aggressive tumours upon co-implantation with a 'foreign body', i.e. a gelatin sponge, and the rate of tumour take ranged from 8% to 58% among QR clones. The enhanced tumorigenicity was due to host cell-mediated reaction to the gelatin sponge (inflammation). Immunoblot analysis and enzyme activity assay revealed a significant inverse correlation between the frequencies of tumour formation, by QR clones and the levels of manganese superoxide dismutase (Mn-SOD, P<0.005) and glutathione peroxidase (GP chi, P<0.01) in the respective tumour clones. Electron spin resonance (ESR) revealed that superoxide-scavenging ability of cell lysates of the QR clone with high [level of Mn-SOD was significantly higher than that with low level of the antioxidative enzyme in the presence of potassium cyanide, an inhibitor for copper-zinc superoxide dismutase (CuZn-SOD) (P<0.001). Minisatellite mutation (MSM) induced by the inflammatory cells in tumour cells were investigated by DNA fingerprint analysis after QR clones had been cocultured with ge[gelatin-sponge-reactive cells. The MSM rate was significantly higher in the subclones with low levels of Mn-SOD and GP chi (P<0.05) than in the subclones with high levels of both enzymes. The MSM of the subclones with low levels of both enzymes was inhibited in the presence of mannitol, a hydroxyl radical scavenger. The content of 8-hydroxydeoxyguanosine (8-OHdG) by which the cellular DNA damage caused by active oxygen species can be assessed was significantly low in the tumours arising from the QR clone with high levels of Mn-SOD and GP chi even if the clone had been co-implanted with gelatin sponge, compared with the arising tumour from the QR clone with low levels of those antioxidative enzymes (P<0.001). In contrast, CuZn-SOD and catalase levels in the six QR clones did not have any correlation with tumour progression parameters. These results suggest that tumour progression is accelerated by inflammation-induced active oxygen species particularly accompanied with declined levels of intracellular antioxidative enzymes in tumour cells.
  • O Inanami, K Takahashi, M Kuwabara
    INTERNATIONAL JOURNAL OF RADIATION BIOLOGY 75 2 155 - 163 1999年02月 [査読有り][通常論文]
     
    Purpose: The relationship between post-irradiation treatment with Trolox, an antioxidant that inhibits lipid peroxidation, and X-ray-induced apoptosis, with regard to signal transduction pathways, was examined in MOLT-4, a human leukaemia cell line. Materials and methods: In MOLT-4 cells treated with Trolox after X-irradiation, viability, DNA fragmentation, expression of p53 BCL-2, BAX, active SAPK/JNK, active caspase-3 and the cleavage of PARP were measured by the trypan blue exclusion test, agarose pel electrophoresis and Western blotting. Results: Stained cells and ladder-like DNA cleavage were observed after X-irradiation. Cell death and DNA fragmentation were significantly inhibited by the post-irradiation treatment with Trolox. The expression of p53 and active SAPK/JNK was increased after X-irradiation, and fragments of PARP and the activated fragment of caspase-3 were produced. Post-irradiation treatment with Trolox attenuated the X-irradiation-induced expression, fragmentation or activation of these apoptosis-related biomolecules. The expression of BCL-2 and BAX, which would occur downstream from p53, was not changed by irradiation and Trolox treatment. Furthermore, cell death was associated with caspase-3 because the ladder-like DNA cleavage was completely inhibited by Ac-DEVD-CHO but not Ac-YVAD-CHO, TLCK and PMSF. Conclusion. Post-irradiation events such as membrane damage induce caspase-3-dependent apoptosis, which might be mediated by the activation of SAPK/JNK and be independent of p53.
  • N Matsuki, A Takanohashi, FM Boffi, O Inanami, M Kuwabara, K Ono
    FREE RADICAL RESEARCH 31 1 1 - 8 1999年 [査読有り][通常論文]
     
    To mimic exercise-induced events such as energetic impairment, free radical generation, and lipid peroxidation in vitro, mouse-derived C2C12 myotubes were submitted to the inhibition of glycolytic and/or oxidative metabolism with ImM iodoacetate (IAA) and/or 2 mM sodium cyanide (CN), respectively, under 5% CO2/95% air up to 180 min. Electron spin resonance (ESR) analysis with a spin-trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) revealed time-course increases in spin adducts from hydroxyl radical (DMPO-OH) and carbon-centered radical (DMPO-R) in the supernatant of C2C12 myotubes treated with the combination of IAA + CN. In this condition, malondialdehyde (MDA) and lactate dehydrogenase (LDH) were released into the supernatant. By the addition of iron-chelating 1 mM deferoxamine to the C2C12 preparation with IAA + CN, both ESR signals of DMPO-OH and DMPO-R were completely abolished, and the release of MDA and LDH were significantly reduced, while cyanide-resistant manganese superoxide dismutase had neglegible effects on these parameters. Hence, a part of the injury of C2C12 myotube under IAA + CN was considered to result from the lipid peroxidation, which was induced by hydroxyl radical generated from iron-catalyzed systems such as the Fenton-type reaction. This in vitro model would be a helpful tool for investigating the free radical-related muscle injury.
  • Osamu Inanami, Kenji Takahashi, Asuka Yoshito, Mikinori Kuwabara
    Antioxidants and Redox Signaling 1 1 113 - 121 1999年 [査読有り][通常論文]
     
    To clarify activation mechanisms of stress-activated protein kinase/C-Jun N-terminal kinase (SAPK/JNK) during oxidative stress, the roles of phosphatidylinositol 3-kinase (PI 3-kinase), concentration of intracellular calcium ([Ca2+]i), and cyclic AMP-dependent kinase (PKA) in hydrogen peroxide (H2O2)-induced SAPK/JNK activation were examined in Chinese hamster V79 cells. SAPK/JNK was dose-dependently activated after H2O2 treatment (from 10 μM to 1 mM), and a PI 3-kinase inhibitor (wortmaninn), intracellular calcium chelator (BAPTA-AM), and PKA activator (dibutyl cyclic AMP and forskolin) inhibited this activation. An increase in [Ca2+]i was observed after treatment with H2O2. Immunoprecipitation revealed that a PI 3-kinase regulatory subunit, p85α, was associated with insulin receptor substance 1 (IRS-1) phosphorylated by H2O2 treatment. Furthermore, the formation of this complex of p85α and phospho-IRS-1 was abolished by the presence of BAPTA-AM but not forskolin. These results indicated that the PI 3-kinase activated through phosphorylation of IRS-1 upstream of SAPK/JNK after H2O2 treatment of V79 cells and that [Ca2+]i was a regulation factor for phosphorylation of IRS-1.
  • Osamu Inanami, Toshio Ohta, Shigeo Ito, Mikinori Kuwabara
    Antioxidants and Redox Signaling 1 4 501 - 508 1999年 [査読有り][通常論文]
     
    The mitogen-activated protein kinases (MAPK), including stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), p38, and extracellular signal-related kinase (ERK), are believed to be important biomolecules in cell proliferation, survival, and apoptosis induced by extracellular stimuli. In Chinese hamster V79 cells exposed to hydrogen peroxide (H2O2), we recently demonstrated that SAPK/JNK was activated by tyrosine kinase and intracellular Ca2+ ([Ca2+]i). In this study, we report that [Ca2+]i release from intracellular stores is important in the activation of SAPK/JNK but not p38 and ERK. H2O2-induced elevation of [Ca2+]i was observed in Ca2+-free medium. Pretreatment with thapsigargin, a Ca2+-ATPase inhibition of endoplasmic reticulum (ER), did not influence H2O2-induced elevation of [Ca2+]i in the absence of external Ca2+. An intracellular Ca2+ chelator (BAPTA-AM) inhibited H2O2-induced phosphorylation of SAPK/JNK, but an extracellular Ca2+ chelator (EDTA) or a Ca2+ entry blocker (NiCl2) did not. Activation of p38 and ERK in V79 cells exposed to H2O2 was observed in the presence of these inhibitors. These results suggest that [Ca2+]i release from intracellular stores such as mitochondria or nuclei but not ER, occurred after H2O2 treatment and Ca2+-dependent tyrosine kinase-induced activation of SAPK/JNK, although [Ca2+]i was unnecessary for the H2O2-induced activation of p38 and ERK.
  • JL Johnson, JW Park, J El Benna, LP Faust, O Inanami, BM Babior
    JOURNAL OF BIOLOGICAL CHEMISTRY 273 52 35147 - 35152 1998年12月 [査読有り][通常論文]
     
    The leukocyte NADPH oxidase catalyzes the reduction of oxygen to superoxide (O-2(radical anion) at the expense of NADPH in phagocytes and B lymphocytes, The enzyme is dormant in resting cells but becomes active when the cells are exposed to appropriate stimuli. During oxidase activation, the highly basic cytosolic oxidase component p47(PHOX) becomes phosphorylated on several serines and migrates to the plasma membrane. We report here that p47(PHOX)-deficient B lymphoblasts expressing the p47(PHOX) S359A/S370A or p47PHOX S359K/S370K double mutation show dramatically reduced levels of enzyme activity and phosphorylation of p47PHOX as compared with the same cells expressing wild type p47PHOX. I, addition, these mutant p47PHOX proteins fails to translocate to the plasma membrane when the cells are stimulated. In contrast, normal phosphorylation and translocation are seen in mutants containing aspartate or glutamate at positions 359 and 370, but oxidase activity is still greatly reduced. These results imply that a negative charge at position 359 and/or 370 is sufficient to allow the phosphorylation and translocation of p47(PHOX) to take place but that features unique to a phosphorylated hydroxyamino acid are required to support O-2(radical anion) production. These findings, plus those from an earlier study (Inanami, O., Johnson, J, L., McAdara, J. K., El Benna, J., Faust, L. P., Newburger, P, E., and Babior, B, M. (1998) J. Biol. Chem. 273, 9539-9543), suggest that oxidase activation requires 1) the sequential phosphorylation of at least two serines on p47(PHOX): Ser-359 or Ser-370, followed by Ser-303 or Ser-304; and 2) the translocation of p47(PHOX) to the membrane at some point after the first phosphorylation takes place.
  • T Asanuma, S Shimokawa, O Inanami, Y Kon, M Kuwabara
    JOURNAL OF VETERINARY MEDICAL SCIENCE 60 12 1311 - 1314 1998年12月 [査読有り][通常論文]
     
    A nuclear magnetic resonance (NMR) spectrometer equipped with a magnet producing a high and extremely uniform magnetic field (7.05 T) was combined with a strong field gradient coil (3.5 mT/cm) and applied to MR microimaging of the mouse brain to visualize its topograph ic al structure. Since the proton-density-weighted condition (long repetition time (TR) and short echo time (TE); TR/TE=3,000 ms/10.4 ms) was found to be the most suitable for imaging the mouse brain, mid-sagittal and coronal sections in 1-mm- or 0.3-mm-thick slices were imaged according to the multislice spin echo sequence with 2 or 8 acquisitions, a 2 kHz pulse width and a 256 x 256 data matrix. As expected, the resolution of MR microimaging was comparable to that of the histological sections. The white matter especially, could be distinguished from the gray matter in same regions of the brain. Coronal sections of the brain also showed that the hippocampal CA1-CA3 regions were distinguishable from the other regions. The results suggested that the present MR microimaging technique might be a useful tool for the study of topological anatomy and submicroscopic research using brains of small laboratory animals.
  • H Nagahata, S Matsuki, H Higuchi, O Inanami, M Kuwabara, K Kobayashi
    VETERINARY JOURNAL 156 1 15 - 21 1998年07月 [査読有り][通常論文]
     
    Bone marrow transplantation (BMT) was performed in a 9-month-old heifer with bovine leucocyte adhesion deficiency (BLAD). Clinical and haematological findings, selected neutrophil function and CD18 expression of neutrophils in a B-2 integrin-deficient heifer were examined. Twelve months after BMT, a small fluorescent region in the CD18-positive area of a flow cytometric profile was demonstrated and estimated to represent 0.3-0.5% of the CD18-positive neutrophils as measured by flow cytometric analysis following immunomagnetic separation. The animal's clinical condition appeared to improve and stabilize over our observation period of 28 months following BMT. Newly expressed CD18 seemed to play an important role in ameliorating the clinical signs of BLAD in this heifer.
  • A Ogawa, S Shuto, O Inanami, M Kuwabara, M Tanaka, T Sasaki, A Matsuda
    BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 8 14 1913 - 1918 1998年07月 [査読有り][通常論文]
     
    The design and synthesis of potential antitumor antimetabolites 2'-deoxy-2'-hydroxylaminouridine (2'-DHAU) and -cytidine (2'-DHAC) are described. We found that 2'-DHAC in neutral solution generated 2'-aminoxy radicals at room temperature. 2'-DHAC inhibited the growth of L1210 and KB cells, with IC50 values of 1.58 and 1.99 mu M, respectively, more potently than 2'-DHAU, with IC50 values of 34.5 and 27.3 mu M, respectively. 2'-DHAC was effective against 9 human cell lines, with IC50 values in the micromolar range. The in vivo antitumor activity of 2'-DHAC was also examined using the mouse leukemia P388 model, which gave a T/C value of 167%. Phosphorylation of 2'-DHAC by uridine/cytidine kinase was essential for its cytotoxicity, as suggested by a competition experiment using several common nucleosides. Inhibition of DNA synthesis was the predominant mechanism of action of 2'-DHAC, although it has a ribo-configuration. (C) 1998 Elsevier Science Ltd. All rights reserved.
  • O Inanami, JL Johnson, JK McAdara, J El Benna, LRP Faust, PE Newburger, BM Babior
    JOURNAL OF BIOLOGICAL CHEMISTRY 273 16 9539 - 9543 1998年04月 [査読有り][通常論文]
     
    The leukocyte NADPH oxidase is an enzyme in phagocytes and B lymphocytes that when activated catalyzes the production of O-2((.) over bar) from oxygen and NADPH, During oxidase activation, serine residues in the C-terminal quarter of the oxidase component p47(PHOX) become extensively phosphorylated, the protein acquiring as many as 9 phosphate residues. In a study of 11 p47(PHOX) mutants, each containing an alanine instead of a serine at a single potential phosphorylation site, we found that all but S379A corrected the defect in O-2((.) over bar) production in Epstein-Barr virus (EBV)-transformed p47(PHOX)-deficient B cells (Faust, L. P., El Benna, J., Babior, B. M., and Chanock, S. J. (1995) J. Clin. Invest. 96, 1499-1505). In particular, O-2((.) over bar) production was restored to these cells by the mutants S303A and S304A. Therefore, apart from serine 379, whose state of phosphorylation in the activated oxidase is unclear, no single potential phosphorylation site appeared to be essential for oxidase activation. We now report that the double mutant p47(PHOX) S303A/S304A was almost completely inactive when ex pressed in EBV-transformed p47(PHOX)-deficient B cells, even though it was expressed in normal amounts in the transfected cells and was able to translocate to the plasma membrane when the cells were stimulated, In contrast, the double mutant p47(PHOX) S303E/S304E was able to support high levels of O-2((.) over bar) production by EBV-transformed p47(PHOX)-deficient B cells. The surprising discovery that the double mutant S303K/S304K was also able to support considerable O-2((.) over bar) production suggests either that the effect of phosphorylation is related to the increase in hydrophilicity around serines 303 and 304 or that activation involves the formation of a metal bridge between the phosphorylated serines and another region of the protein.
  • O Inanami, JL Johnson, BM Babior
    ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 350 1 36 - 40 1998年02月 [査読有り][通常論文]
     
    The leukocyte NADPH oxidase is a multi-subunit enzyme that catalyzes the reduction of oxygen to O-2(-) at the expense of a reduced pyridine nucleotide. We have used site-directed mutagenesis to examine the functional role of the four cysteines in p47(PHOX), one of the subunits of the oxidase. For these experiments, mutant proteins in which a single cysteine was replaced with alanine were expressed in p47(PHOX)-deficient Epstein-Barr virus-transformed B lymphoblasts, and O-2(-) production by these transfected cells was measured. The activity of the mutant C98A was similar to that of wild type, but the maximum rate of O-2(-) production by C196A was significantly larger than seen with wild type, The other two mutants (i.e., C111A and C378A) differed from wild type not only in maximum O-2(-) production, but also in the time required for activation, which was considerably delayed with both of these mutants. The similarity in the time courses of oxidase activation with the C111A and C378A mutants, and the finding that C378A occurs in the sequence CSE, raises the possibility that these cysteines may be involved in redox regulation of oxidase activity. (C) 1998 Academic Press.
  • O Inanami, Y Watanabe, B Syuto, M Nakano, M Tsuji, M Kuwabara
    FREE RADICAL RESEARCH 29 4 359 - 365 1998年 [査読有り][通常論文]
     
    The effect of ad libitum oral-administration of (-)catechin solution on ischemia-reperfusion-induced cell death of hippocampal CA1 in the gerbil was histologically examined. When (-)catechin solution instead of drinking water was orally administered ad libitum for 2 weeks, dose-dependent protection against neuronal death following by transient ischemia and reperfusion was observed. To evaluate the involvement of reduction of reactive-oxygen-species (ROIs) by the antioxidant activity of (-)catechin in this protection, the superoxide scavenging activity of the brain in catechin-treated gerbils was measured by ESR and spin-trapping using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The superoxide scavenging activities of the brains obtained from catechin-treated gerbils were significantly higher than those of catechin-untreated animals. From these results, it was suggested that orally administered (-)catechin was absorbed, passed through the blood-brain barrier and that delayed neuronal death of hippocampal CA1 after ischemia-reperfusion was prevented due to its antioxidant activities.
  • M Kuwabara, O Inanami, K Takahashi, W Hiraoka
    BIOLOGICAL OXIDANTS AND ANTIOXIDANTS: MOLECULAR MECHANISMS AND HEALTH EFFECTS 14 - 21 1998年 [査読有り][通常論文]
  • M Kuwabara, H Ohshima, Y Iida, O Inanami
    JOURNAL OF RADIATION RESEARCH 38 3 153 - 155 1997年09月 [査読有り][通常論文]
  • 稲波 修, 桑原 幹典
    家畜生化学 33 2 11 - 21 家畜生化学会 1996年12月 [査読無し][通常論文]
  • R Sato, O Inanami, Y Tanaka, M Takase, Y Naito
    AMERICAN JOURNAL OF VETERINARY RESEARCH 57 10 1443 - 1446 1996年10月 [査読有り][通常論文]
     
    Objective-To study the effects of oral administration of bovine lactoferrin (LF) on intractable stomatitis in feline immunodeficiency virus (FIV)-positive and FIV-negative cats, and phagocytosis of neutrophils in healthy and ill cats, simultaneously. Animals-7 ill cats with diagnosis of intractable stomatitis (4 FIV positive and 3 FIV negative) and 7 healthy, FIV-negative cats. Procedure-LF (40 mg/kg of body weight) was applied topically to the oral mucosa of cats with intractable stomatitis daily for 14 days and improvement of clinical signs of disease (pain-related response, salivation, appetite, and oral inflammation), expressed by scoring from 1 to 4, were evaluated, Assay of neutrophil phagocytosis was examined before and 2 weeks after starting LF treatment, using nonopsonized hydrophilic polymer particles (2 mu m). Results-Oral administration of LF improved intractable stomatitis in all 4 respects. Phagocytic activity of neutrophils increased after LF treatment. This effect was observed in healthy and ill (FIV positive and FIV negative) cats. Conclusion and Clinical Relevance-Oral administration of if improved intractable stomatitis and concurrently enhanced the host defense system. Topical application of if to oral mucous membrane is useful as a treatment for intractable stomatitis even in FIV-positive cats.
  • T Suzuki, M Goryo, O Inanami, JY Uetsuki, SY Saito, K Kaketa, T Oshima, H Shimizu, S Okabe, T Tanaka, R Kamata, F Shuto, Sato, I, E Tachikawa, M Sakaguchi, H Kobayashi, K Okada
    JOURNAL OF VETERINARY MEDICAL SCIENCE 58 7 647 - 654 1996年07月 [査読有り][通常論文]
     
    Aggregation properties of platelets were examined in Japanese Black cattle with Chediak-Higashi syndrome (CHS) and normal control cattle. Platelet aggregation induced by collagen was decreased in platelets of the cattle with CHS, but not ADP (10-20 mu M), thrombin (0.5-1.0 U/ml) and phorbol-12-myristate 13-acetate (PMA, 3.2 mu M). The aggregation response induced by collagen in CHS platelets lacks the change in shape which usually occurs in normal platelets. Simultaneous stimulation by collagen (10 mu g/ml) + ADP(10 mu M) is effective in restoring collagen-induced aggregating response in CHS platelet, although pretreatment of ADP (10 mu M) could not restore the collagen (10 mu g/ml)-induced aggregating response, suggesting that there is a certain threshold of stimulation intensity above which collagen-induced aggregation of CHS platelet can begin. Control normal platelets, previously exposed to ADP (10 mu M) and collagen (10 mu g/ml), showed no further response to exposure to a third aggregating agent (arachidonic acid, 5 mM). On the other hand, the final agent was able to elicit aggregating responses in CHS platelets, suggesting that the arachidonate aggregating system may be suppressed in CHS cattle, but fully activated in control animals. Furthermore, normal platelets showed a significant decrease in aggregating response to collagen when pretreated with a cyclooxygenase inhibitor, indomethacin (10(-5) M), whereas CHS platelet was insensitive to indomethacin. This indomethacin-treated normal platelet mimicked the CHS collagen-induced aggregation pattern. These data suggest that a signal transduction process from receptor-operated events to arachidonate metabolism is suppressed in collagen-induced CHS platelet aggregation.
  • M Kuwabara, N Inukai, O Inanami, Y Miyake, N Tsunoda, Y Maki, F Sato
    JOURNAL OF VETERINARY MEDICAL SCIENCE 58 2 97 - 101 1996年02月 [査読有り][通常論文]
     
    Hotbred (Thoroughbred) horses were grouped into three classes according to the levels of constant physical exercise (foals, 6 months old; racing horses, 5 years old; horses for breeding, 6-10 years old), and lipid peroxide levels in their sera were measured as thioburbituric acid-reactive substances. No significant differences were observed among them. The superoxide-scavenging abilities of sera were measured next; to examine the antioxidative properties of hotbreds, and were found to be highest in the racing horses. The higher scavenging ability of the racing horses might contribute to keep their lipid peroxide levels as low as those of the other two groups. HPLC analysis of substances in sera suggested that the presence of albumin-bound bilirubin was one of the reasons for the high superoxide-scavenging ability of sera of the racing horses. When the hotbreds were compared with coldbred (crossbred) horses, the lipid peroxide level of hotbreds was higher (7.0 +/- 1.2 nmol/ml) than that of coldbreds (2.6 +/- 0.7 nmol/ml). Comparison of the superoxide-scavenging abilities of sera between hotbreds and coldbreds showed that the hotbreds possessed higher scavenging ability than the coldbreds. These results indicated that the lipid peroxide level in sera of hotbreds was higher than that of coldbreds regardless of the higher superoxide-scavenging abilities of sera.
  • O INANAMI, T ASANUMA, N INUKAI, T JIN, S SHIMOKAWA, N KASAI, M NAKANO, F SATO, M KUWABARA
    NEUROSCIENCE LETTERS 196 1-2 85 - 88 1995年08月 [査読有り][通常論文]
     
    The protective effects of Rooibos tea (RT), Aspalathus linearis, against damage to the central nervous system (CNS) accompanying aging were examined by both the thiobarbituric acid reaction (TBA) and magnetic resonance imaging (MRI) methods in brains of chronically RT-treated rats. Ad libitum administration of RT was begun with 3-month-old Wistar female rats and continued for 21 months, The contents of TBA reactive substances (TBARS) in the frontal cortex, occipital cortex, hippocampus and cerebellum in 24-month-old rats after administration with water were significantly higher than those in young rats (5 weeks old), However, no significant increase of TBARS was observed in RT-administered aged rats. When MR images of the brains of 24-month-old rats with and without RT as well as 5-week-old rats were taken, a decrease of the signal intensity was observed in the cerebral cortex, hippocampus and cerebellum in MR images of aged rats without RT, whereas little change of the signal intensity was observed in MR images of the same regions of 24-month-old rats treated with RT, whose images were similar to those of young rats. These observations suggested that (1) the age-related accumulation of lipid peroxides in the brain was closely related to the morphological changes observed by MRI, and (2) chronic RT-administration prevented age-related accumulation of lipid peroxides in several regions of rat brain.
  • M KUWABARA, S SAWAMURA, O INANAMI, K KOBAYASHI
    RADIATION DAMAGE IN DNA: STRUCTURE/FUNCTION RELATIONSHIPS AT EARLY TIMES 241 - 249 1995年 [査読有り][通常論文]
  • O INANAMI, M KUWABARA
    FREE RADICAL RESEARCH 23 1 33 - 39 1995年 [査読有り][通常論文]
     
