Researcher Profile and Settings

Master

Affiliation (Master)

• Faculty of Veterinary Medicine　Veterinary Medicine　Applied Veterinary Sciences

Affiliation (Master)

• Faculty of Veterinary Medicine　Veterinary Medicine　Applied Veterinary Sciences

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Profile and Settings

Degree

• Ph.D（Hokkaido University）

Profile and Settings

• Profile

  DATE OF BIRTH:
  ６ July 1959
  
  NATIONALITY:
  Japanese
  
  EDUCATION:
  B.S. in Veterinary Medicine, Hokkaido University, March 1982
  M.S. in Radiation Biology, Hokkaido University, March 1984
  Ph.D. in Radiation Biology, Hokkaido University, March 1987
  
  EXPERIENCE:

  Assistant Researcher, April 1987 to August1988
  Laboratory of Physiology
  Tokyo Metropolitan Institute Gerontology
  
  Associate Researcher, October 1988 to December 1992
  Laboratory of Physiology
  Tokyo Metropolitan Institute Gerontology
  
  Assistant Professor, January 1993 to October 1994
  Laboratory of Physiology, Department of Agriculture,
  Iwate University
  
  Visiting Researcher, July 1995 to April 1996
  Laboratory of Biochemistry (Prof. Barnard Babior)
  Department of Molecular and Experimental Medicine,
  Scripps Research Institute, CA, USA
  
  Associate Professor, October 1994 to October1996
  Laboratory of Physiology, Department of Agriculture,
  Iwate University
  
  Associate Professor, September 1996 to April 2007
  Laboratory of Radiation Biology, Graduate School of Veterinary Medicine,
  Hokkaido University
  
  Professor, May 2007 to present

  Laboratory of Radiation Biology, Graduate School of Veterinary Medicine,
  Hokkaido University

• Name (Japanese)

  Inanami

• Name (Kana)

  Osamu

• Name

  200901002930980367

Alternate Names

https://www.vetmed.hokudai.ac.jp/organization/radbiol/

Achievement

Research Interests

• 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   

Research Areas

• Life sciences / Radiology / Radiation Biology
• Life sciences / Veterinary medicine
• Life sciences / Veterinary medicine
• Environmental science/Agricultural science / Environmental policy and society
• Environmental science/Agricultural science / Environmental impact assessment

Research Experience

• 2017/04 - Today Graduate school of Veterinary Medicine, Hokkaido University. Dept. of Applied Veterinary Medicine, Lab. Radiation Biology Professor
• 2007/05 - 2017/03 Hokkaido University Professor
• 1996/09 - 2007/04 Hokkaido University Associate Professor
• 1994/10 - 1996/08 Iwate University Associate Professor
• 1995/07 - 1996/04 Scripps Research Institute Visiting Researcher
• 1987/04 - 1988/08 Tokyo Metropolitan Institute of Gerontology Assistant Researcher
• Tokyo Metropolitan Institute of Gerontology Researcher

Education

• 1982/04 - 1994/03  Hokkaido University  Graduate School of Veterinary Medicine  M.S. (Veterinary Medicine)
•        - 1987  Hokkaido University  Graduate School, Division of Veterinary Medicine
• 1978/04 - 1982/03  Hokkaido University  School of Veterinary Medicine  Veterinary Medicine
•        - 1982  Hokkaido University  Faculty of Veterinary Medicine
•        -   Hokkaido University  Graduate School of Veterinary Medicine  Ph.D. (Veterinary Medicine)

Awards

• 2018 日本アイソトープ協会 放射線安全取扱部会功労表彰
  
• 2003 日本獣医学会賞
  
• 1993 若手奨励賞(オクラホマ医学研究財団)
  

Published Papers

• Mechanism of the Radioresistant Colorectal Cancer Cell Line SW480RR Established after Fractionated X Irradiation.

  Koya Yamashita, Hironobu Yasui, Tomoki Bo, Masaki Fujimoto, Osamu Inanami

  Radiation research 202 (1) 38 - 50 2024/07/01 

  Radioresistant cancer cells are risk factors for recurrence and are occasionally detected in recurrent tumors after radiotherapy. Intratumor heterogeneity is believed to be a potential cause of treatment resistance. Heterogeneity in DNA content has also been reported in human colorectal cancer; however, little is known about how such heterogeneity changes with radiotherapy or how it affects cancer radioresistance. In the present study, we established radioresistant clone SW480RR cells after fractionated X-ray irradiation of human colorectal cancer-derived SW480.hu cells, which are composed of two cell populations with different chromosome numbers, and examined how cellular radioresistance changed with fractionated radiotherapy. Compared with the parental cell population, which mostly comprised cells with higher ploidy, the radioresistant clones showed lower ploidy and less initial DNA damage. The lower ploidy cells in the parental cell population were identified as having radioresistance prior to irradiation; thus, SW480RR cells were considered intrinsically radioresistant cells selected from the parental population through fractionated irradiation. This study presents a practical example of the emergence of radioresistant cells from a cell population with ploidy heterogeneity after irradiation. The most likely mechanism is the selection of an intrinsically radioresistant population after fractionated X-ray irradiation, with a background in which lower ploidy cells exhibit lower initial DNA damage.
• Partial Acquisition of Spectral Projections Accelerates Four-dimensional Spectral-spatial EPR Imaging for Mouse Tumor Models: A Feasibility Study.

  Misa Oba, Mai Taguchi, Yohei Kudo, Koya Yamashita, Hironobu Yasui, Shingo Matsumoto, Igor A Kirilyuk, Osamu Inanami, Hiroshi Hirata

  Molecular imaging and biology 26 (3) 459 - 472 2024/06 

  PURPOSE: Our study aimed to accelerate the acquisition of four-dimensional (4D) spectral-spatial electron paramagnetic resonance (EPR) imaging for mouse tumor models. This advancement in EPR imaging should reduce the acquisition time of spectroscopic mapping while reducing quality degradation for mouse tumor models. PROCEDURES: EPR spectra under magnetic field gradients, called spectral projections, were partially measured. Additional spectral projections were later computationally synthesized from the measured spectral projections. Four-dimensional spectral-spatial images were reconstructed from the post-processed spectral projections using the algebraic reconstruction technique (ART) and assessed in terms of their image qualities. We applied this approach to a sample solution and a mouse Hs766T xenograft model of human-derived pancreatic ductal adenocarcinoma cells to demonstrate the feasibility of our concept. The nitroxyl radical imaging agent 2H,15N-DCP was exogenously infused into the mouse xenograft model. RESULTS: The computation code of 4D spectral-spatial imaging was tested with numerically generated spectral projections. In the linewidth mapping of the sample solution, we achieved a relative standard uncertainty (standard deviation/| mean |) of 0.76 μT/45.38 μT = 0.017 on the peak-to-peak first-derivative EPR linewidth. The qualities of the linewidth maps and the effect of computational synthesis of spectral projections were examined. Finally, we obtained the three-dimensional linewidth map of 2H,15N-DCP in a Hs766T tumor-bearing leg in vivo. CONCLUSION: We achieved a 46.7% reduction in the acquisition time of 4D spectral-spatial EPR imaging without significantly degrading the image quality. A combination of ART and partial acquisition in three-dimensional raster magnetic field gradient settings in orthogonal coordinates is a novel approach. Our approach to 4D spectral-spatial EPR imaging can be applied to any subject, especially for samples with less variation in one direction.
• Feasibility study of multimodal imaging for redox status and glucose metabolism in tumor.

  Kazuhiro Kato, Hironobu Yasui, Hideo Sato-Akaba, Miho C Emoto, Hirotada G Fujii, Maciej M Kmiec, Periannan Kuppusamy, Yuki Mizuno, Yuji Kuge, Masaki Nagane, Tadashi Yamashita, Osamu Inanami

  Free radical biology & medicine 2024/04/02 

  Understanding the tumor redox status is important for efficient cancer treatment. Here, we noninvasively detected changes in the redox environment of tumors before and after cancer treatment in the same individuals using a novel compact and portable electron paramagnetic resonance imaging (EPRI) device and compared the results with glycolytic information obtained through autoradiography using 2-deoxy-2-[18F]fluoro-d-glucose ([18F]FDG). Human colon cancer HCT116 xenografts were used in the mice. We used 3-carbamoyl-PROXYL (3CP) as a paramagnetic and redox status probe for the EPRI of tumors. The first EPRI was followed by the intraperitoneal administration of buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, or X-ray irradiation of the tumor. A second EPRI was performed on the following day. Autoradiography was performed after the second EPRI. After imaging, the tumor sections were evaluated by histological analysis and the amount of reducing substances in the tumor was measured. BSO treatment and X-ray irradiation significantly decreased the rate of 3CP reduction in tumors. Redox maps of tumors obtained from EPRI can be compared with tissue sections of approximately the same cross section. BSO treatment reduced glutathione levels in tumors, whereas X-ray irradiation did not alter the levels of any of the reducing substances. Comparison of the redox map with the autoradiography of [18F]FDG revealed that regions with high reducing power in the tumor were active in glucose metabolism; however, this correlation disappeared after X-ray irradiation. These results suggest that the novel compact and portable EPRI device is suitable for multimodal imaging, which can be used to study tumor redox status and therapeutic efficacy in cancer, and for combined analysis with other imaging modalities.
• Development of a red-shifted photosensitizer for near-infrared photoimmunotherapy of cancer

  Yuto Goto, Kanta Ando, Hideo Takakura, Kohei Nakajima, Masato Kobayashi, Osamu Inanami, Tetsuya Taketsugu, Mikako Ogawa

  Journal of Photochemistry and Photobiology 20 100230 - 100230 2666-4690 2024/04
• Time-dependent changes in retinoids content in liver and adipose tissue after feeding of a vitamin A-deficient diet to mice.

  Mira Kato-Suzuki, Yuko Okamatsu-Ogura, Osamu Inanami, Kazuhiro Kimura

  Experimental animals 2024/02/21 

  Vitamin A is an important nutrient for multiple physiological functions. To elucidate the role of vitamin A in vivo, vitamin A-deficient diets have been often used in mice to establish a vitamin A-deficiency model. However, the information on the appropriate feeding periods and time course of changes in vitamin A content in organs after the start of vitamin A-deficient diet feeding is lacking. This study aimed to assess the retinoids levels in liver and white adipose tissue in mice fed a vitamin A-deficient diet for £8 weeks. High-performance liquid chromatography was used to measure the retinoids levels in liver and white adipose tissue every 2 weeks for £8 weeks. Vitamin A-deficient diet feeding significantly decreased retinol in the liver over 6 weeks, but retinyl palmitate, a main storage form of vitamin A, was not changed over 8 weeks. The plasma retinol level remained constant throughout the experiment. In white adipose tissue, retinyl palmitate gradually decreased over 8 weeks. These results indicate that vitamin A-deficient diet feeding longer than 6 weeks reduced retinol in liver and retinyl palmitate in white adipose tissue over 8 weeks, although it is not enough for the induction of a whole-body vitamin A deficiency.
• Theoretical Design and Synthesis of Caged Compounds Using X‐Ray‐Triggered Azo Bond Cleavage

  Koki Ogawara, Osamu Inanami, Hideo Takakura, Kenichiro Saita, Kohei Nakajima, Sonu Kumar, Naoya Ieda, Masato Kobayashi, Tetsuya Taketsugu, Mikako Ogawa

  Advanced Science 11 (12) e2306586  2198-3844 2024/01/15 

  Abstract Caged compounds are frequently used in life science research. However, the light used to activate them is commonly absorbed and scattered by biological materials, limiting their use to basic research in cells or small animals. In contrast, hard X‐rays exhibit high bio‐permeability due to the difficulty of interacting with biological molecules. With the main goal of developing X‐ray activatable caged compounds, azo compounds are designed and synthesized with a positive charge and long π‐conjugated system to increase the reaction efficiency with hydrated electrons. The azo bonds in the designed compounds are selectively cleaved by X‐ray, and the fluorescent substance Diethyl Rhodamine is released. Based on the results of experiments and quantum chemical calculations, azo bond cleavage is assumed to occur via a two‐step process: a two‐electron reduction of the azo bond followed by N─N bond cleavage. Cellular experiments also demonstrate that the azo bonds can be cleaved intracellularly. Thus, caged compounds that can be activated by an azo bond cleavage reaction promoted by X‐ray are successfully generated.
• Electron Paramagnetic Resonance Implemented with Multiple Harmonic Detections Successfully Maps Extracellular pH In Vivo

  Ririko Nakaoka, Kazuhiro Kato, Kumiko Yamamoto, Hironobu Yasui, Shingo Matsumoto, Igor A. Kirilyuk, Valery V. Khramtsov, Osamu Inanami, Hiroshi Hirata

  Analytical Chemistry 95 (8) 3940 - 3950 0003-2700 2023/02/01 

  Extracellular acidification indicates a metabolic shift in cancer cells and is, along with tissue hypoxia, a hallmark of tumor malignancy. Thus, non-invasive mapping of extracellular pH (pHe) is essential for researchers to understand the tumor microenvironment and to monitor tumor response to metabolism-targeting drugs. While electron paramagnetic resonance (EPR) has been successfully used to map pHe in mouse xenograft models, this method is not sensitive enough to map pHe with a moderate amount of exogenous pH-sensitive probes. Here, we show that a modified EPR system achieves twofold higher sensitivity by using the multiple harmonic detection (MHD) method and improves the robustness of pHe mapping in mouse xenograft models. Our results demonstrate that treatment of a mouse xenograft model of human-derived pancreatic ductal adenocarcinoma cells with the carbonic anhydrase IX (CAIX) inhibitor U-104 delays tumor growth with a concurrent tendency toward further extracellular acidification. We anticipate that EPR-based pHe mapping can be expanded to monitor the response of other metabolism-targeting drugs. Furthermore, pHe monitoring can also be used for the development of improved metabolism-targeting cancer treatments.
• Ligand release from silicon phthalocyanine dyes triggered by X-ray irradiation.

  Hideo Takakura, Shino Matsuhiro, Osamu Inanami, Masato Kobayashi, Kenichiro Saita, Masaki Yamashita, Kohei Nakajima, Motofumi Suzuki, Naoki Miyamoto, Tetsuya Taketsugu, Mikako Ogawa

  Organic & biomolecular chemistry 2022/08/16 

  Ligand release from silicon phthalocyanine (SiPc) dyes triggered by near-infrared (NIR) light is a key photochemical reaction involving caged compounds based on SiPc. Although NIR light is relatively permeable compared with visible light, this light can be attenuated by tissue absorption and scattering; therefore, using light to induce photochemical reactions deep inside the body is difficult. Herein, because X-rays are highly permeable and can produce radicals through the radiolysis of water, we investigated whether the axial ligands of SiPcs can be cleaved using X-ray irradiation. SiPcs with different axial ligands (alkoxy, siloxy, oxycarbonyl, and phenoxy groups) were irradiated with X-rays under hypoxic conditions. We found that the axial ligands were cleaved via reactions with hydrated electrons (e-aq), not OH radicals, generated from water in response to X-ray irradiation, and SiPc with alkoxy groups exhibited the highest cleavage efficiency. A quantitative investigation revealed that X-ray-induced axial ligand cleavage proceeds via a radical chain reaction. The reaction is expected to be applicable to the molecular design of X-ray-activatable functional molecules in the future.
• Preclinical studies for improving radiosensitivity of non-small cell lung cancer cell lines by combining glutaminase inhibition and senolysis.

  Masaki Fujimoto, Ritsuko Higashiyama, Hironobu Yasui, Koya Yamashita, Osamu Inanami

  Translational oncology 21 101431 - 101431 2022/07 [Refereed]
   

  Glutamine metabolism, known as glutaminolysis, is abnormally activated in many cancer cells with KRAS or BRAF mutations or active c-MYC. Glutaminolysis plays an important role in the proliferation of cancer cells with oncogenic mutations. In this study, we characterized radiation-induced cell death, which was enhanced by glutaminolysis inhibition in non-small cell lung cancer A549 and H460 cell lines with KRAS mutation. A clonogenic survival assay revealed that treatment with a glutaminase inhibitor, CB839, enhanced radiosensitivity. X-irradiation increased glutamate production, mitochondrial oxygen consumption, and ATP production, whereas CB839 treatment suppressed these effects. The data suggest that the enhancement of glutaminolysis-dependent energy metabolism for ATP production is important for survival after X-irradiation. Evaluation of the cell death phenotype revealed that glutaminolysis inhibitory treatment with CB839 or a low-glutamine medium significantly promoted the proliferation of β-galactosidase-positive and IL-6/IL-8 secretory cells among X-irradiated tumor cells, corresponding to an increase in the senescent cell population. Furthermore, treatment with ABT263, a Bcl-2 family inhibitor, transformed senescent cells into apoptotic cells. The findings suggest that combination treatment with a glutaminolysis inhibitor and a senolytic drug is useful for efficient radiotherapy.
• USP2 in the ventromedial hypothalamus modifies blood glucose levels by controlling sympathetic nervous activation.

  Mayuko Hashimoto, Masaki Fujimoto, Kohtarou Konno, Ming Liang Lee, Yui Yamada, Koya Yamashita, Chitoku Toda, Michio Tomura, Masahiko Watanabe, Osamu Inanami, Hiroshi Kitamura

  The Journal of neuroscience : the official journal of the Society for Neuroscience 42 (23) 4607 - 4618 2022/04/26 [Refereed]
   

  Ubiquitin-specific protease (USP) 2 participates in glucose metabolism in peripheral tissues such as the liver and skeletal muscle. However, the glucoregulatory role of USP2 in the central nervous system is not well known. In this study, we focus on USP2 in the ventromedial hypothalamus (VMH), which has dominant control over systemic glucose homeostasis. ISH using a Usp2-specific probe showed that Usp2 mRNA is present in VMH neurons, as well as other glucoregulatory nuclei, in the hypothalamus of male mice. Administration of a USP2-selective inhibitor, ML364 (20 ng/head), into the VMH elicited a rapid increase in the circulating glucose level in male mice, suggesting USP2 has a suppressive role on glucose mobilization. ML364 treatment also increased serum norepinephrine concentration, while it negligibly affected serum levels of insulin and corticosterone. ML364 perturbated mitochondrial oxidative phosphorylation in neural SH-SY5Y cells, and subsequently promoted the phosphorylation of AMP-activated protein kinase (AMPK). Consistent with these findings, hypothalamic ML364 treatment stimulated AMPKα phosphorylation in the VMH. Inhibition of hypothalamic AMPK prevented ML364 from increasing serum norepinephrine and blood glucose. Removal of ROS restored the ML364-evoked mitochondrial dysfunction in SH-SY5Y cells and impeded the ML364-induced hypothalamic AMPKα phosphorylation, as well as prevented the elevation of serum norepinephrine and blood glucose levels in male mice. These results indicate hypothalamic USP2 attenuates perturbations in blood glucose levels by modifying the ROS-AMPK-sympathetic nerve axis.Significance statement:Under normal conditions (excluding hyperglycemia or hypoglycemia), blood glucose levels are maintained at a constant level. In this study, we used a mouse model to identify a hypothalamic protease controlling blood glucose levels. Pharmacological inhibition of ubiquitin-specific protease (USP) 2 in the ventromedial hypothalamus (VMH) caused a deviation in blood glucose levels under a non-stressed condition, indicating that USP2 determines the set point of the blood glucose level. Modification of sympathetic nervous activity accounts for the USP2-mediated glucoregulation. Mechanistically, USP2 mitigates the accumulation of ROS in the VMH, resulting in attenuation of the phosphorylation of AMP-activated protein kinase (AMPK). Based on these findings, we uncovered a novel glucoregulatory axis consisting of hypothalamic USP2, ROS, AMPK, and the sympathetic nervous system.
• Eribulin improves tumor oxygenation demonstrated by 18F-DiFA hypoxia imaging, leading to radio-sensitization in human cancer xenograft models

  Tomoki Bo, Hironobu Yasui, Tohru Shiga, Yuki Shibata, Masaki Fujimoto, Motofumi Suzuki, Kei Higashikawa, Naoki Miyamoto, Osamu Inanami, Yuji Kuge

  European Journal of Nuclear Medicine and Molecular Imaging 49 (3) 821 - 833 1619-7070 2022/02
• Redox-Sensitive Mapping of a Mouse Tumor Model Using Sparse Projection Sampling of Electron Paramagnetic Resonance.