    The effects of intravenous administration of ol-phenyl N-tert-butyl nitrone (PEN) on cortical cerebral blood flow (CEF) were examined in Wistar rats under pentobarbital anesthesia and artificial ventilation. The cortical CBF in parietal cortex was measured by laser Doppler flowmetry. Intravenous administrations of 2 mg/kg and 20 mg/kg of PEN dose-dependently produced significant increases in cortical CBF and decreases in systemic blood pressure (BP). To examine whether these increased responses in cortical CBF produced by PEN were associated with the vasodilatation system of nitric oxide (NO), the NO synthase inhibitor L-N-G-nitroarginine (L-NOArg), which is an analog of L-arginine, was used to inhibit the NO-related-vasodilatative system. Since the PEN-induced responses in the cortical CEF were much attenuated in L-NOArg-treated rats (30 mg/kg, iv.), it was inferred that NO-related vasodilatation was strongly associated with the PEN-induced increase in cortical CBF.
  • M KUWABARA, T ASANUMA, O INANAMI, T JIN, S SHIMOKAWA, N KASAI, K KATOR, F SATO
    JOURNAL OF VETERINARY MEDICAL SCIENCE 56 5 933 - 938 1994年10月 [査読有り][通常論文]
     
    Using a homemade MR imaging probe (Helmholtz coil), MR images of brains of 5-week-old and 23- or 24-month-old Wistar rats were taken under a magnetic field of 7.05 T (Tesla). The probe was designed to fit the rat head and made by winding thin copper film round an acrylic tube with a 5-cm i.d., 10-cm length and 2-mm thickness. This was adjusted to resonate with the 300 MHz radiofrequency corresponding to the resonance frequency of H-1 under a magnetic field of 7.05 T. MR images were obtained by T1-weighted and two-dimensional Fourier transformation techniques. The sagittal and coronal sections were imaged in 1-mm-thick slices. The size of the data matrix was 128 phase-encoded steps. Each image was obtained through eight acquisitions. A comparison of the MR images with those semi-microscopically taken at the same position of the coronal section revealed that the cerebral cortex, hippocampus, hypothalamus and thalamus were clearly imaged by this probe. With aging, MR images of cerebral cortices were observed with decreased signal intensities. Enlargement of the third ventricles and hypertrophy of cranical parietal bones were also recognized in sagittal MR images of aged rats. These observations were more marked in males than in females. From these observations it was concluded that this probe was applicable for MR imaging of rat brains under a magnetic field of 7.05 T.
  • O INANAMI, K OHNO, A SATO
    NEUROSCIENCE LETTERS 143 1-2 151 - 154 1992年08月 [査読有り][通常論文]
     
    The effects of i.v. administration of thyrotropin releasing hormone (TRH) on regional cerebral blood flow (rCBF) were examined in both healthy adult (3-5 months old) and healthy aged (24-25 months old) male Wistar rats under halothane anesthesia. The rCBFs in 9 different brain regions - cerebral cortex, caudate putamen, hippocampus, thalamus+hypothalamus, superior colliculus, inferior colliculus, cerebellum, pons, and medulla - were measured by [C-14]iodoantipyrine method. In the adult rats, i.v. administration of TRH (300-mu-g/kg) produced significant increases in rCBFs in cerebral cortex, caudate putamen, hippocampus, thalamus+hypothalamus and superior colliculus. In the aged rats, the rCBFs in all brain regions measured did not change significantly by TRH administration, From these results, it is suggested that the system involved in TRH-induced vasodilatation of cerebral blood vessels was impaired with aging.
  • T ADACHI, O INANAMI, A SATO
    NEUROSCIENCE LETTERS 139 2 201 - 204 1992年05月 [査読有り][通常論文]
     
    The effects of i.v. administration of a nitric oxide (NO) synthase inhibitor, L-N(G)-nitroarginine (L-NOArg), on the increase in cerebral cortical blood flow (cortical BF), following either electrical stimulation of the nucleus basalis of Meynert (NBM), whose cholinergic fibers project to the cortex, or hypercapnia with 10% CO2 inhalation, were studied in anesthetized rats. Cortical BF was measured using laser Doppler flowmetry. The threshold intensity of electrical stimulation of the NBM (0. 5 ms, 50 Hz for 10 s) that induced an increase in regional cortical BF was defined as 1T. The cortical BF was increased on a stimulus intensity dependent manner at 1T-5T intensities tested. L-NOArg was administered cumulatively i.v. starting from 0.3 mg/kg, then 3 mg/kg, and 30 mg/kg. Time interval between each cumulative administration of L-NOArg was approximately 40 min. Three and 30 Mg/kg Of L-NOArg significantly reduced the NBM stimulation-induced increase of cortical BF at intensities of 2T and 3T. The response at an intensity of 5T was reduced only by 30 mg/kg Of L-NOArg to about half the control response. The reduced responses at 2T, 3T, and 5T were reversed following the i.v. administration of a physiological precursor of NO, L-Arg (300 mg/kg). Inhalation of 10% CO2 for 15 s induced an increase in cortical BF which was not influenced by L-NOArg and L-Arg. These results suggest that NO is a necessary factor in the vasodilatation of the cortical BF that is brought about by cholinergic fibers originating in the NBM.
  • T ADACHI, D BIESOLD, O INANAMI, A SATO
    BRAIN RESEARCH 514 1 163 - 166 1990年04月 [査読有り][通常論文]
  • Adachi T, Biesold D, Cao W.-H, Inanami O, Kimura A, Kurosawa M, Ohno K, Sato A, Sato Y
    Therapeutic Research 11 12 92 - 98 1990年 [査読有り][通常論文]
  • WH CAO, O INANAMI, A SATO, Y SATO
    NEUROSCIENCE LETTERS 107 1-3 135 - 140 1989年12月 [査読有り][通常論文]
  • D BIESOLD, O INANAMI, A SATO, Y SATO
    NEUROSCIENCE LETTERS 98 1 39 - 44 1989年03月 [査読有り][通常論文]
  • M KUWABARA, T YAMAMOTO, O INANAMI, F SATO
    PHOTOCHEMISTRY AND PHOTOBIOLOGY 49 1 37 - 41 1989年01月 [査読有り][通常論文]
  • O INANAMI, M KUWABARA, F SATO
    FREE RADICAL RESEARCH COMMUNICATIONS 5 1 43 - 49 1988年 [査読有り][通常論文]
  • Osamu Inanami, Mikinori Kuwabara, Fumiaki Sato
    Free Radical Research 5 1 43 - 49 1988年 [査読有り][通常論文]
     
    Free radicals produced by γirradiation of solid lysozyme were investigated by a technique combining ESR, spin-trapping and enzymatic digestion. MNP and DMPO were used as spin-trapping reagents. The solid lysozyme was first γirradiated and then dissolved in an aqueous solution containing the spin-trapping reagent to stabilize free radicals. The spin adducts of lysozyme were digested to oligopeptides to get ESR spectra having a well-resolved hyperfine structure. The ESR spectra obtained showed that carbon-centered radicals, CH-, at the side chains of amino acids, and thiyl radicals, -CH2--S-, at disulfide bridges were produced in γirradiated solid lysozyme. © 1988 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.
  • O INANAMI, M KUWABARA, F SATO
    RADIATION RESEARCH 112 1 36 - 44 1987年10月 [査読有り][通常論文]
  • M KUWABARA, O INANAMI, D ENDOH, F SATO
    BIOCHEMISTRY 26 9 2458 - 2465 1987年05月 [査読有り][通常論文]
  • O INANAMI, M KUWABARA, D ENDOH, F SATO
    RADIATION RESEARCH 108 1 1 - 11 1986年10月 [査読有り][通常論文]
  • Kuwabara M, Inanami O, Sato F
    International journal of radiation biology and related studies in physics, chemistry, and medicine 49 5 829 - 844 1986年05月 [査読有り][通常論文]
  • M KUWABARA, O INANAMI, F SATO
    INTERNATIONAL JOURNAL OF RADIATION BIOLOGY 49 5 829 - 844 1986年05月 [査読有り][通常論文]
  • Inanami O, Kuwabara M, Hayashi M, Yoshii G, Syuto B, Sato F
    International journal of radiation biology and related studies in physics, chemistry, and medicine 49 1 47 - 56 1986年01月 [査読有り][通常論文]

書籍

  • 獣医放射線学教育研究会, 稲波 修, 浅沼 武敏, 久保 喜平, 中山 智宏, 林 正信, 藤田 道郎, 宮原 和郎 
    近代出版 2015年 (ISBN: 9784874022139)
  • Anders Lund, Masaru Shiotani 
    Springer 2014年10月 (ISBN: 3319092154) 773
  • 内藤 裕二, 豐國 伸哉, 吉川 敏一 
    診断と治療社 2014年 (ISBN: 9784787821188)
  • 内藤 裕二, 豐國 伸哉, 吉川 敏一 
    診断と治療社 2008年 (ISBN: 9784787816566)
  • Effects of the combination of thrombopoietin of other cytokines on survival of X-irradiated CD34+ Megakaryocytic progenitor cells from human placental and umbilical cord blood.
    Bull. Health Science, Hirosaki 2002年
  • Antioxidant activity of quinone-derivatives from freeze-dried powder of the acidians, In The Biology of Ascidians
    Springer, Tokyo 2001年
  • Biodefense against active oxygens and free radicals induced by oxidative stress.
    Kodansha and Springer 1998年
  • Radiation- and reactive oxygen-induced cell death and signal transduction in cultured mammalian cells.
    AOCS Press 1998年
  • An ESR study of superoxide generation in stimulated neutrophils from a calf with bovine leukocyte adhension deficiency(BLAD).
    Springer 1997年

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    山本久美子, 安井博宣, 房知輝, 山盛徹, 平岡和佳子, 山崎俊栄, 山崎俊栄, 山田健一, 山田健一, 稲波修
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    永根大幹, 永根大幹, 安井博宣, 山盛徹, 稲波修, 山下匡, クプサミー ペリアナン
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    電子スピンサイエンス学会年会講演要旨集 2017年11月
  • 放射線照射されたがん細胞のセミキノンラジカルならびにFe‐Sクラスター由来するESRシグナルの挙動  [通常講演]
    稲波修, 山本久美子, 山盛徹, 平岡和佳子
    電子スピンサイエンス学会年会講演要旨集 2017年11月
  • がん由来培養細胞のミトコンドリアに由来するESRスペクトルの解析  [通常講演]
    山本久美子, 酒井友里, 房知輝, 平岡和佳子, 山盛徹, 稲波修
    電子スピンサイエンス学会年会講演要旨集 2017年11月
  • ヒト子宮頸がん由来HeLa細胞の鉄‐硫黄(Fe‐S)クラスターの20K Xバンド電子スピン共鳴法による評価  [通常講演]
    山本久美子, 酒井友里, 房知輝, 平岡和佳子, 山盛徹, 稲波修
    日本放射線影響学会大会抄録(Web) 2017年10月
  • 放射線によるストレス誘発性早期老化に伴う活性酸素種産生に対しNADPH oxidase 4が与える影響の解析  [通常講演]
    酒井友里, 山盛徹, 房知輝, 山本久美子, 吉川容司, 吾郷哲朗, 稲波修
    日本放射線影響学会大会抄録(Web) 2017年10月
  • ミトコンドリアダイナミクスが細胞の放射線感受性に与える影響の解明  [通常講演]
    房知輝, 山盛徹, 酒井友里, 山本久美子, 稲波修
    日本放射線影響学会大会抄録(Web) 2017年10月
  • ミトコンドリア形態制御機構が細胞の放射線感受性に与える影響の解明  [通常講演]
    房知輝, 山盛徹, 酒井友里, 山本久美子, 稲波修
    日本獣医学会学術集会講演要旨集 2017年08月
  • 放射線照射がミトコンドリア電子伝達系酸化還元関連分子に与える影響の電子スピン共鳴法を用いた評価  [通常講演]
    山本久美子, 酒井友里, 房知輝, 平岡和佳子, 山盛徹, 稲波修
    日本獣医学会学術集会講演要旨集 2017年08月
  • 放射線照射後の血管内皮細胞における一酸化窒素産生の亢進は固形腫瘍を再酸素化する  [通常講演]
    永根大幹, 永根大幹, 安井博宣, 山盛徹, 山盛徹, KUPPUSAMY Periannan, 稲波修
    放射線生物研究 2017年06月
  • 放射線によるストレス誘発性早期老化に伴う活性酸素種産生へのNOX4の関与  [通常講演]
    酒井友里, 山盛徹, 房知輝, 山本久美子, 吉川容司, 吾郷哲朗, 稲波修
    日本酸化ストレス学会学術集会プログラム・抄録集 2017年06月
  • トビケラウォッチ 水生昆虫をもちいた河川の環境放射能モニタリング(第4報)  [通常講演]
    松尾友貴, 上野大介, 染谷孝, 水川葉月, 稲波修, 長坂洋光, 藤野毅, 渡邉泉, 龍田希, 仲井邦彦
    環境化学討論会要旨集(CD-ROM) 2017年06月
  • 腫瘍内酸素イメージングのための同位体標識ニトロキシルラジカル混合溶液の特性評価  [通常講演]
    久保田晴江, 安井博宣, 松元慎吾, 稲波修, KIRILYUK Igor, KHRAMTSOV Valery V, 平田拓
    電子スピンサイエンス学会年会講演要旨集 2016年11月
  • X線照射後のヒト子宮頸がん由来HeLa細胞におけるセミキノンラジカルおよびFe‐SクラスターのESRによる評価  [通常講演]
    山本久美子, 池中良徳, 一瀬貴大, 石塚真由美, 安井博宣, 平岡和佳子, 鵜飼光子, 山盛徹, 稲波修
    電子スピンサイエンス学会年会講演要旨集 2016年11月
  • 親脂質性Tetraphenylphosphonium誘導体は放射線によるがん細胞の致死作用を増強する  [通常講演]
    稲波修, 安井博宣, 西村英里, 山本久美子, 鈴木基史, 酒井友里, 房知輝, 山盛徹, 山崎俊栄, 山田健一, 山田健一
    電子スピンサイエンス学会年会講演要旨集 2016年11月
  • DNA修復酵素APエンドヌクレアーゼ1のミトコンドリアでの過剰発現による酸化ストレス抵抗性発現メカニズムの解析  [通常講演]
    山盛徹, 玉置光, 稲波修
    日本酸化ストレス学会学術集会プログラム・抄録集 2016年08月
  • 放射線によるストレス誘発性早期老化に伴う活性酸素種産生におけるNOXファミリータンパク質の寄与の検討  [通常講演]
    酒井友里, 山盛徹, 日吉美恵, 鈴木基史, 稲波修
    日本獣医学会学術集会講演要旨集 2016年08月
  • 放射線照射後のミトコンドリア融合制御タンパク質動態とその調節機構の解析  [通常講演]
    髭白侑香, 山盛徹, 房知輝, 鈴木基史, 酒井友里, 山本久美子, 稲波修
    日本獣医学会学術集会講演要旨集 2016年08月
  • X線照射したヒト子宮頸がん由来HeLa細胞におけるミトコンドリア機能を中心としたエネルギー代謝応答の解析  [通常講演]
    山本久美子, 池中良徳, 一瀬貴大, 石塚真由美, 安井博宣, 鵜飼光子, 山盛徹, 稲波修
    日本獣医学会学術集会講演要旨集 2016年08月
  • ヒト子宮頸部がんHeLa細胞における放射線照射後のミトコンドリア応答  [通常講演]
    稲波修, 山本久美子, 池中良徳, 一瀬貴大, 石塚真由美, 安井博宣, 鵜飼光子, 山盛徹
    日本酸化ストレス学会学術集会プログラム・抄録集 2016年08月
  • トビケラウォッチ水生昆虫をもちいた河川の環境放射能モニタリング(第3報)  [通常講演]
    大坪栄二郎, 上野大介, 染谷孝, 水川葉月, 稲波修, 長坂洋光, 藤野毅, 渡邉泉, 大葉隆, 龍田希, 仲井邦彦
    環境化学討論会要旨集(CD-ROM) 2016年06月
  • X線照射はミトコンドリア生合成を亢進することなく細胞内ミトコンドリア含量を増大させる  [通常講演]
    山盛徹, 笹川朋哉, 市居修, 房知輝, 昆泰寛, 稲波修
    日本放射線影響学会大会抄録(Web) 2016年
  • 新規Chk1阻害剤MK‐8776の放射線増感剤としての効果の検討  [通常講演]
    鈴木基史, 山盛徹, 稲波修
    日本放射線影響学会大会抄録(Web) 2016年
  • がん細胞の放射線応答における転写調節因子Id1の役割  [通常講演]
    安井博宣, 安井博宣, 竹内麻依, 山盛徹, 松本英樹, 高橋昭久, 稲波修
    日本放射線影響学会大会抄録(Web) 2016年
  • HeLa細胞における放射線照射後のミトコンドリアのセミキノンラジカルとFe‐Sクラスターの電子スピン共鳴法による評価  [通常講演]
    稲波修, 山本久美子, 房知輝, 酒井友里, 鈴木基史, 平岡和佳子, 安井博宣, 山盛徹
    日本放射線影響学会大会抄録(Web) 2016年
  • 放射線照射後のDrp1リン酸化を実行する責任キナーゼの解明  [通常講演]
    房知輝, 山盛徹, 髭白侑香, 酒井友里, 鈴木基史, 稲波修
    日本放射線影響学会大会抄録(Web) 2016年
  • 酸素感受性同位体ニトロキシルラジカルの特性評価実験  [通常講演]
    久保田晴江, 安井博宣, 松元慎吾, 三宅祐輔, 稲波修, KIRILYUK I. A, KHRAMTSOV V. V, 平田拓
    電子スピンサイエンス学会年会講演要旨集 2015年11月
  • X線照射したがん細胞のESRオキシメトリーを用いた酸素消費率を指標としたミトコンドリア機能の解析  [通常講演]
    山本久美子, 山本久美子, 安井博宣, 山盛徹, 中村秀夫, 鵜飼光子, 稲波修
    電子スピンサイエンス学会年会講演要旨集 2015年11月
  • ミトコンドリア指向性化合物における分子骨格と放射線増感作用との相関性に関する研究  [通常講演]
    安井博宣, 西村英里, 山盛徹, 山本久美子, 鵜飼光子, 山崎俊栄, 山田健一, 山田健一, 稲波修
    放射線ワークショップ講演論文集 2015年10月
  • MPS1阻害によるスピンドル形成チェックポイントの抑制がX線の殺細胞効果に与える影響の解析  [通常講演]
    鈴木基史, 山盛徹, 安井博宣, 稲波修
    放射線ワークショップ講演論文集 2015年10月
  • 1‐(3‐C‐ethynyl‐β‐D‐ribo‐pentofuranosyl)cytosine(ECyd)併用による陽子線増感治療の実現に向けた基礎研究  [通常講演]
    前田憲一郎, 安井博宣, 山盛徹, 松浦妙子, 高尾聖心, 鈴木基史, 松田彰, 稲波修, 白土博樹
    放射線ワークショップ講演論文集 2015年10月
  • MPS1を介したスピンドル形成チェックポイントが哺乳類動物細胞の化学療法剤感受性に与える影響の解析  [通常講演]
    鈴木基史, 山盛徹, 安井博宣, 稲波修
    日本獣医学会学術集会講演要旨集 2015年08月
  • 放射線照射後のミトコンドリア分裂におけるDrp1リン酸化の意義  [通常講演]
    房知輝, 山盛徹, 池悟志, 鈴木基史, 酒井友里, 安井博宣, 稲波修
    日本獣医学会学術集会講演要旨集 2015年08月
  • 転写調節因子Id1ノックダウンが放射線感受性に与える影響とそのメカニズムの解明  [通常講演]
    竹内麻依, 安井博宣, 鈴木基史, 酒井友里, 山盛徹, 稲波修
    日本獣医学会学術集会講演要旨集 2015年08月
  • 阿武隈川および阿賀川水系における淡水魚137Csの生態学的半減期の推定  [通常講演]
    仲井邦彦, 上野大介, 水川葉月, 藤野毅, 川田暁, 泉茂彦, 渡邉泉, 長坂洋光, 松村徹, 龍田希, 稲波修
    環境化学討論会要旨集(CD-ROM) 2015年06月
  • 親脂質性triphenylphosphonium(TPP)化合物によるがん細胞における放射線増感作用  [通常講演]
    稲波修, 西村英里, 安井博宣, 山盛徹, 山本久美子, 鵜飼光子, 山崎俊栄, 山田健一, 山田健一
    日本酸化ストレス学会学術集会プログラム・抄録集 2015年05月
  • 金粒子含有ナノゲルによる小胞体ストレスを介した放射線増感作用  [通常講演]
    安井博宣, 武内亮, 永根大幹, 山盛徹, 池中良徳, 昆泰寛, 室谷憲紀, 大石基, 長崎幸夫, 稲波修
    日本放射線影響学会大会講演要旨集 2014年09月
  • Drp1依存性ミトコンドリア分裂が細胞の放射線応答に与える影響の評価とそのメカニズムの解析  [通常講演]
    山盛徹, 池悟志, 笹川朋哉, 鈴木基史, 酒井友里, 安井博宣, 稲波修
    日本放射線影響学会大会講演要旨集 2014年09月
  • ミトコンドリア指向性ニトロキシドが放射線感受性に及ぼす影響  [通常講演]
    西村英里, 安井博宣, 永根大幹, 笹川朋哉, 山盛徹, 山崎俊栄, 山田健一, 稲波修
    日本酸化ストレス学会学術集会プログラム・抄録集 2014年08月
  • Drp1を介したミトコンドリア形態変化が細胞の放射線感受性に与える影響の評価とそのメカニズムの解析  [通常講演]
    池悟志, 山盛徹, 鈴木基史, 酒井友里, 安井博宣, 稲波修
    日本獣医学会学術集会講演要旨集 2014年08月
  • ミトコンドリア指向性ニトロキシドが放射線感受性に及ぼす影響  [通常講演]
    西村英里, 安井博宣, 永根大幹, 笹川朋哉, 山盛徹, 山田健一, 山崎俊栄, 稲波修
    日本獣医学会学術集会講演要旨集 2014年08月
  • MPS1を介したスピンドル形成チェックポイントが哺乳類動物細胞の放射線/化学療法剤感受性に与える影響の解析  [通常講演]
    鈴木基史, 山盛徹, 安井博宣, 稲波修
    日本獣医学会学術集会講演要旨集 2014年08月
  • DNA修復酵素APエンドヌクレアーゼ1の発現抑制による細胞外マトリックス関連遺伝子発現への影響とそれに伴う細胞機能の変化  [通常講演]
    酒井友里, 山盛徹, 安井博宣, 稲波修
    日本獣医学会学術集会講演要旨集 2014年08月
  • トビケラウォッチ―被災地における水生昆虫をもちいた放射能モニタリング  [通常講演]
    平野剛史, 上野大介, 染谷孝, 長坂洋光, 楢崎幸範, 稲波修, 水川葉月, 藤野毅, 渡邉泉, 龍田希, 仲井邦彦
    環境化学討論会要旨集(CD-ROM) 2014年05月
  • DNA修復酵素APエンドヌクレアーゼ1の発現抑制による細胞外マトリックス関連遺伝子発現および細胞機能への影響  [通常講演]
    酒井友里, 山盛徹, 安井博宣, 稲波修
    日本生化学会大会(Web) 2014年
  • パルスEPRイメージングを用いたがん代謝標的薬剤ジクロロ酢酸処理後の腫瘍内酸素環境の経時的解析  [通常講演]
    安井博宣, 齋藤圭太, 西田直哉, 松元慎吾, 山盛徹, KRISHNA Murali C, 稲波修
    電子スピンサイエンス学会年会講演要旨集 2013年10月
  • プリオンタンパク質凝集体のQバンド電子スピン二重共鳴法(Q‐band DEER)による構造解析  [通常講演]
    稲波修, 山盛徹, 安井博宣, 堀内基広, 平岡和佳子, 古川貢, 中村敏和
    電子スピンサイエンス学会年会講演要旨集 2013年10月
  • EPR分光法による鏡像異性体のタンパク質結合性の観測  [通常講演]
    天坂光男, 三宅祐輔, 安井博宣, 稲波修, WANG Xialoei, 一刀かおり, XU Shu, 有本博一, 平田拓
    電子スピンサイエンス学会年会講演要旨集 2013年10月
  • 電子常磁性共鳴分光による生体内pHの測定  [通常講演]
    GOODWIN Jonathan, 谷内勝哉, 永根大幹, 安井博宣, 三宅祐輔, 稲波修, BOBKO Andrey, KHRAMTSOV Valery, 平田拓
    電子スピンサイエンス学会年会講演要旨集 2013年10月
  • 放射線により引き起こされるeNOS活性化に対するATMの関与  [通常講演]
    永根大幹, 安井博宣, 山盛徹, 丹羽光一, 稲波修
    日本放射線影響学会大会講演要旨集 2013年10月
  • 放射線生物作用の初期過程  [通常講演]
    桑原幹典, 稲波修
    日本放射線影響学会大会講演要旨集 2013年10月
  • チャイニーズハムスターV79細胞における硫化水素による放射線防護効果の検討  [通常講演]
    稲波修, 山本栄輔, 永根大幹, 安井博宣, 山盛徹
    日本放射線影響学会大会講演要旨集 2013年10月
  • 電子スピン共鳴法を用いた酸素イメージングによる腫瘍内間欠的低酸素の描出とその意義  [通常講演]
    安井博宣, 松元慎吾, 齋藤圭太, 山盛徹, KRISHNA Murali, 稲波修
    日本放射線影響学会大会講演要旨集 2013年10月
  • X線照射により引き起こされる細胞内ミトコンドリア量変動メカニズムの解析  [通常講演]
    笹川朋哉, 山盛徹, 永根大幹, 安井博宣, 稲波修
    日本放射線影響学会大会講演要旨集 2013年10月
  • X線照射により引き起こされる細胞内ミトコンドリア形態変化と核形態異常に対するDrp1の関与  [通常講演]
    笹川朋哉, 山盛徹, 永根大幹, 安井博宣, 稲波修
    日本獣医学会学術集会講演要旨集 2013年08月
  • 放射線による内皮型一酸化窒素合成酵素活性化におけるATMおよびHSP90の関与  [通常講演]
    永根大幹, 安井博宣, 山盛徹, 丹羽光一, 稲波修
    日本獣医学会学術集会講演要旨集 2013年08月
  • プリオンタンパク質凝集体の電子スピン共鳴‐スピンラベル法による解析  [通常講演]
    稲波修, 山盛徹, 安井博宣, 堀内基広, 平岡和佳子, 古川貢, 中村敏和
    日本獣医学会学術集会講演要旨集 2013年08月
  • 赤血球膜の不安定性を示すウシαスペクトリンの局所的構造変化に関する研究  [通常講演]
    藤本桃子, 山盛徹, 安井博宣, 永根大幹, 大津航, 木崎皓太, 稲葉睦, 稲波修
    日本獣医学会学術集会講演要旨集 2013年08月
  • ―トビケラウオッチ―被災地における水生昆虫を用いた放射能モニタリング  [通常講演]
    平田和沙, 平野剛史, 上野大介, 染谷孝, 長坂洋光, 楢崎幸範, 稲波修, 龍田希, 仲井邦彦
    環境化学討論会要旨集(CD-ROM) 2013年07月
  • 放射線誘導性一酸化窒素の生成機構および放射線感受性の調節に関する研究  [通常講演]
    永根大幹, 安井博宣, 山盛徹, 中村秀夫, 稲波修
    日本酸化ストレス学会学術集会プログラム・抄録集 2013年06月
  • 低レベル放射能汚染による健康リスクのリスクコミュニケーションの試み  [通常講演]
    仲井邦彦, 龍田希, 阿部和眞, 稲波修, 黒川修行, 柳沼梢, 大葉隆, 坂田あゆみ, 有馬隆博
    日本衛生学雑誌 2013年03月
  • In vivo ESR oxymetryを用いた放射線照射による腫瘍の再酸素化動態の解析  [通常講演]
    永根大幹, 安井博宣, 山盛徹, 亀谷宏美, 中村秀夫, 稲波修
    電子スピンサイエンス学会年会講演要旨集 2012年11月
  • がんの放射線治療効率化に向けたESR技術の適用  [通常講演]
    安井博宣, 松元慎吾, 伊藤慎治, 山盛徹, 兵藤文紀, 市川和洋, 中村秀夫, 内海英雄, KRISHNA Murali C, 稲波修
    電子スピンサイエンス学会年会講演要旨集 2012年11月
  • EPRスペクトロスコピーによる腫瘍の細胞外pH測定  [通常講演]
    谷内勝哉, GOODWIN Jonathan, 永根大幹, 三宅祐輔, 安井博宣, 稲波修, BOBKO Andrey, KHARAMTSOV Valery V, 平田拓
    電子スピンサイエンス学会年会講演要旨集 2012年11月
  • 生体内酸素分圧イメージングに向けた試薬の継続投与の実験  [通常講演]
    青野尚平, 永根大幹, 三宅祐輔, 奥村大地, 安井博宣, 稲波修, 平田拓
    電子スピンサイエンス学会年会講演要旨集 2012年11月
  • チャイニーズハムスターV79細胞における硫化水素による放射線防護作用の検討  [通常講演]
    山本栄輔, 山盛徹, 永根大幹, 永瀧正人, 西田直哉, 安井博宣, 稲波修
    電子スピンサイエンス学会年会講演要旨集 2012年11月
  • 放射線誘導バイスタンダー効果ががん細胞の細胞外基質分解能に及ぼす影響  [通常講演]
    永瀧正人, 山盛徹, 安井博宣, 稲波修
    日本放射線影響学会大会講演要旨集 2012年09月
  • 放射線照射により生成される一酸化窒素は固形腫瘍の再酸素化に必須である。  [通常講演]
    永根大幹, 安井博宣, 山盛徹, 趙松吉, 久下裕司, 中村秀夫, 稲波修
    日本獣医学会学術集会講演要旨集 2012年08月
  • 固形腫瘍内における放射線誘導性再酸素化の動態の評価  [通常講演]
    永根大幹, 安井博宣, 趙松吉, 久保直樹, 玉木長良, 久下裕司, 稲波修
    核医学 2012年08月
  • チャイニーズハムスターV79細胞における硫化水素による放射線防護効果の検討  [通常講演]
    山本栄輔, 山盛徹, 永根大幹, 永瀧正人, 西田直也, 安井博宣, 稲波修
    日本獣医学会学術集会講演要旨集 2012年08月
  • 放射線照射後に生成される一酸化窒素は固形腫瘍の再酸素化に関与する  [通常講演]
    永根大幹, 安井博宣, 山盛徹, 趙松吉, 久下裕司, 中村秀夫, 稲波修
    日本酸化ストレス学会学術集会プログラム・抄録集 2012年06月
  • ミトコンドリアに由来する酸化ストレス障害に対するリジン脱アセチル化酵素の寄与とその役割  [通常講演]
    山盛徹, 稲田桂, 女池俊介, 永瀧正人, 安井博宣, 稲波修
    日本酸化ストレス学会学術集会プログラム・抄録集 2012年06月
  • パルスQ‐bandシステムを利用した生体系物質のPELDORによる距離測定の試み  [通常講演]
    古川貢, 平岡和佳子, 稲波修, 中村敏和
    電子スピンサイエンス学会年会講演要旨集 2011年11月
  • Methyl‐pyruvateによるミトコンドリア機能の修飾と放射線増感効果  [通常講演]
    西田直哉, 安井博宣, 山盛徹, 稲波修
    日本放射線影響学会大会講演要旨集 2011年11月
  • 腫瘍組織に特徴的な間欠的低酸素のイメージングと放射線抵抗性への関与  [通常講演]
    安井博宣, 松元慎吾, 新坊弦也, KRISHNA Murali, 山盛徹, MITCHELL James, 稲波修
    日本放射線影響学会大会講演要旨集 2011年11月
  • 放射線誘導バイスタンダー効果が細胞の浸潤能に与える影響  [通常講演]
    永瀧正人, 山盛徹, 安井博宣, 稲波修
    日本放射線影響学会大会講演要旨集 2011年11月
  • X線照射によるアポトーシスはミトコンドリアが主体となりおこる  [通常講演]
    馬嶋秀行, 犬童寛子, 稲波修, 幸村知子, 中川靖一, 松本謙一郎, 小澤俊彦
    日本放射線影響学会大会講演要旨集 2011年11月
  • 免疫組織化学と電子スピン共鳴法を用いた腫瘍における放射線照射後の再酸素化機構に関する研究  [通常講演]
    永根大幹, 安井博宣, 山盛徹, 中村秀夫, 稲波修
    日本放射線影響学会大会講演要旨集 2011年11月
  • 放射線により引き起こされるミトコンドリア由来ROS産生のメカニズム  [通常講演]
    山盛徹, 安井博宣, 稲波修
    日本放射線影響学会大会講演要旨集 2011年11月
  • 小胞体ストレスがDNA損傷修復機構に及ぼす影響とそれを利用した放射線増感の可能性の検討  [通常講演]
    女池俊介, 山盛徹, 安井博宣, 稲波修
    日本放射線影響学会大会講演要旨集 2011年11月
  • インビボ電子スピン共鳴法を用いた放射線照射後の再酸素化機構に関する研究  [通常講演]
    永根大幹, 安井博宣, 山盛徹, 中村秀夫, 稲波修
    日本獣医学会学術集会講演要旨集 2011年08月
  • ラット脳腫瘍における間欠的低酸素の可視化と放射線感受性に与える影響  [通常講演]
    安井博宣, 新坊弦也, 山盛徹, 永根大幹, 趙松吉, 久下祐司, 稲波修
    日本獣医学会学術集会講演要旨集 2011年08月
  • 高磁場BOLD‐fMRIで観察された侵害刺激に対するラット大脳皮質帯状回における情動応答のc‐Fos発現の免疫組織化学的評価による検討  [通常講演]
    古賀智之, 佐藤裕之, 安井博宣, 乙黒兼一, 日高勇一, 木曽哲男, 高橋正泰, 井上達雄, 藤川昭彦, 稲波修, 伊藤茂男, 浅沼武敏
    日本獣医学会学術集会講演要旨集 2011年08月
  • p66shc CH2ドメインを介したタンパク質相互作用が細胞の放射線感受性に与える影響  [通常講演]
    山盛徹, 松下明子, 女池俊介, 永瀧正人, 安井博宣, 稲波修
    日本獣医学会学術集会講演要旨集 2011年08月
  • Guanine nucleotide exchange factor‐H1はMKN45細胞においてビンクリスチンによる浸潤能の促進を媒介する  [通常講演]
    永瀧正人, 山盛徹, 女池俊介, 安井博宣, 稲波修
    日本獣医学会学術集会講演要旨集 2011年08月
  • 核酸代謝拮抗剤TAS106による相同組換え修復阻害作用を介した放射線増感効果  [通常講演]
    女池俊介, 山盛徹, 安井博宣, 松田彰, 森松正美, 福島正和, 山崎靖人, 稲波修
    日本獣医学会学術集会講演要旨集 2011年08月
  • マウス扁平上皮癌SCCVII細胞におけるmethyl‐pyruvateを用いた放射線増感効果とそのメカニズムの解析  [通常講演]
    西田直哉, 安井博宣, 山盛徹, 稲波修
    日本獣医学会学術集会講演要旨集 2011年08月
  • 細胞周期との関連性からみたX線照射に起因する活性酸素種の遅発性産生機構  [通常講演]
    安井博宣, 山盛徹, 和田悠佑, 山住雅之, 稲波修
    日本酸化ストレス学会学術集会プログラム・抄録集 2011年06月
  • X線照射により引き起こされるミトコンドリア由来活性酸素種生成メカニズムの解析  [通常講演]
    山盛徹, 安井博宣, 山住雅之, 中村吉就, 中村秀夫, 桑原幹典, 稲波修
    日本酸化ストレス学会学術集会プログラム・抄録集 2011年06月
  • 新規核酸誘導体のアポトーシス増強ならびにDNA修復阻害を介した放射線増感作用  [通常講演]
    稲波修
    日本医学放射線学会総会抄録集 2011年02月
  • p66shcはHSP72との相互作用を介して細胞の放射線感受性を調節する  [通常講演]
    山盛徹, 松下明子, 女池俊介, 永瀧正人, 安井博宣, 稲波修
    生化学 2011年
  • ビンクリスチンはGEF‐H1/RhoA/ROCK/Myosin light chainシグナルを介してMKN45細胞のアメーバ様運動を促進する  [通常講演]
    永瀧正人, 山盛徹, 女池俊介, 安井博宣, 稲波修
    生化学 2011年
  • X線照射後のがん細胞におけるESRオキシメトリーによる酸素消費率を指標としたミトコンドリア機能解析  [通常講演]
    安井博宣, 山盛徹, 中村秀夫, 山住雅之, 稲波修
    電子スピンサイエンス学会年会講演要旨集 2010年11月
  • 間欠的低酸素が腫瘍細胞の放射線抵抗性に与える影響  [通常講演]
    新坊弦也, 安井博宣, 山盛徹, 稲波修
    日本放射線影響学会大会講演要旨集 2010年10月
  • X線照射により誘導されるミトコンドリア由来活性酸素種生成上昇メカニズムの解析  [通常講演]
    山盛徹, 安井博宣, 中村秀夫, 山住雅之, 稲波修
    日本放射線影響学会大会講演要旨集 2010年10月
  • X線照射されたA549細胞における遅発性細胞内活性酸素種産生と細胞周期との関連性  [通常講演]
    和田悠佑, 山盛徹, 安井博宣, 山住雅之, 稲波修
    日本放射線影響学会大会講演要旨集 2010年10月
  • 核酸代謝拮抗剤TAS106による相同組換え修復阻害作用を介した放射線増感効果  [通常講演]
    女池俊介, 山盛徹, 安井博宣, 松田彰, 森松正美, 福島正和, 山崎靖人, 稲波修
    日本放射線影響学会大会講演要旨集 2010年10月
  • 高濃度VincristineはROCK経路活性化を介してアメーバ様浸潤を誘導する  [通常講演]
    永瀧正人, 山盛徹, 稲波修
    日本獣医学会学術集会講演要旨集 2010年09月
  • X線照射後に遅れて起きる細胞内活性酸素種の増加反応  [通常講演]
    稲波修, 安井博宣, 山住雅之, 桑原幹典, 山盛徹
    日本酸化ストレス学会学術集会プログラム・抄録集 2010年06月
  • 低酸素環境を標的としたがん治療の新たな展開  [通常講演]
    安井博宣, 山盛徹, 女池俊介, 永瀧正人, 飯塚大輔, 桑原幹典, 稲波修
    放射線生物研究 2010年03月
  • 獣医放射線学  [通常講演]
    稲波修
    日本獣医学会学術集会講演要旨集 2010年
  • X線照射はA549細胞においてミトコンドリア機能亢進と遅発性細胞内活性酸素種産生を誘導する  [通常講演]
    山住雅之, 山盛徹, 稲波修
    日本放射線影響学会大会講演要旨集 2009年11月
  • マウス扁平上皮がんSCC VII細胞におけるエネルギー代謝と放射線感受性  [通常講演]
    西田直哉, 山盛徹, 安井博宣, 稲波修
    日本放射線影響学会大会講演要旨集 2009年11月
  • 8‐Amino‐adenosineによる放射線誘導細胞死の増感効果  [通常講演]