  Kota Kimura, Nami Iguchi, Hitomi Nakano, Hironobu Yasui, Shingo Matsumoto, Osamu Inanami, Hiroshi Hirata

  Antioxidants & redox signaling 36 (1-3) 57 - 69 2022/01 [Refereed]
   

  Aims: This work aimed to establish an accelerated imaging system for redox-sensitive mapping in a mouse tumor model using electron paramagnetic resonance (EPR) and nitroxyl radicals. Results: Sparse sampling of EPR spectral projections was demonstrated for a solution phantom. The reconstructed three-dimensional (3D) images with filtered back-projection (FBP) and compressed sensing image reconstruction were quantitatively assessed for the solution phantom. Mouse xenograft models of a human-derived pancreatic ductal adenocarcinoma cell line, MIA PaCa-2, were also measured for redox-sensitive mapping with the sparse sampling technique. Innovation: A short-lifetime redox-sensitive nitroxyl radical (15N-labeled perdeuterated Tempone) could be measured to map the decay rates of the EPR signals for the mouse xenograft models. Acceleration of 3D EPR image acquisition broadened the choices of nitroxyl radical probes with various redox sensitivities to biological environments. Conclusion: Sparse sampling of EPR spectral projections accelerated image acquisition in the 3D redox-sensitive mapping of mouse tumor-bearing legs fourfold compared with conventional image acquisition with FBP.
• EPR Characterisation of Phthalocyanine Radical Anions in Near‐Infrared Photocleavage of the Hydrophilic Axial Ligand of a Photoimmunotherapeutic Reagent, IR700

  Osamu Inanami, Wakako Hiraoka, Yuto Goto, Hideo Takakura, Mikako Ogawa

  ChemPhotoChem 2367-0932 2021/11/15 [Refereed]
  
• Electron Donors Rather Than Reactive Oxygen Species Needed for Therapeutic Photochemical Reaction of Near-Infrared Photoimmunotherapy.

  Takuya Kato, Ryuhei Okada, Yuto Goto, Aki Furusawa, Fuyuki Inagaki, Hiroaki Wakiyama, Hideyuki Furumoto, Dagane Daar, Baris Turkbey, Peter L Choyke, Hideo Takakura, Osamu Inanami, Mikako Ogawa, Hisataka Kobayashi

  ACS pharmacology & translational science 4 (5) 1689 - 1701 2021/10/08 [Refereed]
   

  Near-infrared photoimmunotherapy (NIR-PIT) employs molecularly targeted antibodies conjugated with a photoabsorbing silicon-phthalocyanine dye derivative which binds to cancer cells. Application of NIR light following binding of the antibody-photoabsorber conjugates (APCs) results in ligand release on the dye, dramatic changes in solubility of the APC-antigen complex, and rapid, irreversible cell membrane damage of cancer cells in a highly selective manner, resulting in a highly immunogenic cell death. Clinically, this process results in edema after treatment mediated by reactive oxygen species (ROS). Based on the chemical and biological mechanism of NIR-PIT cytotoxicity and edema formation, in order to minimize acute inflammatory edema without compromising therapeutic effects, l-sodium ascorbate (l-NaAA) was administered to quench harmful ROS and accelerate the ligand release reaction. l-NaAA suppressed acute edema by reducing ROS after NIR-PIT yet did not alter the therapeutic effects. NIR-PIT could be performed safely under existence of l-NaAA without side effects caused by unnecessary ROS production.
• Metformin preferentially enhances the radio-sensitivity of cancer stem-like cells with highly mitochondrial respiration ability in HMPOS.

  Tatsuya Deguchi, Kenji Hosoya, Shango Kim, Yusuke Murase, Kumiko Yamamoto, Tomoki Bo, Hironobu Yasui, Osamu Inanami, Mahiro Okumura

  Molecular therapy oncolytics 22 143 - 151 2021/09/24 [Refereed]
   

  Metformin has many anti-cancer effects, alone or in combination with radiation. However, the mechanism underlying its radio-sensitized effect is still unclear, especially for cancer stem-like cells (CSCs). Here, the radio-sensitized effect of metformin was investigated, and its mechanism was revealed in CSCs derived from canine osteosarcoma cell line (HMPOS), a canine osteosarcoma cell line. Spheroid cells (SCs) were used as CSCs-rich cells derived from sphere formation, and SCs were compared with normal adherent culture cells (ACs). The radio-sensitizing effect of metformin using clonogenic assay and tumor growth in mice xenograft model were evaluated, and the mechanism of its radio-sensitization focusing on mitochondrial function was revealed. Metformin significantly enhanced radio-sensitivity of SCs through its inhibition of the mitochondrial function, as shown by decreased oxygen consumption, decreased mitochondrial membrane potential, and decreased ATP production. Additionally, SCs had a higher ability of mitochondrial respiration than ACs, which may have caused difference of their sensitivity of metformin and irradiation. In conclusion, mitochondrial function might play an important role in the sensitivity of metformin and irradiation, and drugs that target mitochondrial respiration, such as metformin, are promising radio-sensitizers to target CSCs.
• LAT1 inhibitor JPH203 sensitizes cancer cells to radiation by enhancing radiation-induced cellular senescence.

  Tomoki Bo, Sho Kobayashi, Osamu Inanami, Junichi Fujii, Osamu Nakajima, Tsunekata Ito, Hironobu Yasui

  Translational oncology 14 (11) 101212 - 101212 2021/08/27 [Refereed]
   

  L-type amino acid transporter 1 (LAT1) is important for transporting neutral amino acids into cells. LAT1 expression is correlated with cancer malignancy, suggesting that LAT1 is a promising target for cancer therapy. JPH203, a potential novel drug targeting LAT1, has been shown to suppress tumor growth in various cancer cell lines. However, a combination study of JPH203 and radiation therapy has not been reported. Here, we examined the effects of JPH203 on radiosensitivity after irradiation in A549 and MIA Paca-2 cells. We showed that X-irradiation increased cellular neutral amino acid uptake via LAT1 in both cell lines. JPH203 inhibited the radiation-induced increase in neutral amino acid uptake. We demonstrated that JPH203, at minimally toxic concentrations, significantly sensitized cancer cells to radiation. JPH203 significantly downregulated mTOR activity and enhanced cellular senescence post-irradiation without reducing ATP and GSH levels. These results indicate that LAT1 inhibition by JPH203 sensitizes cancer cells to radiation by enhancing cellular senescence via mTOR downregulation. Thus, JPH203 may be a potent anti-cancer drug in combination with radiation therapy.
• DNA damage response in vascular endothelial senescence: Implication for radiation-induced cardiovascular diseases.

  Masaki Nagane, Hironobu Yasui, Periannan Kuppusamy, Tadashi Yamashita, Osamu Inanami

  Journal of radiation research 62 (4) 564 - 573 2021/04/28 [Refereed]
   

  A post-exposure cohort study in Hiroshima and Nagasaki reported that low-dose exposure to radiation heightened the risk of cardiovascular diseases (CVD), such as stroke and myocardial infarction, by 14-18% per Gy. Moreover, the risk of atherosclerosis in the coronary arteries reportedly increases with radiation therapy of the chest, including breast and lung cancer treatment. Cellular senescence of vascular endothelial cells (ECs) is believed to play an important role in radiation-induced CVDs. The molecular mechanism of age-related cellular senescence is believed to involve genomic instability and DNA damage response (DDR); the chronic inflammation associated with senescence causes cardiovascular damage. Therefore, vascular endothelial cell senescence is believed to induce the pathogenesis of CVDs after radiation exposure. The findings of several prior studies have revealed that ionizing radiation (IR) induces cellular senescence as well as cell death in ECs. We have previously reported that DDR activates endothelial nitric oxide (NO) synthase, and NO production promotes endothelial senescence. Endothelial NO synthase (eNOS) is a major isoform expressed in ECs that maintains cardiovascular homeostasis. Therefore, radiation-induced NO production, a component of the DDR in ECs, may be involved in CVDs after radiation exposure. In this article, we describe the pathology of radiation-induced CVD and the unique radio-response to radiation exposure in ECs.
• Characterization of a novel nicotinamide adenine dinucleotide-cytochrome b5 reductase mutation associated with canine hereditary methemoglobinemia.

  Yayoi Otsuka-Yamasaki, Osamu Inanami, Haruka Shino, Reeko Sato, Masahiro Yamasaki

  The Journal of veterinary medical science 83 (2) 315 - 321 2021/03/05 [Refereed]
   

  Hereditary methemoglobinemia associated with nicotinamide adenine dinucleotide-cytochrome b5 reductase (b5R) deficiency is a rare autosomal recessive disorder in animals. Recently, nonsynonymous b5R gene (CYB5R3) variants have been reported to be associated with canine and feline hereditary methemoglobinemia. However, the underlying molecular mechanisms of canine and feline methemoglobinemia caused by these nonsynonymous variants have not yet been reported. Previously, we reported a Pomeranian dog family with hereditary methemoglobinemia, carrying CYB5R3 mutation of an A>C transition at codon 194 in exon 7, replacing an isoleucine residue with leucine (p.Ile194Leu). In this study, we investigated the enzymatic and structural properties of the soluble form of wild-type and Ile194Leu canine b5Rs to characterize the effects of this missense mutation. Our results showed that the kinetic properties of the mutant enzyme were not affected by this amino acid substitution. The secondary structure of the wild-type and Ile194Leu b5Rs detected by circular dichroism showed a similar pattern. However, the mutant enzyme exhibited decreased heat stability and increased susceptibility to trypsin hydrolysis. Moreover, the thermostability and unfolding measurements indicated that the mutant enzyme was more sensitive to temperature-dependent denaturation than the wild-type b5R. We concluded from these results that unstable mutant enzyme properties with normal enzymatic activity would be associated with hereditary methemoglobinemia in the Pomeranian dog family.
• Using the larvae of caddisfly as a biomonitor to assess the spatial distribution and effective half-life of radiocesium in riverine environments in Fukushima, Japan

  Yuki Matsuo, Kunihiko Nakai, Nozomi Tatsuta, Osamu Inanami, Kumiko Yamamoto, Hazuki Mizukawa, Hiromitsu Nagasaka, Futoshi Mizutani, Youichi Chisaki, Toshiki Aiba, Takashi Ohba, Izumi Watanabe, Hiromi Nabeshi, Taiki Higuchi, Yuki Koga, Hideaki Matsumoto, Kou Nishimuta, Hideki Miyamoto, Tomokazu Haraguchi, Noriko Ryuda, Daisuke Ueno

  Physics Open 6 2021/02 [Refereed]
   

  The environmental monitoring survey using this organisms was called “Caddisfly Watch” and this activity has involved both scientists and local people for collecting them. A simple method is needed for the continuous monitoring of radiocesium (137Cs) contamination in riverine environments after the 2011 accident at the Fukushima Dai-ichi Nuclear Power Plant (FDNPP) in Japan. In a program called “Caddisfly Watch”, we used larvae of the caddisfly Stenopsyche marmorata (Trichoptera: Stenopsychidae) to monitor the spatial distribution and estimate effective half-life (Teff) of 137Cs pollution in riverine environments. Caddisfly larvae showed that the highest concentration of 137Cs among several aquatic organisms and no apparent variation between growth stage. In addition, caddisfly larvae reflected 137Cs concentrations in suspended particulate matter in their gut, and that showed no seasonal variation, better reproducibility, and significant correlation with those in sediment. Results indicate that caddisfly larvae can be used as a biological sampler of suspended particulate matters. The Teff values of 137Cs concentrations in caddisfly larvae estimated by single component decay function model showed significant fit. The Teff values in Kuma, Maeda, downstream Niida, upstream Niida, Ohkawa, and Ukedo river showed 2.8, 5.7, 3.1, 6.7, 0.6, and 4.8 years (34, 68, 38, 80, 6.9, and 58 months), respectively. The results of declining trend in this study were similar to those in previous reported in Fukushima. Further continuous observations using this simple approach of “Caddisfly Watch” make it possible to predict the future of the contamination with radioactive Cs in the river environment.
• Radiation-induced abnormal centrosome amplification and mitotic catastrophe in human cervical tumor HeLa cells and murine mammary tumor EMT6 cells.

  Masaki Fujimoto, Tomoki Bo, Kumiko Yamamoto, Hironobu Yasui, Tohru Yamamori, Osamu Inanami

  Journal of clinical biochemistry and nutrition 67 (3) 240 - 247 2020/11 [Refereed]
   

  Mitotic catastrophe is a form of cell death linked to aberrant mitosis caused by improper or uncoordinated mitotic progression. Abnormal centrosome amplification and mitotic catastrophe occur simultaneously, and some cells with amplified centrosomes enter aberrant mitosis, but it is not clear whether abnormal centrosome amplification triggers mitotic catastrophe. Here, to investigate whether radiation-induced abnormal centrosome amplification is essential for induction of radiation-induced mitotic catastrophe, centrinone-B, a highly selective inhibitor of polo-like kinase 4, was utilized to inhibit centrosome amplification, since polo-like kinase 4 is an essential kinase in centrosome duplication. When human cervical tumor HeLa cells and murine mammary tumor EMT6 cells were irradiated with 2.5 Gy of X-rays, cells with morphological features of mitotic catastrophe and the number of cells having >2 centrosomes increased in both cell lines. Although centrinone-B significantly inhibited radiation-induced abnormal centrosome amplification in both cell lines, such treatment did not change cell growth and significantly enhanced mitotic catastrophe in HeLa cells exposed to X-rays. In contrast, inhibition of centrosome amplification reduced cell growth and mitotic catastrophe in EMT6 cells exposed to X-rays. These results indicated that the role of radiation-induced abnormal centrosome amplification in radiation-induced mitotic catastrophe changes, depending on the cell type.
• Mitochondrial fission promotes radiation-induced increase in intracellular Ca2+ level leading to mitotic catastrophe in mouse breast cancer EMT6 cells.

  Tomoki Bo, Tohru Yamamori, Kumiko Yamamoto, Masaki Fujimoto, Hironobu Yasui, Osamu Inanami

  Biochemical and biophysical research communications 522 (1) 144 - 150 0006-291X 2020/01/29 [Refereed][Not invited]
   

  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.
• Nucleoside analogs as a radiosensitizer modulating DNA repair, cell cycle checkpoints, and apoptosis.

  Hironobu Yasui, Daisuke Iizuka, Wakako Hiraoka, Mikinori Kuwabara, Akira Matsuda, Osamu Inanami

  Nucleosides, nucleotides & nucleic acids 39 (1-3) 439 - 452 1525-7770 2020 [Refereed][Not invited]
   

  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.
• Inhibition of ubiquitin-specific protease 2 causes accumulation of reactive oxygen species, mitochondria dysfunction, and intracellular ATP decrement in C2C12 myoblasts.

  Mayuko Hashimoto, Natsuko Saito, Haru Ohta, Kumiko Yamamoto, Asuka Tashiro, Kosuke Nakazawa, Osamu Inanami, Hiroshi Kitamura

  Physiological reports 7 (14) e14193  2019/07 [Refereed][Not invited]
   

  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.
• Differentiation of bone marrow-derived cells toward thermogenic adipocytes in white adipose tissue induced by the β3 adrenergic stimulation.

  Takeshi Yoneshiro, Woongchul Shin, Ken Machida, Keigo Fukano, Ayumi Tsubota, Yong Chen, Hironobu Yasui, Osamu Inanami, Yuko Okamatsu-Ogura, Kazuhiro Kimura

  FASEB journal : official publication of the Federation of American Societies for Experimental Biology 33 (4) 5196 - 5207 0892-6638 2019/04 [Refereed][Not invited]
   

  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.
• The Adjuvant Effect of Squalene, an Active Ingredient of Functional Foods, on Doxorubicin-Treated Allograft Mice.

  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 0163-5581 2019 [Refereed][Not invited]
   

  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.
• In Vivo Extracellular pH Mapping of Tumors Using Electron Paramagnetic Resonance.

  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 0003-2700 2018/12/04 [Refereed][Not invited]
   

  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.
• Genotoxic Responses of Mitochondrial Oxygen Consumption Rate and Mitochondrial Semiquinone Radicals in Tumor Cells

  Kumiko Yamamoto, Hironobu Yasui, Tomoki Bo, Tohru Yamamori, Wakako Hiraoka, Toshihide Yamasaki, Ken-ichi Yamada, Osamu Inanami

  APPLIED MAGNETIC RESONANCE 49 (8) 837 - 851 0937-9347 2018/08 [Refereed][Not invited]
   

  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.
• Evaluation of mitochondrial redox status and energy metabolism of X-irradiated HeLa cells by LC/UV, LC/MS/MS and ESR.

  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 1071-5762 2018/06 [Refereed][Not invited]
   

  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.
• A 750-MHz Electronically Tunable Resonator Using Microstrip Line Couplers for Electron Paramagnetic Resonance Imaging of a Mouse Tumor-Bearing Leg.

  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 0018-9294 2018/05 [Refereed][Not invited]
   

  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.
• Ataxia-Telangiectasia Mutated (ATM) Kinase Regulates eNOS Expression and Modulates Radiosensitivity in Endothelial Cells Exposed to Ionizing Radiation

  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 1938-5404 2018/05/01 [Refereed][Not invited]
   

  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.
• Preclinical study on hypoxic radiosensitizing effects of glycididazole in comparison with those of doranidazole in vitro and in vivo

  Hironobu Yasui, Nobuo Kubota, Junko Nishizumi, Yuri Sakai, Tohru Yamamori, Osamu Inanami

  Oncology Letters 15 (2) 1993 - 1998 1792-1082 2018/02/01 [Refereed][Not invited]
   

  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.
• Calmodulin-dependent protein kinase II (CaMKII) mediates radiation-induced mitochondrial fission by regulating the phosphorylation of dynamin-related protein 1 (Drp1) at serine 616.