    女池俊介, 山盛徹, 安井博宣, 松田彰, 稲波修
    日本放射線影響学会大会講演要旨集 2009年11月
  • 金粒子含有ナノゲル存在下でのX線照射による細胞死の増強作用とその機序の解析  [通常講演]
    武内亮, 安井博宣, 山盛徹, 中村隆仁, 大石基, 長崎幸夫, 稲波修
    日本放射線影響学会大会講演要旨集 2009年11月
  • ヒト胃腺がん細胞MKN45における浸潤能と微小管重合状態の関連性  [通常講演]
    永瀧正人, 山盛徹, 稲波修
    生化学 2009年09月
  • 制癌剤処理細胞における細胞内活性酸素種の増加と浸潤能力との関連性について  [通常講演]
    永瀧正人, 山盛徹, 稲波修
    日本酸化ストレス学会学術集会プログラム・抄録集 2009年06月
  • 経口吸着薬AST‐120(クレメジン)は虚血性急性腎不全(ARF)ラット腎ミトコンドリアからのフリーラジカル産生を抑制する  [通常講演]
    大和田滋, 平山暁, 植田敦志, 伊藤俊輔, 後藤寿美恵, 西島冬彦, 稲波修
    日本酸化ストレス学会学術集会プログラム・抄録集 2009年06月
  • 放射線照射したA549細胞に見られる遅延性細胞内活性酸素種の上昇に関する研究  [通常講演]
    山住雅之, 山盛徹, 稲波修
    日本酸化ストレス学会学術集会プログラム・抄録集 2009年06月
  • 電磁波に応答する金ナノ粒子含有ナノゲルの設計と評価  [通常講演]
    長崎幸夫, 中村隆仁, 大石基, 神事裕太, 松石清人, 武内亮, 安井博宣, 稲波修
    Drug Delivery System 2009年06月
  • 電磁波ナノ治療を目指した金コロイド含有PEG化ナノゲルの調製  [通常講演]
    中村隆仁, 田村篤志, 大石基, 神事裕太, 松石清人, 武内亮, 安井博宣, 稲波修, 長崎幸夫
    高分子学会予稿集(CD-ROM) 2009年05月
  • 痛みのimaging,MRIを用いての試み  [通常講演]
    浅沼武敏, 稲波修, 安井博宣, 桑原幹典
    日本獣医学会学術集会講演要旨集 2009年03月
  • 金ナノ粒子含有ナノゲルによる新規治療技術へのアプローチ  [通常講演]
    中村隆仁, 田村篤志, 大石基, 安井博宣, 武内亮, 稲波修, 長崎幸夫
    高分子ゲル研究討論会講演要旨集 2009年01月
  • Functional MRIによる痛みの定量化  [通常講演]
    浅沼武敏, 佐藤裕之, 稲波修, 桑原幹典
    日本獣医学会学術集会講演要旨集 2009年
  • A549細胞における8‐amino‐adenosineと放射線の併用による抗腫瘍効果の検討  [通常講演]
    女池俊介, 山盛徹, 安井博宣, 松田彰, 稲波修
    日本獣医学会学術集会講演要旨集 2009年
  • マウスプリオンタンパク質ヒスチジン186周辺の銅イオン結合部位に関する構造解析  [通常講演]
    稲波修, 渡辺康子, 五十嵐学, 伊藤公人, 堀内基広, 桑原幹典, 平岡和佳子
    日本獣医学会学術集会講演要旨集 2009年
  • 金ナノ粒子存在下でのX線照射による細胞増殖死の増強作用  [通常講演]
    武内亮, 安井博宣, 長崎幸夫, 大石基, 中村隆仁, 稲波修
    日本放射線影響学会大会講演要旨集 2008年11月
  • ネフローゼ症候群顕性蛋白尿発症過程におけるミトコンドリア機能異常のESR解析  [通常講演]
    平山暁, 植田敦志, 青柳一正, 古武弥成, 稲波修, 大和田滋
    電子スピンサイエンス学会年会講演要旨集 2008年10月
  • Feline immunodeficiency virus(FIV)感染初期におけるネコの好中球機能の評価ならびにウシラクトフェリン投与の影響  [通常講演]
    小林沙織, 坂下豪, 永井恭子, 関隆志, 稲波修, 安田準, 佐々木重荘, 佐藤れえ子
    日本獣医学会学術集会講演要旨集 2008年09月
  • PBNによるPC12細胞に対する神経様突起誘導のメカニズムに関する検討  [通常講演]
    伊藤望, 小倉亜希, 渡辺康子, 浅沼武敏, 稲波修
    日本獣医学会学術集会講演要旨集 2008年09月
  • C6担癌ラットを用いた新規低酸素性放射線増感剤PR‐350(ドラニダゾール)の脳腫瘍に対する薬効評価  [通常講演]
    木野潤一, 浅沼武敏, 安井博宣, 久保田信雄, 辻谷典彦, 桑原幹典, 稲波修
    日本獣医学会学術集会講演要旨集 2008年09月
  • 放射線誘導細胞死におけるサバイビンの役割  [通常講演]
    稲波修, 小倉亜希, 安井博宣, 飯塚大輔, 浅沼武敏, 桑原幹典
    日本獣医学会学術集会講演要旨集 2008年03月
  • DNA損傷チェックポイントを介した五味子のガン治療増感作用  [通常講演]
    吉田真奈美, 乾直人, 安井博宣, 浅沼武敏, 稲波修, 吉田雅昭, 安藤英広, 近藤誠三, 半田修, 内藤裕二, 吉川敏一, 西田浩志, 小西徹也
    日本薬学会年会要旨集 2008年03月
  • G2期チェックポイント阻害による放射線誘導細胞致死効果のメカニズム  [通常講演]
    飯塚大輔, 稲波修, 安井博宣, 小倉亜希, 桑原幹典
    放射線生物研究 2007年12月
  • 固形腫瘍内低酸素細胞に対する核酸代謝拮抗剤TAS106の抑制効果  [通常講演]
    安井博宣, 桑原幹典, 小倉亜希, 浅沼武敏, 松田彰, 稲波修
    日本放射線影響学会大会講演要旨集 2007年11月
  • MCF‐7細胞におけるカスパーゼ3過剰発現によるX線誘導アポトーシスの増強効果  [通常講演]
    女池俊介, 小倉亜希, 浅沼武敏, 桑原幹典, 稲波修
    日本放射線影響学会大会講演要旨集 2007年11月
  • ヒト胃癌細胞MKN45の腹腔内播種におけるRNA合成阻害薬ECydの薬効評価  [通常講演]
    永瀧正人, 安井博宣, 浅沼武敏, 松田彰, 桑原幹典, 稲波修
    日本獣医学会学術集会講演要旨集 2007年08月
  • プリオンタンパク質C末端領域の新たなCu2+結合構造の同定  [通常講演]
    稲波修, 渡邊康子, 堀内基広, 平岡和佳子, 下山雄平, 桑原幹典
    日本獣医学会学術集会講演要旨集 2007年08月
  • プリオンタンパク質のpH感受性における塩橋とヒスチジン残基の役割  [通常講演]
    渡邊康子, 平岡和佳子, 下山雄平, 堀内基広, 桑原幹典, 稲波修
    日本獣医学会学術集会講演要旨集 2007年08月
  • 抗酸化剤のラット脳腫瘍成長と浸潤に対する神経線維防護効果のMRIファイバートラクトグラフィ法による非侵襲的評価法  [通常講演]
    浅沼武敏, RHEAL Towner, YASHIGE Kotake, ROBERT Floyd, 桑原幹典, 稲波修
    日本獣医学会学術集会講演要旨集 2007年08月
  • ウシラクトフェリンの局所接種による抗腫瘍効果  [通常講演]
    山田裕一, 小林沙織, 稲波修, 桑原幹典, 安田準, 佐藤れえ子
    日本獣医学会学術集会講演要旨集 2007年03月
  • 好中球CR3(CD11b/CD18)発現低下を示した先天性好中球機能異常症の犬に対するウシラクトフェリン(bLF)投与の影響  [通常講演]
    阿部裕哉, 小林沙織, 稲波修, 桑原幹典, 安田準, 佐々木重荘, 佐藤れえ子
    日本獣医学会学術集会講演要旨集 2007年03月
  • Site‐Directed Spin Labeling(SDSL)法を用いたマウスプリオンタンパク質C末端領域の新たなCu2+結合構造の同定  [通常講演]
    稲波修, 渡邊康子, 平岡和佳子, 下山雄平, 稲垣冬彦, 桑原幹典
    生化学 2007年
  • プリオンタンパク質β‐シート領域のpH感受性における塩橋とヒスチジン残基の役割  [通常講演]
    渡邊康子, 平岡和佳子, 下山雄平, 堀内基広, 桑原幹典, 稲波修
    生化学 2007年
  • 腫瘍細胞における抗アポトーシス因子サバイビンを分子標的とした放射線致死効果増強機構の解析  [通常講演]
    小倉亜希, 安井博宣, 桑原幹典, 稲波修
    生化学 2007年
  • プリオンたんぱく質の塩橋形成部位に関する部位特異的ESRスピンラベル法による解析  [通常講演]
    玉山博敏, 渡辺康子, 音嶋優紀, 稲波修, 桑原幹典, 下山雄平
    電子スピンサイエンス学会年会講演要旨集 2006年11月
  • プリオンタンパク質のpH依存構造変化のESRシミュレーションによる解析  [通常講演]
    音嶋優紀, 渡邊康子, 玉山博敏, 稲波修, 下山雄平
    電子スピンサイエンス学会年会講演要旨集 2006年11月
  • プリオンタンパク質の抗酸化機能の解明  [通常講演]
    渡邊康子, 稲波修, 井上哲, 飯塚大輔, 堀内基広, 桑原幹典
    電子スピンサイエンス学会年会講演要旨集 2006年11月
  • ドロマイトの抗菌メカニズムに関する検討  [通常講演]
    駒井仁史, 村瀬敏之, 大槻公一, 若林一夫, 山名英明, 稲波修, 桑原幹典
    日本獣医学会学術集会講演要旨集 2006年08月
  • Site‐Directed Spin Labeling(SDSL)法によるプリオンタンパク質pH感受性領域の解析  [通常講演]
    渡邊康子, 稲波修, 堀内基広, 下山雄平, 中村秀夫, 桑原幹典
    日本獣医学会学術集会講演要旨集 2006年08月
  • 固形腫瘍内の低酸素細胞に対するエチニルシチジン(ECyd)の効果  [通常講演]
    安井博宣, 稲波修, 浅沼武敏, 松田彰, 桑原幹典
    日本獣医学会学術集会講演要旨集 2006年08月
  • アデノウイルスベクターによる変異サバイビン導入腫瘍細胞における放射線誘導アポトーシスの増強効果  [通常講演]
    小倉亜希, 稲波修, 桑原幹典
    日本獣医学会学術集会講演要旨集 2006年08月
  • ―基礎と臨床との対話―低LET放射線は生物学的手法の併用によって高LET放射線を超えられるか 抗がん剤による増感 エチニルシチジンの放射線増感効果  [通常講演]
    桑原幹典, 稲波修, 安井博宣, 飯塚大輔, 松田彰
    癌の臨床 2006年04月
  • Site‐directed spin‐label法によるマウスプリオンたんぱく質の構造解析  [通常講演]
    稲波修, 渡邊康子
    電子スピンサイエンス 2006年03月
  • キナーゼ活性阻害剤による放射線誘発抗アポトーシスたんぱく質の発現抑制  [通常講演]
    飯塚大輔, 稲波修, 安井博宣, 桑原幹典
    日本放射線影響学会大会講演要旨集 2006年
  • ヒト肺がん由来A549細胞での放射線によるTRAIL誘導性アポトーシスの増感効果  [通常講演]
    高橋桃子, 稲波修, 辻谷典彦, 久保田信雄, 桑原幹典
    日本放射線影響学会大会講演要旨集 2006年
  • Site‐directed spin label(SDSL)法によるp47phoxリン酸化の解析  [通常講演]
    鈴木友子, 稲波修, 桑原幹典, 下山雄平, 稲垣冬彦, 平岡和佳子
    電子スピンサイエンス学会年会講演要旨集 2005年10月
  • プリオンH95‐H110領域の新たなCu(II)結合構造―Site‐directed spin‐label(SDSL)法を用いた双極子‐双極子相互作用による距離情報解析―  [通常講演]
    稲波修, 橋田修吉, 渡邊康子, 平岡和佳子, 下山雄平, 中村秀夫, 稲垣冬彦, 桑原幹典
    電子スピンサイエンス学会年会講演要旨集 2005年10月
  • プリオンα‐ヘリックスおよびβ‐シート領域のpH依存性構造変化のSite‐Directed Spin Labeling(SDSL)法による解析  [通常講演]
    渡邊(浅野)康子, 渡邊(浅野, 康子, 稲波修, 飯塚大輔, 下山雄平, 中村秀夫, 桑原幹典
    電子スピンサイエンス学会年会講演要旨集 2005年10月
  • 核酸代謝きっ抗剤エチニルシチジンによる細胞死誘導機構  [通常講演]
    飯塚大輔, 稲波修, 安井博宣, 浅沼武敏, 松田彰, 桑原幹典
    日本獣医学会学術集会講演要旨集 2005年08月
  • プリオンタンパク質の銅イオンの新たな結合様式  [通常講演]
    稲波修, 堀内基広, 平岡和佳子, 稲垣冬彦, 下山雄平, 中村秀夫, 桑原幹典
    日本獣医学会学術集会講演要旨集 2005年08月
  • マウスはい線維芽細胞NIH3T3細胞におけるサバイビンの細胞増殖能に及ぼす影響  [通常講演]
    小倉亜希, 稲波修, 桑原幹典
    日本獣医学会学術集会講演要旨集 2005年08月
  • NGF分化誘導PC12細胞の虚血性細胞死の抑制にはCREB活性の維持とカルパインの過剰活性の抑制が必要  [通常講演]
    中島崇行, 若狭崇, 稲波修, 桑原幹典, 河原剛一
    日本獣医学会学術集会講演要旨集 2005年08月
  • PKCδの好中球活性酸素生成,細胞骨格制御ならびにどん食能における役割  [通常講演]
    脇研二, 稲波修, 山盛徹, 永幡肇, 桑原幹典
    日本獣医学会学術集会講演要旨集 2005年08月
  • エチニルシチジン(ECyd)による移植固形腫ようの放射線増感作用  [通常講演]
    安井博宣, 稲波修, 浅沼武敏, 中島崇行, 中島美穂子, 飯塚大輔, 松田彰, 桑原幹典
    日本獣医学会学術集会講演要旨集 2005年08月
  • プリオンタンパク質過剰発現細胞における酸化ストレス応答  [通常講演]
    井上哲, 稲波修, 飯塚大輔, 堀内基広, 桑原幹典
    日本獣医学会学術集会講演要旨集 2005年08月
  • 両親媒性スピントラップ剤LPBNSHの銅誘導性肝炎に対する活性酸素傷害防護能力の評価  [通常講演]
    浅沼武敏, 安井博宣, 稲波修, 上村健人, 脇研二, 高橋桃子, GREGORY Durand, ANGE Polidori, BERNARD Pucci, 桑原幹典
    日本獣医学会学術集会講演要旨集 2005年08月
  • マウスプリオンタンパク質α‐ヘリックス領域のSite‐Directed Spin Labeling(SDSL)法を用いた解析  [通常講演]
    渡辺康子, 稲波修, 堀内基広, 下山雄平, 中村秀夫, 桑原幹典
    日本獣医学会学術集会講演要旨集 2005年08月
  • 高磁場BOLD‐fMRIによるラットにおけるカプサイシン痛覚の可視化と鎮痛薬の評価  [通常講演]
    上村健人, 浅沼武敏, 稲波修, 佐藤昌泰, 桑原幹典
    日本獣医学会学術集会講演要旨集 2005年08月
  • 血管内皮細胞のシグナル伝達と活性酸素  [通常講演]
    丹羽光一, 稲波修
    放射線生物研究 2005年03月
  • アデノウイルスベクターを用いた変異サバイビン過剰発現によるX線誘導アポトーシスの増感作用  [通常講演]
    小倉亜希, 稲波修, 桑原幹典
    日本放射線影響学会大会講演要旨集 2005年
  • MKN45異種移植固形腫瘍に対するエチニルシチジン(ECyd)の放射線増感作用  [通常講演]
    安井博宣, 稲波修, 浅沼武敏, 松田彰, 桑原幹典
    日本放射線影響学会大会講演要旨集 2005年
  • ヒト肺腺がん由来A549細胞における放射線誘発性アポトーシスのTRAILによる増感機構  [通常講演]
    高橋桃子, 稲波修, 飯塚大輔, 桑原幹典
    日本放射線影響学会大会講演要旨集 2005年
  • ヒト巨核球前駆細胞に対するカテキン(EGCg)の放射線防護効果  [通常講演]
    門前暁, 盛孝男, 高橋賢次, 阿部由直, 稲波修, 桑原幹典, 柏倉幾郎
    日本放射線影響学会大会講演要旨集 2005年
  • ネコ免疫不全症ウイルス(FIV)感染におけるウシラクトフェリンの抗炎症作用  [通常講演]
    佐藤れえ子, 小林沙織, 稲波修, 佐藤淳, 山田裕一, 内藤善久, 佐々木重荘
    ミルクサイエンス 2004年12月
  • Site‐directed spin label(SDSL)法によるマウスプリオンタンパク質の銅イオン結合部位周辺の構造解析  [通常講演]
    稲波修, 橋田修吉, 飯塚大輔, 堀内基広, 平岡和佳子, 稲垣冬彦, 桑原幹典
    電子スピンサイエンス学会年会講演要旨集 2004年11月
  • Feline Immunodeficiency Virus(FIV)感染ネコ末梢血単核球におけるコンカナバリンAによるインターフェロンγ誘導に及ぼすラクトフェリンの影響  [通常講演]
    小林沙織, 佐藤れえ子, 稲波修, 桑原幹典, 重茂克彦, 佐藤淳, 内藤善久
    日本獣医学会学術集会講演要旨集 2004年08月
  • プリオンの部位特異的スピンラベル法(SDSL‐ESR)による構造変化の解析  [通常講演]
    稲波修, 橋田修吉, 飯塚大輔, 井上哲, 堀内基広, 桑原幹典
    日本獣医学会学術集会講演要旨集 2004年08月
  • 大脳皮質虚血耐性現象とAkt活性との関係について  [通常講演]
    中島崇行, 岩淵禎弘, 宮崎浩之, 大熊康修, 稲波修, 桑原幹典, 野村靖幸, 河原剛一
    日本獣医学会学術集会講演要旨集 2004年08月
  • 放射線照射と膜透過性ペプチドTAT融合変異サバイビン局所投与の併用によるマウス大腸癌由来Colon26細胞移植腫ようの増殖遅延効果  [通常講演]
    網谷誠, 稲波修, 浅沼武敏, 安井博宣, 小倉亜希, 桑原幹典
    日本獣医学会学術集会講演要旨集 2004年08月
  • 高磁場BOLD‐fMRIによるラットにおける痛覚の分析法の確立  [通常講演]
    浅沼武敏, 稲波修, 佐藤昌泰, 上村健人, 桑原幹典
    日本獣医学会学術集会講演要旨集 2004年08月
  • 放射線あるいは温熱誘発アポトーシスにおよぼす抗酸化物剤の影響  [通常講演]
    近藤隆, さい正国, FERIL R B JR, 脇研二, 稲波修, 桑原幹典
    日本獣医学会学術集会講演要旨集 2004年08月
  • NADPHオキシダーゼ活性化機構とウシ分娩後の好中球機能低下  [通常講演]
    稲波修, 脇研二, 山盛徹, 浅沼武敏, 中島崇行, 永幡肇, 桑原幹典
    獣医生化学 2004年04月
  • ネコ末梢血単核球におけるコンカナバリンAによるインターフェロンγ誘導に及ぼすラクトフェリンの影響  [通常講演]
    小林沙織, 佐藤れえ子, 稲波修, 山盛徹, 桑原幹典, 大和修, 重茂克彦, 佐藤淳, 内藤善久
    日本獣医学会学術集会講演要旨集 2004年03月
  • 胃がん株化細胞におけるPurvalanol AによるG2チェックポイントを介した放射線誘導アポトーシス増強作用  [通常講演]
    飯塚大輔, 稲波修, 安井博宣, 桑原幹典
    日本獣医学会学術集会講演要旨集 2004年03月
  • ウシ好中球の活性酸素生成機構の解析と炎症制御への応用  [通常講演]
    稲波修
    日本獣医学会学術集会講演要旨集 2004年03月
  • X線照射後の低酸素ならびにドラニダゾールのアポトーシスシグナル伝達経路への影響  [通常講演]
    浜洲拓, 稲波修, 横山浩治, 辻谷典彦, 桑原幹典
    日本獣医学会学術集会講演要旨集 2004年03月
  • 変異サバイビン発現細胞における放射線ならびに制がん剤によるアポトーシス  [通常講演]
    小倉亜希, 稲波修, 桑原幹典
    日本獣医学会学術集会講演要旨集 2004年03月
  • キナーゼ活性阻害剤による胃がん株化細胞の放射線誘発アポトーシス増強効果とその機構  [通常講演]
    飯塚大輔, 稲波修, 安井博宣, 桑原幹典
    日本放射線影響学会大会講演要旨集 2004年
  • ヒト腺癌細胞におけるデスレセプターアゴニストの誘導するアポトーシスのX線照射による増強  [通常講演]
    浜洲拓, 稲波修, 桑原幹典
    日本放射線影響学会大会講演要旨集 2004年
  • X線照射されたマウスはい線維芽細胞NIH3T3細胞における変異サバイビン過剰発現によるアポトーシス増感作用  [通常講演]
    小倉亜希, 稲波修, 桑原幹典
    日本放射線影響学会大会講演要旨集 2004年
  • Colon26マウス移植固形腫ようにおけるエチニルシチジン(ECyd)の放射線増感作用  [通常講演]
    安井博宣, 稲波修, 浅沼武敏, 中島崇行, 飯塚大輔, 松田彰, 桑原幹典
    日本放射線影響学会大会講演要旨集 2004年
  • PI3KによるNADPH oxidase細胞質因子p47phoxのリン酸化制御機構  [通常講演]
    山盛徹, 稲波修, 永幡肇, 桑原幹典
    獣医生化学 2003年10月
  • ヒト末梢血に含まれる放射線感受性の異なる巨核球前駆細胞の存在  [通常講演]
    柏倉幾郎, 阿部由直, 稲波修, 高橋恒夫, 桑原幹典
    日本放射線影響学会大会講演要旨集 2003年10月
  • X線誘導アポトーシスシグナル伝達経路への低酸素及び放射線増感剤ドラニダゾールの影響  [通常講演]
    浜洲拓, 稲波修, 辻谷典彦, 横山浩治, 桑原幹典
    日本放射線影響学会大会講演要旨集 2003年10月
  • 新規ヌクレオシド増感剤TAS106によるG2/M期チェックポイント機構とアポトーシス抑制因子を標的とした放射線増感  [通常講演]
    稲波修, 飯塚大輔, 岩原明子, 山盛徹, 昆泰寛, 北里健二, 松田彰, 桑原幹典
    日本放射線影響学会大会講演要旨集 2003年10月
  • 胃がん株化細胞におけるCdc2キナーゼ活性阻害剤Purvalanol Aによる放射線誘導アポトーシス増感作用  [通常講演]
    飯塚大輔, 稲波修, 桑原幹典
    日本放射線影響学会大会講演要旨集 2003年10月
  • 西ナイルウイルスの感染におけるJNKの役割  [通常講演]
    赤穂芳彦, 水谷哲也, 三好洋嗣, 小林正之, 白戸憲也, 江下優樹, 木村享史, 山盛徹, 稲波修
    日本獣医学会学術集会講演要旨集 2003年09月
  • マロン酸誘導脳傷害の早期段階におけるJNKの阻害効果~拡散強調画像法による可視化~  [通常講演]
    たぶ康一, 浅沼武敏, 脇研二, 中島崇行, 稲波修, 桑原幹典
    日本獣医学会学術集会講演要旨集 2003年09月
  • エチニルシチジン(ECyd)とX線併用によるColon26マウス移植固形腫ようの増殖抑制効果  [通常講演]
    安井博宣, 稲波修, 浅沼武敏, 中島崇行, 飯塚大輔, 松田彰, 桑原幹典
    日本獣医学会学術集会講演要旨集 2003年09月
  • Phosphoinositide 3‐kinaseにより調節される食細胞NADPH oxidase細胞質因子p47phoxリン酸化のシグナル伝達機構  [通常講演]
    山盛徹, 稲波修, 桑原幹典
    日本獣医学会学術集会講演要旨集 2003年09月
  • JNK阻害によりマロン酸誘導脳障害は軽減される‐MRIによる非侵襲的観察  [通常講演]
    たぶのき康一, 浅沼武敏, 昆泰寛, 稲波修, 桑原幹典
    日本獣医学会学術集会講演要旨集 2003年03月
  • ウシ分娩後における末梢血好中球の活性酸素生成能の低下機構  [通常講演]
    脇研二, 稲波修, 矢用健一, 山田豊, 桑原幹典
    日本獣医学会学術集会講演要旨集 2003年03月
  • マクロファージIL‐1β産生におけるNADPHオキシダーゼ由来活性酸素種の役割  [通常講演]
    稲波修, 小野耕介, 山盛徹, 桑原幹典
    日本獣医学会学術集会講演要旨集 2003年03月
  • 周産期のどん食細胞機能に着目した乳房炎の発症機序の解明とその制御  [通常講演]
    稲波修
    動物衛生試験研究成績・計画概要集 平成14年度 2003年
  • V 病態‐基礎 1 低分子量G蛋白質Racを介したp38 MAPKによる好中球NADPH oxidaseの活性化制御  [通常講演]
    山盛徹, 稲波修, 住本英樹, 永幡肇, 桑原幹典
    磁気共鳴と医学 2002年09月
  • V 病態‐基礎 1 好中球NADPHオシキダーゼ活性化におけるERKの役割  [通常講演]
    脇研二, 稲波修, 山盛徹, 永幡肇, 桑原幹典
    磁気共鳴と医学 2002年09月
  • 放射線照射ヒト末梢血由来巨核球前駆細胞の分化増殖に対するTPOとIL‐3の相乗効果  [通常講演]
    柏倉幾郎, 稲波修, 高橋恒夫, 桑原幹典
    日本放射線影響学会大会講演要旨集 2002年08月
  • 光増感物質を用いたゴルジ装置損傷を起点とするアポトーシスシグナルの解析  [通常講演]
    緒方麻衣子, 稲波修, 高橋賢次, 山盛徹, 中島美穂子, 平岡和佳子, 桑原幹典
    日本放射線影響学会大会講演要旨集 2002年08月
  • 新規抗ガン剤エチニルシチジン(ECyd)とX線同時併用によるV79細胞のアポトーシス誘導機構  [通常講演]
    飯塚大輔, 稲波修, 高橋賢次, 松田彰, 桑原幹典
    日本放射線影響学会大会講演要旨集 2002年08月
  • 新規抗がん剤エチニルシチジン(ECyd)とX線同時併用によるアポトーシス誘導におけるサバイビンの役割  [通常講演]
    稲波修, 飯塚大輔, 山盛徹, 昆泰寛, 松田彰, 桑原幹典
    日本放射線影響学会大会講演要旨集 2002年08月
  • Preconditioning処置の有無による大脳皮質虚血後のAktのリン酸化動態の変化について  [通常講演]
    中島崇行, 宮崎浩之, 大熊康修, 稲波修, 桑原幹典, 野村靖幸, 河原剛一
    日本獣医学会学術集会講演要旨集 2002年08月
  • FSE‐DWI法の作成とその適用によるα‐phenyl‐N‐tert‐butylnitoroneの中枢神経防護効果の可視化  [通常講演]
    たぶ康一, 浅沼武敏, 栗林秀人, 今野明弘, 昆泰寛, 稲波修, 桑原幹典
    日本獣医学会学術集会講演要旨集 2002年08月
  • ヒト白血病由来株化細胞MOLT‐4における2‐クロロデオキシアデノシン誘発アポトーシスのタンパク質合成依存性  [通常講演]
    高橋恵理子, 稲波修, 松田彰, 桑原幹典
    日本獣医学会学術集会講演要旨集 2002年08月
  • アポトーシスシグナルに及ぼす低酸素の影響とドラニダゾールの効果  [通常講演]
    浜洲拓, 稲波修, 辻谷典彦, 横山浩治, 桑原幹典
    日本獣医学会学術集会講演要旨集 2002年08月
  • ヒトスジシマカ由来の培養細胞,C6/36における大腸菌に対する応答  [通常講演]
    小林正之, 水谷哲也, 江下優樹, 稲波修, 山盛徹, 後藤明子, 赤穂芳彦, 桑原幹典, 高島郁夫
    日本獣医学会学術集会講演要旨集 2002年08月
  • BOLD法によるα‐phenyl‐N‐tert‐butylnitoroneの脳機能防護効果の評価  [通常講演]
    浅沼武敏, たぶ康一, 稲波修, 桑原幹典
    日本獣医学会学術集会講演要旨集 2002年08月
  • マイクロビームによるアポトーシスシグナルのトリガー損傷の解析  [通常講演]
    稲波修
    KEK Proceedings 2002年05月
  • 新規制がん剤による胃腺がん細胞のアポトーシス放射線増感  [通常講演]
    稲波修
    日本獣医学会学術集会講演要旨集 2002年03月
  • 酸化ストレスによる血管内皮細胞のp53発現とアポトーシスにおけるPKCδとカルシウムの役割  [通常講演]
    丹羽光一, 稲波修, 桑原幹典
    日本獣医学会学術集会講演要旨集 2002年03月
  • ゴルジ装置をターゲットとした光増感剤によるアポトーシス誘導機構  [通常講演]
    緒方麻衣子, 稲波修, 高橋賢次, 山盛徹, 中島美穂子, 桑原幹典
    日本獣医学会学術集会講演要旨集 2002年03月
  • Preconditioningによる大脳皮質虚血後のc‐Junならびにリン酸化CREB発現動態の変化について  [通常講演]
    岩淵禎弘, 中島崇行, 河原剛一, 宮崎浩之, 大熊康修, 野村靖幸, 稲波修, 桑原幹典
    日本生理学雑誌 2002年01月
  • 放射線照射によるヒト白血病由来株化細胞MOLT‐4細胞のNADHオキシダーゼの活性化  [通常講演]
    稲波修, 高橋賢次, 桑原幹典
    日本放射線影響学会大会講演要旨集 2001年09月
  • Amifostineによるヒト巨核球前駆細胞に対する増殖促進および放射線防護効果  [通常講演]
    柏倉幾郎, 村上美穂, 高木良成, 高橋恒夫, 稲波修, 桑原幹典
    日本放射線影響学会大会講演要旨集 2001年09月
  • エチニルシチジン(ECyd)存在下でX線照射した胃腺癌由来株化細胞MKN 45細胞のアポトーシス誘導  [通常講演]
    岩原明子, 稲波修, 高橋賢次, 昆泰寛, 松田彰, 桑原幹典
    日本放射線影響学会大会講演要旨集 2001年09月
  • エチニルシチジン(ECyd)存在下でX線照射したチャイニーズハムスターV79細胞の放射線誘発細胞死の増感効果  [通常講演]
    飯塚大輔, 稲波修, 高橋賢次, 松田彰, 桑原幹典
    日本放射線影響学会大会講演要旨集 2001年09月
  • 高磁場MRIを用いた中枢神経防護効果の観察  [通常講演]
    石橋佑規, 浅沼武敏, 山田英二, 稲波修, 昆泰寛, 桑原幹典
    日本獣医学会学術集会講演要旨集 2001年09月
  • エチニルシチジン(ECyd)存在下でX線照射した胃腺癌由来株化細胞MKN45細胞のアポトーシス誘導  [通常講演]
    岩原明子, 稲波修, 高橋賢次, 昆泰寛, 松田彰, 桑原幹典
    日本獣医学会学術集会講演要旨集 2001年09月
  • 酸化ストレス誘発性アポトーシスにおけるミトコンドリア膜電位変化とチトクロームc放出の関与  [通常講演]
    丹羽光一, 稲波修, 桑原幹典
    日本獣医学会学術集会講演要旨集 2001年09月
  • ヒト白血病由来株MOLT‐4細胞のタンパク質合成依存性放射線誘発アポトーシスのメカニズム  [通常講演]
    高橋賢次, 稲波修, 桑原幹典
    日本獣医学会学術集会講演要旨集 2001年09月
  • 低分子量G蛋白質Racを介したp38 MAPKによる好中球NADPH oxidaseの活性化制御  [通常講演]
    山盛徹, 稲波修, 住本英樹, 永幡肇, 桑原幹典
    日本獣医学会学術集会講演要旨集 2001年09月
  • 1H‐MRSを用いた中枢神経防護薬PBNの非侵襲的評価法  [通常講演]
    浅沼武敏, 石橋佑規, 山田英二, 稲波修, 昆泰寛, 堤耀広, 桑原幹典
    日本獣医学会学術集会講演要旨集 2001年09月
  • Preconditioning処置の有無による大脳皮質虚血後のCREBのリン酸化動態の変化について  [通常講演]
    中島崇行, 岩淵禎弘, 宮崎浩之, 大熊康修, 稲波修, 桑原幹典, 野村靖幸, 河原剛一
    日本獣医学会学術集会講演要旨集 2001年09月
  • 好中球NADPHオキシダーゼ活性化におけるERKの役割  [通常講演]
    脇研二, 稲波修, 山盛徹, 永幡肇, 桑原幹典
    日本獣医学会学術集会講演要旨集 2001年09月
  • フリーラジカルと神経系 フリーラジカルによる生体障害 フリーラジカルとアポトーシス  [通常講演]
    桑原幹典, 稲波修
    Clinical Neuroscience 2001年05月
  • ウシ好中球のどん食殺菌機構におけるp38 MARKとPKCの役割  [通常講演]
    山盛徹, 稲波修, 永幡肇, 桑原幹典
    磁気共鳴と医学 2001年03月
  • マロン酸誘導ラット脳虚血モデルの高磁場下ADCマッピング PBNによる脳神経傷害防護作用の研究への適用  [通常講演]
    石橋佑規, 浅沼武敏, 稲波修, 昆泰寛, 桑原幹典
    日本獣医学会学術集会講演要旨集 2001年03月
  • 活性酸素・フリーラジカルの生成機構と疾病  [通常講演]
    桑原幹典, 稲波修, 浅沼武敏, 山盛徹, 昆泰寛, 永幡肇, 樋口豪紀
    日本獣医学会学術集会講演要旨集 2001年03月
  • 血管内皮細胞のアポトーシスと生存シグナルにおけるカルシウムの役割  [通常講演]
    丹羽光一, 稲波修, 山盛徹, 太田利男, 狩野猛, 桑原幹典
    日本獣医学会学術集会講演要旨集 2001年03月
  • どん食細胞のフリーラジカル生成とCR3(CD11b/CD18)分子欠損病態における動態  [通常講演]
    永幡肇, 樋口豪紀, 稲波修, 桑原幹典
    日本獣医学会学術集会講演要旨集 2001年03月
  • 血管内皮細胞の透過性変化における細胞内Ca2+とMAPキナーゼの役割  [通常講演]
    丹羽光一, 稲波修, 太田利男, 桑原幹典, 狩野猛
    日本生理学雑誌 2001年01月
  • フラボノイド投与マウスにおける脳損傷軽減化のメカニズム  [通常講演]
    桑原幹典, 稲波修
    三島海雲記念財団研究報告書 2000年12月
  • 酸化ストレスによる細胞内シグナル活性化とカルシウムイオンの役割  [通常講演]
    稲波修, 丹羽光一, 太田利男, 桑原幹典
    日本獣医学会学術集会講演要旨集 2000年09月
  • ニワトリのテレピン油誘導タンパク質がヘテロフィル細胞の活性酸素産生能に及ぼす影響  [通常講演]
    岩崎健, 稲波修, 内田英二, 三好健二郎, 森松正美, 首藤文栄, 桑原幹典, 新山雅美
    日本獣医学会学術集会講演要旨集 2000年09月
  • 酸化ストレス負荷による血管内皮細胞の透過性変化におけるMAPキナーゼの役割  [通常講演]
    丹羽光一, 稲波修, 太田利男, 狩野猛, 桑原幹典
    日本獣医学会学術集会講演要旨集 2000年09月
  • 抗酸化剤の照射後処理による放射線誘発アポトーシスの抑制効果  [通常講演]
    榊原慶祐, 桑原幹典, 稲波修
    日本獣医学会学術集会講演要旨集 2000年09月
  • 低酸素細胞増感剤エタニダゾールによる放射線誘発アポトーシスの増感効果  [通常講演]
    杉原潔, 稲波修, 飯田義晴, 桑原幹典
    日本獣医学会学術集会講演要旨集 2000年09月
  • パーキンソン症候群モデルラット脳の高磁場下ADCマッピング画像と1H局所スペクトロスコピー(MRS)による脳機能解析  [通常講演]
    浅沼武敏, 高橋賢次, 山田英二, 辻雅久, 稲波修, 桑原幹典
    日本獣医学会学術集会講演要旨集 2000年09月
  • タマネギおよびニンニクに含まれる有機チオ硫酸化合物のイヌ赤血球酸化傷害機序 II フリーラジカルの関与  [通常講演]
    紺谷有子, 大和修, 稲波修, 桑原幹典, 山崎真大, 前出吉光
    日本獣医学会学術集会講演要旨集 2000年09月
  • ヒトさい帯血および末梢血由来CD34陽性巨核球前駆細胞の放射線感受性とサイトカインの作用  [通常講演]
    柏倉幾郎, 村上美穂, 高木良成, 高橋恒夫, 高橋賢次, 稲波修, 桑原幹典
    日本放射線影響学会大会講演要旨集 2000年08月
  • 低酸素細胞増感剤エタニダゾールによるアポトーシスの増感効果  [通常講演]
    杉原潔, 稲波修, 飯田義晴, 桑原幹典
    日本放射線影響学会大会講演要旨集 2000年08月
  • MOLT‐4細胞のX線誘発アポトーシスにおけるカスパーゼ‐8ならびにカスパーゼ‐9の関与  [通常講演]
    高橋賢次, 稲波修, 桑原幹典
    日本放射線影響学会大会講演要旨集 2000年08月
  • ウシ血管内皮細胞のH2O2誘発アポトーシス誘導におけるシグナリングとそのレドックス制御  [通常講演]
    丹羽光一, 稲波修, 太田利男, 桑原幹典
    日本放射線影響学会大会講演要旨集 2000年08月
  • 酸化ストレスと細胞内シグナル伝達 MAPキナーゼとカルシウムの役割  [通常講演]
    稲波修, 太田利男, 昆泰寛, 平岡和佳子, 桑原幹典
    放射線科学 2000年04月
  • DNAおよび二本鎖polyA/polyUにおいて炭素ビームの直接および擬直接作用により誘発される損傷  [通常講演]
    桑原幹典, 稲波修, 飯田義晴, 渡辺大介, 馬嶋秀行, 村上健, 安西和紀, 小沢俊彦
    NIRS-M (National Inst. of Radiological Sciences) 2000年04月
  • ヒト末梢血由来巨核球前駆細胞の放射線感受性 (第3報)  [通常講演]
    柏倉幾郎, 村上美穂, 早瀬幸俊, 高木良成, 高橋賢次, 稲波修, 桑原幹典, 高橋恒夫
    日本薬学会年会要旨集 2000年03月
  • fMLPによるNADPHオキシダーゼ活性化とアクチン重合におけるPI3キナーゼとプロテインキナーゼCの役割  [通常講演]
    稲波修, さい玉東, 山盛徹, 丹羽光一, 永幡肇, 桑原幹典
    日本獣医学会学術集会講演要旨集 2000年03月
  • ヒト白血病由来株化細胞の放射線誘発アポトーシスにおけるカスパーゼファミリー活性化経路の解析  [通常講演]
    高橋賢次, 飯田義晴, 稲波修, 桑原幹典
    日本獣医学会学術集会講演要旨集 2000年03月
  • フリーラジカルの細胞生物学 アポトーシス伝達経路におけるフリーラジカル  [通常講演]
    稲波修, 高橋賢次, 飯田義晴, 桑原幹典
    月刊細胞 2000年02月
  • 放射線誘発アポトーシスに対する抗酸化剤の影響  [通常講演]
    稲波修, 高橋賢次, 桑原幹典
    磁気共鳴と医学 2000年
  • 牛好中球による活性酸素生成およびどん食の細胞内情報伝達経路におけるp38MAPKとPKCの役割  [通常講演]
    山盛徹, 稲波修, 永幡肇, 桑原幹典
    日本獣医学会学術集会講演要旨集 1999年09月
  • β2インテグリン欠損ウシ好中球におけるアポトーシス発現遅延  [通常講演]
    永幡肇, 近藤知子, 樋口豪紀, 黒沢隆, 稲波修, 桑原幹典
    日本獣医学会学術集会講演要旨集 1999年09月
  • ヒト白血病由来細胞の2‐クロロデオキシアデノシン誘発アポトーシスにおけるFas/Fas ligand活性化の関与  [通常講演]
    野村夕希子, 稲波修, 高橋賢次, 松田彰, 桑原幹典
    日本獣医学会学術集会講演要旨集 1999年09月
  • 光動力的作用誘発V79細胞アポトーシス‐カルシウム依存性チトクロームC遊離を引き金とするカスペース3活性化の関与  [通常講演]
    稲波修, 吉戸あすか, 高橋賢次, 平岡和佳子, 桑原幹典
    日本獣医学会学術集会講演要旨集 1999年09月
  • スピントラップ剤α‐phenyl N‐tert‐butylnitroneによる放射線誘発アポトーシス防護効果のメカニズム  [通常講演]
    高橋賢次, 稲波修, 飯田義晴, 桑原幹典
    日本放射線影響学会大会講演要旨集 1999年08月
  • H2O2によるV79細胞のカルシウム依存性SAPK/JNK活性化機構の解析  [通常講演]
    稲波修, 太田利男, 吉戸あすか, 高橋賢次, 伊藤茂男, 桑原幹典
    日本放射線影響学会大会講演要旨集 1999年08月
  • 光動力的作用誘発V79細胞アポトーシス‐カルシウム依存性チトクロームC遊離を引き金とするカスペース3活性化の関与  [通常講演]
    桑原幹典, 吉戸あすか, 高橋賢次, 平岡和佳子, 稲波修
    日本放射線影響学会大会講演要旨集 1999年08月
  • 直接・擬直接作用条件下で重粒子線照射により誘発されるDNA鎖切断前駆ラジカルの検出と同定  [通常講演]
    桑原幹典, 渡辺大介, 飯田義晴, 稲波修, 峯岸安津子, 馬嶋秀行, 村上健, 安西和紀, 小沢俊彦
    NIRS-M (National Inst. of Radiological Sciences) 1999年04月
  • 電子スピン共鳴法を用いたラクトフェリンによるマクロファージのNO生成能に関する研究  [通常講演]
    稲波修, 佐藤れえ子, 内藤善久, 桑原幹典
    日本獣医学会学術集会講演要旨集 1999年03月
  • ラット実験的中枢神経変性のMRIによる評価法の確立  [通常講演]
    高塚公章, 浅沼武敏, 稲波修, 桑原幹典
    日本獣医学会学術集会講演要旨集 1999年03月
  • catechin経口投与によるLECラット肝炎の遅延効果  [通常講演]
    浅沼武敏, 昆泰宏, 稲波修, 桑原幹典
    日本獣医学会学術集会講演要旨集 1999年03月
  • 非侵襲的生体機能測定 診断への応用 4. 核磁気共鳴スペクトロスコピー(MRS)と核磁気共鳴イメージン法(MRI)の生体機能および画像診断  [通常講演]
    桑原幹典, 浅沼武敏, 稲波修, 数坂昭夫, 入口紀男
    日本獣医学会学術集会講演要旨集 1999年03月
  • スピントラップ剤α‐Phenyl‐N‐tert‐butylnitrone(PBN)によりスナネズミ海馬に誘導されるストレス誘導タンパク質の解析  [通常講演]
    辻雅久, 稲波修, 桑原幹典
    日本獣医学会学術集会講演要旨集 1999年03月
  • 二重鎖PolyA/PolyUにおける放射線誘発ラジカルの解析  [通常講演]
    渡辺大輔, 稲波修, 沢村貞史, 小沢俊彦, 桑原幹典
    磁気共鳴と医学 1999年
  • MOLT‐4によるX線誘発アポトーシスと細胞内カルシウムの関与  [通常講演]
    高橋賢次, 稲波修, 飯田義晴, 辻雅久, 桑原幹典
    磁気共鳴と医学 1999年
  • V79線維芽細胞における酸化ストレスに対する細胞内情報伝達応答  [通常講演]
    稲波修, 高橋賢次, 吉戸あすか, 桑原幹典
    磁気共鳴と医学 1999年
  • チャイニーズハムスター肺線維芽細胞V79におけるフェオフォーバイドaの光増感作用による細胞損傷の解析  [通常講演]
    吉戸あすか, 平岡和佳子, 稲波修, 桑原幹典
    磁気共鳴と医学 1999年
  • X線誘発アポトーシスに対する脂質酸化抑制剤Troloxの照射後処理の効果  [通常講演]
    高橋賢次, 稲波修, 飯田義晴, 桑原幹典
    日本放射線影響学会大会講演要旨集 1998年11月
  • 8‐oxoguanineを含む二本鎖オリゴヌクレオチド凍結水溶液におけるγ線誘発ピペリジン感傾性塩基損傷  [通常講演]
    飯田義晴, 稲波修, 小井詰史朗, 井上英夫, 大塚栄子, 桑原幹典
    日本放射線影響学会大会講演要旨集 1998年11月
  • ヒトリンパ芽球由来MOLT‐4細胞における放射線応答とアポトーシス  [通常講演]
    稲波修, 高橋賢次, 飯田義晴, 桑原幹典
    日本放射線影響学会大会講演要旨集 1998年11月
  • 二重鎖PolyA/PolyUにおける・OH誘発ラジカルのスピントラッピング法による解析  [通常講演]
    渡辺大輔, 稲波修, 沢村貞史, 小沢俊彦, 桑原幹典
    日本放射線影響学会大会講演要旨集 1998年11月
  • 2重鎖polyA/polyU(DNAモデル)における放射線誘発ラジカルのESR解析  [通常講演]
    桑原幹典, 渡辺大輔, 沢村貞史, 稲波修, 小沢俊彦
    ESR討論会講演要旨集 1998年10月
  • ヒト白血病由来株化細胞の放射線誘発アポトーシスにおけるカルシウムの関与  [通常講演]
    高橋賢次, 稲波修, 飯田義晴, 辻雅久, 桑原幹典
    日本獣医学会学術集会講演要旨集 1998年08月
  • か粒球におけるインテグリンファミリーの活性酸素生成能に及ぼす影響  [通常講演]
    山盛徹, 稲波修, 樋口豪紀, 永幡肇, 桑原幹典
    日本獣医学会学術集会講演要旨集 1998年08月
  • ヒト白血病由来細胞における2‐クロロデオキシアデノシンのアポトーシス関連情報伝達系への影響  [通常講演]
    野村夕希子, 稲波修, 高橋賢次, 松田彰, 桑原幹典
    日本獣医学会学術集会講演要旨集 1998年08月
  • 慢性肉芽腫症とNADPHオキシダーゼ  [通常講演]
    稲波修, JOHNSON J L, PARK J W, BABIOR B M, 桑原幹典
    日本獣医学会学術集会講演要旨集 1998年08月
  • ヒトさい帯血巨核球前駆細胞の放射線感受性  [通常講演]
    柏倉幾郎, 村上美穂, 早瀬幸俊, 高木良成, 稲波修, 桑原幹典, 高橋恒夫
    日本薬学会年会要旨集 1998年03月
  • 牛か粒球粘着不全症(BLAD)の単球におけるINF‐γ/LPSによる一酸化窒素生成  [通常講演]
    稲波修, 山盛徹, 永幡肇, 犬丸茂樹, 桑原幹典, KOTAKE Y
    日本獣医学会学術集会講演要旨集 1998年03月
  • 麻酔下マウスの頭部NMRマイクロイメージング  [通常講演]
    浅沼武敏, 下川繁三, 稲波修, 桑原幹典
    日本獣医学会学術集会講演要旨集 1998年03月
  • 軽種馬ならびに重種馬の新生時期における酸化ストレスと抗酸化能  [通常講演]
    和田正一郎, 稲波修, 角田修男, 三宅陽一, 桑原幹典
    日本獣医学会学術集会講演要旨集 1998年03月
  • エネルギー代謝を阻害したC2C12筋管細胞で発生するフリーラジカルの検出  [通常講演]
    藤原俊介, 松木直章, 高野橋麻子, BOFFI F M, 稲波修, 桑原幹典, 小野憲一郎
    日本獣医学会学術集会講演要旨集 1998年03月
  • ラット肝臓におけるLPS誘発NO生成とその加齢変化  [通常講演]
    稲波修, 浅沼武敏, 桑原幹典, KOTAKE Y
    磁気共鳴と医学 1998年
  • 水和層の放射線誘発DNA損傷生成における役割  [通常講演]
    桑原幹典, 大島秀基, 飯田義晴, 稲波修
    磁気共鳴と医学 1998年
  • ヒト白血病リンパ芽球由来株化培養細胞MOLT‐4の放射線誘発アポトーシスにおけるSAPKとICEプロテアーゼの活性化  [通常講演]
    高橋賢次, 稲波修, 飯田義晴, 辻雅久, 桑原幹典
    日本放射線影響学会大会講演要旨集 1997年10月
  • 酸化ストレス誘発細胞死におけるSAPK/JNKの活性化機構  [通常講演]
    稲波修, 吉戸あすか, 高橋賢次, 平岡和佳子, 桑原幹典
    日本放射線影響学会大会講演要旨集 1997年10月
  • チャイニーズハムスター肺線維芽細胞における光増感剤フェオフォーバイドaによる細胞損傷の解析  [通常講演]
    吉戸あすか, 平岡和佳子, 高橋賢次, 稲波修, 桑原幹典
    日本獣医学会学術集会講演要旨集 1997年09月
  • 酸化ストレス誘発細胞死におけるSAPK/JNKの活性化機構  [通常講演]
    稲波修, 吉戸あすか, 高橋賢次, 辻雅久, 平岡和佳子, 桑原幹典
    日本獣医学会学術集会講演要旨集 1997年09月
  • ヒト白血病リンパ芽球由来株化培養細胞の放射線誘発アポトーシスにおけるICEプロテアーゼとSAPK/JNKの関与  [通常講演]
    高橋賢次, 掛樋真彰, 吉戸あすか, 飯田義晴, 稲波修, 桑原幹典
    日本獣医学会学術集会講演要旨集 1997年09月
  • 肝腫よう・肺腫ようマウスのマルチスライス二次元MR画像をもとにした三次元MR画像の作成と病巣部の可視化  [通常講演]
    大蔵佳織, 浅沼武敏, 山本強, 佐藤裕二, 下川繁三, 稲波修, 桑原幹典
    日本獣医学会学術集会講演要旨集 1997年03月
  • ラットにおけるLPS誘発NO生成とその加齢変化 電子スピン共鳴法を用いた評価法  [通常講演]
    稲波修, 浅沼武敏, 有川二郎, KOTAKE Y, 桑原幹典
    日本獣医学会学術集会講演要旨集 1997年03月
  • Spin Trapping法の応用 白血球粘着蛋白質欠損症ホルスタイン牛(BLAD)好中球を用いた活性酸素生成細胞内情報伝達機構の研究  [通常講演]
    桑原幹典, 郷司典子, 稲波修, 樋口豪紀, 永幡肇
    磁気共鳴と医学 1997年
  • 好中球どん食時におけるO-2ならびにOHラジカル生成とその局在に関する研究  [通常講演]
    稲波修, 桑原幹典
    家畜生化学 1996年12月
  • 牛血清オプソニンの活性発現機構  [通常講演]
    竹内由香, 塚田隆興, 麦島明香, 小島万季, 稲波修, 首藤文栄
    日本獣医学会学術集会講演要旨集 1996年08月
  • ウシ血清中の糖結合性高分子タンパク質の多様性  [通常講演]
    麦島明香, 中尾敦子, 竹内由香, 鬼柳麗子, 稲波修, 首藤文栄
    日本獣医学会学術集会講演要旨集 1996年08月
  • ウマ血清中のキチン結合性蛋白質の性状  [通常講演]
    鈴木雅子, 麦島明香, 竹内由香, 稲波修, 首藤文栄
    日本獣医学会学術集会講演要旨集 1996年08月
  • ウシ血清CRP濃度は発情により上昇する  [通常講演]
    塚田隆興, 竹内由香, 三宅陽一, 稲波修, 首とう文栄
    日本獣医学会学術集会講演要旨集 1996年08月
  • β‐catechinの脳虚血‐再潅流による細胞死への効果  [通常講演]
    渡辺康子, 稲波修, 首藤文栄, 中野昌俊, 桑原幹典
    日本獣医学会学術集会講演要旨集 1996年08月
  • NADPHオキシダーゼ活性化におけるp47phoxのリン酸化の意義  [通常講演]
    稲波修, MCADARA J K, FAUST L R P, BABIOR B M
    日本獣医学会学術集会講演要旨集 1996年08月
  • アフリカツメガエルの3種のきゅう覚受容器における糖蛋白質の比較  [通常講演]
    鈴木恵子, 谷口和之, 平秀晴, 小川和重, 稲波修, 首藤文栄
    日本獣医学会学術集会講演要旨集 1996年08月
  • 血清タンパク質の鶏卵への移行  [通常講演]
    鳥居恭司, 麦島明香, 新山雅美, 市川舜, 鷲盛精, 稲波修, 首藤文栄
    日本獣医学会学術集会講演要旨集 1996年08月
  • 牛白血球粘着不全症(BLAD)好中球の活性酸素生成異常機構  [通常講演]
    郷司典子, 桑原幹典, 佐藤文昭, 稲波修, 樋口豪紀, 永幡肇
    日本獣医学会学術集会講演要旨集 1996年03月
  • ESRの応用 (I) 新生仔ウシ好中球の活性酸素産生機構について  [通常講演]
    稲波修, 佐々木正徳, 小島万季, 岡田啓司, 首藤文栄, 桑原幹典
    磁気共鳴と医学 1996年
  • ESRの応用 (I) 多形核白血球のスーパーオキシド産生能における低分子量GTP結合蛋白質の関与  [通常講演]
    小島万季, 稲波修, 鈴木恵子, 平岡和佳子, 首藤文栄, 桑原幹典
    磁気共鳴と医学 1996年
  • ESRの応用 (II) 軽種馬(サラブレッド)血清中の脂質過酸化量とスーパーオキシド除去活性  [通常講演]
    桑原幹典, 犬養尚子, 稲波修, 三宅陽一, 角田修男, 牧与志幸, 佐藤文昭
    磁気共鳴と医学 1996年
  • ヨシ血清中のキチン結合生蛋白質の性状  [通常講演]
    中尾敦子, 竹内由香, 小島万季, 稲波修, 首藤文栄
    日本獣医学会学術集会講演要旨集 1995年09月
  • PBN(N‐tert‐butyl‐α‐phenylnitrone)による遅発性脳神経細胞死の抑制  [通常講演]
    渡辺康子, 稲波修, 桑原幹典, 谷口和之, 首藤文栄
    日本獣医学会学術集会講演要旨集 1995年09月
  • 神経細胞内指向性分子の調製  [通常講演]
    四谷芳, 小島万季, 稲波修, 首藤文栄
    日本獣医学会学術集会講演要旨集 1995年09月
  • 多形核白血球のスーパーオキシド産生系における低分子量GTP結合蛋白質の関与  [通常講演]
    小島万季, 稲波修, 平岡和佳子, 桑原幹典, 首藤文栄
    日本獣医学会学術集会講演要旨集 1995年09月
  • 乳牛の血清CRPの消長  [通常講演]
    塩川嗣章, 森松正美, 稲波修, 岡田啓司, 志賀あき郎, 首藤文栄
    家畜生化学 1995年03月
  • ウシ血清中キチン結合タンパク質の精製と分析  [通常講演]
    中尾敦子, 四谷芳, 森松正美, 岡田啓司, 稲波修, 首藤文栄
    家畜生化学 1995年03月
  • ボツリヌス菌C3の多形核白血球に対する作用  [通常講演]
    小島万季, 稲波修, 佐々木正徳, 平岡和佳子, 桑原幹典, 佐藤れえ子, 内藤善久, 駒込理佳, 首藤文栄
    日本獣医学会学術集会講演要旨集 1995年03月
  • ラクトフェリンのネコ好中球機能に及ぼす影響 1. 健康ネコの好中球機能に及ぼす影響  [通常講演]
    田中有希, 佐藤れえ子, 稲波修, 高瀬光徳, 島村誠一, 添田聡, 佐藤淳, 内藤善久
    日本獣医学会学術集会講演要旨集 1995年03月
  • 子牛多形核白血球のcytochrome b558について  [通常講演]
    佐々木正徳, 稲波修, 首藤文栄, 岡田啓司, 上野郁子, 桑原幹典
    日本獣医学会学術集会講演要旨集 1995年03月
  • ラクトフェリンのネコ好中球機能に及ぼす影響 2. 口内炎ネコの好中球機能に及ぼす影響  [通常講演]
    佐藤れえ子, 田中有希, 稲波修, 高瀬光徳, 島村誠一, 添田聡, 佐藤淳, 内藤善久
    日本獣医学会学術集会講演要旨集 1995年03月
  • 軽種馬と重種馬における血清脂質過酸とスーパーオキサイド(O2)除去作用を有する血清成分について  [通常講演]
    犬養尚子, 稲波修, 三宅陽一, 角田修男, 佐藤文昭, 桑原幹典
    日本獣医学会学術集会講演要旨集 1995年03月
  • 好中球の機能と感染防御 ネコ好中球の活性酸素産生系に及ぼすラクトフェリンならびにラクトフェリシンの効果  [通常講演]
    稲波修, 佐々木正徳, 田中有希, 佐藤れえ子, 内藤善久, 桑原幹典
    日本獣医学会学術集会講演要旨集 1994年08月
  • ラット各組織における抗酸化物質の比較  [通常講演]
    田中昭広, 稲波修, 谷口和之, 志賀ろう郎, 菅原伯, 桑原幹典
    日本獣医学会学術集会講演要旨集 1994年03月
  • フリーラジカル Nitric oxideと脳血流  [通常講演]
    稲波修, 足立健彦, さき山陽一郎, 佐藤昭夫
    神経精神薬理 1994年03月
  • 子牛の好中球のスーパーオキシド生成機能低下の解明  [通常講演]
    稲波修, 佐々木正徳, 岡田啓司, 志賀たき郎, 菅原伯, 桑原幹典
    日本獣医学会学術集会講演要旨集 1994年03月
  • 牛の好中球のスーパーオキシド生成機構について  [通常講演]
    佐々木正徳, 稲波修, 岡田啓司, 志賀たき郎, 菅原伯, 桑原幹典
    日本獣医学会学術集会講演要旨集 1994年03月
  • NOが関与する生体調節系 特に脳循環について  [通常講演]
    稲波修
    大気汚染学会講演要旨集 1994年
  • 放射線による染色体蛋白質損傷とその意義  [通常講演]
    稲波修, 桑原幹典
    放射線生物研究 1993年09月
  • 新生子牛における血清過酸化脂質とスーパーオキサイドディスムターゼ活性 子牛の酸化ストレスに対する高感受性  [通常講演]
    稲波修, 岡田啓司, 佐藤直人, 志賀たつ郎, 菅原伯, 桑原幹典
    日本獣医学会学術集会講演要旨集 1993年08月
  • 実験動物頭部MRIプローブの作製とそれを用いたラット頭部MRI  [通常講演]
    浅沼武敏, 神隆, 下川繁三, 山下匡, 笠井憲雪, 佐藤文昭, 桑原幹典, 稲波修
    日本獣医学会学術集会講演要旨集 1993年08月
  • きゅう球における一酸化窒素(NO)合成酵素の局在について  [通常講演]
    中島崇行, 稲波修, 小川和重, 谷口和之
    日本獣医学会学術集会講演要旨集 1993年08月
  • マイネルト核電気刺激による大脳皮質血流増加反応に一酸化窒素(NO)が関与する  [通常講演]
    足立健彦, 稲波修, 佐藤昭夫
    基礎老化研究 1992年09月
  • ラット中隔野刺激による海馬の血流増加反応  [通常講演]
    CAO W‐H, 稲波修, 西園寺麗, 佐藤昭夫
    自律神経 1990年06月
  • TRH投与による麻酔ラット脳血流量の増加反応  [通常講演]
    稲波修, 佐藤昭夫
    生理学研究所年報 1988年
  • OHによる核酸の損傷生成機構 ヌクレオシド,ヌクレオチド,ポリヌクレオチドにおける共通性と相違  [通常講演]
    桑原幹典, 稲波修
    放射線化学 1987年09月