  Tomoki Bo, Tohru Yamamori, Motofumi Suzuki, Yuri Sakai, Kumiko Yamamoto, Osamu Inanami

  Biochemical and biophysical research communications 495 (2) 1601 - 1607 0006-291X 2018/01/08 [Refereed][Not invited]
   

  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.
• “Caddisfly watch,” a biomonitoring program using Stenopsyche larvae to determine radioactive cesium contamination in rivers following the Fukushima nuclear disaster

  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 1860-188X 2018/01/01 [Refereed][Not invited]
   

  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.
• NADPH oxidase 4 mediates ROS production in radiation-induced senescent cells and promotes migration of inflammatory cells.

  Yuri Sakai, Tohru Yamamori, Yoji Yoshikawa, Tomoki Bo, Motofumi Suzuki, Kumiko Yamamoto, Tetsuro Ago, Osamu Inanami

  Free radical research 52 (1) 92 - 102 1071-5762 2018/01 [Refereed][Not invited]
   

  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.
• Familial Congenital Methemoglobinemia in Pomeranian Dogs Caused by a Missense Variant in the NADH-Cytochrome B5 Reductase Gene

  H. Shino, Y. Otsuka-Yamasaki, T. Sato, K. Ooi, O. Inanami, R. Sato, M. Yamasaki

  Journal of Veterinary Internal Medicine 32 (1) 165 - 171 1939-1676 2018/01/01 [Refereed][Not invited]
   

  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.
• Quantitative pO(2) Mapping Using Electron Paramagnetic Resonance Imaging in Orthotopic Murine Glioma

  Hironobu Yasui, Tatsuya Kawai, Shingo Matsumoto, Keita Saito, Nallathamby Devasahayam, James B. Mitchell, Kevin Camphausen, Osamu Inanami, Murali C. Krishna

  FREE RADICAL BIOLOGY AND MEDICINE 112 106 - 106 0891-5849 2017/11
• MK-8776, a novel Chk1 inhibitor, exhibits an improved radiosensitizing effect compared to UCN-01 by exacerbating radiation-induced aberrant mitosis

  Motofumi Suzuki, Tohru Yamamori, Tomoki Bo, Yuri Sakai, Osamu Inanami

  TRANSLATIONAL ONCOLOGY 10 (4) 491 - 500 1936-5233 2017/08 [Refereed][Not invited]
   

  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.
• Feasibility of in vivo three-dimensional T-2(*) mapping using dicarboxy-PROXYL and CW-EPR-based single-point imaging

  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 0968-5243 2017/06 [Refereed][Not invited]
   

  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.
• Analysis of the mechanism of radiation-induced upregulation of mitochondrial abundance in mouse fibroblasts

  Tohru Yamamori, Tomoya Sasagawa, Osamu Ichii, Mie Hiyoshi, Tomoki Bo, Hironobu Yasui, Yasuhiro Kon, Osamu Inanami

  JOURNAL OF RADIATION RESEARCH 58 (3) 292 - 301 0449-3060 2017/05 [Refereed][Not invited]
   

  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.
• Lipophilic triphenylphosphonium derivatives enhance radiation-induced cell killing via inhibition of mitochondrial energy metabolism in tumor cells

  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 0304-3835 2017/04 [Refereed][Not invited]
   

  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.
• Metabolic analysis of radioresistant medulloblastoma stem-like clones and potential therapeutic targets

  Lue Sun, Takashi Moritake, Kazuya Ito, Yoshitaka Matsumoto, Hironobu Yasui, Hidehiko Nakagawa, Aki Hirayame, Osamu Inanami, Koji Tsuboi

  PLOS ONE 12 (4) e0176162  1932-6203 2017/04 [Refereed][Not invited]
   

  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 弘前大学出版会 6 (1) 1 - 5 2423-9097 2017/02 [Refereed][Not invited]
  
• Quantitative imaging of pO(2) in orthotopic murine gliomas: hypoxia correlates with resistance to radiation

  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 1071-5762 2017 [Refereed][Not invited]
   

  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.
• A Nucleoside Anticancer Drug, 1-(3-C-Ethynyl-beta-D-Ribo-Pentofuranosyl)Cytosine, Induces Depth-Dependent Enhancement of Tumor Cell Death in Spread-Out Bragg Peak (SOBP) of Proton Beam

  Kenichiro Maeda, Hironobu Yasui, Tohru Yamamori, Taeko Matsuura, Seishin Takao, Motofumi Suzuki, Akira Matsuda, Osamu Inanami, Hiroki Shirato

  PLOS ONE 11 (11) e0166848  1932-6203 2016/11 [Refereed][Not invited]
   

  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.
• Evaluation of the relative biological effectiveness of spot-scanning proton irradiation in vitro

  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 0449-3060 2016/06 [Refereed][Not invited]
   

  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.
• Effect of MPS1 Inhibition on Genotoxic Stress Responses in Murine Tumour Cells

  Motofumi Suzuki, Tohru Yamamori, Hironobu Yasui, Osamu Inanami

  ANTICANCER RESEARCH 36 (6) 2783 - 2792 0250-7005 2016/06 [Refereed][Not invited]
   

  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.
• Inhibition of the mitochondrial fission protein dynamin-related protein 1 (Drp1) impairs mitochondrial fission and mitotic catastrophe after x-irradiation

  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 1059-1524 2015/12 [Refereed][Not invited]
   

  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.
• Roles of ROS and PKC-beta II in ionizing radiation-induced eNOS activation in human vascular endothelial cells

  Kimimasa Sakata, Takashi Kondo, Natsumi Mizuno, Miki Shoji, Hironobu Yasui, Tohru Yamamori, Osamu Inanami, Hiroki Yokoo, Naoki Yoshimura, Yuichi Hattori

  VASCULAR PHARMACOLOGY 70 55 - 65 1537-1891 2015/07 [Refereed][Not invited]
   

  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.
• Downregulation of the DNA repair enzyme apurinic/apyrimidinic endonuclease 1 stimulates transforming growth factor-beta 1 production and promotes actin rearrangement

  Yuri Sakai, Tohru Yamamori, Hironobu Yasui, Osamu Inanami

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 461 (1) 35 - 41 0006-291X 2015/05 [Refereed][Not invited]
   

  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.
• Activation of eNOS in endothelial cells exposed to ionizing radiation involves components of the DNA damage response pathway

  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 0006-291X 2015/01 [Refereed][Not invited]
   

  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.
• 3-Methyl pyruvate enhances radiosensitivity through increasing mitochondria-derived reactive oxygen species in tumor cell lines

  Naoya Nishida, Hironobu Yasui, Masaki Nagane, Tohru Yamamori, Osamu Inanami

  JOURNAL OF RADIATION RESEARCH 55 (3) 455 - 463 0449-3060 2014/05 [Refereed][Not invited]
   

  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.
• Radiosensitization of tumor cells through endoplasmic reticulum stress induced by PEGylated nanogel containing gold nanoparticles

  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 0304-3835 2014/05 [Refereed][Not invited]
   

  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.
• In vivo tumour extracellular pH monitoring using electron paramagnetic resonance: the effect of X- ray irradiation

  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 0952-3480 2014/04 [Refereed][Not invited]
   

  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.
• The Role of HSP90 and ATM in the Radiation-Induced eNOS Activation

  Masaki Nagane, Hironobu Yasui, Tohru Yamamori, Yuri Sakai, Koichi Niwa, Osamu Inanami

  FREE RADICAL BIOLOGY AND MEDICINE 65 S102 - S102 0891-5849 2013/11
• Metabolic-targeted Treatment with Dichloroacetate Transiently Induces Tumor Hypoxia in Murine Squamous Cell Carcinoma Model

  Hironobu Yasui, Keita Saito, Naoya Nishida, Shingo Matsumoto, Tohru Yamamori, Murali Cherukuri Krishna, Osamu Inanami

  FREE RADICAL BIOLOGY AND MEDICINE 65 S26 - S26 0891-5849 2013/11
• ER stress suppresses DNA double-strand break repair and sensitizes tumor cells to ionizing radiation by stimulating proteasomal degradation of Rad51

  Tohru Yamamori, Shunsuke Meike, Masaki Nagane, Hironobu Yasui, Osamu Inanami

  FEBS LETTERS 587 (20) 3348 - 3353 0014-5793 2013/10 [Refereed][Not invited]
   

  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.
• Radiation-induced nitric oxide mitigates tumor hypoxia and radioresistance in a murine SCCVII tumor model

  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 0006-291X 2013/08 [Refereed][Not invited]
   

  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.
• 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

  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 (一社)日本衛生学会 68 (Suppl.) S176 - S176 0021-5082 2013/03 [Refereed][Not invited]
  
• The prospective application of a hypoxic radiosensitizer, doranidazole to rat intracranial glioblastoma with blood brain barrier disruption

  Hironobu Yasui, Taketoshi Asanuma, Junichi Kino, Tohru Yamamori, Shunsuke Meike, Masaki Nagane, Nobuo Kubota, Mikinori Kuwabara, Osamu Inanami

  BMC CANCER 13 106  1471-2407 2013/03 [Refereed][Not invited]
   

  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.
• Vincristine enhances amoeboid-like motility via GEF-H1/RhoA/ROCK/Myosin light chain signaling in MKN45 cells

  Masato Eitaki, Tohru Yamamori, Shunsuke Meike, Hironobu Yasui, Osamu Inanami

  BMC CANCER 12 469  1471-2407 2012/10 [Refereed][Not invited]
   

  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.
• Roles of mitochondria-generated reactive oxygen species on X-ray-induced apoptosis in a human hepatocellular carcinoma cell line, HLE

  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 1071-5762 2012/08 [Refereed][Not invited]
   

  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.
• Ionizing radiation induces mitochondrial reactive oxygen species production accompanied by upregulation of mitochondrial electron transport chain function and mitochondrial content under control of the cell cycle checkpoint

  Tohru Yamamori, Hironobu Yasui, Masayuki Yamazumi, Yusuke Wada, Yoshinari Nakamura, Hideo Nakamura, Osamu Inanami

  FREE RADICAL BIOLOGY AND MEDICINE 53 (2) 260 - 270 0891-5849 2012/07 [Refereed][Not invited]
   

  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.
• Oral administration of bovine lactoferrin upregulates neutrophil functions in a dog with familial beta 2-integrin-related neutrophil dysfunction

  Saori Kobayashi, Yuya Abe, Osamu Inanami, Shinichi Oda, Koji Yamauchi, Careen Hankanga, Jun Yasuda, Reeko Sato

  VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 143 (1-2) 155 - 161 0165-2427 2011/09 [Refereed][Not invited]
   

  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.
• 8-Aminoadenosine Enhances Radiation-induced Cell Death in Human Lung Carcinoma A549 Cells

  Shunsuke Meike, Tohru Yamamori, Hironobu Yasui, Masato Eitaki, Akira Matsuda, Osamu Inanami

  JOURNAL OF RADIATION RESEARCH 52 (4) 456 - 463 0449-3060 2011/07 [Refereed][Not invited]
   

  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.
• A nucleoside anticancer drug, 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine (TAS106), sensitizes cells to radiation by suppressing BRCA2 expression

  Shunsuke Meike, Tohru Yamamori, Hironobu Yasui, Masato Eitaki, Akira Matsuda, Masami Morimatsu, Masakazu Fukushima, Yasundo Yamasaki, Osamu Inanami

  MOLECULAR CANCER 10 92  1476-4598 2011/07 [Refereed][Not invited]
   

  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.
• Phosphorylation of p66shc mediates 6-hydroxydopamine cytotoxicity

  Tohru Yamamori, Ayano Mizobata, Yoshiro Saito, Yasuomi Urano, Osamu Inanami, Kaikobad Irani, Noriko Noguchi

  FREE RADICAL RESEARCH 45 (3) 342 - 350 1071-5762 2011/03 [Refereed][Not invited]
   

  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.
• Induction of neurite outgrowth by alpha-phenyl-N-tert-butylnitrone through nitric oxide release and Ras-ERK pathway in PC12 cells

  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 1071-5762 2010/06 [Refereed][Not invited]
   

  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.
• A novel copper(II) coordination at His186 in full-length murine prion protein

  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 0006-291X 2010/04 [Refereed][Not invited]
   

  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
• Super paramagnetic iron oxide MRI shows defective Kupffer cell uptake function in non-alcoholic fatty liver disease

  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 0017-5749 2010/02 [Refereed][Not invited]
   

  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.
• Canine neutrophil dysfunction caused by downregulation of beta 2-integrin expression without mutation

  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 0165-2427 2009/08 [Refereed][Not invited]
   

  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.
• Redox regulation in radiation-induced cytochrome c release from mitochondria of human lung carcinoma A549 cells

  Aki Ogura, Shigeru Oowada, Yasuhiro Kon, Aki Hirayama, Hironobu Yasui, Shunsuke Meike, Saori Kobayashi, Mikinori Kuwabara, Osamu Inanami

  CANCER LETTERS 277 (1) 64 - 71 0304-3835 2009/05 [Refereed][Not invited]
   

  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.
• 3P-016 Fibril formation of mouse priori mutants(Protein:Structure,The 47th Annual Meeting of the Biophysical Society of Japan)

  Shibuya Masaki, Watanabe Yasuko, Kubota Kazuhisa, Takahashi Keiko, Nakagawa Hidehiko, Miyata Naoki, Inanami Osamu, Hiraoka Wakako

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 49 S153  2009
• Dynamics and Local Ordering of Spin-Labeled Prion Protein: An ESR Simulation Study of a Highly PH-Sensitive Site

  Yun-Wei Chiang, Yuki Otoshima, Yasuko Watanabe, Osamu Inanami, Yuhei Shimoyama

  JOURNAL OF BIOMOLECULAR STRUCTURE & DYNAMICS 26 (3) 355 - 365 0739-1102 2008/12 [Refereed][Not invited]
   

  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.
• Inhibition of HIF-1 alpha by the anticancer drug TAS106 enhances X-ray-induced apoptosis in vitro and in vivo

  H. Yasui, A. Ogura, T. Asanuma, A. Matsuda, I. Kashiwakura, M. Kuwabara, O. Inanami

  BRITISH JOURNAL OF CANCER 99 (9) 1442 - 1452 0007-0920 2008/10 [Refereed][Not invited]
   

  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.
• Visualization of the protective ability of a free radical trapping compound against rat C6 and F98 gliomas with diffusion tensor fiber tractography

  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 1053-1807 2008/09 [Refereed][Not invited]
   

  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
• Regulation of Cell Survival and Death Signals Induced by Oxidative Stress

  Mikinori Kuwabara, Taketoshi Asanuma, Koichi Niwa, Osamu Inanami

  JOURNAL OF CLINICAL BIOCHEMISTRY AND NUTRITION 43 (2) 51 - 57 0912-0009 2008/09 [Refereed][Not invited]
   

  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.
• A BOLD-fMRI study of cerebral activation induced by injection of algesic chemical substances into the anesthetized rat forepaw

  Taketoshi Asanuma, Hironobu Yasui, Masayasu Sato, Osamu Inanami, Mikinori Kuwabara

  JAPANESE JOURNAL OF VETERINARY RESEARCH 56 (2) 99 - 107 0047-1917 2008/08 [Refereed][Not invited]
   

  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.
• Purvalanol A induces apoptosis and downregulation of antiapoptotic proteins through abrogation of phosphorylation of JAK2/STAT3 and RNA polymerase II

  Daisuke Iizuka, Aki Ogura, Mikinori Kuwabara, Osamu Inanami

  ANTI-CANCER DRUGS 19 (6) 565 - 572 0959-4973 2008/07 [Refereed][Not invited]
   

  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.
• Proinsutin C-peptide abrogates type-1 diabetes-induced increase of renal endothelial nitric oxide synthase in rats

  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 1520-7552 2008/05 [Refereed][Not invited]
   

  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.
• Effect of bovine lactoferrin on functions of activated feline peripheral blood mononuclear cells during chronic feline immunodeficiency virus infection

  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 0916-7250 2008/05 [Refereed][Not invited]
   

  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.
• The antiproliferative effect of bovine lactoferrin on canine mammary gland tumor cells

  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 0916-7250 2008/05 [Refereed][Not invited]
   

  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.
• Neuron is the primary target of Ca2+ paradox-type insult-induced cell injury in neuron/astrocyte co-cultures

  Tatsuro Kosugi, Koichi Kawahara, Motoki Tanaka, Yasuko Watanabe, Osamu Inanami

  NEUROCHEMISTRY INTERNATIONAL 52 (4-5) 887 - 896 0197-0186 2008/03 [Refereed][Not invited]
   

  "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.
• Individual differences in the radiosensitivity of hematopoietic progenitor cells detected in steady-state human peripheral blood

  Asami Oriya, Kenji Takahashi, Osamu Inanami, Toshiaki Miura, Yoshinao Abe, Mikinori Kuwabara, Ikuo Kashiwakura

  JOURNAL OF RADIATION RESEARCH 49 (2) 113 - 121 0449-3060 2008/03 [Refereed][Not invited]
   

  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.
• X irradiation combined with TNF alpha-related apoptosis-inducing ligand (TRAIL) reduces hypoxic regions of human gastric adenocarcinoma xenografts in SCID mice

  Momoko Takahashi, Hironobu Yasui, Aki Ogura, Taketoshi Asanuma, Nobuo Kubota, Michihiko Tsujitan, Mikinori Kuwabara, Osamu Inanami

  JOURNAL OF RADIATION RESEARCH 49 (2) 153 - 161 0449-3060 2008/03 [Refereed][Not invited]
   

  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.
• Instability of familial spongiform encephalopathy-related prion mutants

  Yasuko Watanabe, Wakako Hiraoka, Yuhei Shimoyama, Motohiro Horiuchi, Mikinori Kuwabara, Osamu Inanami

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 366 (1) 244 - 249 0006-291X 2008/02 [Refereed][Not invited]
   

  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.
• Radiation-induced apoptosis of tumor cells is facilitated by inhibition of the interaction between Survivin and Smac/DIABLO

  Aki Ogura, Yasuko Watanabe, Daisuke Iizuka, Hironobu Yasui, Makoto Amitani, Saorl Kobayashi, Mikinori Kuwabara, Osamu Inanami

  CANCER LETTERS 259 (1) 71 - 81 0304-3835 2008/01 [Refereed][Not invited]
   

  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.
• 1P-015 pH-induced conformational change in full-length mouse prion mutants(The 46th Annual Meeting of the Biophysical Society of Japan)