その他活動・業績

特許

受賞

  • 2003年 日本獣医学会賞
  • 1993年 若手奨励賞(オクラホマ医学研究財団)

共同研究・競争的資金等の研究課題

  • がん特異的代謝システムを標的とした新規セノリティックテラピー開発のための基盤研究
    日本学術振興会:科学研究費助成事業 基盤研究(A)
    研究期間 : 2020年04月 -2025年03月 
    代表者 : 稲波 修, 平岡和佳子, 岡松優子, 滝口満喜, 平田拓, 安井博宣
  • 電子スピン共鳴分光による悪性腫瘍の酸素及びpHの三次元イメージング法の開発
    日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2019年04月 -2022年03月 
    代表者 : 平田 拓, 稲波 修, 安井 博宣, 松元 慎吾
  • 鉄介在性細胞死フェロトーシス制御による新たながん放射線治療戦略の構築
    日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2019年04月 -2022年03月 
    代表者 : 安井 博宣, 稲波 修, 平田 拓, 久下 裕司
  • 侵害受容体を介した病態痛発現メカニズムの解明と疼痛緩和への応用
    日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2018年04月 -2022年03月 
    代表者 : 太田 利男, 高橋 賢次, 稲波 修, 今川 敏明
     
    炎症や癌などの病態や刺激性化学物質によって生じる病態痛の分子基盤として、近年種々の侵害受容チャネルの関与が示唆されている。痛み刺激を受容する代表的なチャネル分子としてTRPV1及びTRPA1チャネルがあり、申請者らはこれまで、これら侵害受容チャネルに対する薬物応答やその制御作用について検討してきた。本年度は炎症性疼痛や侵害受容性疼痛の発現に重要な役割を果たしている侵害受容性TRPA1チャネルと低閾値型Caチャネルとの機能的関連性について、知覚神経細胞を用いて解析した。その結果、TRPA1チャネルが低閾値型Caチャネルを介したCaイオンによって調節性に制御されていることを明らかにした。野生型マウス及びTRPA1遺伝子欠損マウスから知覚神経細胞を単離し、細胞内Ca濃度変化を蛍光イオンイメージング法を用いて検討した。静止レベルより若干高いカリウム濃度溶液(15mM)により知覚神経細胞の細胞内Ca濃度が増加し、この反応は低閾値型Caチャネル阻害薬によって抑制され、このカリウム濃度でのCa増加に低閾値型Caチャネルの活性が関与することが分かった。更に、リアルタイムPCR及びウエスタンブロット解析により知覚神経細胞にはTRPA1に加えて、低閾値型Caチャネルが発現していることが示された。選択的TRPA1阻害薬は15mMカリウムによるCa増加反応を可逆的に抑制した。一方、TRPA1阻害薬によるこの抑制作用は、TRPA1遺伝子欠損マウス由来細胞では見られなかった。起炎性物質の適用による亜急性炎症病態モデル動物では15mMカリウムによる細胞内Ca増加反応は増大し、その作用はTRPA1の活性増加に依存していた。これらの成績から、知覚神経では、軽度脱分極反応による細胞内Ca増加により、TRPA1チャネル活性が増強されることから、両チャネルが機能的な相互作用していることが示唆された。また、両チャネルを介する作用は炎症病態における疼痛伝達の増大に寄与することが示された。
  • 老化誘導がん治療を実現する質量分析イメージング診断法の開発
    日本学術振興会:科学研究費助成事業 挑戦的研究(萌芽)
    研究期間 : 2019年06月 -2021年03月 
    代表者 : 安井 博宣, 稲波 修, 東川 桂, 孫 略, 久下 裕司
  • 質量分析イメージングとESRイメージングを用いたがん治療抵抗性代謝因子の解明
    日本学術振興会:科学研究費助成事業 挑戦的研究(萌芽)
    研究期間 : 2018年06月 -2021年03月 
    代表者 : 稲波 修, 平田 拓
     
    本研究の大きな目標はがんの微小環境を代謝を中心とした空間的・時間的な評価を可能とする方法・技術を確立することにある。当該年度の研究進捗状況としてはまず、その基礎となるインビトロでの代謝評価法を確立することである。既に申請者はATP、ADP、AMP、NADHのHPLCを用いた評価法や脂質過酸化の質量分析法を用いた評価法については既に確立していたが、酸素消費率(OCR)の評価法や癌の糖代謝にともなう細胞外酸性化速度(ECAR)については研究当初は検討を開始している最中であった。本年度はインビボで重要となる培養系でのミトコンドリアの酸素消費率とpHについて電子スピン共鳴法(ESR)を用いて中心的に研究を進めてきた。その結果、OCRについては5,9,14,18,23,27,32,36-octa-n-butoxy-2,3-naphthalocyanine (LiNc-BuO)を用いて、ECARについては2-(4-((2-(4-amino-4-carboxybutanamido)-3-(carboxymethylamino)-3-oxoproylthio)methyl)phenyl)-4-pyrrolidino-2,5,5-triethyl-2,5-dihydro-1H-imidazol-1-oxylを用いたESRを用いた方法論をヒト子宮頸癌HeLa細胞、ヒト大腸がん細胞SW480ならびにヒト肺がん細胞A549細胞を用いて開発した。更にはプレリミナリーではあるが研究分担者の平田らのグループと動物実験も開始し、担癌マウスでのECARのイメージングによる評価系をマウス頭頸部がん移植系で確立しつつある(Anal. Chem. 2018, 90, 13938-13945)。次年度はESRによるpHイメージングの生物学的意義とあわせて酸素(OCR)イメージングについても進めて行く予定である。
  • がん特異的グルタミン代謝とアセテート代謝を標的とした新規がん治療法の開発
    文部科学省:科学研究費補助金(基盤研究B)
    研究期間 : 2019年 -2021年 
    代表者 : 稲波 修
  • 超音波メカノバイオロジーによる神経軸索の伸展制御
    日本学術振興会:科学研究費助成事業 挑戦的研究(萌芽)
    研究期間 : 2018年06月 -2020年03月 
    代表者 : 滝口 満喜, 佐々木 東, 稲波 修
     
    低強度パルス超音波を利用して中枢神経の治癒力を高め、脊髄損傷治療法を開発する第1段階として、中枢神経再生に最も重要である神経細胞の軸索伸展を低強度パルス超音波で制御するための検討をin vitroで行なった。 脊髄損傷が難しい理由の1つには、ミエリン由来やグリア瘢痕由来因子による神経軸索再生阻害が知られている。そこで、本年度はミエリン由来軸索再生阻害因子としてもっともよく知られている、Nogo-Aを神経細胞に作用させ、その後に低強度パルス超音波を照射して、その影響を観察した。 その結果、1回の低強度パルス超音波照射によって神経細胞当たりの神経突起の総和が優位に長くなった。また、Nogo-Aによって萎縮した神経細胞が、1回の低強度パルス超音波照射により大きくなる傾向が認められた。これらの結果は低強度パルス超音波が神経軸索再生阻害因子の効果を減弱させ、神経細胞を賦活しうること、また神経突起を再伸展させる作用があることを示している。 本年度の研究成果により、低強度パルス超音波は神経軸索再生阻害因子存在下でも神経再生に寄与する可能性が示され、今後の展開が期待される。
  • 放射線曝露個体に最適な治療法の開発
    日本学術振興会:科学研究費助成事業 基盤研究(A)
    研究期間 : 2016年04月 -2020年03月 
    代表者 : 柏倉 幾郎, 稲波 修, 渡邉 純, 中井 雄治, 吉野 浩教, 細田 正洋, 床次 眞司, 山村 仁, 山盛 徹, 三浦 富智, 千葉 満, 岩岡 和輝
     
    平成30年度は下記の4項目を検討課題として設定した。 ① 蛍光ガラス線量計を用いた人体ファントムを作製し、シミュレーションから求めた吸収線量の妥当性を検討する。② 曝露後の個体の線量分布評価方法と、障害予測バイオマーカーへの生体内分子の活用と両者の関連性把握。③ 線量当量と放射線感受性の個体差、それに応じた放射線障害を軽減する治療法の関連性検討。④ 致死を回避した曝露個体の白血球や発がん等の観察と変動する生体内分子の探索を継続。これらに加えて、昨年度検討した国内承認薬の中ではロミプロスチム(RP)が極めて高い致死回避効果を示した事から、RPの放射線障害軽減の作用機序についてより詳細に検討する。 その結果、①での検討成果を論文化し、②の検討では線量評価マーカーの探索から、成果の一部を「放射線被ばくの検出方法」(特願2018-223161、平成30年11月29日)として特許出願した。③については酸化ストレスを制御する転写因子であるNrf2標的遺伝子の変動を解析し、RP投与個体の脾臓細胞においてFth1、Nqo1、Gclc、Gclmの発現が有意に増加することを見出した。④については、高精度質量分析計による血清成分の解析から照射後18日目に有意に増加し、RP投与個体で有意に低下する10種類のタンパク質を同定した。これらの成果の一部は、国際学術誌にて発表し(Sci Rep, 2018; Free Radical Bio Med, 2019)、さらに16th International Congress of Radiation Research(ICRR)2019 (Aug 25-29, Manchester)で発表予定である。
  • 日本学術振興会:科学研究費助成事業 挑戦的萌芽研究
    研究期間 : 2012年04月 -2014年03月 
    代表者 : 仲井 邦彦, 稲波 修
     
    福島第一原子力発電所の事故は、低レベルながら広い地域で放射性物質による環境汚染を引き起こした。低レベル汚染地区では、食品等を介した内部被ばくである。このため、低レベル汚染地区にて、出産した女性を対象に、母乳と胎盤を用いたモニタリング調査を実施した。また、陰膳による放射性化学物質の摂取の実態調査を行った。その結果、生体試料から事故由来の放射性セシウムは検出されず、陰膳のレベルも十分に低かった。これらの知見を含め、市民を対象に勉強会を開催し、リスクコミュニケーションに有用な知見を検討した。その結果、モニタリング結果の返却や勉強会は不安解消に効果があることが質問票調査からが示された。
  • 文部科学省:科学研究費補助金(挑戦的萌芽研究)
    研究期間 : 2012年 -2012年 
    代表者 : 稲波 修
  • 文部科学省:科学研究費補助金(基盤研究(C))
    研究期間 : 2010年 -2012年 
    代表者 : 永幡 肇, 樋口 豪紀, 丹治 保典, 稲波 修, 園元 謙二
     
    本研究は、乳腺感染の効果的な制御を目標に、乳腺感染の病態を把握するとともに修飾物質としての乳酸菌およびその産物としてのバクテリオシンの乳腺内投与による乳腺内への白血球誘導とその機能増強による乳腺感染の制御を意図したものである。乳腺感染牛および正常泌乳牛の白血球機能および細胞情報伝達系の解析を実施するとともに有用微生物に対する乳腺の反応性を検討した。黄色ブドウ球菌に自然感染した乳房炎牛の血液および乳腺中の白血球機能および関連遺伝子の発現解析から、乳中好中球のL-セレクチンおよびCD18発現は血液のそれらに比較して有意に低下していた。またリンパ球の表面抗原であるCD4,CD8の発現は感染分房のそれらは正常ならびに血液のそれらに比較して増加していた。感染牛の血好中球の活性酸素生成(O_2-)能およびリンパ球の幼若化反応の低下が認められた。血液および乳中からの分離白血球のサイトカイン関連遺伝子の発現:インターロイキン(IL)1-β,腫瘍壊死因子(TNF-α),IL-6,IL-8,IL-12のmRNAの増加が検出された。これらの成績から黄色ブドウ球菌による乳房炎牛では免疫抑制的な面と防御対応型の反応が進行している可能性が示唆された。また有用な修飾物質としての乳酸菌の施用条件、乳腺への施用に伴う白血球反応および感染防御の把握を目的として正常および潜在性乳房炎分房へ調整乳酸菌(食品グレード...
  • 文部科学省:科学研究費補助金(基盤研究(B))
    研究期間 : 2009年 -2011年 
    代表者 : 稲波 修, 稲葉 睦, 堀内 基広, 桑原 幹典, 山盛 徹
     
    平成22年度は初年度で確立した部位特異的スピンラベル法による2点間距離計測技術をプリオン凝集体の構造解析に適用を広げることを目的に研究を推進した。測定系の確立は遺伝的プリオン病の変異体D178Nのαヘリックス2上のT188にスピンプローブ(R1)を導入したD178N/T188R1で行い、連続波ESRにより凝集体では1.8nmであることを明らかにした。pH4.0で1M塩酸グアニジン処理すると凝集体構造を形成することはAFMによるフィラメント構造の観察により確かめられた。本年度は引き続き解析を進め、オクタリピートの最後部分でランダムコイル構造のD178N/S97R1、αヘリックス1のD178N/D144R1、αヘリックス2のD178N/R204R1とD178N/Y225R1について同様に検討した。どの場所でも凝集体のスピン間の距離は1.5nm~2.0nmの間の値が持続波ESR法で検出された。パルスESR法でも計測範囲の短距離限界を示し、周期的で規則正しい構造ではなく、凝集体中の同じアミノ酸残基の分子間の距離は1.5nm~2.0nm程度の間隔を持つランダムに凝集体を形成している可能性を示していると類推された。既に他の研究者によって電子顕微鏡像から得られたシミュレーションでは規則正しい対称構造モデルが提唱されており、オクタリピートを除いたブリオンタンパク質凝集体の構造について、同様方...
  • 文部科学省:科学研究費補助金(挑戦的萌芽研究)
    研究期間 : 2009年 -2010年 
    代表者 : 稲波 修, 滝口 満喜, 山盛 徹
     
    本研究では、p34^-サイクリンB1によるリン酸化部位である34番目のスレオニン(T)をアラニン(A)へ置換したT34A変異サバイビンのN末端に膜透過性ペプチドTAT配列を付加した融合蛋白質を作成し、腫瘍治療におけるTATペプチドによる蛋白質導入技術の有効性と、T34A変異サバイビンのcolon26マウス移植固形腫瘍モデルへの局所投与と放射線照射の併用による抗腫瘍効果について検討した。TATペプチドを付加したサバイビン蛋白質は、腫瘍組織への直接投与で、短時間に腫瘍細胞内へ導入された。X線2Gyの単独照射は、有意な腫瘍の増殖遅延を示さなかった。また、TAT融合T34A変異サバイビンの腫瘍細胞への単独投与も、有意な増殖遅延を示さなかった。一方、X線2GyとTAT融合T34A変異サバイビンの併用は顕著な増殖遅延を示した。組織学的な検索によりX線2GyとTAT融合T34A変異サバイビンの併用による抗腫瘍効果はアポトーシスの誘導によるものであることが示された。さらに、併用により腫瘍組織中の増殖細胞数の減少が観察されたことより、アポトーシス誘導は増殖細胞に特異的に起こることが示唆された。細胞周期関連蛋白質の検出から放射線照射によるG2/M期停止が示唆された。これらの結果から、TAT融合T34A変異サバイビンとX線2Gyの併用による抗腫瘍効果はX線照射で生じる細胞周期チェックポ...
  • 文部科学省:科学研究費補助金(基盤研究(A))
    研究期間 : 2007年 -2009年 
    代表者 : 稲葉 睦, 山本 雅之, 高桑 雄一, 佐藤 耕太, 安居院 高志, 大塚 弥生, 稲波 修, 渡邊 信久, 山本 雅之, 高桑 雄一, 大田 寛, 梅村 孝司, 日笠 喜朗
     
    細胞膜構造の機能的構築・形成メカニズムの解明と疾患分子病態解明への応用を目標に、「膜骨格はいかにして作られるのか」を具体的目的に据えた解析を行った。膜タンパク質AE1と先天性溶血性貧血の原因となるその変異体を利用した小胞体ERにおける足場タンパク質アンキリンとの相互作用、ERからの小胞輸送を規定する特定のアミノ酸配列の解明等により、小胞体における膜骨格ユニット形成の可能性を示した。
  • 文部科学省:科学研究費補助金(基盤研究(A))
    研究期間 : 2006年 -2009年 
    代表者 : 堀内 基広, 落合 謙爾, 瓜生 匡秀, 大島 正伸, 稲波 修, 木村 和弘, 長谷部 理絵, 宋 昌絃, 落合 謙爾, 瓜生 匡秀, 大島 正伸, 古岡 秀文
     
    DNAマイクロアレイ法を用いて、プリオン感染早期から発現が変動する宿主遺伝子の探索を行い、発現がプリオン感染早期から上昇する遺伝子の病態機序への関与を解析した。その結果、LPSレセプターとして知られるCd14分子が病態進行に関与すること、神経保護作用を有するケモカインとして知られるCxcl10が病気の進行に抑制的に働くことを明らかにした。また、ミクログリアの活性化が病態進行に抑制的に働くことが示唆された。
  • 文部科学省:科学研究費補助金(基盤研究(B))
    研究期間 : 2006年 -2009年 
    代表者 : 太田 利男, 今川 敏明, 稲波 修, 今川 敏明, 稲波 修
     
    侵害受容体であるバニロイド受容体(TRPV1)遺伝子をブタ及びヒトからクローニングし、HEK293細胞へ異所性発現させ、本受容体の機能解析のためインビトロ定量系を確立した。この定量系を用いた結果、バニロイド受容体はカプサイシン、熱及び酸で活性化するのみならず、細胞外ナトリウムによっても制御を受けていることを明らかにした。また、炎症性メディエーターであるセロトニンやヒスタミンによる痛覚過敏の発現にはバニロイド受容体の過活性化が関与していることを示した。マスタードオイルやメチルサリチル酸はバニロイド受容体に対する新規アゴニストとして作用すること、また後者はこの受容体を脱感作させることにより鎮痛作用を示すことを見出した。これらの結果から、バニロイド受容体が痛覚過敏発現に重要なターゲット分子であることが明らかになった。
  • 文部科学省:科学研究費補助金(基盤研究(B))
    研究期間 : 2007年 -2008年 
    代表者 : 稲波 修, 平岡 和佳子, 下山 雄平, 稲葉 睦, 堀内 基広, 桑原 幹典, 浅沼 武敏, 平岡 和佳子, 下山 雄平
     
    本研究はブリオン病の原因であるブリオンタンパク質の凝集体変化を新しい方法を用いて明らかにし、今後のプリオン病の病態解明や治療薬の評価方法を開発することを目的とした。このプリオン凝集体への変化を起こすには細胞内低pH器官であるエンドソームで起きることから、pHの低下が必須であると考えられていることからpH7.0の生理条件からpH4.0に変化させたときの構造変化を明らかにした。電子スピン共鳴法、ならびに原子間力顕微鏡を用いて家族性プリオン病でよく見られる変異体プリオンD177NはpH4.0、変性下に置くことで変異を持っていない野生型よりも効率よく線維状の凝集体が起きることが示され。凝集の引き金となる領域がタンパク中心部のβシート付近にあることが明らかとなった。今回の結果はプリオン病の原因タンパク質の構造変化を明らかにしただけでなく、今回用いた新たな方法はプリオン関連病の治療薬の評価系に用いることが可能であると考えられる。
  • 文部科学省:科学研究費補助金(基盤研究(B))
    研究期間 : 2006年 -2008年 
    代表者 : 柏倉 幾郎, 高橋 賢次, 稲波 修, 阿部 由直, 稲波 修
     
    放射線非照射及び照射CD34陽性細胞(ヒト造血幹・前駆細胞)を、臍帯血由来間葉系幹細胞様ストローマ細胞と共培養することにより、サイトカイン単独での培養に比べ有意に高い細胞増殖及び未分化維持といった造血支持能が示された。サイトカインを共培養開始16 時間後に添加すると、照射細胞はストローマ非存在下培養では著しく造血が低下したが、共培養では同時添加した場合と同等の造血が認められた。この時、共培養上清中のヒアルロン酸は顕著に増加し、一方硫酸化グリコサミノグリカンはストローマ非存在下放射線照射細胞単独培養で有意に増加した。同様に幾つかのサイトカイン産生も放射線照射細胞単独培養で増加した。以上の結果から、間葉系幹細胞様ストローマ細胞は放射線曝露ヒト造血幹・前駆細胞の造血再生に有用であることが明らかとなった。この時、造血幹/前駆細胞とストローマ細胞との接触刺激が重要であると共に、細胞外マトリックス成分産生が大きく関与している可能性が示唆された。また、本研究の関連成果が2 件の特許出願に繋がった。
  • 文部科学省:科学研究費補助金(萌芽研究)
    研究期間 : 2006年 -2007年 
    代表者 : 稲波 修, 太田 利男, 下山 雄平
     
    マウス組み換えプリオンタンパク質を作成し、部位特異的にN96、Q97、W98、T106、V111、D144およびT190をシステイン残基に改変した変異タンパク質を調製した。これら変異プリオンタンパク質にスピンプローブと反応させ部位特異的ラベルを行った。得られた試料はESR測定を行った。N96C、Q97C、W98C、T106CならびにV111Cから得られたESRスペクトルは、Cu2+添加により、常磁性金属とNオキシルとの相互作用による線幅の広がりによるシグナル強度の減弱が観察された。さらにRedfield theoryを用いることによりCu^<2+>とN96C、Q97C、W98C、T106CならびにV111CCのスピンプローブとの距離はそれぞれ9.3A、11.9A、12.7A、9.2Aならびに12.5Aであると計算された。この距離情報からH95とH110のヒスチジン残基にCu^<2+>が配位していることが強く示唆された。また、C末側のCu^<2+>結合部位を同定するためにY156ならびにT189にスピンプローブを導入し、Cu^<2+>との距離を同様に計測したところ、それぞれの距離は6.8Åならびに11.6Åであった。この距離情報からH186にCu^<2+>が配位している可能性が強く示唆された。こうしたCu^<2+>が配位した蛋白質の構造についてはNMRやX線解析が適用できないこ...
  • 文部科学省:科学研究費補助金(基盤研究(C))
    研究期間 : 2005年 -2007年 
    代表者 : 永幡 肇, 稲波 修, 桐沢 力雄, 林 正信, 桑原 幹典
     
    乳牛における乳房炎の発症機構の解析と効果的な制御を目的として、感染防御に重要な役割を担う白血球の機能を解析するとともに、展開として免疫機能への刺激増強に関与する候補物質を用いて機能修飾を検討し以下の成績を得た。乳腺感染の免疫学的応答について、正常牛および黄色ブドウ球菌(SA)感染牛を用いて、血液および乳腺中の白血について表面抗原の解析および刺激に対する応答性を評価した。主な特徴としては、SA感染により白血球に発現する表面抗原分子(CD18,L-selectin、CD4&8)は正常対照のそれらに比較して機能対応型の変化とその応答性が示された。また好中球のFcR受容体を介する酸素代謝能およびリンパ球の幼若化能の低反応性が示されたことから、感染に伴う防御機構の低下が示唆された。炎症に付随するサイトカイン関連遺伝子(IL-1β,TNF-α,IL8)の増高が認められた。好中球の異物排除に関与する補体(CR3)と免疫グロブリン(IgG)に対する受容体(IgG-FcR)への刺激に伴う活性酸素生成とその情報伝達系への解析から、共刺激に伴うリン酸化および機能増強が正常対照で認められた。免疫機能修飾の上から、候補物質(lactoferrinとその分解産物、数種のprobiotics)に対する乳腺の機能的応答性、関連遺伝子発現、異物排除活性の検討からその特性を評価し効果的な炎症制御に沿った可能性の...
  • 文部科学省:科学研究費補助金(基盤研究(B))
    研究期間 : 2005年 -2006年 
    代表者 : 稲波 修, 堀内 基広, 苅和 宏明, 稲葉 睦, 桑原 幹典
     
    BSEやCJDなどのプリオン病では、構造異常を呈する異常型プリオンタンパク質(PrPSc)が正常型PrPCと相互作用し、その高次構造を異常型に変換させ、病態を引き起こすと考えられている。このPrPの構造変化機構には、酸性エンドソームが関与していると考えられている。本研究ではsite-directed spin labeling (SDSL)法を用い、αヘリックス1(H1)の裏側、βシート1(S1)とβシート2(S2)の3つのpH感受性領域をESRによる解析で見出した。近年、molecular dynamics (MD) simulationを用いた研究により、酸性pHが引き起こすPrPSc様のβ-sheet-rich構造への転換にアスパラギン酸177(Dl77)とヒスチジン186(H186)が重要な役割を持つと報告されたが、実際には実験的な証明はなされていない。本研究ではさらにD177とR163が形成する塩橋とH176とH186の2つのヒスチジン残基について、β-sheet2(S2)のpH感受性に対する影響をSDSL-ESR法で解析した。その結果、D177が形成する塩橋の形成がS2の中性付近でのβシート構造の維持に重要であることが明らかとなったが、近傍のH176の存在がこの構造安定性にも重要であることが見いだされた。また、H186のCJD変異についてはS2の構造安定化には寄与...
  • 文部科学省:科学研究費補助金(萌芽研究)
    研究期間 : 2005年 -2006年 
    代表者 : 桑原 幹典, 稲波 修, 平岡 和佳子, 浅沼 武敏
     
    近年、ヒトにおける痛み応答に関与する部位には、PETとfMRIによる研究から、第二体性感覚野、島皮質、anterior cingulated cortex(ACC:前帯状皮質)、反対側の第一体性感覚野および視床部が関与する事が知られるようになった。興味あることに、allodyniaなどの異常な痛みに対してACC領域以外の領域では血流量やfMRI-BOLD信号が上昇するのに対してACC領域では血流量やBOLD信号が低下することがある。さらに、ACC領域では痛みの強さや場所に関連した血流量の変化は起こらず、痛みによる不快感や嫌悪感などの「感情」に関する応答が起こる事が知られている。本研究では、ヒトACCに該当するラット脳cingulate cortex(CG)領域におけるホルマリン刺激に対する痛みに対する評価を刺激後24分間と比較的長く観察することにした。ホルマリン刺激を行った全ての個体において、第一体性感覚野領域のBOLD信号は、刺激直後から2分間の間にピークを迎えていた。その後、観察された24分後までBOLD信号は低下することなく4-5%程度のBOLD信号の増加を示していた。興味あることに、CG領域での時間変化は実験群の半数以上において、ホルマリン刺激後1分半程度にピークを示し、9%程度の信号上昇が観察され、刺激後信号が2%以下に一度低下した。その後、刺激後16分から再度信号...
  • 文部科学省:科学研究費補助金(基盤研究(A))
    研究期間 : 2004年 -2006年 
    代表者 : 稲葉 睦, 山本 雅之, 陰山 聡一, 堀内 基広, 稲波 修, 梅村 孝司
     