  Shibuya Masaki, Watanabe Yasuko, Inanami Osamu, Hiraoka Wakako

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 48 S23  2008
• Enhancement of cell death by TNF alpha-related apoptosis-inducing ligand (TRAIL) in human lung carcinoma A549 cells exposed to X rays under hypoxia

  Momoko Takahashi, Osamu Inanami, Nobuo Kubota, Michihiko Tsujitan, Hironobu Yasui, Aki Ogura, Mikinori Kuwabara

  JOURNAL OF RADIATION RESEARCH 48 (6) 461 - 468 0449-3060 2007/11 [Refereed][Not invited]
   

  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.
• Costimulatory effects of complement receptor type 3 and Fc receptor for IgG (Fc gamma R) on superoxide production and signal transduction in bovine neutrophils

  Hajime Nagahata, Hidetoshi Higuchi, Osamu Inanami, Mikinori Kuwabara

  JOURNAL OF VETERINARY MEDICAL SCIENCE 69 (10) 993 - 997 0916-7250 2007/10 [Refereed][Not invited]
   

  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.
• Treatment combining X-irradiation and a ribonucleoside anticancer drug, TAS106, effectively suppresses the growth of tumor cells transplanted in mice

  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 0360-3016 2007/05 [Refereed][Not invited]
   

  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.
• Purvalanol A enhances cell killing by inhibiting up-regulation of CDC2 kinase activity in tumor cells irradiated with high doses of X rays

  Daisuke Iizuka, Osamu Inanami, Ikuo Kashiwakura, Mikinori Kuwabara

  RADIATION RESEARCH 167 (5) 563 - 571 0033-7587 2007/05 [Refereed][Not invited]
   

  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.
• Effects of leptin and tumor necrosis factor-alpha on degranulation and superoxide production of polymorphonuclear neutrophils from Holstein cows

  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 0916-7250 2007/02 [Refereed][Not invited]
   

  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.
• Oral administration of Antioxidant Biofactor (AOB (TM)) ameliorates ischemia/reperfusion-induced neuronal death in the gerbil

  Hironobu Yasui, Taketoshi Asanuma, Yasuko Watanabe, Kenji Waki, Osamu Inanami, Mikinori Kuwabara

  BIOFACTORS 29 (2-3) 113 - 121 0951-6433 2007 [Refereed][Not invited]
   

  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.
• A new Amphiphilic derivative, N-{[4-(lactobionamido)methyl]benzylidene}1,1-dimethyl-2-(octylsulfanyl)ethylamine N-oxide, has a protective effect against copper-induced fulminant hepatitis in Long-Evans Cinnamon rats at an extremely low concentration compared with its original form alpha-phenyl-N-(tert-butyl) nitrone

  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 1612-1872 2007 [Refereed][Not invited]
   

  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.
• Lipid raft disruption prevents apoptosis induced by 2-chloro-2 '-deoxyadenosine (Cladribine) in leukemia cell lines

  Eriko Takahashi, Osamu Inanami, Toshio Ohta, Akira Matsuda, Mikinori Kuwabara

  LEUKEMIA RESEARCH 30 (12) 1555 - 1561 0145-2126 2006/12 [Refereed][Not invited]
   

  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.
• Identification of pH-sensitive regions in the mouse prion by the cysteine-scanning spin-labeling ESR technique

  Yasuko Watanabe, Osamu Inanami, Motohiro Horiuchi, Wakako Hiraoka, Yuhei Shimoyama, Fuyuhiko Inagaki, Mikinori Kuwabara

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 350 (3) 549 - 556 0006-291X 2006/11 [Refereed][Not invited]
   

  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.
• Synthesis and characterization of a practically better DEPMPO-type spin trap, 5-(2,2-dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1-pyrroline N-oxide (CYPMPO)

  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 1071-5762 2006/11 [Refereed][Not invited]
   

  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.
• Regeneration of megakaryocytopoiesis and thrombopoiesis in vitro from X-irradiated human hematopoietic stem cells

  Ikuo Kashiwakura, Osamu Inanami, Yoshinao Abe, Kei Satoh, Tsuneo A. Takahashi, Mikinori Kuwabara

  RADIATION RESEARCH 166 (2) 345 - 351 0033-7587 2006/08 [Refereed][Not invited]
   

  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
• Effects of overexpression and antisense RNA expression of Orf17, a MutT-type enzyme

  Mika Hori, Taketoshi Asanuma, Osamu Inanami, Mikinori Kuwabara, Hideyoshi Harashima, Hiroyuki Kamiya

  BIOLOGICAL & PHARMACEUTICAL BULLETIN 29 (6) 1087 - 1091 0918-6158 2006/06 [Refereed][Not invited]
   

  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.
• The effects of (-)-epigallocatechin-3-gallate on the proliferation and differentiation of human megakaryocytic progenitor cells

  Satoru Monzen, Takao Mori, Kenji Takahashi, Yoshinao Abe, Osamu Inanami, Mikinori Kuwabara, Ikuo Kashiwakura

  JOURNAL OF RADIATION RESEARCH 47 (2) 213 - 220 0449-3060 2006/06 [Refereed][Not invited]
   

  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.
• Involvement of protein kinase C delta in the activation of NADPH oxidase and the phagocytosis of neutrophils

  K Waki, O Inanami, T Yamamori, H Nagahata, M Kuwabara

  FREE RADICAL RESEARCH 40 (4) 359 - 367 1071-5762 2006/04 [Refereed][Not invited]
   

  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.
• Inhibition of cell proliferation by SARS-CoV infection in Vero E6 cells

  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 0928-8244 2006/03 [Refereed][Not invited]
   

  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.
• Effects of ceramide inhibition on radiation-induced apoptosis in human leukemia MOLT-4 cells

  E Takahashi, O Inanami, T Asanuma, M Kuwabara

  JOURNAL OF RADIATION RESEARCH 47 (1) 19 - 25 0449-3060 2006/03 [Refereed][Not invited]
   

  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.
• Dual inhibition of protein phosphatase-1/2A and calpain rescues nerve growth factor-differentiated PC12 cells from oxygen-glucose deprivation-induced cell death

  T Nakajima, T Wakasa, Y Okuma, O Inanami, Y Nomura, M Kuwahara, K Kawahara

  JOURNAL OF NEUROSCIENCE RESEARCH 83 (3) 459 - 468 0360-4012 2006/02 [Refereed][Not invited]
   

  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.
• Reactive oxygen species mediate shear stress-induced fluid-phase endocytosis in vascular endothelial cells

  K Niwa, J Sakai, T Karino, H Aonuma, T Watanabe, T Ohyama, O Inanami, M Kuwabara

  FREE RADICAL RESEARCH 40 (2) 167 - 174 1071-5762 2006/02 [Refereed][Not invited]
   

  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.
• Magnetic resonance imaging of alveolar echinococcosis experimentally induced in the rat lung

  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 0916-7250 2006/01 [Refereed][Not invited]
   

  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.
• 2P085 Phosphorylation-induced conformational change of p47^ : CW ESR and Four-Pulse DEER study by a site-directed spin labeling (SDSL)(30. Protein function (II),Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

  Hiraoka Wakako, Suzuki Tomoko, Hara Hideyuki, Kuwabara Mikinori, Inanami Osamu

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 46 (2) S317  2006
• Conformational change in full-length mouse prion: A site-directed spin-labeling study

  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 0006-291X 2005/09 [Refereed][Not invited]
   

  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.
• Different radiosensitive megakaryocytic progenitor cells exist in steady-state human peripheral blood

  Kashiwakura, I, O Inanami, Y Abe, TA Takahashi, M Kuwabara

  RADIATION RESEARCH 164 (1) 10 - 16 0033-7587 2005/07 [Refereed][Not invited]
   

  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.
• X irradiation induces the proapoptotic state independent of the loss of clonogenic ability in Chinese hamster V79 cells

  D Iizuka, O Inanami, A Matsuda, Kashiwakura, I, T Asanuma, M Kuwabara

  RADIATION RESEARCH 164 (1) 36 - 44 0033-7587 2005/07 [Refereed][Not invited]
   

  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.
• Reduction of concanavalin A-induced expression of interferon-gamma by bovine lactoferrin in feline peripheral blood mononuclear cells

  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 0165-2427 2005/05 [Refereed][Not invited]
   

  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.
• Post-irradiation hypoxic incubation of X-irradiated MOLT-4 cells reduces apoptotic cell death by changing the intracellular redox state and modulating SAPK/JNK pathways

  T Hamasu, O Inanami, M Tsujitani, K Yokoyama, E Takahashi, Kashiwakura, I, M Kuwabara

  APOPTOSIS 10 (3) 557 - 567 1360-8185 2005/05 [Refereed][Not invited]
   

  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.
• Enhanced induction of apoptosis by combined treatment of human carcinoma cells with X rays and death receptor agonists

  T Hamasu, O Inanami, T Asanuma, M Kuwabara

  JOURNAL OF RADIATION RESEARCH 46 (1) 103 - 110 0449-3060 2005/03 [Refereed][Not invited]
   

  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.
• 1P027 Phosphorylation-induced conformational change of NADPH oxidase subunitp47^ : A site-directed spin labeling (SDSL) study

  Suzuki T., Inanami O., Kuwabara M., Inagaki F., Hiraoka W.

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 45 S38  2005
• A novel anticancer ribonucleoside, 1-(3-C-Ethynyl-beta-D-ribo-pentofuranosyl)cytosine, enhances radiation-induced cell death in tumor cells

  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 0033-7587 2004/12 [Refereed][Not invited]
   

  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.
• Activation of c-kit by stem cell factor induces radioresistance to apoptosis through ERK-dependent expression of survivin in HL60 cells

  SJ Hosseinimehr, O Inanami, T Hamasu, M Takahashi, Kashiwakura, I, T Asanuma, M Kuwabara

  JOURNAL OF RADIATION RESEARCH 45 (4) 557 - 561 0449-3060 2004/12 [Refereed][Not invited]
   

  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.
• Effects of antioxidants on X-ray- or hyperthermia-induced apoptosis in human lymphoma U937 cells

  ZG Cui, T Kondo, LB Feril, K Waki, O Inanami, M Kuwabara

  APOPTOSIS 9 (6) 757 - 763 1360-8185 2004/11 [Refereed][Not invited]
   

  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.
• Phosphoinositide 3-kinase regulates the phosphorylation of NADPH oxidase component p47(phox) by controlling cPKC/PKC6 but not Akt

  T Yamamori, O Inanami, H Nagahata, M Kuwabara

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 316 (3) 720 - 730 0006-291X 2004/04 [Refereed][Not invited]
   

  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.
• Protection against malonate-induced ischemic brain injury in rat by a cell-permeable peptidic c-Jun N-terminal kinase inhibitor, (L)-H1V-TAT(48-57)-PP-JBD(20), observed by the apparent diffusion coefficient mapping magnetic resonance imaging method

  T Asanuma, O Inanami, K Tabu, K Waki, Y Kon, M Kuwabara

  NEUROSCIENCE LETTERS 359 (1-2) 57 - 60 0304-3940 2004/04 [Refereed][Not invited]
   

  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.
• Decreased apoptosis of beta(2)- integrin-deficient bovine neutrophils

  H Nagahata, H Higuchi, H Teraoka, K Takahashi, O Inanami, M Kuwabara

  IMMUNOLOGY AND CELL BIOLOGY 82 (1) 32 - 37 0818-9641 2004/02 [Refereed][Not invited]
   

  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.
• Suppression of malonate-induced ischemic brain injury in rat by a cell-permeable petptidic JNK inhibitor: MRI and ADC mapping study

  M Kuwabara, T Asanuma, O Inanami

  SFRR: PROCEEDINGS OF THE XII BIENNIAL MEETING OF THE SOCIETY FOR FREE RADICAL RESEARCH INTERNATIONAL 257 - 260 2004 [Refereed][Not invited]
   

  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.
• Redox regulation of PI3K/Akt and p53 in bovine aortic endothelial cells exposed to hydrogen peroxide

  K Niwa, O Inanami, T Yamamori, T Ohta, T Hamasu, M Kuwabara

  ANTIOXIDANTS & REDOX SIGNALING 5 (6) 713 - 722 1523-0864 2003/12 [Refereed][Not invited]
   

  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.
• Magnetic resonance imaging and immunoblot analyses in rats with experimentally induced cerebral alveolar echinococcosis

  T Asanuma, Y Matsumoto, M Takiguchi, O Inanami, M Nakao, K Nakaya, A Ito, A Hashimoto, M Kuwabara

  COMPARATIVE MEDICINE 53 (6) 649 - 656 1532-0820 2003/12 [Refereed][Not invited]
   

  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.
• Ca (2+)-dependent and caspase-3-independent apoptosis caused by damage in golgi apparatus due to 2,4,5,7-tetrabromorhodamine 123 bromide-induced photodynamic effects

  M Ogata, O Inanami, M Nakajima, T Nakajima, W Hiraoka, M Kuwabara

  PHOTOCHEMISTRY AND PHOTOBIOLOGY 78 (3) 241 - 247 0031-8655 2003/09 [Refereed][Not invited]
   

  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.
• Induction of apoptosis through the activation of SAPK/JNK followed by the expression of death receptor fas in X-irradiated cells

  M Kuwabara, K Takahashi, O Inanami

  JOURNAL OF RADIATION RESEARCH 44 (3) 203 - 209 0449-3060 2003/09 [Refereed][Not invited]
   

  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.
• Protective effects of thrombopoietin and stem cell factor on X-irradiated CD34(+) megakaryocytic progenitor cells from human placental and umbilical cord blood

  Kashiwakura, I, O Inanami, K Takahashi, TA Takahashi, M Kuwabara, Y Takagi

  RADIATION RESEARCH 160 (2) 210 - 216 0033-7587 2003/08 [Refereed][Not invited]
   

  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.
• Extracellular signal-regulated kinase 1/2 is involved in the activation of NADPH oxidase induced by FMLP receptor but not by complement receptor 3 in rat neutrophils

  K Waki, O Inanami, T Yamamori, M Kuwabara

  FREE RADICAL RESEARCH 37 (6) 665 - 671 1071-5762 2003/06 [Refereed][Not invited]
   

  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.
• The plasma superoxide scavenging activity in canine cancer and hepatic disease

  R Sato, O Inanami, B Syuto, J Sato, M Kuwabara, Y Naito

  JOURNAL OF VETERINARY MEDICAL SCIENCE 65 (4) 465 - 469 0916-7250 2003/04 [Refereed][Not invited]
   

  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.
• Characterization of JNK-like protein derived from a mosquito cell line, C6/36

  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 0962-1075 2003/02 [Refereed][Not invited]
   

  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.
• Roles of protein kinase C delta in the accumulation of P53 and the induction of apoptosis in H2O2-treated bovine endothelial cells

  K Niwa, O Inanami, T Yamamori, T Ohta, T Hamasu, T Karino, M Kuwabara

  FREE RADICAL RESEARCH 36 (11) 1147 - 1153 1071-5762 2002/11 [Refereed][Not invited]
   

  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.
• Relationship between the activation of cyclic AMP responsive element binding protein and ischemic tolerance in the penumbra region of rat cerebral cortex.

  Takayuki Nakajima, Sadahiro Iwabuchi, Hiroyuki Miyazaki, Yasunobu Okuma, Osamu Inanami, Mikinori Kuwabara, Yasuyuki Nomura, Koichi Kawahara

  Neuroscience letters 331 (1) 13 - 6 0304-3940 2002/10/04 [Refereed][Not invited]
   

  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.
• Assessment of neuroprotective ability of a spin trap, alpha-phenyl-N-tert-butylnitrone, against malonate-induced ischemic injury of rat brain by apparent water diffusion coefficient mapping

  T Asanuma, H Ishibashi, A Konno, Y Kon, O Inanami, M Kuwabara

  NEUROSCIENCE LETTERS 329 (3) 281 - 284 0304-3940 2002/09 [Refereed][Not invited]
   

  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.
• Effects of the combination of thrombopoietin with cytokines on the survival of X-irradiated CD34(+) megakaryocytic progenitor cells from normal human peripheral blood

  Kashiwakura, I, O Inanami, M Murakami, TA Takahashi, M Kuwabara, Y Takagi

  RADIATION RESEARCH 158 (2) 202 - 209 0033-7587 2002/08 [Refereed][Not invited]
   

  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.
• Relationship between p38 mitogen-activated protein kinase and small GTPase Rac for the activation of NADPH oxidase in bovine neutrophils

  T Yamamori, O Inanami, H Sumimoto, T Akasaki, H Nagahata, M Kuwabara

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 293 (5) 1571 - 1578 0006-291X 2002/05 [Refereed][Not invited]
   

  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.
• Hypoxia and etanidazole alter radiation-induced apoptosis in HL60 cells but not in MOLT-4 cells

  O Inanami, K Sugihara, T Okui, M Hayashi, M Tsujitani, M Kuwabara

  INTERNATIONAL JOURNAL OF RADIATION BIOLOGY 78 (4) 267 - 274 0955-3002 2002/04 [Refereed][Not invited]
   

  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.
• Radiation-chemical properties of the hypoxic cell radiosensitizer doranidazole (PR-350)

  M Kuwabara, Y Iida, O Inanami, S Sawamura, K Yokoyama, M Tsujitani

  JOURNAL OF RADIATION RESEARCH 43 (1) 77 - 88 0449-3060 2002/03 [Refereed][Not invited]
   

  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.
• Protein synthesis-dependent apoptotic signalling pathway in X-irradiated MOLT-4 human leukaemia cell line

  K Takahashi, O Inanami, M Hayashi, M Kuwabara

  INTERNATIONAL JOURNAL OF RADIATION BIOLOGY 78 (2) 115 - 124 0955-3002 2002/02 [Refereed][Not invited]
   

  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.
• Effects of amifostine on the proliferation and differentiation of megakaryocytic progenitor cells

  Kashiwakura, I, M Murakami, O Inanami, Y Hayase, TA Takahashi, M Kuwabara, Y Takagai

  EUROPEAN JOURNAL OF PHARMACOLOGY 437 (1-2) 19 - 25 0014-2999 2002/02 [Refereed][Not invited]
   

  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.
• Induction of neurite outgrowth in PC12 cells by alpha-phenyl-N-tert-butylnitron through activation of protein kinase C and the Ras-extracellular signal-regulated kinase pathway

  M Tsuji, O Inanami, M Kuwabara

  JOURNAL OF BIOLOGICAL CHEMISTRY 276 (35) 32779 - 32785 0021-9258 2001/08 [Refereed][Not invited]
   

  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.
• PI3-K mediates polymerization of actin in FMLP-Stimulated HL-60 cells

  YD Cui, O Inanami, T Yamamori, M Kuwabara

  PROGRESS IN BIOCHEMISTRY AND BIOPHYSICS 28 (3) 377 - 380 1000-3282 2001/06 [Refereed][Not invited]
   