    1)AHSP遺伝子の5'上流領域プロモーター領域の解析から、赤芽球系分化誘導時には、翻訳開始点上流-328〜-286bpに位置する3カ所の転写因子GATA結合モチーフがAHSPの転写活性化に必要な最小要素であることが明らかになった。EMSA解析等の結果を加え、赤芽球系前駆細胞におけるAHSPの転写にはGATA-1の作用が必須なことが明らかになった。同時に、PrP^侵襲によるAHSPの発現抑制には、GATA-1による転写制御が関連することが推定された。2)プリオン感染マウス由来脳乳剤、あるいはPrP^持続感染神経細胞株ScN2a存在下にMELhide8細胞を培養し、AHSP等の発現を解析した。しかし、赤芽球系分化誘導時、AHSPをはじめ、α-グロビン、β-グロビン、GATA-1、EKLF、NF-E2などの転写にはいずれも何ら影響が認められず、また、長期継代後にもMELhide8細胞にはPrP^の蓄積が全くみられなかった。これらの結果から、PrP^はMELhide8細胞に対する直接作用をもたないことが明らかになった。3)MELhide8細胞におけるAHSP mRNA発現量、ならびに上記GATA依存性のAHSPプロモーターレポーター遺伝子発現は、IL-6の添加で濃度依存性に抑制された。IL-1βにも類似作用が認められたが、その効果はIL-6に比して低...
  • 文部科学省:科学研究費補助金(基盤研究(B))
    研究期間 : 2004年 -2006年 
    代表者 : 河原 剛一, 米山 満, 稲波 修, 内貴 猛, 山内 芳子
     
    生体には呼吸や心拍など多くのリズム現象が存在している。生体を器官、組織、細胞などから構成される階層性を有するシステムとして捉えると、その最小機能単位である細胞においても様々なリズム現象が認められる。本研究では、細胞全体、細胞質内およびオルガネラであるミトコンドリア・クリステ内という、異なった階層において認められる複数リズム間相互作用、それらリズムの協調と破綻のメカニズムの解明を目指した。研究成果は以下のようにまとめられる。(1)培養開始から4日目(4DIV)の心筋細胞の拍動リズムは細胞間で同期しており、細胞内Ca^<2+>振動も細胞間で同期していることを明らかに出来た。(2)物理的に接触が認められない心筋細胞間でも心筋細胞間での細胞内Ca^<2+>振動は同期していることを明らかに出来た。(3)細胞内Ca^<2+>振動の心筋細胞間同期は、ギャップ結合をDSAやheptanolによって薬理的に阻害しても持続すること、およびBDMで拍動を完全に停止させても持続することが分かった。(4)P2 purinoceptorの阻害薬であるsuramin付加によって、細胞内Ca^<2+>振動の心筋細胞間同期が崩壊することが分かった。(5)以上のことから、細胞内Ca^<2+>振動の心筋細胞間同期に細胞外情報伝達系としてのATP-puriniceptor系が関与している可能性を明らかに出来た。(6...
  • 文部科学省:科学研究費補助金(基盤研究(C))
    研究期間 : 2004年 -2006年 
    代表者 : 佐藤 れえ子, 寺口 進, 内藤 善久, 安田 準, 稲波 修
     
    1)ラクトフェリン(LF)の経口投与がFIV感染ネコのリンパ球機能に与える影響健康ネコと各ステージのFIV感染ネコの末梢血リンパ球の幼若化能にLFが及ぼす影響を検索した.LHは経口投与では、感染ネコならびに健康ネコの両方で、そのリンパ球幼若化能を低下させた.次ぎに分離した末梢血リンパ球にLFを添加培養した場合には、感染ネコと健康ネコの両方で経口投与の場合と同様に、その幼若化能を抑制することが明らかとなった.2)ConAおよびウシLFがネコ末梢血単核球におけるサイトカインmRNAの発現に及ぼす影響分離したFIV陰性ならびに陽性ネコ末梢血単核球にConAを添加後、IFN-γ、IL-1β、TNF-αおよびIL-12 p40 mRNAの発現を半定量的RT-PCR法にて測定した。ConA刺激4時間後に初めてIFN-γ mRNAの強い発現の誘導が認められたが、IL-1β、TNF-αおよびIL-12P40mRNA発現は、ConAによって増強されることはなかった。次に、上述の半定量的RT-PCRの結果を確かめるために、ConA誘導IFN-γ mRNAの発現の定量を行うリアルタイムRT-PCR法を確立した。FIV陰性ネコ群においても、ConA刺激によるIFN-γの発現誘導は全ての個体の末梢血単核球において観察された,またConA刺激後のさまざまな時間にウシLFを末梢血単核球へ添加したところ、C...
  • 文部科学省:科学研究費補助金(萌芽研究)
    研究期間 : 2004年 -2006年 
    代表者 : 太田 利男, 稲波 修
     
    神経や組織の炎症等による傷害後に発生するモルヒネ抵抗性の難治性慢性疼痛として知られる神経因性疼痛は、その発生メカニズムが未だ十分に解明されていない。炎症時には様々な炎症性内因性物質が放出され、これが痛覚過敏を引き起こすと考えられている。そこで本年度は、炎症メディエーターであるセロトニンによる疼痛過敏機序について調べ、以下の成績を得た。セロトニンは5HT2Aおよび5HT7受容体を活性化させ、Aキナーゼ、Cキナーゼを介して、TRPV1受容体を活性化(感作)することが分かった。また、アジュバンドにより惹起した炎症ラットの後根神経節細胞では5HT2A及び5HT7受容体遺伝子の発現量が増加することを見出した。これらの結果より、炎症性痛覚過敏の発生は、炎症時に血小板や肥満細胞から放出されるセロトニンがTRPV1分子の活性化、あるいはセロトニン受容体サブセットの発現量増大が関与していることを明らかにした。
  • 文部科学省:科学研究費補助金(基盤研究(B))
    研究期間 : 2003年 -2005年 
    代表者 : 太田 利男, 稲波 修
     
    本研究の目的は、胃腸管内在ニューロンにおける容量性Ca流入機構の性質とその制御メカニズムを解明することである。そのため、初代培養した内在ニューロンを用いて、ストア内Ca濃度に影響を与える薬物によって生じるCa流入の性質やそのキネティクス解析、細胞内メッセンジャーによる制御及びCa流入経路としてのイオンチャネル分子の同定を試み、以下の成績を得た。1)新生ラット内在神経の初代培養系を用いて、細胞内Caシグナルを可視化する実験系を確立した。2)ATPは細胞内ストアからのCa遊離、容量性Ca流入及びP2X2受容体を介した[Ca^<2+>]i増加反応を引き起こすことを明らかにした。4)ブラジキニンによる[Ca^<2+>]i増加反応はB2受容体を介した細胞内CaストアからのCa遊離に加えて、容量性Ca流入機構が関与することが示された。また、その反応にはグリア細胞からのPGE2放出が関与していることを見出した。5)PC12細胞に発現させたTRPC5蛋白質はGTP結合型受容体と関連したイオンチャネルを形成することを明らかにした。6)新規Ca流入チャネルであるTRPV1を遺伝子クローニングし、HEK293細胞へ発現させ機能解析を行った。TRPV1蛋白質はバニロイドアゴニスト、酸および熱により活性化されるポリモーダル受容体チャネルであることが示された。本研究により壁内神経細胞における容量性Ca流...
  • 文部科学省:科学研究費補助金(基盤研究(B))
    研究期間 : 2003年 -2004年 
    代表者 : 稲波 修, 佐藤 基佳, 稲葉 睦, 桑原 幹典, 浅沼 武敏, 太田 利男
     
    本研究は乳房炎が頻発する産後直後の免疫機能低下の原因を好中球機能を中心に検索するとともに、乳房炎機能を明らかにすることを目的とする。そのためにウシ好中球機能の活性化のためのシグナル伝達機構の解明を行い、周産期での好中球機能さらには母胎側の好中球機能に影響を与える因子の同定と機能を明らかにする。本年度は(1)好中球のNADPHオキシダーゼを制御するシグナル伝達機構として現在まで本研究で同定したcPKC、p38MAPKやERKなどのMAPK、さらにPI3Kの関与に加えて、novel PKC (nPKC)に属するPKCdeltaの機能について検討した。(2)また、昨年に引き続き、胎盤や乳汁中に好中球機能に変動を与える因子が存在しないかどうか検索することを試み、新たなNADPHオキシダーゼ発現に抑制的な作用を持つタンパク質の同定を試みた。(3)新たなNADPHオキシダーゼ発現抑制タンパク質の一つとしてラクトフェリンが同定され、ウシラクトフェリンは乳汁中に多量に含まれることから、その機能検索を進め、ラットマクロファージと単球を用い、LPSあるいはConA刺激によって誘導されるINF-γ、IL-1β、TNF-α、IL-12p40ならびにiNOSのmRNA発現に及ぼす影響を検討した。その結果、(1)PKCdelta阻害剤rottlerinを処理すると、OZによるウシ好中球のNADPHオキシ...
  • 文部科学省:科学研究費補助金(基盤研究(B))
    研究期間 : 2002年 -2004年 
    代表者 : 永幡 肇, 林 正信, 桐沢 力雄, 稲波 修, 桑原 幹典
     
    周産期乳牛の生体防御機構の解析と機能修飾に関する研究を実施し以下の成績を得た。(1)周産期乳牛の好中球化学発光反応およびp38MAPK活性より、分娩時の好中球CR3およびFcrR刺激反応は有意に低下しており、細胞の情報伝達系と密接に関連していることを明らかにした。(2)白血球機能に及ぼすサイトカイン1L-1β,1L-6,TNF-αの影響1L-1β,TNF-αは好中球の活性酸素生成およびT-リンパ球を活性化し、アポトーシスを早期に誘導することを認めた。刺激に伴う好中球のアポトーシス,アネキシンV発現、細胞内[Ca^<2+>]、濃度およびCaspase-3活性の関連をウシ好中球で明らかにした。(3)好中球のCR3-FcrRの共刺激に伴う機能と情報伝達解析により、共刺激により、O_2^-生成および伝達系(リン酸化)の増強を明らかにした。同産期好中球では、その反応が低率化することを認めた。(4)乳牛へのビタミンE・Se投与と細胞濃度への影響。血清E・Seの分娩時の低下および投与により血清Seは48時間後、Eは6時間でピークに達した。抗酸化活性は投与後1〜3週間で有意に高値を示し機能修飾が認められた。(5)ウシ乳腺へのラクトフェリン(Lf)投与により、白血球機能および乳腺の感染防御効果の向上が示唆された。
  • 文部科学省:科学研究費補助金(萌芽研究)
    研究期間 : 2002年 -2003年 
    代表者 : 稲波 修, 太田 利男, 桑原 幹典
     
    PBNはスピントラップ剤として開発されたニトロン系化合物であるが、NGFなどの神経栄養因子と類似したPC12細胞に対する神経様突起誘導能を持っていることが既に報告されている。本研究では、他のスピントラップ剤にも類似の効果があるか、さらにはこのニトロン化合物の神経突起誘導のメカニズムを明らかにすることを目的に研究を行った。現在まで著者らはこのPBNによるPC12細胞の神経様突起形成のメカニズムは、TrkAとShcの活性化、Grb2のShcへの結合を引き起こさないが、Rasのレベルで活性化することによることを示している。本研究は、PBNの誘導体であるPOBN、S-PBNとDMPOについてその分化能を評価し、さらにRasのシステイン残基がPBNの標的になっているのではないかと考え、RasのCys-51、Cys-80、Cys-118,Cys-181ならびにCys-184をそれぞれセリンに改変したほ乳類細胞発現ベクターを用いた過剰発現系で評価した。いくつかのスピントラップ剤を用いてPC12細胞の神経様突起形成能を比較したところ、PBN>POBN>S-PBNの順で神経様突起形成能が強い傾向にあることを示し、DMPOは全く活性を示さなかった。このことは、用いるトラップ剤が疎水性であるほど神経様突起形成能が強い傾向にあること示唆している。また、Ras(WT)を過剰発現させたPC12細胞ではP...
  • 文部科学省:科学研究費補助金(基盤研究(B))
    研究期間 : 2002年 -2003年 
    代表者 : 稲葉 睦, 滝口 満喜, 稲波 修, 前出 吉光, 山本 雅之, 高桑 雄一, 斉藤 昌之
     
    本研究は、赤芽球系細胞における赤血球膜骨格組み込みの"ユニット工法仮説"実証を目的とする。骨髄赤芽球系細胞、赤芽球系培養細胞を用い、遺伝子導入とタンパク質の生化学的・形態学的解析で以下の成果を得た。●赤芽球系培養細胞K562を用いて、膜骨格と相互作用する主要膜タンパク質、バンド3、グライコフォリンC、アンキリン、プロテイン4.1、ならびにそれらの変異体の安定発現系を構築し、細胞内局在と赤芽球分化誘導時の挙動や細胞分化・増殖に及ぼす影響を検討した。膜内在性であるバンド3とグライコフォリンCはともにER/Golgiを経て細胞膜に移行した。これらと膜骨格を連結すると考えられるアンキリンとプロテイン4.1も同様にERとGolgiに局在が認められ、これは赤芽球系分化誘導後にも変化しなかった。また、内因性のアンキリンやプロテイン4.1、さらにスペクトリンも主にER/Golgiに分布が認められた。したがって、膜骨格組み込みは赤芽球系細胞の細胞膜ではなく、ERで生じる現象であることが明らかになった。●同様の解析で、K562細胞におけるバンド3分子の恒常的発現により細胞増殖速度が約70%に低下すること、誘導剤による赤芽球系分化速度も低下する現象が見出された。しかし、誘導可能な赤芽球までのステージでは、細胞骨格タンパク質の局在や細胞形態に対する影響は認められなかった。●骨髄赤芽球系前駆細胞ではス...
  • 文部科学省:科学研究費補助金(基盤研究(B))
    研究期間 : 2001年 -2002年 
    代表者 : 稲葉 睦, 小野 憲一郎, 印牧 美佐生, 小川 博之, 今川 和彦, 小林 正人, 前出 吉光, 稲波 修
     
    A.肉質との連鎖に関する解析1.前年度に、バンド3欠損症原因遺伝子異常保因と肉質との連鎖について、脂肪酸不飽和度との関連性が示唆された。これに関してさらに詳細に検討した結果、直接の連鎖の存在は否定された。腎尿細管形成不全症(CL-16欠損症)についても明確な連鎖は認められなかった。さらに、両遺伝子とも近傍領域における直接肉質に関連すると考えられる遺伝子の存在は明確ではなかった。B.疾患分子病態の解析上記のように、疾患遺伝子異常と産肉形質の直接の連鎖が否定されたことは、疾患病態が産肉形質のうえからは良好な効果をもたらす可能性が示唆するものであり、詳細な分子病態の解明が今後の育種戦略策定に必須となる。1.バンド3欠損症については、バンド3導入培養細胞を用い、この細胞における細胞内pHの低下が生じる(細胞内酸性化に機能する)ことをとおして細胞外液pH変化を生じることを実証した。したがってバンド3の欠損、あるいは部分欠損は体液の酸塩基平衡を酸性化に傾け、体細胞代謝に影響するものと考えられる。2.CL-16欠損症については、そのバリア機能を中心に検討した。MDCK細胞でCL-16発現系を構築し、その局在と傍細胞輸送への影響を、蛍光抗体法、電気抵抗測定で解析した。CL-16は細胞膜の細胞同士が隣接する部位、特にタイトジャンクション領域に分布し、発現量増加に伴い、培養細胞モノレイヤーの電気...
  • 文部科学省:科学研究費補助金(萌芽的研究, 萌芽研究)
    研究期間 : 2001年 -2002年 
    代表者 : 桑原 幹典, 佐藤 れえ子, 稲波 修
     
    本研究はヒトエイズのネコのホモログであるFIVにおいて、FIVのウイルス増殖の際生じる活性酸素の上昇に呼応して活性化するNFκBなどの転写因子にレドックス制御が関与するかどうか、抗酸化物質で抑制されるのか、さらには、それが、実際の疾患に対して、抗酸化物質が治療効果を増強できるのかを明らかにする目的で基礎的な検討を行った。まず、酸化ストレスによって転写因子NFκBやAP-1などの活性化のアッセイ系を確立し、抗酸化剤の効果をインビトロで評価できる確立した。モデル系として牛血管内皮細胞とMOLT-4細胞を用いて、過酸化水素処理あるいは放射線照射による転写因子NFκ-BとAP1の活性活性化のゲルシフトアッセイ法をもちいて検討した。。その結果、過酸化水素1mM処理あるいは放射線照射でスーパーシフトしたバンドがそれぞれ観察され、抗NFκB抗体あるいは抗c-Jun抗体処理で消失した。また、これら転写因子活性化に対する抗酸化剤の効果ではαフェニルブチルニトロン(PBN)が酸化ストレス誘導AP-1の活性化を抑制することが明かとなった。更に、RAWマクロファージ細胞でのLPS誘発NFκ-Bの活性化において、同じ抗酸化物質であるカテキンでは影響を受けず、LPS誘発AP-1がSAPK/JNKの活性化のレベルで抑制されることにより、活性化抑制を起こしていることを示しており、抗酸化物質の種類によってレド...
  • 文部科学省:科学研究費補助金(基盤研究(B))
    研究期間 : 2000年 -2002年 
    代表者 : 昆 泰寛, 岩永 敏彦, 稲波 修, 遠藤 大二
     
    ホルモンは生体の生理活性物質としてごく微量でその機能を具現化する。レニン-アンギオテンシン系(RAS)は血圧調節と水・電解質代謝の調節に働く。RASのトリガー的酵素であるレニンは主に腎臓から分泌されるが、他組織にも存在し、従来考えられなかった調節系の存在が報告されている。本研究の目的は、局所免疫機構に関与するホルモンシステムを特にRASを中心に解明することにある。レニンノックアウトマウスの形態学的観察を行い、以下の結果を得た。ホモ個体の腎臓は種々の程度に水腎症の形態を呈し、腎臓内血管は内皮細胞の肥厚と、平滑筋細胞の増殖を特徴とした。血圧は著しく低下していた。腎臓内血管壁には神経が増加していた。しかし、腎臓外レニン産生臓器、特に外分泌組織に顕著な形態学的変化は認められなかった。これらのことから、レニンは腎臓の形態形成には必須の因子であるが、腎臓外においては未知の因子が代償することが考えられた。免疫系に関しては、レニンをノックアウトすることによって胸腺萎縮を示す例も観察されたことから、免疫系に対し何らかの作用がある可能性が伺われた。さらに、精巣における局所免疫防御に関連する因子としての一酸化窒素(NO)に着目し、その合成酵素であるiNOSの発現を免疫組織化学的ならびに分子生物学的に観察した。その結果、iNOSは微小精巣環境とくに血液-精巣関門を構成する一因子としての役割を果たすも...
  • 文部科学省:科学研究費補助金(特定領域研究(A))
    研究期間 : 2001年 -2001年 
    代表者 : 稲波 修, 下山 雄平, 桑原 幹典
     
    NADPHオキシダーゼは好中球に存在し、スーパーオキサイドを生成する酵素として知られている。このNADPHオキシダーゼは感染防御にとって非常に重要であると認識されており、細胞膜に局在するp22^とgp91^、細胞質に局在するp47^、p67^ならびにracから構成されることが知られている。活性化に際しては、FcRやFMLP刺激によって活性化したprotein kinase C(PKC)がp47^のC末端領域のリン酸化を促し、リン酸化p47^の二つのSH3ドメイン(SH3(N),SH3(C)とプロリンリッチ領域(PRR)が、それぞれp22^とp67^と結合し、膜に会合し、活性を表すと考えられている。本研究では、システイン残基をターゲットとした3-malemide-proxylをスピンプローブとして用いたスピンラベル法を用い、リン酸化に伴うp47^の構造変化を解析した。wild typep47^のESRスペクトルはラベルされたN-oxylの早い運動を示すunimmobilizedコンポーネントと運動の抑制を示すimmobilizedコンポーネントが観察された。C378Aの変異蛋白ではunimmobilizedコンポーネントのみが消失した。またC196Aの変異蛋白...
  • 文部科学省:科学研究費補助金(特定領域研究(C))
    研究期間 : 2001年 -2001年 
    代表者 : 稲波 修, 桑原 幹典
     
    ガン細胞特異的にRNA合成阻害起こすことで細胞死を起こすようにデザインされた新規ヌクレオシド系抗がん剤エチニルシチジン(ECyd)が放射線増感作用のメカニズムを調べる目的で本研究は行われた。細胞はp53野生型のヒト胃腺ガン細胞由来MKN-45細胞ならびにp53変異型のチャイニーズハムスター肺線維芽細胞由来V79細胞を用いて検討した。両細胞とも放射線照射単独ではアポトーシスを引き起こさないが、ECydをそれ単独では細胞毒性を示さない濃度のECyd存在下で照射するとアポトーシスを効率よく誘導できることが明らかとなった。このアポトーシス誘導はカスパーゼ阻害剤Z-VAD-FMKならびにキモトリプシン様セリンプロテアーゼ阻害剤TPCKで有意に阻害された。また、細胞周期との関連をフローサイトメトリー法で解析したところ、放射線照射のみではG2期停止が両細胞で起きるのに対して、ECyd存在下ではこのG2期停止機構が破壊され、G2期からアポトーシスが誘引されていることが明らかとなった。以上の結果は細胞のp53のステータスに関わらず、ECydは放射線によるアポトーシスによる細胞死を増感し、そのメカニズムとしてG2期停止の解除によってカスパーゼならびにキモトリプシン様プロテアーゼが関与していることが示された。また、メカニズムとしてどのようにネクローシス様細胞死をアポトーシスによる細胞死に変換するに...
  • 文部科学省:科学研究費補助金(基盤研究(C))
    研究期間 : 2000年 -2001年 
    代表者 : 稲波 修, 滝口 満喜, 松田 彰, 桑原 幹典
     
    本研究は、アポトーシスによる細胞死を効率的に引き起こすことを目指したヌクレオシド系増感剤を開発し、獣医領域で問題となるネコ白血病、イヌリンパ腫などの治療のための制がん剤の開発を目的とする。モデル細胞としてヒト胃癌細胞MKN-45細胞とMOLT-4細胞の制がん剤による細胞死の機構を検討した。制がん剤としては1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine(Ecyd)と2'-chrolo-deoxyadenosine(Cl-dAde)を化学合成し、それぞれ検討した。その結果、両細胞ともそれぞれの制がん剤でカスパーゼファミリーの活性化を伴うアポトーシスを効率よく引き起こすことが明らかとなった。そのメカニズムには細胞によって差があり、MKN-45細胞についてはp53依存性のアポトーシスであり、MOLT-4の場合はp53非依存性、Fas活性化依存性のアポトーシスであることが明らかとなった。MKN-45細胞の場合は、蛋白合成に非依存性のカスパーゼ3依存性のアポトーシスを、MOLT-4細胞の場合は、アポトーシス誘導に蛋白質合成を必要とし、FASの活性化、カスパーゼ8を活性化し、カスパーゼ3に依存するアポトーシスを引き起こすことが明らかとなった。この結果は、細胞の種類に依存してアポトーシスを引き起こすシグナル経路の違いを意味している。また、...
  • 文部科学省:科学研究費補助金(基盤研究(B))
    研究期間 : 2000年 -2001年 
    代表者 : 桑原 幹典, 太田 利男, 松田 彰, 稲波 修, 岡田 幸助, 佐藤 れえ子
     
    本研究は、アポトーシスによる細胞死を効率的に引き起こすことを目指したヌクレオシド系増感剤を開発し、獣医領域で問題となるネコ白血病、イヌリンパ腫などの治療のための制がん剤の開発を目的とする。本年度は、計画に用いるモデル細胞としてヒト胃癌細胞MKN45細胞、チャイニーズハムスターV79細胞ならびに白血病由来細胞MOLT-4細胞の制がん剤による細胞死の機構を検討した。制がん剤としては1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine(Ecyd)と2'-chrolo-deoxyadenosine(Cl-dAde)を化学合成し、それぞれ検討した。その結果、両細胞ともそれぞれの制がん剤でカスパーゼファミリーの活性化を伴うアポトーシスを効率よく引き起こすことが明らかとなった。そのメカニズムには細胞によって差があり、MKN45細胞についてはp53依存性のアポトーシスであり、MOLT-4の場合はp53非依存性、Fas活性化依存性のアポトーシスであることが明らかとなった。また、MKN-45細胞の場合は蛋白合成非依存性でカスパーゼ3依存性アポトーシスであること、MOLT-4細胞の場合は、アポトーシス誘導に蛋白質合成を必要とし、FASの活性化、カスバーゼ8を活性化し、カスパーゼ3を活性化しアポトーシスを引き起こすことも明らかとなった。これら結果は、細...
  • 文部科学省:科学研究費補助金(基盤研究(B))
    研究期間 : 2000年 -2001年 
    代表者 : 稲葉 睦, 辻本 元, 稲波 修, 高桑 雄一, 山本 雅之, 松木 直章, 小野 憲一郎
     