  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.
• Isolation, characterization, and cDNA cloning of chicken turpentine-induced protein, a new member of the scavenger receptor cysteine-rich (SRCR) family of proteins

  K Iwasaki, M Morimatsu, O Inanami, E Uchida, B Syuto, M Kuwabara, M Niiyama

  JOURNAL OF BIOLOGICAL CHEMISTRY 276 (12) 9400 - 9405 0021-9258 2001/03 [Refereed][Not invited]
   

  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.
• ESR detection of intraphagosomal superoxide in polymorphonuclear leukocytes using 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide

  O Inanami, T Yamamori, TA Takahashi, H Nagahata, M Kuwabara

  FREE RADICAL RESEARCH 34 (1) 81 - 92 1071-5762 2001 [Refereed][Not invited]
   

  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.
• Superoxide generation from human polymorphonuclear leukocytes by liposome-encapsulated hemoglobin

  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 1073-1199 2001 [Refereed][Not invited]
   

  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.
• p38 MAPK and Ca2+ contribute to hydrogen peroxide-induced increase of permeability in vascular endothelial cells but ERK does not

  K Niwa, O Inanami, T Ohta, S Ito, T Karino, M Kuwabara

  FREE RADICAL RESEARCH 35 (5) 519 - 527 1071-5762 2001 [Refereed][Not invited]
   

  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.
• FMLP-induced formation of F-actin in HL60 cells is dependent on PI3-K but not on intracellular Ca2+, PKC, ERK or p38 MAPK

  YD Cui, O Inanami, T Yamamori, K Niwa, H Nagahata, M Kuwabara

  INFLAMMATION RESEARCH 49 (12) 684 - 691 1023-3830 2000/12 [Refereed][Not invited]
   

  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+.
• Unique response of double-stranded oligonucleotides containing a single 8-oxo-7,8-dihydroguanine to gamma rays in the frozen aqueous state at 77 K

  Y Iida, O Inanami, H Inoue, E Ohtsuka, M Kuwabara

  RADIATION RESEARCH 154 (5) 582 - 589 0033-7587 2000/11 [Refereed][Not invited]
   

  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.
• Spin-trapping detection of superoxides in polymorphonuclear leukocytes stimulated with serum-opsonized zymosan

  M Kuwabara, TA Takahashi, H Nagahata, O Inanami

  JAPANESE JOURNAL OF VETERINARY RESEARCH 48 (1) 3 - 13 0047-1917 2000/05 [Refereed][Not invited]
   

  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.
• Neuroprotective effect of alpha-phenyl-N-tert-butylnitrone in gerbil hippocampus is mediated by the mitogen-activated protein kinase pathway and heat shock proteins

  M Tsuji, O Inanami, M Kuwabara

  NEUROSCIENCE LETTERS 282 (1-2) 41 - 44 0304-3940 2000/03 [Refereed][Not invited]
   

  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.
• Radiation sensitivity of megakaryocyte colony-forming cells in human placental and umbilical cord blood

  Kashiwakura, I, M Kuwabara, O Inanami, M Murakami, Y Hayase, TA Takahashi, Y Takagi

  RADIATION RESEARCH 153 (2) 144 - 152 0033-7587 2000/02 [Refereed][Not invited]
   

  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.
• 2-Chloro-2 '-deoxyadenosine induces apoptosis through the Fas/Fas ligand pathway in human leukemia cell line MOLT-4

  Y Nomura, O Inanami, K Takahashi, A Matsuda, M Kuwabara

  LEUKEMIA 14 (2) 299 - 306 0887-6924 2000/02 [Refereed][Not invited]
   

  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.
• Roles of p38 MAPK, PKC and PI3-K in the signaling pathways of NADPH oxidase activation and phagocytosis in bovine polymorphonuclear leukocytes

  T Yamamori, O Inanami, H Nagahata, YD Cui, M Kuwabara

  FEBS LETTERS 467 (2-3) 253 - 258 0014-5793 2000/02 [Refereed][Not invited]
   

  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.
• Effects of BAPTA-AM and forskolin on apoptosis and cytochrome c release in photosensitized Chinese hamster V79 cells.

  Inanami O, Yoshito A, Takahashi K, Hiraoka W, Kuwabara M

  Photochemstry and Photobiology 70 (4) 650 - 655 1751-1097 1999/10 [Refereed][Not invited]
  
• Effects of BAPTA-AM and forskolin on apoptosis and cytochrome c release in photosensitized Chinese hamster V79 cells

  O Inanami, A Yoshito, K Takahashi, W Hiraoka, M Kuwabara

  PHOTOCHEMISTRY AND PHOTOBIOLOGY 70 (4) 650 - 655 0031-8655 1999/10 [Refereed][Not invited]
   

  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.
• Effects of intracellular calcium chelator BAPTA-AM on radiation-induced apoptosis regulated by activation of SAPK/JNK and caspase-3 in MOLT-4 cells

  K Takahashi, O Inanami, M Kuwabara

  INTERNATIONAL JOURNAL OF RADIATION BIOLOGY 75 (9) 1099 - 1105 0955-3002 1999/09 [Refereed][Not invited]
   

  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.
• Lipid peroxides and antioxidants in serum of neonatal calves

  O Inanami, A Shiga, KJ Okada, R Sato, Y Miyake, M Kuwabara

  AMERICAN JOURNAL OF VETERINARY RESEARCH 60 (4) 452 - 457 0002-9645 1999/04 [Refereed][Not invited]
   

  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.
• Inflammatory cell-mediated tumour progression and minisatellite mutation correlate with the decrease of antioxidative enzymes in murine fibrosarcoma cells

  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 0007-0920 1999/02 [Refereed][Not invited]
   

  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.
• Attenuation of caspase-3-dependent apoptosis by Trolox post-treatment of X-irradiated MOLT-4 cells

  O Inanami, K Takahashi, M Kuwabara

  INTERNATIONAL JOURNAL OF RADIATION BIOLOGY 75 (2) 155 - 163 0955-3002 1999/02 [Refereed][Not invited]
   

  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.
• Hydroxyl radical generation and lipid peroxidation in C2C12 myotube treated with iodoacetate and cyanide

  N Matsuki, A Takanohashi, FM Boffi, O Inanami, M Kuwabara, K Ono

  FREE RADICAL RESEARCH 31 (1) 1 - 8 1071-5762 1999 [Refereed][Not invited]
   

  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.
• Hydrogen Peroxide-Induced Activation of SAPK/JNK Regulated by Phosphatidylinositol 3-Kinase in Chinese Hamster V79 Cells

  Osamu Inanami, Kenji Takahashi, Asuka Yoshito, Mikinori Kuwabara

  Antioxidants and Redox Signaling 1 (1) 113 - 121 1523-0864 1999 [Refereed][Not invited]
   

  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.
• Elevation of Intracellular Calcium Ions Is Essential for the H2O2-Induced Activation of SAPK/JNK but Not for That of p38 and ERK in Chinese Hamster V79 Cells

  Osamu Inanami, Toshio Ohta, Shigeo Ito, Mikinori Kuwabara

  Antioxidants and Redox Signaling 1 (4) 501 - 508 1523-0864 1999 [Refereed][Not invited]
   

  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.
• Activation of p47(PHOX), a cytosolic subunit of the leukocyte NADPH oxidase - Phosphorylation of Ser-359 or Ser-370 precedes phosphorylation at other sites and is required for activity

  JL Johnson, JW Park, J El Benna, LP Faust, O Inanami, BM Babior

  JOURNAL OF BIOLOGICAL CHEMISTRY 273 (52) 35147 - 35152 0021-9258 1998/12 [Refereed][Not invited]
   

  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.
• Visualization of the topographical structure of the anesthetized mouse brain by MR microimaging

  T Asanuma, S Shimokawa, O Inanami, Y Kon, M Kuwabara

  JOURNAL OF VETERINARY MEDICAL SCIENCE 60 (12) 1311 - 1314 0916-7250 1998/12 [Refereed][Not invited]
   

  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.
• Bone marrow transplantation in a Holstein heifer with bovine leucocyte adhesion deficiency

  H Nagahata, S Matsuki, H Higuchi, O Inanami, M Kuwabara, K Kobayashi

  VETERINARY JOURNAL 156 (1) 15 - 21 1090-0233 1998/07 [Refereed][Not invited]
   

  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.
• Nucleosides and nucleotides. 176. 2 '-deoxy-2 '-hydroxylaminocylidine: A new antitumor nucleoside that inhibits DNA synthesis although it has a ribonucleoside structure

  A Ogawa, S Shuto, O Inanami, M Kuwabara, M Tanaka, T Sasaki, A Matsuda

  BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 8 (14) 1913 - 1918 0960-894X 1998/07 [Refereed][Not invited]
   

  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.
• Activation of the leukocyte NADPH oxidase by phorbol ester requires the phosphorylation of p47(PHOX) on serine 303 or 304

  O Inanami, JL Johnson, JK McAdara, J El Benna, LRP Faust, PE Newburger, BM Babior

  JOURNAL OF BIOLOGICAL CHEMISTRY 273 (16) 9539 - 9543 0021-9258 1998/04 [Refereed][Not invited]
   

  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.
• The leukocyte NADPH oxidase subunit p47(PHOX) the role of the cysteine residues

  O Inanami, JL Johnson, BM Babior

  ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 350 (1) 36 - 40 0003-9861 1998/02 [Refereed][Not invited]
   

  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.
• Oral administration of (-)catechin protects against ischemia-reperfusion-induced neuronal death in the gerbil

  O Inanami, Y Watanabe, B Syuto, M Nakano, M Tsuji, M Kuwabara

  FREE RADICAL RESEARCH 29 (4) 359 - 365 1071-5762 1998 [Refereed][Not invited]
   

  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.
• The increased formation of alkali-labile sites by a spin-trapping agent alpha-phenyl-N-tert-butylnitrone in gamma-irradiated frozen aqueous solution of DNA

  M Kuwabara, H Ohshima, Y Iida, O Inanami

  JOURNAL OF RADIATION RESEARCH 38 (3) 153 - 155 0449-3060 1997/09 [Refereed][Not invited]
  
• Oral administration of bovine lactoferrin for treatment of intractable stomatitis in feline immunodeficiency virus (FIV)-positive and FIV-negative cats

  R Sato, O Inanami, Y Tanaka, M Takase, Y Naito

  AMERICAN JOURNAL OF VETERINARY RESEARCH 57 (10) 1443 - 1446 0002-9645 1996/10 [Refereed][Not invited]
   

  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.
• Inhibition of collagen-induced platelet aggregation in Japanese black cattle with inherited platelet disorder, Chediak-Higashi syndrome

  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 0916-7250 1996/07 [Refereed][Not invited]
   

  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.
• Lipid peroxide levels and superoxide-scavenging abilities of sera obtained from hotbred (Thoroughbred) horses

  M Kuwabara, N Inukai, O Inanami, Y Miyake, N Tsunoda, Y Maki, F Sato

  JOURNAL OF VETERINARY MEDICAL SCIENCE 58 (2) 97 - 101 0916-7250 1996/02 [Refereed][Not invited]
   

  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.
• THE SUPPRESSION OF AGE-RELATED ACCUMULATION OF LIPID PEROXIDES IN RAT-BRAIN BY ADMINISTRATION OF ROOIBOS TEA (ASPALATHUS-LINEARIS)

  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 0304-3940 1995/08 [Refereed][Not invited]
   

  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.
• ALPHA-PHENYL N-TERT-BUTYL NITRONE (PBN) INCREASES THE CORTICAL CEREBRAL BLOOD-FLOW BY INHIBITING THE BREAKDOWN OF NITRIC-OXIDE IN ANESTHETIZED RATS

  O INANAMI, M KUWABARA

  FREE RADICAL RESEARCH 23 (1) 33 - 39 1071-5762 1995 [Refereed][Not invited]
   

  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.
• MAGNETIC-RESONANCE-IMAGING OF YOUNG AND AGED RAT BRAINS UNDER A MAGNETIC-FIELD OF 7.05-T

  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 0916-7250 1994/10 [Refereed][Not invited]
   

  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.
• RESPONSES OF REGIONAL CEREBRAL BLOOD-FLOW TO INTRAVENOUS ADMINISTRATION OF THYROTROPIN-RELEASING-HORMONE IN AGED RATS

  O INANAMI, K OHNO, A SATO

  NEUROSCIENCE LETTERS 143 (1-2) 151 - 154 0304-3940 1992/08 [Refereed][Not invited]
   

  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.
• NITRIC-OXIDE (NO) IS INVOLVED IN INCREASED CEREBRAL CORTICAL BLOOD-FLOW FOLLOWING STIMULATION OF THE NUCLEUS BASALIS OF MEYNERT IN ANESTHETIZED RATS

  T ADACHI, O INANAMI, A SATO

  NEUROSCIENCE LETTERS 139 (2) 201 - 204 0304-3940 1992/05 [Refereed][Not invited]
   

  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.
• RESPONSES OF REGIONAL CEREBRAL BLOOD-FLOW FOLLOWING FOCAL ELECTRICAL-STIMULATION OF THE NUCLEUS BASALIS OF MEYNERT AND THE MEDIAL SEPTUM USING THE [C-14] IODOANTIPYRINE METHOD IN RATS

  T ADACHI, O INANAMI, K OHNO, A SATO

  NEUROSCIENCE LETTERS 112 (2-3) 263 - 268 0304-3940 1990/05 [Refereed][Not invited]
  
• STIMULATION OF THE NUCLEUS BASALIS OF MEYNERT AND SUBSTANTIA INNOMINATA PRODUCES WIDESPREAD INCREASES IN CEREBRAL BLOOD-FLOW IN THE FRONTAL, PARIETAL AND OCCIPITAL CORTICES

  T ADACHI, D BIESOLD, O INANAMI, A SATO

  BRAIN RESEARCH 514 (1) 163 - 166 0006-8993 1990/04 [Refereed][Not invited]
  
• Regulation of regional blood flow in cerebral cortex and hippocampus by cholinergic fibers originating in the nucleus basalis of Meynert and septal complex in the basal forebrain

  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 [Refereed][Not invited]
  
• STIMULATION OF THE SEPTAL COMPLEX INCREASES LOCAL CEREBRAL BLOOD-FLOW IN THE HIPPOCAMPUS IN ANESTHETIZED RATS

  WH CAO, O INANAMI, A SATO, Y SATO

  NEUROSCIENCE LETTERS 107 (1-3) 135 - 140 0304-3940 1989/12 [Refereed][Not invited]
  
• STIMULATION OF THE NUCLEUS BASALIS OF MEYNERT INCREASES CEREBRAL CORTICAL BLOOD-FLOW IN RATS

  D BIESOLD, O INANAMI, A SATO, Y SATO

  NEUROSCIENCE LETTERS 98 (1) 39 - 44 0304-3940 1989/03 [Refereed][Not invited]
  
• MECHANISM OF PHOTOSENSITIZATION BY PHEOPHORBIDE-A STUDIED BY PHOTOHEMOLYSIS OF ERYTHROCYTES AND ELECTRON-SPIN RESONANCE SPECTROSCOPY

  M KUWABARA, T YAMAMOTO, O INANAMI, F SATO

  PHOTOCHEMISTRY AND PHOTOBIOLOGY 49 (1) 37 - 41 0031-8655 1989/01 [Refereed][Not invited]
  
• CONTRIBUTION OF CHOLINERGIC VASODILATORS ON THE INCREASE IN CEREBRAL CORTICAL BLOOD-FLOW RESPONSES TO THE INTRAVENOUS ADMINISTRATION OF THYROTROPIN RELEASING HORMONE IN ANESTHETIZED RATS

  O INANAMI, K MEGURO, K OHNO, A SATO

  NEUROSCIENCE LETTERS 88 (2) 184 - 188 0304-3940 1988/05 [Refereed][Not invited]
  
• ELECTRON-SPIN-RESONANCE AND SPIN-TRAPPING STUDY OF FREE-RADICALS IN GAMMA-IRRADIATED SOLID LYSOZYME

  O INANAMI, M KUWABARA, F SATO

  FREE RADICAL RESEARCH COMMUNICATIONS 5 (1) 43 - 49 8755-0199 1988 [Refereed][Not invited]
  
• Esr and spin-trapping study of free radicals in γirradiated solid lysozyme

  Osamu Inanami, Mikinori Kuwabara, Fumiaki Sato

  Free Radical Research 5 (1) 43 - 49 1071-5762 1988 [Refereed][Not invited]
   

  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.
• OH-INDUCED FREE-RADICALS IN 3'-UMP AND POLY(U) - SPIN-TRAPPING AND RADICAL CHROMATOGRAPHY

  O INANAMI, M KUWABARA, F SATO

  RADIATION RESEARCH 112 (1) 36 - 44 0033-7587 1987/10 [Refereed][Not invited]
  
• SPIN TRAPPING OF PRECURSORS OF THYMINE DAMAGE IN X-IRRADIATED DNA

  M KUWABARA, O INANAMI, D ENDOH, F SATO

  BIOCHEMISTRY 26 (9) 2458 - 2465 0006-2960 1987/05 [Refereed][Not invited]
  
• OH-INDUCED FREE-RADICALS IN URIDINE STUDIED BY A METHOD COMBINING ELECTRON-SPIN-RESONANCE, SPIN-TRAPPING, AND LIQUID-CHROMATOGRAPHY

  O INANAMI, M KUWABARA, D ENDOH, F SATO

  RADIATION RESEARCH 108 (1) 1 - 11 0033-7587 1986/10 [Refereed][Not invited]
  
• OH-induced free radicals in purine nucleosides and their homopolymers: e.s.r. and spin-trapping with 2-methyl-2-nitrosopropane.

  Kuwabara M, Inanami O, Sato F

  International journal of radiation biology and related studies in physics, chemistry, and medicine 49 (5) 829 - 844 0020-7616 1986/05 [Refereed][Not invited]
  
• OH-INDUCED FREE-RADICALS IN PURINE NUCLEOSIDES AND THEIR HOMOPOLYMERS - ELECTRON-SPIN-RESONANCE AND SPIN-TRAPPING WITH 2-METHYL-2-NITROSOPROPANE

  M KUWABARA, O INANAMI, F SATO

  INTERNATIONAL JOURNAL OF RADIATION BIOLOGY 49 (5) 829 - 844 0955-3002 1986/05 [Refereed][Not invited]
  
• Reaction of the hydrated electron with histone H1 and related compounds studied by e.s.r. and spin-trapping.