    ●培養細胞系の確立:レトロウイルスベクターを用いて、K562、およびHEK293細胞における正常牛赤血球型バンド3(bebWT)、ならびにバンド3完全欠損症における変異バンド3(bebRX)、またそれらのEGFP融合タンパク質の安定発現系を構築した。さらに、これらの細胞系でバンド3に加えアンキリンのバンド3結合領域(AnkN90)を発現する細胞株を確立した。●上記の各細胞を用い、bebWTが細胞膜に輸送され安定化すること、一方、bebRXはレトログレードトランスポートを含む細胞内小胞輸送を経て細胞質ユビキチン・プロテアソーム系により合成後速やかに分解されること、さらにbebWTとbebRXが同時に存在すると、それらの相互作用によりbebWTもが分解され安定性が低下する、即ち、bebRXがbebWTの発現に対してドミナントネガティブに作用することを明らかにした。諸種の小胞輸送阻害剤、あるいは形態学的観察により、これらがER、ならびにGolgiにおいて生じることを示した。同様に、バンド3とアンキリンの相互作用・結合はER、あるいはGolgiにおいて既に生じることを明らかにした。一方で、アンキリンはバンド3非存在下でも小胞に結合することも明らかとなり、この結合を介して膜骨格を形成し得るものと考えられた。●正常個体、欠損症個体由来の骨髄細胞におけるバンド3、アンキリン、スペクトリン、...
  • 文部科学省:科学研究費補助金(基盤研究(C))
    研究期間 : 2000年 -2001年 
    代表者 : 柏倉 幾郎, 村上 美穂, 稲波 修, 桑原 幹典, 高橋 恒夫, 高木 良成
     
    研究課題「放射線曝露ヒト造血細胞の血球産生機能回復を促進する至適サイトカインの作用」の平成12年〜13年度における研究の結果、以下の点が明らかになった。1.ヒト臍帯血に含まれるCD34陽性巨核球前駆細胞(megakaryocyte colony-forming units ; CFU-Meg)は、他の骨髄系前駆細胞である赤血球前駆細胞、白血球前駆細胞、混合コロニー形成細胞に比べ放射線感受性が高い。2.ヒト臍帯血CFU-Megのうちその60%〜70%は未熟なCFU-Megであり、また分化が進んだ成熟CFU-Megに比べより放射線感受性が高い。一方、ヒト末梢血CFU-Megはその多くが成熟CFU-Megであるが、末梢血CFU-Megは臍帯血CFU-Megに比べ放射線感受性が高い。3.放射線に曝露された臍帯血CFU-Megに対し、thrombopoietin(TPO)、interleukin-3(IL-3)及びstem cell factor(SCF)の組合せが最も強い放射線障害軽減・修復作用を示した。末梢血CFU-Megでは、TPOとIL-3の組合せが最も効果的であった。4.放射線照射ヒト臍帯血CD34陽性CFU-Megに対するTPOとSCFの併用による放射線障害軽減作用は、細胞内ERKを活性化し、カスパーゼ-3に依存するアポトーシスを抑制する事に起因している。5.ヒト末梢血CD...
  • 文部科学省:科学研究費補助金(基盤研究(B))
    研究期間 : 1999年 -2001年 
    代表者 : 永幡 肇, 辻 正義, 林 正信, 稲波 修, 桑原 幹典, 樋口 豪紀
     
    牛白血球粘着不全症(BLAD)の病態と生態防御修飾機構に関し,検討をすすめ以下の知見を得た。おもな点を示す。(1)BLADのCD18欠損好中球の接着分子L-セレクチンの発現と機能について,低発現と関連した情報伝達系の特徴を明らかにした。(2)CD18欠損好中球の抗体依存性細胞傷害活性の特徴を明らかにし,代償的に発現増強が生じることを示した。(3)ESRによる好中球の活性酸素種を検索し,O_2は好中球ファゴソーム内で,OHは細胞外で産生されることを示した。(4)好中球のF-actinはP1-3キナーゼに依存し,NADPH酵素は,PKCと細胞内Ca^<2+>濃度の増加に強く依存していることを提示した。(5)ウシ好中球の細胞内情報伝達において,p38MAPKとP1-3が,NADPH活性と食作用に関与することを明らかにした。遺伝的CD11b/CD18欠損的好中球の生体防御機構(修飾)の特徴を明らかにし得たことより,CD3欠損病態の把握が可能となった。また,比較医学の上から,ヒトのLAD疾患の理解にも寄与しうることが示された。
  • 文部科学省:科学研究費補助金(特定領域研究(A))
    研究期間 : 2000年 -2000年 
    代表者 : 稲波 修, 桑原 幹典
     
    NADPHオキシダーゼは好中球・B細胞に存在し、スーパーオキサイドを生成する酵素として知られている。このオキシダーゼコンポーネントの欠失、突然変異は慢性肉芽腫症(CGD)として知られ、重篤な免疫疾患を示すことから、NADPHオキシダーゼは感染防御にとって非常に重要であると認識されている。この酵素は細胞膜に局在する p22^Tとgp91^、細胞質に局在するp47phox、p67phoxならびにracから構成されることが知られている。活性化に際しては、FcRやFMLP刺激によって活性化したprotein kinase C(PKC)がp47^のC末端領域のリン酸化を促し、リン酸化p47^の二つのSH3ドメイン(SH3A,SH3B)とプロリンリッチ領域(PRR)が、それぞれp22^とP67^と結合し、膜に会合し、活性を表すと考えられている。また、p38MAPK、ERKなどのMAP kinaseもこの活性化に関与することを本年度の結果として明らかにされた^<1-3)>。本研究ではシステイン残基をターゲットとしたスピンラベル法を用い、リン酸化に伴うp47^の構造変化を部位特異的スピンラベル法を用い解析した。3-maleimide-proxylをスピンプローブとして用い、pGEX-1λT発現系で作成したp4...
  • 文部科学省:科学研究費補助金(基盤研究(C))
    研究期間 : 1997年 -1998年 
    代表者 : 稲波 修, 永幡 肇, 桑原 幹典
     
    本研究は慢性肉芽腫症(CGD)を中心とした遺伝性白血球機能障害の情報伝達から見た解析と、それを通じて好中球の殺菌能として重要なNADPHオキシダーゼの活性化機構を明らかにすることを目的とした。本研究で明らかに出来た事として、次のようなものである。第一に正常好中球のNADPHオキシダーゼ活性化と貪食機構に関してp38MAPキナーゼという、最近発見された新しいキナーゼの関与を明らかにした。第二に好中球の活性酸素生成が欠損しているヒト慢性肉芽腫症(CGD)の細胞を用いた研究で,NADPHオキシダーゼコンポーネントであるp47phox発現ベクターを発現させることでNADPHオキシダーゼ活性を回復させる事に成功した。これは遺伝子治療の基礎的研究として意義があると考えられる。またこのp47phoxの部位特異的突然変異法を用いた詳細な研究で、p47phoxの11個のセリン残基のうちS303.S304.S359.S370のリン酸化がNADPHオキシダーゼ活性化の引き金になること、スピンラベル法を用いた研究でp47phoxがリン酸化する際に、そのC末端領域の構造変化が起きることを明らかにした。第三にCGDと類似の活性酸素生成能が欠損しているウシ顆粒球粘着不全症(BLAD)の好中球はlgGや補体受容体刺激による活性酸素生成能は著しく低下しているが、その情報伝達系にPKCが関係するNADPHオキシ...
  • 文部科学省:科学研究費補助金(基盤研究(B))
    研究期間 : 1997年 -1998年 
    代表者 : 桑原 幹典, 田島 誉士, 木村 和弘, 稲波 修, 永幡 肇, 首籐 文栄
     
    本研究はウシ白血球粘着不全症(BLAD)を中心としたウシ遺伝性白血球機能障害の情報伝達から見た解析と、それを通じて好中球の殺菌能として重要なNADPHオキシダーゼの活性化機構を明らかにすることを目的とした。本研究で明らかに出来た事として、次のようなものである。第一に、正常好中球のNADPHオキシダー活性化と貧食機構に関してp38MAPキナーゼという最近発見された新しいキナーゼの関与を明らかにした。第二に、BLADの好中球はIgGや補体受容体刺激による活性酸素生成能は著しく低下しているが、その情報伝達系にPKCが関係するNADPHオキシダーゼ活性化システムの寄与が大きくなることを明らかにした。第三に、BLADに正常ウシの骨髄の移植を試みたところ、28ケ月に渡りCD18の好中球での発現が観察され、臨床的症状の改善が見られた。第四に、BLADの類似の好中球の活性酸素生成が欠損しているヒト慢性肉芽腫症(CGD)の細胞を用いた研究で、NADPHオキシダーゼコンポーネントであるp47phoxの部位特異的突然変異法を用いた詳細な研究で、p47phoxの11個のセリン残基のうち4つのセリン残基のリン酸化がNADPHオキシダーゼ活性化の引き金になることを明らかにした。第五に、この様な血球細胞からの活性酸素は生体系では、新生児期の様々な疾病、炎症、虚血時の傷害と深く結びついて考えられているが、そ...
  • 文部科学省:科学研究費補助金(基盤研究(B))
    研究期間 : 1997年 -1998年 
    代表者 : 桑原 幹典, 入口 紀男, 下川 繁三, 昆 泰寛, 有川 二郎, 稲浪 修, 山本 強
     
    申請者らは、MRIを実験用小動物(マウス・ラット)に適用するため、最小画像構成単位からS/N比の良い信号を得る必要性上高磁場(7.05テスラ、プロトン共鳴周波数300MHz)および強グラジエントMRIを行った。そのため、強グラジエントコイルを内蔵するOxford社の超伝導マグネットに自作の鞍型Helmholtzコイルをセットし、東芝ワークステーションにてイメージングソフトSISCOを走らせ、スライス厚1mmのMR画像を撮像した。撮像はスビンエコー二次元フーリェ変換法により行った。次いで、この撮像法をマルチスライススピンエコー法に適用し、ラットおよびマウス腹部を心臓から腎臓まで50枚のスライス画像として撮像し、それをUNIXコンピューターに取り込み、ボリュームレンダリングソフトによる基本計算処理に基づき、三次元MR画像の再構成を行った。腹部に含まれる各種臓器をビジュアライズするため、3%食用ゼラチンで固めた造影剤(フェリセルツ)で胃を充填し、画像化された胃との空間位置関係から各種臓器を判定し、それぞれを擬似カラー化した後、三次元MR画像の再構成を行った。さらに、ボリュームレンダリングソフトにトランピアレンス処理のプログラムを書き加え、筋肉など臓器以外の組織を透明化し、擬似カラー化した各種臓器のみが立体的に画像化されるものを作成した。この方法をモデル疾患病態へ適用するため、大腸癌...
  • 文部科学省:科学研究費補助金(基盤研究(C))
    研究期間 : 1997年 -1998年 
    代表者 : 永幡 肇, 桑原 幹典, 稲波 修, 寺岡 宏樹
     
    牛白血球粘着不全症(BLAD)の病態および骨髄移植について研究を実施し下記の成果を得た。(1) CD18欠損好中球のFc受容体を介する貧食能、活性酸素生成および細胞内Ca^<2+>シグナリングを検討した。Fc受容体を介する好中球機能はCD18の存在に依存していることを明らかにした。(2) BLAD牛において,CD18欠損好中球の肺胞気管支領域および体表擦過領域への移出を検討した。CD18欠損好中球の上記部位への血管外遊出を認め,その特徴を明らかにした。(3) 正常好中球をCD18欠損好中球保有(BLAD)牛へ,輸注し,好中球の化学発光反応を追跡した。末梢血液中の滞留時間は2〜5時間であることを示した。(4) BLAD牛へ正常骨髄を移植(BMT)し,経時的に臨床,血液,好中球機能およびCD18細胞を検索した。BMT後12カ月でCD18陽性^+が0.3〜0.5%認められた。臨床所見はBMT後28カ月間安定であった。新しく生着し発現したCD18^+細胞が重要な役割を演じているものと考察され,骨髄移植の成功所見を提示し得た。
  • 文部科学省:科学研究費補助金(基盤研究(B))
    研究期間 : 1996年 -1997年 
    代表者 : 首藤 文榮, 稲波 修
     
    ボツリヌス中毒症を分子レベルで的確に治療するための方法原理を確立する目的で、毒素分子の重鎖と抗軽鎖抗体のキメラ分子を作製し、その分子特性を解析して、分子治療法の可否を判断する。1) 抗軽鎖抗体F(ab′)_2と毒素重鎖によるキメラ分子の合成軽鎖抗体F(ab′)_2と毒素の重鎖とを会合させたキメラ分子を合成した。このキメラ分子の比中和活性は、素材として用いた抗軽鎖抗体の約2.5倍であった。2) モノクロナール抗体を用いたキメラ分子の調整毒素の軽鎖に結合して中和活性を示すモノクロナール抗体を調製し、毒素重鎖と会合させてキメラ分子を合成したところ、回収率が10%に向上し、比中和活性もポリクローナル抗体を使ったものより約3倍高くなった。同型重鎖の連用による抗体産生を避けるために、異型毒素の重鎖をキャリアーとして用いた場合も、ほぼ同じ中和活性を持つキメラ分子が得られた。3) 毒素結合フラグメントの調製と体内動態の解析結合フラグメントは、重鎖と同じ体内動態を示したが、毒素阻害活性は異なっていた。4) 結合フラグメントと抗軽鎖抗体とのキメラ分子の調製抗軽鎖ボリクローナル抗体を結合フラグメントと会合させたが、会合率が0.1%以下であった。比中和活性は、遊離のものに比べて約1.5倍であった。このことから、キャリアーには重鎖のN末端が必要なことが分かった。5) フュージョンプロテインの調製の試み...
  • 文部科学省:科学研究費補助金(基盤研究(C))
    研究期間 : 1996年 -1996年 
    代表者 : 稲波 修, 桑原 幹典, 首藤 文榮
     
    慢性肉芽腫症の基礎的解析を行い、その遺伝子治療の基礎的データを蓄積するために本研究は行われた。そのために、スクリプス研究所Babior博士より慢性肉芽腫症患者由来のB細胞をEBウィルスの株化した細胞の提供をうけた。本細胞はNADPHオキシダーゼコンポーネントの細胞性成分であるp47phoxが欠損していることが明らかにされている。このB細胞は正常のものと違い、ホルボールで刺激してもNADPHオキシダーゼの活性化は全く観察されなかった。また欠損しているp47phoxを過剰発現させるために発現ベクターとしてEBOpLPPにp47phoxのcDNAを組み込み、この細胞に導入したところ、活性の回復が観察され、発現されたp47phoxのC末端領域の11個のセリンがリン酸化にされていることが示された。この実験結果はp47phoxはNADPHオキシダーゼ活性化に必須のものであり、p47phoxがリン酸化されることでNADPHオキシダーゼが活性化の引き金であることが明らかにされた。以上のことからリン酸化の重要性をさらに確かめるために、303と304の位置の2個のセリン残基をアラニン残基に変異を起こさせたcDNAを発現ベクターに組み込んで発現させると活性は全く見られなくなった。さらにセリンがリン酸化させたものと類似の荷電を持つグルタミン酸やアスパラギン酸残基にこの303+304を置き換えてやると...
  • 文部科学省:科学研究費補助金(一般研究(C), 基盤研究(C))
    研究期間 : 1995年 -1996年 
    代表者 : 桑原 幹典, 稲波 修
     
    本研究では、マクロファージの成熟過程に於ける、生体防御機能に重要な役割を表す一酸化窒素(NO)の生成能力を調べる目的で、インターフェロンγ、リポポリサッカライド(LPS)、ラクトフェリンによる成熟過程によるNO合成酵素(iNOS)の誘導とその活性を評価した。方法はiNOSの誘導はwestern blotting法、NOの測定は生じたNOをMGD-Feと反応させることにより、NOを安定化させ、電子スピン共鳴法により測定した。またグリース反応も用いて定量した。チオグリコレートで誘導したマウスの腹腔マクロファージを単離し、大腸菌由来のリポポリサッカライド(LPS)とインターフェロンγ(Inf-γ)存在下で培養したところ、経時的に培養液中にNOの蓄積を示すNO_2^-+NO_2^-の増加が刺激後24時間まで時間依存的に増加することが示めされた。更にMGD-FeでNOを直接測定してもNO_2^-+NO_2^-とパラレルに増加することが示され、このMGD-Feを用いたスピン捕捉法がNOの定量に用いることが可能であることが示された。またiNOSの特異的抗体を用いたウェスタンブロッティング法によりiNOSの発現そのものを調べたところ、時間依存的に蛋白質としても発現していることが明らかとなった。炎症の際に顆粒球よりラクトフェリンが差生されるが、この免疫学的意義を検索するためにLFのマクロファー...
  • 文部科学省:科学研究費補助金(一般研究(C))
    研究期間 : 1995年 -1995年 
    代表者 : 谷口 和之, 稲波 修, 小川 和重
     
    嗅球は嗅覚の一次中枢で主嗅球および副嗅球から成り、主嗅球は嗅上皮から、副嗅球は鋤鼻器からの投射を受ける。嗅上皮および鋤鼻器の感覚細胞は双極性のニューロンで、その軸索はそれぞれ主嗅球および副嗅球の表層に至って嗅神経層および鋤鼻神経層となってからその深部で糸球体とよばれる特殊な構造を形成する。嗅覚情報はこの糸球体で嗅球の固有ニューロンである僧帽細胞および房飾細胞へと伝達される。固有ニューロンの機能は糸球体を取り巻く傍糸球細胞、嗅球全体に散在する短軸索細胞、嗅球最深層に密在する顆粒細胞など、各種介在ニューロンによる調節を受ける。本研究ではまず、NO合成酵素と相同であるといわれるNADPH-ディアフォラーゼを酵素組織化学により、NO合成酵素はその抗体を用いた免疫組織化学により、またNO合成酵素に対するmRNAはそれと相補的な合成オリゴヌクレオチドを用いたin situ hybridizationにより検索した。NADPH-ディアフォラーゼの反応は短軸索細胞と副嗅球の顆粒細胞に強く認められ、また糸球体、傍糸球細胞、主嗅球の顆粒細胞にも中等度に認められた。一方、NO合成酵素に対する免疫組織化学およびin situ hybridizationでは反応およびシグナルはNADPH-ディアフォラーゼの場合とほぼ同様であったが、糸球体には認められなかった。従って、糸球体におけるNADPH-ディアフ...
  • 文部科学省:科学研究費補助金(一般研究(C))
    研究期間 : 1995年 -1995年 
    代表者 : 首藤 文栄, 首藤 文榮, 稲波 修
     
    ボツリヌス中毒症は、致命率の高い疾患である。ボツリヌス毒素は、呼吸筋の神経終末から細胞内に取り込まれ、アセチルコリンの放出を阻害して呼吸麻痺を引き起こす。毒素分子がいったん細胞内に取り込まれると、もはや抗体は結合できなくなるから、抗血清の効果は殆ど期待できなくなる。これがボツリヌス中毒症の致命率を高めている原因の一つである。ボツリヌス神経毒素は重鎖と軽鎖からなり、重鎖が神経終末の特異的結合部位に結合して軽鎖を細胞内に送り込み、軽鎖は重鎖と離れて毒性を発現する。重鎖に抗軽鎖抗体を担がせれば、毒素と同じ経路で中和抗体を障害部位に送り込むことができ、障害部位に存在する毒素を中和できるであろうとの考えからこの研究を始めた。キメラ分子の作製には、化学合成と遺伝子工学による方法とがあるが、この研究では化学合成を試みた。まず、精製毒素を重鎖と軽鎖に分離し、一方では抗毒素血清から抗軽鎖抗体を精製した。抗軽鎖抗体をペプシンで消化してF(ab')_2を得、これをシステアミンで還元した後、0°Cで重鎖と会合させた。この反応では数種類の会合体が得られ、分子サイズは300-25kDaに亘っていた。会合体のなかから分子サイズ約15kDaのものを分離し、分子構成を解析した。この画分には毒素の重鎖とFabの重鎖および軽鎖に相当するコンポーネントが含まれていた。この画分の毒素中和活性は、加工されていない毒素の...
  • 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 : 稲波 修
     
    好中球の殺菌機構において,細菌の貧食に際しての接触刺激によって生成されるスーパーオキシド(O2-)が重要な役割を演じていると考えられている.好中球のO2-産生機能の低下は、感染防護機構の点から重要であると考えられている。家畜においては、特にその新生期においては、肺炎や下痢などの感染症が頻発し、この様な好中球を中心とする非特異性免疫能の低下が類推される。本研究では、まず人と牛の好中球機能の比較をおこない、さらに子牛の好中球機能のなかでO2-の生成能を成牛と比較することで,その機構を明らかにすることを目的とした。この目的のために細胞内の伝達系であるC-Kinase(PKC)を刺激するphorbol 12-myristate 13-acetate(PMA)で好中球を刺激した際のO2-産生能について検討した。まず、人と成牛の比較においてオプソニン化ザイモザンによるC3b受容体刺激とホルボールエステルによる細胞内PKC刺激に伴うスーパーオキサイド生成能の比較から、牛では人と比較してC3受容体に対する反応性が低下していることが明らかとなった。またヒト好中球の走化性因子で活性酸素生成を増加させるFMLPで牛の好中球を刺激しても、全く活性酸素の生成を示さないことから、牛好中球にはFMLPの受容体が存在しないことが示唆された。さらに成牛と新生子牛の好中球の比較において、生後3時間以内に採取した...
  • 文部科学省:科学研究費補助金(一般研究(C))
    研究期間 : 1994年 -1994年 
    代表者 : 谷口 和之, 稲波 修, 小川 和重
     
    本研究では主に嗅覚系におけるプログラム細胞死の発現を形態学的に検出するため種々の検索を行い,以下の結果を得た.1.嗅覚系のプログラム細胞死には-酸化窒素(NO)が関与するとされているため,NO合成酵素の組織内分布を遺伝子組織化学的に検索した.本研究では合成オリゴヌクレオチドを非放射性に標識したプローブを用いて遺伝子組織化学的検索を行った.その結果,NO合成酵素は主嗅球および副嗅球の短軸索細胞,さらに副嗅球の顆粒細胞に局在していることが明らかとなった.また本研究を遂行中にNO合成酵素に対する抗血清が市販されるようになったため,この抗血清を入手して免疫組織化学による検索も行った所,遺伝子組織化学と同一の所見を得た.2.プログラム細胞死ではまず核のDNAが断片化するため,DNAの3′-末端をTUNEL法によって標識し,各嗅覚受容器においてプログラム細胞死を起こしている細胞を検索した.その結果,細胞死の頻度は嗅上皮で高く,マセラ器および鋤鼻器で低く,嗅覚受容器間で細胞交代の速度は異なることが明らかとなった.3.プログラム細胞死により,細胞膜の糖鎖にも変化が起きることが予想されたため,各嗅覚受容器をレクチン組織化学的に検索した.その結果,嗅上皮でBSL-I,PHA-E,PHA-L,マセラ器ではBSL-IとPHA-Lが変性中の細胞を検出したが鋤鼻器ではこのような細胞は検出されなかった.従...
  • 文部科学省:科学研究費補助金(一般研究(B))
    研究期間 : 1988年 -1989年 
    代表者 : 佐藤 昭夫, 佐藤, 昭夫, 佐藤 昭夫, 稲波 修, 佐藤 優子
     
    マイネルトの基底核や中隔野に起始するコリン作動性線維は、それぞれ大脳皮質や海馬に投射しており、高齢者やアルツハイマ-患者において変成脱落していることが報告されているが、このコリン作動性線維の機能的意味については明らかにされていない。本研究は、マイネルト核と中隔野に起始するコリン作動性線維の大脳皮質および海馬の局所血流調節への関与及びその機序を検討することを目的とした。人工呼吸下の麻酔ラットを用いて、脳局所血流の測定には、経時的に測定可能なレ-ザ-ドップラ-法あるいは脳の様々な部位の局所血流が同時に測定できるヨ-ドアンチピリン法を用いた。一側のマイネルト核あるいは正中線に近接している中隔野を電気刺激あるいは化学刺激したところ、刺激と同側の大脳皮質局所血流あるいは両側海馬の局所血流がそれぞれ増加した。しかし、刺激中その他の部位では有意な血液の増加は見られなかった。これらのマイネルト核や中隔野刺激によって起こる大脳皮質や海馬の血流増加反応は、いずれもアセチルコリン受容体遮断薬の静脈内投与によって著しく減弱した。また、大脳皮質ならびに海馬内の細胞外液中のアセチルコリンを微小透析法によって測定したところ、それぞれマイネルト核刺激ならびに中隔野刺激によって増加した。以上の事実から、マイネルト核から大脳皮質に、中隔野から海馬にそれぞれ投射するコリン作動性線維が興奮すると、大脳皮質と海馬にア...
  • 部位特異的スピンラベル/ESRによる距離計測法を応用した人獣共通感染症関連タンパク質の構造解析
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  • Study on free radicals in Biological System

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    キーワード : 放射線、放射性同位元素、放射線単位、線量測定、防護、生物影響、人体に及ぼす影響、放射線感受性、放射線防護関連法令
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    キーワード : 放射線生物学、放射線の生物影響、放射線計測
  • 獣医科学基礎科目B 環境放射線生物学
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    開講年度 : 2018年
    課程区分 : 学士課程
    開講学部 : 全学教育
    キーワード : 放射線、放射能、放射線被ばく、放射線医療、原子炉、環境放射能、放射性廃棄物処理・処分、廃炉工学、オープン教材
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    課程区分 : 博士後期課程
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    キーワード : 画像診断 X線 超音波 CT MRI 核医学 造影検査 放射線防護
  • 獣医科学特論演習
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 獣医学院
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    キーワード : 画像診断 X線 超音波 CT 造影検査 放射線防護
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