  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 0020-7616 1986/01 [Refereed][Not invited]
  
• RADIOLYSIS OF CARBOHYDRATES AS STUDIED BY ELECTRON-SPIN-RESONANCE AND SPIN-TRAPPING .2. GLYCEROL-D8, XYLITOL, DULCITOL, D-SORBITOL AND D-MANNITOL

  M KUWABARA, ZY ZHANG, O INANAMI, G YOSHII

  RADIATION PHYSICS AND CHEMISTRY 24 (2) 217 - 227 1984 [Refereed][Not invited]
  

MISC

• Novel cancer therapeutics aiming to control cell senescence

  安井博宣, 小澤拓実, 藤本政毅, 稲波修  月刊細胞  55-  (2)  115  -118  2023  

  細胞老化(Senescence)は放射線や抗がん剤治療などのストレスによって細胞が増殖を恒久的に停止する現象である。老化細胞は増殖を停止しながらも,サイトカインなど生理活性物質を放出し,炎症や腫瘍の再発といった副作用を引き起こすことが知られている。従って,老化細胞を選択的に除去する方法(セノリシス)を確立することは治療上重要であり,近年,Bcl-2ファミリーなど内因性アポトーシス抑制因子の阻害剤をはじめ様々な治療薬(セノリティック薬)が開発されている。本稿では,筆者らがこれまでに明らかにしてきたがん治療で生じる老化細胞に対する選択的除去に関する研究結果について概説する。(著者抄録)
• 生体ラジカル検出とラジカル関連疾患に関する研究

  稲波修  電子スピンサイエンス  (40)  2023
• Basic research toward cancer senotherapy, a senescent cell-targeted cancer treatment

  安井博宣, 小澤拓実, 藤本政毅, 稲波修  月刊Precision Medicine  6-  (13)  1096  -1100  2023  

  細胞老化(Senescence)は放射線や抗がん剤治療などのストレスによって細胞が増殖を恒久的に停止する現象である。老化細胞は増殖を停止しながらも,サイトカインなど生理活性物質を放出し,炎症や腫瘍の再発といった副作用を引き起こすことが知られている。従って,老化細胞を選択的に除去する方法(セノリシス)を確立することは治療上重要であり,近年,Bcl-2ファミリーなど内因性アポトーシス抑制因子の阻害剤をはじめ様々な治療薬(セノリティック薬)が開発されている。本稿では,筆者らがこれまでに明らかにしてきたがん治療で生じる老化細胞に対する選択的除去に関する研究結果について概説する。(著者抄録)
• 細胞老化の制御に基づく新

  安井 博宣, 稲波 修  細胞  5555-  115  -118  2022  [Not refereed]
  
• グルタミン代謝阻害とセノリシスを組み合わせた新しい放射線治療の可能性—Possibility of a new radiotherapy combining glutamine metabolism inhibition and senolysis

  藤本 政毅, 安井 博宣, 稲波 修  アグリバイオ = Agricultural biotechnology  5-  (13)  1159  -1164  2021/12
• Mechanism of Radiosensitization in Tumor Cells by Nanoparticles

  稲波修, 安井博宣  化学工業  72-  (4)  238  -243  2021  [Not refereed]
  
• Possibility of a new radiotherapy combining glutamine metabolism inhibition and senolysis

  藤本政毅, 安井博宣, 稲波修  月刊細胞  53-  (12)  774  -777  2021  [Not refereed]
   

  グルタミン代謝はがん遺伝子変異を持つ多くのがん細胞において著しく亢進しており、一方では、この様ながん細胞において、グルタミン代謝阻害によって老化様増殖停止が誘導されることが知られている。また、老化様細胞からサイトカイン、ケモカイン、成長因子ならびにプロテアーゼなど炎症、転移、増殖に関連するSenescence-Associated Secretory Phenotype(SASP)因子の分泌亢進によって、治療に対して悪影響を及ぼすことも明らかになってきた。近年、薬剤によって老化様増殖停止をアポトーシスへ変換し、老化様細胞の影響を低減する治療法が加齢関連疾患のために考案され、セノリティックテラピーとして注目されている。本稿では、放射線治療においてグルタミン代謝阻害剤とセノリティック薬の組み合わせ処置によって、老化様増殖停止をアポトーシスに転換させ、副作用の少ない新規治療法の可能性について紹介する。(著者抄録)
• Basic research of radiation therapy targeting cancer stem-like cells in veterinary field

  出口辰弥, 細谷謙次, 安井博宣, 稲波修, 奥村正裕  放射線生物研究  55-  (4)  342  -354  2020  [Not refereed]
  
• Mitochondrial dynamics after irradiation are involved in radiosensitivity

  房知輝, 房知輝, 山盛徹, 安井博宣, 稲波修  放射線生物研究  55-  (4)  355  -369  2020  [Not refereed]
  
• [Imaging of Tumor-Specific Hypoxia Dynamics and Its Significance in Radiation Biology].

  Hironobu Yasui, Shingo Matsumoto, Osamu Inanami, Murali Cherukuri Krishna  Igaku butsuri : Nihon Igaku Butsuri Gakkai kikanshi = Japanese journal of medical physics : an official journal of Japan Society of Medical Physics  40-  (1)  13  -18  2020  [Refereed][Not invited]
   

  Hypoxia has been known to be a feature associated with tumor radioresistance. So far, clinical strategies to overcome chronic hypoxia due to the limitation of the oxygen diffusion have been designed. However, intermittent or acute/cycling hypoxia, whose frequency can range between a few cycles per minutes to hours, is receiving increased attention, because this type of hypoxia has been reported to have an influence on tumor malignancy as well as treatment resistance via increased expression of pro-survival pathways. Therefore, a priori information on fluctuating hypoxia can be important in clinical treatment planning, but complicated dynamics makes it difficult to elucidate biological significance of intermittent hypoxia.Here, we illustrate the use of pulsed electron spin resonance imaging (ESRI) as a novel imaging method to directly monitor fluctuating oxygenation i.e. cycling hypoxia in transplanted tumors. A common resonator platform for both ESRI and magnetic resonance imaging (MRI) provided pO2 maps with anatomical guidance without positional movement. Oxygen images every 3 min in pO2 could visualize the rapid oxygen fluctuation and distinguish the cycling hypoxia and chronic hypoxia. Furthermore, we have examined the vascular renormalization process by longitudinally pO2 mapping during treatments with a multi-tyrosine kinase inhibitor sunitinib. Transient improvement in tumor oxygenation and the decrease of cycling tumor hypoxia were visualized by ESRI 2 to 4 days following antiangiogenic treatments. Radiation treatment during this time period of improved oxygenation by antiangiogenic therapy resulted in a synergistic delay in tumor growth.In conclusion, this ESRI technique combined with MRI, may offer a powerful clinical tool to noninvasively detect variable hypoxic status in tumors and to identify a window of vascular renormalization to maximize the effects of combination therapy with antiangiogenic drugs.
• Radiation-induced nitric oxide production in endothelial cells re-oxygenates solid tumor

  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  2017/06  [Not refereed][Not invited]
  
• 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

  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)  24th-  2015
• 低酸素環境を標的としたがん治療の新たな展開

  安井博宣, 安井博宣, 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  45-  (1)  2010
• 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

  飯塚 大輔, 稲波 修, 安井 博宣  放射線生物研究  42-  (4)  444  -455  2007/12  [Refereed]
  
• 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

  渡邊康子, 渡邊康子, 平岡和佳子, 下山雄平, 堀内基広, 堀内基広, 桑原幹典, 桑原幹典, 稲波修, 稲波修  生化学  2007
• Radiosensitization of tumor cells by ethynylcytidine

  桑原 幹典, 稲波 修, 安井 博宣  Japanese journal of cancer clinics  52-  (1)  31  -36  2006/04  [Not refereed][Not invited]
   

  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
• 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

  稲波修, 渡邊康子  電子スピンサイエンス  (6)  2006
• 血管内皮細胞のシグナル伝達と活性酸素

  丹羽 光一, 稲波 修  放射線生物研究  40-  (1)  1  -13  2005/03  [Not refereed][Not invited]
  
• 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 (ラクトフェリン研究--基礎から応用への掛け橋)

  佐藤 れえ子, 小林 沙織, 稲波 修  ミルクサイエンス  53-  (4)  296  -298  2004
• Regenerative induction of megakaryopoiesis and thrombopoiesis in vitro from X-irradiated CD34^+ cells prepared from human placental and umbilical cord blood

  Kashiwakura Ikuo, Kuwabara Mikinori, Inanami Osamu, Takahashi Tuneo A., Abe Yoshinao  Bulletin of Health Sciences Hirosaki  3-  17  -24  2004
• Activation mechanism of NADPH oxidase and impaired function of bovine neutrophil after parturition

  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  41-  (1)  1  -13  2004
• 268 A Novel Anticancer Ribonucleoside, TAS106 Radiosensitizes Apoptosis through Survivin Down-regulation and G2/M Checkpoint Abrogation in MKN45 Cells(Bases of radiotherapy, Abstracts of the 46th Annual Meeting of the Japan Radiation Research Society) :

  INANAMI Osamu, IIZUKA Daisuke, IWAHARA Akiko, YAMAMORI Tohru, KON Yasuhiro, KITAZATO Kenji, MATSUDA Akira, KUWABARA Mikinori  Journal of radiation research  44-  (4)  452  -452  2003
• 239 Inhibition of Cdc2 kinase activity by Purvalanol A sensitizes X rays-induced apoptosis in gastric tumor cells(Apoptosis related, Abstracts of the 46th Annual Meeting of the Japan Radiation Research Society) :

  IIZUKA Daisuke, INANAMI Osamu, KUWABARA Mikinori  Journal of radiation research  44-  (4)  445  -445  2003
• Effects of combinaions of thrombopoietin with other cytokines on the survival of X-irradiated CD34^+ megakaryocytic progenitor cells from human placental and umbilical cord blood

  Kashiwakura Ikuo, Inanami Osamu, Takahashi Tuneo A., Kuwabara Mikinori  Bulletin of Health Sciences Hirosaki  2-  53  -60  2003
• The molecular mechanisms regulating the phosphorylation of the NADPH oxidase component p47^phox by phosphoinositide 3-kinase.

  山盛徹, 稲波修, 永幡肇, 桑原幹典  獣医生化学  40-  (2)  63  -76  2003
• Hypoxia and Etanidazole Alter Radiation-Induced Apoptosis in HL60 Cells but Not in MOLT-4 Cells

  Inanami Osamu, Sugihara Kiyoshi, Okui Toyo, Hayashi Masanobu, Tsujitani Mitsuhiko, Kuwabara Mikinori  北海道立衛生研究所報 = Report of the Hokkaido Institute of Public Health  52-  114  -114  2002/11/29  

  培養細胞において放射線照射誘発アポトーシスに対する低酸素状態での照射の影響を検討した。HL60及びMolt4細胞を空気中及び低酸素下で15GyのX線を照射した。放射線増感剤としてEtanidazoleを用いた。アポトーシスの検出は核の形態観察と電気泳動によるラダーの検出により行った。HL60細胞では低酸素下での照射ではアポトーシスによる細胞死が減少し、カスパーゼ8、9及び3の活性誘導も減少した。低酸素下でEtanidazoleはX線誘発アポトーシスとカスパーゼの活性を高めた。しかし、Molt4細胞ではEtanidazoleの影響は認められなかった。この2細胞におけるX線誘発DNA二重鎖切断（DSB）は低酸素下での照射ではともに空気中での照射に比べて有意に減少した。低酸素下でEtanidazoleはX線誘発DSBを増加させた。これらの結果からHL60においてはX線誘発アポトーシスはDSBによって引導されること、Molt4では他の損傷が引き金になってアポトーシスが誘発されることが示唆された。
• 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

  稲波修  KEK Proceedings  (2002-4)  2002
• 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

  脇研二, 稲波修, 山盛徹, 永幡肇, 桑原幹典  磁気共鳴と医学  13-  2002
• 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

  桑原幹典, 稲波修  Clinical Neuroscience  19-  (5)  2001
• ウシ好中球のどん食殺菌機構におけるp38 MARKとPKCの役割

  山盛徹, 稲波修, 永幡肇, 桑原幹典  磁気共鳴と医学  12-  2001
• アポトーシス伝達経路におけるフリーラジカル (特集 フリーラジカルの細胞生物学)

  稲波 修, 高橋 賢次, 飯田 義晴  細胞  32-  (2)  47  -51  2000/02  [Not refereed][Not invited]
  
• Oxidative stress and intracellular signal transduction. Role of MAP kinase and calcium.

  稲波修, 太田利男, 昆泰寛, 平岡和佳子, 桑原幹典  放射線科学  43-  (4)  2000
• 放射線誘発アポトーシスに対する抗酸化剤の影響

  稲波修, 高橋賢次, 桑原幹典  磁気共鳴と医学  11-  2000
• 二重鎖PolyA/PolyUにおける放射線誘発ラジカルの解析

  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  10-  1999
• 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

  吉戸あすか, 平岡和佳子, 稲波修, 桑原幹典  磁気共鳴と医学  10-  1999
• 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

  稲波修, 高橋賢次, 吉戸あすか, 桑原幹典  磁気共鳴と医学  10-  1999
• 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

  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  10-  1999
• Roles of water molecules in the hydration layer of DNA in the formation of radiation-induced DNA damage.

  桑原幹典, 大島秀基, 飯田義晴, 稲波修  磁気共鳴と医学  9-  1998
• Effects of Aging on Nitric Oxide Production in LPS-treated Wistar Rats.

  稲波修, 浅沼武敏, 桑原幹典, KOTAKE Y  磁気共鳴と医学  9-  1998
• Radiation- and reactive oxygen-induced cell death and signal transduction in cultured mammalian cells

  M Kuwabara, O Inanami, K Takahashi, W Hiraoka  BIOLOGICAL OXIDANTS AND ANTIOXIDANTS: MOLECULAR MECHANISMS AND HEALTH EFFECTS  14  -21  1998  [Refereed][Not invited]
  
• Analysis of signal transduction mechanisms leading to the reactive oxygen generation by employing neutrophils from a Holstein calf with bovine leukocyte adhesion deficiency(BLAD).

  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  8-  1997
• 好中球貪食時におけるO-2ならびにOHラジカル生成とその局在に関する研究

  稲波 修, 桑原 幹典  家畜生化学  33-  (2)  11  -21  1996/12  [Not refereed][Not invited]
   

  資料形態 : テキストデータ プレーンテキスト
• The small molecular weight GTP-binding proteins(Rho) regulate the superoxide production from polymorphonuclear leukocytes.

  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  7-  1996
• The superoxide production from opsonized-zymosan- and PMA-stimulated polymorphonuclear leukocyte in neonatal calf.

  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  7-  1996
• Isolation and Charatrerization of Chitin-Binding Protein in Bovine Serum.

  NAKAO ATSUKO, YOTSUYA KAORU, MORIMATSU MASAMI, OKADA KEIJI, INANAMI OSAMU, SHUTO BUN'EI  家畜生化学  32-  (1)  31  -36  1995/03  [Not refereed][Not invited]
  
• The function of C-reactive protein on milk production and fertilization in the cow.

  SHIOKAWA TSUGUAKI, MORIMATSU MASAMI, INANAMI OSAMU, OKADA KEIJI, SHIGA AKIO, SHUTO BUN'EI  家畜生化学  32-  (1)  37  -41  1995/03  [Not refereed][Not invited]
  
• The yields of free radicals induced by monochromatic soft X-rays with energy of the K-absorption edge of bromine in BrdU/dThd complexes

  M KUWABARA, S SAWAMURA, O INANAMI, K KOBAYASHI  RADIATION DAMAGE IN DNA: STRUCTURE/FUNCTION RELATIONSHIPS AT EARLY TIMES  241  -249  1995  [Refereed][Not invited]
  
• Free radical. Nitric oxide and cerebral blood flow.

  稲波修, 足立健彦, さき山陽一郎, 佐藤昭夫  神経精神薬理  16-  (3)  1994
• 放射線による染色体蛋白質損傷とその意義

  稲波修, 桑原幹典  放射線生物研究  28-  (3)  1993
• Vasodilative responses in the hippocampus induced by electrical and chemical stimulation of septal complex.

  CAO W-H, 稲波修, 西園寺麗, 佐藤昭夫  自律神経  27-  (3)  1990
• Mechanisms of OH-induced damage formation in nucleic acids: Commonality and disparity in nucleoside, nucleotide and polynucleotide.

  桑原幹典, 稲波修  放射線化学  (44)  1987

Books etc

• 放射線生物学

  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 (ISBN: 9784874022139)
• Applications of EPR in Radiation Research

  Anders Lund, Masaru Shiotani 
  Springer 2014/10 (ISBN: 3319092154) 773

  
• 酸化ストレスの医学

  内藤 裕二, 豐國 伸哉, 吉川 敏一 
  診断と治療社 2014 (ISBN: 9784787821188)
• 酸化ストレスの医学

  内藤 裕二, 豐國 伸哉, 吉川 敏一 
  診断と治療社 2008 (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
• Antioxidant activity of quinone-derivatives from freeze-dried powder of the acidians, In The Biology of Ascidians

  Springer, Tokyo 2001
• Biodefense against active oxygens and free radicals induced by oxidative stress.

  Kodansha and Springer 1998
• Radiation- and reactive oxygen-induced cell death and signal transduction in cultured mammalian cells.

  AOCS Press 1998
• An ESR study of superoxide generation in stimulated neutrophils from a calf with bovine leukocyte adhension deficiency(BLAD).

  Springer 1997

Presentations

• 腫瘍内におけるDNA量の不均一性が放射線分割照射後の放射線抵抗性がん細胞の出現につながる(Intratumor heterogeneity in DNA content leads to the emergence of radioresistant cancer cells after fractionated radiotherapy)

  山下 晃矢, 安井 博宣, 房 知輝, 藤本 政毅, 稲波 修

  日本放射線影響学会大会講演要旨集  2023/11  (一社)日本放射線影響学会
  
• マウス扁平上皮癌におけるジクロロ酢酸を放出する自己組織化ナノ粒子の放射線増感作用について(The Radiosensitizing Effect of Self-Assembled Nanoparticle Releasing Dichloroacetic Acid in a Mouse Model of Squamous Cell Carcinoma)

  山田 さと, 安井 博宣, 長崎 幸夫, 稲波 修

  日本放射線影響学会大会講演要旨集  2023/11  (一社)日本放射線影響学会
  
• マウス扁平上皮がん細胞SCCVIIにおける、Pluronic L64で修飾したC60フラーレン(C60/L64)の放射線増感効果(Radiosensitizing effect of C60 fullerenes modified with Pluronic L64(C60/L64) in mouse squamous cell carcinoma SCC VII cells)

  臼田 史仁, 加藤 千博, 安井 博宣, 王 ぎょく博, 山本 拓矢, 稲波 修

  日本放射線影響学会大会講演要旨集  2023/11  (一社)日本放射線影響学会
  
• 放射線によるDNA損傷は遅延性のミトコンドリア由来の細胞内活性酸素種を上昇させるか?

  稲波修, 加藤千博, 安井博宣, 後藤悠人, 小川美香子

  日本酸化ストレス学会学術集会プログラム・抄録集  2023
• スフィンゴミエリン合成酵素2は虚血性筋障害を促進する

  水垣ひなの, 永根大幹, 永根大幹, 赤羽英夫, PERIANNAN Kuppusamy, 安井博宣, 稲波修, 相原尚之, 上家潤一, 水野谷航, 山下匡

  日本酸化ストレス学会学術集会プログラム・抄録集  2023
• Analysis of X-ray responsiveness of phthalocyanine derivatives for development of caged compounds that can be used deep in vivo

  山下柾, 松廣志乃, 高倉栄男, 中島孝平, 稲波修, 小川美香子

  日本薬学会年会要旨集(Web)  2023
• Investigation of a reaction mechanism of photoimmunotherapy using ESR

  後藤悠人, 高倉栄男, 中島孝平, 稲波修, 小川美香子

  日本薬学会年会要旨集(Web)  2023
• 母親のビタミンA欠乏食摂取は子のベージュ脂肪細胞の出現を抑制する

  加藤美羅, 岡松優子, 塚田杏樹, 二川瑛実, 稲波修, 木村和弘

  日本獣医学会学術集会講演要旨集  2023
• Partial acquisition of spectral projections accelerates four-dimensional spectral-spatial EPR imaging for mouse tumor models: A feasibility study

  大場光紗, 田口真衣, 工藤洋平, 山下昴矢, 安井博宣, 松元慎吾, KIRILYUK Igor A., 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  2023
• Comparison of electron paramagnetic resonance imaging and immunohistochemical staining for the possibility of detecting ferroptosis in tumors

  加藤千博, 安井博宣, 赤羽英夫, 江本美穂, 藤井博匡, 永根大幹, 山下匡, 稲波修

  電子スピンサイエンス学会年会講演要旨集  2023
• Analysis of radiation reaction mechanisms of hypervalent iodine compounds using ESR for the development of radiation-responsive caged compounds.

  稲波修, 榎本将聖, 我喜屋祥, 中島孝平, 小川美香子

  電子スピンサイエンス学会年会講演要旨集  2023
• Analysis of azo cleavage reaction for development of X-ray induced caged compounds

  小河原浩輝, 高倉栄男, 中島孝平, 稲波修, 小川美香子

  日本薬学会年会要旨集(Web)  2023
• Analysis of X-ray response of hypervalent iodine compounds for development of X-ray caged compounds

  榎本将聖, 高倉栄男, 中島考平, 稲波修, 小川美香子

  日本薬学会年会要旨集(Web)  2023
• X線照射および抗がん剤によって生じるがん老化細胞に対するYM155のセノリシス作用に関する研究(Senolytic effects of YM155, a survivin inhibitor, on X-irradiation- and etoposide-induced senescence in human colon HCT116 cancer cells)

  小澤 拓実, 安井 博宣, 加藤 千博, 山下 晃矢, 藤本 政毅, 稲波 修

  日本放射線影響学会大会講演要旨集  2022/09  (一社)日本放射線影響学会
  
• 新しい可搬型EPRI装置を用いた腫瘍内レドックスイメージングによる放射線治療応答の非侵襲的解析(Noninvasive analysis of radioresponse by redox imaging in tumors by using a novel portable EPRI device)

  加藤 千博, 安井 博宣, 赤羽 英夫, 藤井 博匡, 江本 美穂, 永根 大幹, 山下 匡, 稲波 修

  日本放射線影響学会大会講演要旨集  2022/09  (一社)日本放射線影響学会
  
• スフィンゴミエリン合成酵素2は重症下肢虚血での骨格筋再生を抑制する

  水垣ひなの, 永根大幹, 水野谷航, 赤羽英夫, 安井博宣, 稲波修, 藤井博匡, 相原尚之, 上家潤一, 山下匡

  日本酸化ストレス学会学術集会プログラム・抄録集  2022
• Analysis of X-ray-triggered reaction of phthalocyanine derivatives for development of caged compound activated in deep tissue

  後藤悠人, 松廣志乃, 山下柾, 高倉栄男, 中島孝平, 稲波修, 小川美香子

  電子スピンサイエンス学会年会講演要旨集  2022
• Study on detection of biological radicals and its related diseases

  稲波修

  電子スピンサイエンス学会年会講演要旨集  2022
• Comparison and investigation of the cell-killing effects of photoimmunotherapy (PIT) and photodynamic therapy (PDT) in hypoxic cancer cells

  白川晴香, 後藤悠人, 中島孝平, 高倉栄男, 安井博宣, 小川美香子, 稲波修

  電子スピンサイエンス学会年会講演要旨集  2022
• Function of sphingomyelin synthase 2 in ischemic muscle disease

  水垣ひなの, 永根大幹, 赤羽英夫, MACIEJ Kmiec, PERIANNAN Kuppusamy, 水野谷航, 安井博宣, 稲波修, 相原尚之, 上家潤一, 山下匡

  電子スピンサイエンス学会年会講演要旨集  2022
• Analysis of changes in cancer treatment response in the same individual by noninvasive redox imaging with a novel portable EPRI device

  加藤千博, 安井博宣, 赤羽英夫, 藤井博匡, 江本美穂, 永根大幹, 山下匡, 稲波修

  電子スピンサイエンス学会年会講演要旨集  2022
• Analysis of X-ray response of hypervalent iodine compounds for development of X-ray caged compounds

  榎本将聖, 高倉栄男, 中島孝平, 稲波修, 小川美香子

  バイオメディカル分析科学シンポジウム講演要旨集  2022
• Analysis of X-ray responsiveness of phthalocyanine derivatives for development of caged compounds

  山下柾, 松廣志乃, 高倉栄男, 中島孝平, 稲波修, 小川美香子

  バイオメディカル分析科学シンポジウム講演要旨集  2022
• Development of X-ray induced caged compounds using azo bond cleavage

  小河原浩輝, 高倉栄男, 中島孝平, 稲波修, 小川美香子

  バイオメディカル分析科学シンポジウム講演要旨集  2022
• Analysis of therapeutic effects and photochemical reaction of a new red-shifted photosensitizer for photoimmunotherapy

  後藤悠人, 安藤完太, 中島孝平, 高倉栄男, 稲波修, 小川美香子

  バイオメディカル分析科学シンポジウム講演要旨集  2022
• HCT116細胞におけるエトポシドによって生じる細胞老化の選択除去法に関する研究

  小澤拓実, 安井博宣, 加藤千博, 山下晃矢, 藤本政毅, 稲波修

  日本獣医学会学術集会講演要旨集  2022
• 低酸素条件下のがん細胞に対する光免疫療法(PIT)の有効性の評価

  白川晴香, 後藤悠人, 中島孝平, 高倉栄男, 安井博宣, 小川美香子, 稲波修

  日本獣医学会学術集会講演要旨集  2022
• 可搬型EPRI装置を用いた腫瘍内レドックスイメージングによるがん治療応答解析

  加藤千博, 安井博宣, 赤羽英夫, 藤井博匡, 江本美穂, 永根大幹, 山下匡, 稲波修

  日本獣医学会学術集会講演要旨集  2022
• イヌ悪性黒色腫細胞株における放射線照射で生じる上皮間葉転換とDNAメチル化の関与

  田邊裕晶, 安井博宣, 山崎淳平, 山崎淳平, 木之下怜平, 山下晃矢, 加藤千博, 稲波修

  日本獣医学会学術集会講演要旨集  2022
• 虚血性筋障害におけるスフィンゴミエリン合成酵素2の役割

  水垣ひなの, 永根大幹, 佐藤沙菜, 水野谷航, 赤羽英夫, MACIEJ Kmiec, PERIANNAN Kuppusamy, 安井博宣, 稲波修, 相原尚之, 上家潤一, 山下匡

  日本生化学会大会(Web)  2022
• 腸内細菌を介した酸素ナノバブル水による抗腫瘍作用

  永根大幹, 永根大幹, 鈴木利美奈, 水垣ひなの, 丹羽智瑛, 内山淳平, 稲波修, 赤羽英夫, 安井博宣, 山下匡

  日本生化学会大会(Web)  2022
• セノリティック薬ABT263のアポトーシス誘導およびSASP因子分泌抑制によるおよびSASP因子分泌抑制によるがん放射線治療による副作用の軽減効果の可能性

  藤本 政毅, 東山 りつ子, 安井 博宣, 山下 晃矢, 稲波 修

  日本獣医学会学術集会講演要旨集  2021/09  (公社)日本獣医学会
  
• X線照射およびCA9阻害による癌細胞の増殖抑制に関するメカニズムの検討

  山下 僚太, 安井 博宣, 山下 晃矢, 藤本 政毅, 稲波 修

  日本獣医学会学術集会講演要旨集  2021/09  (公社)日本獣医学会
  
• 培養がん細胞の治療を模倣した放射線照射に伴う放射線抵抗性獲得機構の解析

  山下 晃矢, 房 知輝, 藤本 政毅, 安井 博宣, 辻 雅久, 稲波 修

  日本獣医学会学術集会講演要旨集  2021/09  (公社)日本獣医学会
  
• 酸素ナノバブル水の抗腫瘍作用に関する基礎的研究

  永根大幹, 丹羽智瑛, 内山淳平, 稲波修, 山下匡

  日本酸化ストレス学会学術集会プログラム・抄録集  2021
• ヒト肺腺がん由来A549細胞においてグルタミノリシス阻害は放射線誘発性の細胞老化を亢進する

  藤本政毅, 東山りつ子, 安井博宣, 山下晃矢, 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  2021
• Redox Potential of Single Octarepeat from Prion Protein with Divalent Metals

  Wakako Hiraoka, Osamu Inanami, Shuhei Murakami

  Free Radical Biology and Medicine  2020/11  Elsevier BV
  
• 光免疫治療に資するX線誘導による生体深部腫瘍での選択的分子解離の探索(Explore of X-ray induced selective molecular degradation for photoimmuno-therapy targeting deep tissues)

  横谷 明徳, 藤井 健太郎, 渡辺 立子, 平戸 未彩紀, 中川 桂一, 稲波 修, 武次 徹也, 小川 美香子

  日本放射線影響学会大会講演要旨集  2020/10  (一社)日本放射線影響学会
  
• 分割照射によって出現した放射線抵抗性大腸がんSW480RR株化細胞の抵抗性メカニズムの解析(Analysis of radiation-resistant colorectal cancer SW48ORR cell line induced by fractionated radiation therapy)

  山下 晃矢, 房 知輝, 藤本 政毅, 安井 博宣, 稲波 修

  日本放射線影響学会大会講演要旨集  2020/10  (一社)日本放射線影響学会
  
• 18F-DiFA PET/CTイメージングを用いたエリブリンによる腫瘍内低酸素解除効果の解析と放射線増感作用の検討(Eribulin improves tumor oxygenation demonstrated by 18F-DiFA hypoxia imaging, leading to radio-sensitization in human breast cancer xenograft model)

  房 知輝, 安井 博宣, 志賀 哲, 柴田 悠貴, 藤本 政毅, 鈴木 基史, 東川 桂, 宮本 直樹, 稲波 修, 久下 裕司

  日本放射線影響学会大会講演要旨集  2020/10  (一社)日本放射線影響学会
  
• 放射線誘発性分裂期崩壊における中心体過剰複製の役割(The role of abnormal centrosome amplification in radiation-induced mitotic catastrophe in cancer cell)

  藤本 政毅, 房 知輝, 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  2020/10  (一社)日本放射線影響学会
  
• 放射線とナノメディシン-放射線生物・治療学におけるナノ粒子の役割- 金属ナノ粒子によるがん放射線増感のメカニズムについて DNA鎖が金ナノゲル粒子による放射線増感作用の主要な標的か?(Radiation and Nanomedicine-Roles of nano-particles in radiation biology and therapeutics Radiosensitization by metal nanoparticles in tumor cells: Is DNA primarily target for radiosensitizaion by gold nanoparticles?)

  稲波 修, 安井 博宣, 山盛 徹, 長崎 幸夫

  日本放射線影響学会大会講演要旨集  2020/10  (一社)日本放射線影響学会
  
• Mitochondrial dynamics after irradiation are involved in radiosensitivity

  房知輝, 房知輝, 山盛徹, 安井博宣, 稲波修

  放射線生物研究  2020  放射線生物研究会
  
• Basic research of radiation therapy targeting cancer stem-like cells in veterinary field

  出口辰弥, 細谷謙次, 安井博宣, 稲波修, 奥村正裕

  放射線生物研究  2020  放射線生物研究会
  
• ヒト肺腺がんA549細胞におけるグルタミノリシス阻害は放射線誘導性細胞老化を増強する(Inhibition of glutaminolysis enhances X-irradiation-induced cellular senescence in human lung adenocarcinoma A549 cells)

  東山 りつ子, 安井 博宣, 房 知輝, 山本 久美子, 藤本 政毅, 稲波 修

  日本放射線影響学会大会講演要旨集  2019/11  (一社)日本放射線影響学会
  
• ヒト肺がん由来A549細胞における解糖系の放射線応答性に関する研究(Radiation response of glycolysis in human lung cancer cells A549)

  福島 佑一郎, 安井 博宜, 藤本 政毅, 山本 久美子, 房 知輝, 稲波 修

  日本放射線影響学会大会講演要旨集  2019/11  (一社)日本放射線影響学会
  
• 固形腫瘍株化細胞におけるエネルギー代謝の放射線応答の解析(The evaluation of radiation response of energy metabolism in solid tumor cell lines)

  山本 久美子, 安井 博宣, 藤本 政毅, 福島 佑一郎, 房 知輝, 稲波 修

  日本放射線影響学会大会講演要旨集  2019/11  (一社)日本放射線影響学会
  
• ヒト肺腺がん由来A549細胞においてグルタミノリシス依存性のエネルギー代謝の阻害は放射線の効果を増強する(Inhibition of glutaminolysis in energy metabolism enhances radiation sensitivity in human lung adenocarcinoma A549 cells)

  藤本 政毅, 房 知輝, 山本 久美子, 安井 博宣, 稲波 修

  日本放射線影響学会大会講演要旨集  2019/11  (一社)日本放射線影響学会
  
• 筋芽細胞におけるUSP2によるミトコンドリア機能制御  [Not invited]

  太田葉瑠, 橋本茉由子, 齋藤菜津子, 山本久美子, 中澤康裕, 稲波修, 北村浩

  日本獣医学会学術集会講演要旨集  2019/08
• ヒト肺がん由来A549細胞における解糖系の放射線応答性に関する研究  [Not invited]

  福島佑一郎, 安井博宜, 藤本政毅, 山本久美子, 房知輝, 稲波修

  日本獣医学会学術集会講演要旨集  2019/08
• 種々のがん細胞におけるミトコンドリア電子伝達系の放射線応答の解析  [Not invited]

  山本久美子, 安井博宣, 藤本政毅, 房知輝, 稲波修

  日本獣医学会学術集会講演要旨集  2019/08
• ヒト肺腺がん由来A549細胞におけるグルタミノリシスを標的とする放射線増感効果  [Not invited]

  藤本政毅, 房知輝, 山本久美子, 安井博宣, 稲波修

  日本獣医学会学術集会講演要旨集  2019/08
• Glutamine代謝阻害がX線照射による早期細胞老化およびアポトーシスの誘導に与える影響の検討  [Not invited]

  東山りつ子, 安井博宣, 房知輝, 山本久美子, 藤本政毅, 稲波修

  日本獣医学会学術集会講演要旨集  2019/08
• ミトコンドリア分裂はCa2+制御を通じて放射線による分裂期崩壊誘導に寄与する

  房 知輝, 山盛 徹, 山本 久美子, 藤本 政毅, 安井 博宣, 稲波 修

  日本獣医学会学術集会講演要旨集  2019/08  (公社)日本獣医学会
  
• Centrinonne-Bによる中心体複製阻害が放射線誘発分裂期崩壊に与える影響

  藤本 政毅, 山盛 徹, 房 知輝, 山本 久美子, 稲波 修

  日本放射線影響学会大会講演要旨集  2018/11  (一社)日本放射線影響学会
  
• Application of ESR pO₂ imaging to evaluation of tumor radiosensitivity in intracranial glioma mouse model  [Not invited]

  安井博宣, 河合辰哉, 松元慎吾, 齋藤圭太, CAMPHAUSEN Kevin, KRISHNA Murali, 稲波修

  電子スピンサイエンス学会年会講演要旨集  2018/11
• The evaluation of mitochondrial electron transport chain function in cancer cells using ESR oximetry  [Not invited]

  山本久美子, 安井博宣, 房知輝, 藤本政毅, 稲波修

  電子スピンサイエンス学会年会講演要旨集  2018/11
• 新規ミトコンドリア呼吸機能評価法を用いたがん細胞に対する放射線影響の評価  [Not invited]

  山本久美子, 安井博宣, 房知輝, 山盛徹, 稲波修

  日本獣医学会学術集会講演要旨集  2018/08
• ミトコンドリア分裂抑制が細胞の放射線感受性低下を引き起こすメカニズムの解析  [Not invited]

  房知輝, 山盛徹, 山本久美子, 稲波修

  日本獣医学会学術集会講演要旨集  2018/08
• 放射線照射および脂溶性TPP⁺化合物ががん細胞のミトコンドリア機能に与える影響の評価  [Not invited]

  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

  日本酸化ストレス学会学術集会プログラム・抄録集  2018/04
• 放射線は内皮型一酸化窒素合成酵素の活性化により血管内皮細胞の老化を誘導する  [Not invited]

  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

  日本酸化ストレス学会学術集会プログラム・抄録集  2018/04
• Centrinonne‐Bによる中心体複製阻害が放射線誘発分裂期崩壊に与える影響  [Not invited]

  藤本政毅, 山盛徹, 房知輝, 山本久美子, 稲波修

  日本放射線影響学会大会抄録(Web)  2018
• 電子スピン共鳴酸素イメージングによる脳室内移植グリオーマの放射線感受性評価  [Not invited]

  安井博宣, 安井博宣, 河合辰哉, 松元慎吾, 松元慎吾, 齋藤圭太, CAMPHAUSEN Kevin, 稲波修, KRISHNA Murali

  日本放射線影響学会大会抄録(Web)  2018
• DNA損傷応答は内皮型一酸化窒素合成酵素の活性化を介して血管内皮細胞を老化させる  [Not invited]

  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

  日本放射線影響学会大会抄録(Web)  2018
• 放射線により引き起こされる分裂期崩壊に対するミトコンドリア分裂の寄与メカニズムの解析  [Not invited]

  房知輝, 山盛徹, 山本久美子, 稲波修

  日本放射線影響学会大会抄録(Web)  2018
• ESR法による新規ミトコンドリア機能評価法を用いたがん細胞の放射線応答の解析  [Not invited]

  山本久美子, 安井博宣, 房知輝, 山盛徹, 稲波修

  日本放射線影響学会大会抄録(Web)  2018
• Oxygen partial pressure imaging of tumor model mice with isotopically labeled nitroxyl radicals  [Not invited]

  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  2017/11
• Development of pH mapping for tumor tissues using EPR spectroscopy  [Not invited]

  市川裕貴, KOMAROV Denis A, 山本久美子, 稲波修, 松元慎吾, 平田拓

  電子スピンサイエンス学会年会講演要旨集  2017/11
• Analysis of Mitochondrial Semiquinone Radicals and Fe‐S clusters in Tumor Cells Exposed to Ionizing Radiation  [Not invited]

  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  2017/11
• The analysis of the ESR spectra derived from mitochondria in cancer cells  [Not invited]

  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  2017/11
• 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  [Not invited]

  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)  2017/10
• 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  [Not invited]

  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)  2017/10
• 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  [Not invited]

  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)  2017/10
• 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  [Not invited]

  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  2017/08
• 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  [Not invited]

  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  2017/08
• 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  [Not invited]

  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  2017/06
• 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  [Not invited]

  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  2017/06
• 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)  [Not invited]

  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)  2017/06
• 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  [Not invited]

  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  2016/11
• 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  [Not invited]

  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  2016/11
• 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  [Not invited]

  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  2016/11
• 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  [Not invited]

  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  2016/08
• 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  [Not invited]

  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  2016/08
• 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  [Not invited]

  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  2016/08
• 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  [Not invited]

  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  2016/08
• 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  [Not invited]

  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  2016/08
• 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)  [Not invited]

  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)  2016/06
• 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  [Not invited]

  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
• 新規Chk1阻害剤MK‐8776の放射線増感剤としての効果の検討  [Not invited]

  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
• がん細胞の放射線応答における転写調節因子Id1の役割  [Not invited]

  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
• 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  [Not invited]

  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
• 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  [Not invited]

  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
• 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  [Not invited]

  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  2015/11
• 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  [Not invited]

  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  2015/11
• 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  [Not invited]

  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  2015/10
• 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  [Not invited]

  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  2015/10
• 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  [Not invited]

  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  2015/10
• 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  [Not invited]

  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  2015/08
• 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  [Not invited]

  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  2015/08
• 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  [Not invited]

  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  2015/08
• 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  [Not invited]

  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)  2015/06
• 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  [Not invited]

  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  2015/05
• 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  [Not invited]

  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  2014/09
• 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  [Not invited]

  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  2014/09
• 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  [Not invited]

  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  2014/08
• 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  [Not invited]

  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  2014/08
• 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  [Not invited]

  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  2014/08
• 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  [Not invited]

  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  2014/08
• 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  [Not invited]

  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  2014/08
• 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  [Not invited]

  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)  2014/05
• 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

  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
• 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  [Not invited]

  酒井友里, 山盛徹, 安井博宣, 稲波修

  日本生化学会大会(Web)  2014
• Metabolic-targeted Treatment with Dichloroacetate Transiently Induces Tumor Hypoxia in Murine Squamous Cell Carcinoma Model

  Hironobu Yasui, Keita Saito, Naoya Nishida, Shingo Matsumoto, Tohru Yamamori, Murali Cherukuri Krishna, Osamu Inanami

  FREE RADICAL BIOLOGY AND MEDICINE  2013/11  ELSEVIER SCIENCE INC
  
• The Role of HSP90 and ATM in the Radiation-Induced eNOS Activation

  Masaki Nagane, Hironobu Yasui, Tohru Yamamori, Yuri Sakai, Koichi Niwa, Osamu Inanami

  FREE RADICAL BIOLOGY AND MEDICINE  2013/11  ELSEVIER SCIENCE INC
  
• 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  [Not invited]

  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  2013/10
• 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  [Not invited]

  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  2013/10
• EPR分光法による鏡像異性体のタンパク質結合性の観測  [Not invited]

  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  2013/10
• 電子常磁性共鳴分光による生体内pHの測定  [Not invited]

  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  2013/10
• 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  [Not invited]

  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  2013/10
• 放射線生物作用の初期過程  [Not invited]

  桑原幹典, 稲波修

  日本放射線影響学会大会講演要旨集  2013/10
• 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  [Not invited]

  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  2013/10
• 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  [Not invited]

  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  2013/10
• 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  [Not invited]

  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  2013/10
• 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  [Not invited]

  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  2013/08
• 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  [Not invited]

  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  2013/08
• 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  [Not invited]

  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  2013/08
• 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  [Not invited]

  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  2013/08
• ―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  [Not invited]

  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)  2013/07
• 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  [Not invited]

  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  2013/06
• 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  [Not invited]

  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  2013/03
• -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

  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
• The Effect of Boron Neutron Capture Reaction on the Cell Survival of Tumor Cells Preconditioned with Intermittent Hypoxia  [Not invited]

  Yasui H, Nagae M, Masunaga S, Yamamori T, Inanami O

  Progress Report 2012, The Research Reactor Institute, Kyoto University  2013
• 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

  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
• 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

  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
• Radiation-Induced Nitric Oxide Contributes to the Tumor Reoxygenation in SCCVII Tumors  [Not invited]

  Masaki Nagane, Hironobu Yasui, Tohru Yamamori, Songji Zhao, Yuji Kuge, Nagara Tamaki, Hiromi Kameya, Hideo Nakamura, Osamu Inanami

  FREE RADICAL BIOLOGY AND MEDICINE  2012/11  ELSEVIER SCIENCE INC
  
• 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  [Not invited]

  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  2012/11
• がんの放射線治療効率化に向けたESR技術の適用  [Not invited]

  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  2012/11
• EPRスペクトロスコピーによる腫瘍の細胞外pH測定  [Not invited]

  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  2012/11
• 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  [Not invited]

  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  2012/11
• 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  [Not invited]

  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  2012/11
• 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  [Not invited]

  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  2012/09
• Ionizing Radiation Induces Mitochondrial Reactive Oxygen Species Production accompanied by Upregulation of Mitochondrial Electron Transport Chain Function and Mitochondrial Content under Control of the Cell Cycle Checkpoint  [Not invited]

  T. Yamamori, H. Yasui, M. Yamazumi, Y. Wada, H. Nakamura, O. Inanami

  FREE RADICAL BIOLOGY AND MEDICINE  2012/09  ELSEVIER SCIENCE INC
  
• 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。  [Not invited]

  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  2012/08
• 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  [Not invited]

  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  2012/08
• 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  [Not invited]

  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  2012/08
• 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  [Not invited]

  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  2012/06
• 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  [Not invited]

  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  2012/06
• 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  [Not invited]

  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  2011/11
• 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  [Not invited]

  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  2011/11
• 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  [Not invited]

  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  2011/11
• 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  [Not invited]

  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  2011/11
• 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  [Not invited]

  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  2011/11
• 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  [Not invited]

  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  2011/11
• 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  [Not invited]

  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  2011/11
• 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  [Not invited]

  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  2011/11
• 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  [Not invited]

  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  2011/08
• 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  [Not invited]

  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  2011/08
• 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  [Not invited]

  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  2011/08
• 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  [Not invited]

  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  2011/08
• 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  [Not invited]

  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  2011/08
• 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  [Not invited]

  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  2011/08
• 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  [Not invited]

  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  2011/08
• 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  [Not invited]

  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  2011/06
• 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  [Not invited]

  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  2011/06
• 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  [Not invited]

  稲波修

  日本医学放射線学会総会抄録集  2011/02
• 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  [Not invited]

  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
• 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  [Not invited]

  永瀧正人, 山盛徹, 女池俊介, 安井博宣, 稲波修

  生化学  2011
• 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  [Not invited]

  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  2010/11
• 間欠的低酸素が腫瘍細胞の放射線抵抗性に与える影響  [Not invited]

  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  2010/10
• 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  [Not invited]

  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  2010/10
• 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  [Not invited]

  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  2010/10
• 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  [Not invited]

  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  2010/10
• 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  [Not invited]

  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  2010/09
• 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  [Not invited]

  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  2010/06
• 低酸素環境を標的としたがん治療の新たな展開  [Not invited]

  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  2010/03
• 獣医放射線学  [Not invited]

  稲波修

  日本獣医学会学術集会講演要旨集  2010
• 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  [Not invited]

  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  2009/11
• 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  [Not invited]

  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  2009/11
• 8‐Amino‐adenosineによる放射線誘導細胞死の増感効果  [Not invited]

  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  2009/11
• 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  [Not invited]

  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  2009/11
• 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  [Not invited]

  永瀧正人, 山盛徹, 稲波修

  生化学  2009/09
• 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  [Not invited]

  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  2009/06
• 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  [Not invited]

  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  2009/06
• 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  [Not invited]

  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  2009/06
• 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  [Not invited]

  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  2009/06
• 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  [Not invited]

  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)  2009/05
• 痛みのimaging,MRIを用いての試み  [Not invited]

  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  2009/03
• 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  [Not invited]

  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  2009/01
• Redox regulation in radiation-induced apoptosis-related cytochrome c release from mitochondria  [Not invited]

  O. Inanami, A. Ogura, M. Yamazumi, T. Yamamori, W. Hiraoka

  FREE RADICAL RESEARCH  2009  TAYLOR & FRANCIS LTD
  
• Functional MRIによる痛みの定量化  [Not invited]

  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
• 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  [Not invited]

  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
• 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  [Not invited]

  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
• 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  [Not invited]

  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  2008/11
• 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  [Not invited]

  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  2008/10
• 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  [Not invited]

  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  2008/09
• 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  [Not invited]

  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  2008/09
• 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  [Not invited]

  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  2008/09
• 放射線誘導細胞死におけるサバイビンの役割  [Not invited]

  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  2008/03
• 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  [Not invited]

  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  2008/03
• 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  [Not invited]

  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  2007/12
• 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  [Not invited]

  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  2007/11
• 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  [Not invited]

  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  2007/11
• 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  [Not invited]

  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  2007/08
• 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  [Not invited]

  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  2007/08
• 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  [Not invited]

  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  2007/08
• 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  [Not invited]

  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  2007/08
• ウシラクトフェリンの局所接種による抗腫瘍効果  [Not invited]

  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  2007/03
• 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  [Not invited]

  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  2007/03
• The protective effect of mouse prion protein from metal-catalyzed oxidative stress  [Not invited]

  Yasuko Asano Watanabe, Tetsu Inoue, Motohiro Horiuchi, Mikinori Kuwabara, Osamu Inanami

  FREE RADICAL BIOLOGY AND MEDICINE  2007  ELSEVIER SCIENCE INC
  
• 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

  渡邊康子, 渡邊康子, 平岡和佳子, 下山雄平, 堀内基広, 堀内基広, 桑原幹典, 桑原幹典, 稲波修, 稲波修

  生化学  2007
• 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  [Not invited]

  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
• 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  [Not invited]

  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
• 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  [Not invited]

  小倉亜希, 安井博宣, 桑原幹典, 稲波修

  生化学  2007
• 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  [Not invited]

  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  2006/11
• 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  [Not invited]

  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  2006/11
• プリオンタンパク質の抗酸化機能の解明  [Not invited]

  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  2006/11
• ドロマイトの抗菌メカニズムに関する検討  [Not invited]

  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  2006/08
• 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  [Not invited]

  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  2006/08
• 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  [Not invited]

  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  2006/08
• 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  [Not invited]

  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  2006/08
• Bovine lactoferrin reduces concanavalin A-induced interferon-gamma expression of peripheral blood mononuclear cells in FIV-negative and FIV-positive cats  [Not invited]

  S. Kobayashi, R. Sato, O. Inanami, T. Yamamori, M. Kuwabara, Y. Naito, M. Takase

  BIOCHEMISTRY AND CELL BIOLOGY-BIOCHIMIE ET BIOLOGIE CELLULAIRE  2006/06  NATL RESEARCH COUNCIL CANADA-N R C RESEARCH PRESS
  
• Radiosensitization of Tumor Cells by Ethynylcytidine  [Not invited]

  桑原幹典, 稲波修, 安井博宣, 飯塚大輔, 松田彰

  癌の臨床  2006/04
• 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  [Not invited]

  稲波修, 渡邊康子

  電子スピンサイエンス  2006/03
• A new amphipathic derivative, LPBNSH, has a protective effect against copper-induced fulminant hepatitis in LEC rats at an extremely low concentration compared with its original form alpha-phenyl-N-tert-butylnitrone  [Not invited]

  Hironobu Yasui, Taketoshi Asanuma, Osamu Inanami, Gregory Durand, Ange Polidori, Yasuhiro Kon, Bernard Pucci, Mikinori Kuwabara

  FREE RADICAL BIOLOGY AND MEDICINE  2006  ELSEVIER SCIENCE INC
  
• Enhancement efficacy of overexpression with Survivin-T34A, T34E, T34D and D53A for radiation-induced apoptosis in various cell lines  [Not invited]

  Aki Ogura, Osamu Inanami, Daisuke Iizuka, Hironobu Yasui, Mikinori Kuwabara

  FREE RADICAL BIOLOGY AND MEDICINE  2006  ELSEVIER SCIENCE INC
  
• The role of the N-terminal octarepeat domain in the antioxidant activity of mouse prion protein  [Not invited]

  Yasuko Asano Watanabe, Osamu Inanami, Tetsu Inoue, Daisuke Iizuka, Mikinori Kuwabara

  FREE RADICAL BIOLOGY AND MEDICINE  2006  ELSEVIER SCIENCE INC
  
• The radiosensitizatizing effects in human carcinoma in vitro and in vivo by TNF-alpha-related apoptosis-inducing ligand (TRAIL)  [Not invited]

  Momoko Takahashi, Osamu Inanami, Michihiko Tsujitani, Nobuo Kubota, Mikinori Kuwabara

  FREE RADICAL BIOLOGY AND MEDICINE  2006  ELSEVIER SCIENCE INC
  
• Sensitization to radiation-induced cell killing by the inhibition of Cdc2 kinase activity with purvalanol A  [Not invited]

  Daisuke Iizuka, Osamu Inanami, Hironobu Yasui, Mikinori Kuwabara

  FREE RADICAL BIOLOGY AND MEDICINE  2006  ELSEVIER SCIENCE INC
  
• Synthesis and characterization of a better DMPO-type spin trap, 5-(2,2-dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1-pyrroline n-oxide (CYPMPO)  [Not invited]

  Osamu Inanami, Masato Kamibayashi, Shigeru Oowada, Hiroaki Kameda, Taiichi Okada, Shunsaku Ohta, Toshihiko Ozawa, Keisuke Makino, Masashi Shimmei, Yashige Kotake

  FREE RADICAL BIOLOGY AND MEDICINE  2006  ELSEVIER SCIENCE INC
  
• 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

  渡邊康子, 稲波修, 稲波修, 堀内基広, 下山雄平, 下山雄平, 中村秀夫, 中村秀夫, 桑原幹典

  日本獣医学会学術集会講演要旨集  2006
• 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  [Not invited]

  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
• 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  [Not invited]

  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
• Site‐directed spin label(SDSL)法によるp47^phoxリン酸化の解析  [Not invited]

  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  2005/10
• 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―  [Not invited]

  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  2005/10
• 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  [Not invited]

  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  2005/10
• 1P027 Phosphorylation-induced conformational change of NADPH oxidase subunitp47^ : A site-directed spin labeling (SDSL) study  [Not invited]

  Suzuki T, Inanami O, Kuwabara M, Inagaki F, Hiraoka W

  Biophysics  2005/10  The Biophysical Society of Japan
  
• 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  [Not invited]

  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  2005/08
• プリオンタンパク質の銅イオンの新たな結合様式  [Not invited]

  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  2005/08
• 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  [Not invited]

  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  2005/08
• 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  [Not invited]

  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  2005/08
• 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  [Not invited]

  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  2005/08
• 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  [Not invited]

  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  2005/08
• 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  [Not invited]

  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  2005/08
• 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  [Not invited]

  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  2005/08
• 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  [Not invited]

  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  2005/08
• 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  [Not invited]

  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  2005/08
• 血管内皮細胞のシグナル伝達と活性酸素  [Not invited]

  丹羽光一, 稲波修

  放射線生物研究  2005/03
• 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

  渡辺康子, 渡辺康子, 稲波修, 稲波修, 稲波修, 堀内基広, 堀内基広, 下山雄平, 下山雄平, 中村秀夫, 中村秀夫, 桑原幹典

  日本獣医学会学術集会講演要旨集  2005
• 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  [Not invited]

  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
• 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  [Not invited]

  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
• 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  [Not invited]

  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
• 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  [Not invited]

  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
• 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  [Not invited]

  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  2004/12
• ラット大脳皮質虚血耐性現象とAkt活性との関係について  [Not invited]

  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  2004/12  日本病態生理学会
  
• 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  [Not invited]

  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  2004/11
• 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  [Not invited]

  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  2004/08
• 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  [Not invited]

  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  2004/08
• 大脳皮質虚血耐性現象とAkt活性との関係について  [Not invited]

  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  2004/08
• 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  [Not invited]

  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  2004/08
• 高磁場BOLD‐fMRIによるラットにおける痛覚の分析法の確立  [Not invited]

  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  2004/08
• 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  [Not invited]

  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  2004/08
• Decreased apoptosis of beta(2)-integrin-deficient bovine neutrophils (vol 82, pg 32, 2004)  [Not invited]

  H Nagahata, H Higuchi, H Teraoka, K Takahashi, O Inanami, O Kuwabara

  IMMUNOLOGY AND CELL BIOLOGY  2004/06  BLACKWELL PUBLISHING ASIA
  
• Activation mechanism of NADPH oxidase and impaired function of bovine neutrophil after parturition  [Not invited]

  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  2004/04
• 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  [Not invited]

  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  2004/03
• 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  [Not invited]

  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  2004/03
• 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  [Not invited]

  稲波修

  日本獣医学会学術集会講演要旨集  2004/03
• 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  [Not invited]

  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  2004/03
• 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  [Not invited]

  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  2004/03
• Suppression of malonate-induced ischemic brain injury in rat by a cell-permeable peptidic JNK inhibitor: MRI and ADC mapping study  [Not invited]

  M Kuwabara, T Asanuma, O Inanami

  FREE RADICAL BIOLOGY AND MEDICINE  2004  PERGAMON-ELSEVIER SCIENCE LTD
  
• 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

  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
• 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

  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
• 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  [Not invited]

  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
• 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  [Not invited]

  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
• 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  [Not invited]

  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
• 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  [Not invited]

  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
• The molecular mechanisms regulating the phosphorylation of the NADPH oxidase component p47^phox by phosphoinositide 3‐kinase.  [Not invited]

  山盛徹, 稲波修, 永幡肇, 桑原幹典

  獣医生化学  2003/10
• 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  [Not invited]

  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  2003/10
• 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  [Not invited]

  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  2003/10
• 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  [Not invited]

  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  2003/10
• 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  [Not invited]

  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  2003/10
• 西ナイルウイルスの感染におけるJNKの役割  [Not invited]

  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  2003/09
• 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~  [Not invited]

  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  2003/09
• 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  [Not invited]

  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  2003/09
• Phosphoinositide 3‐kinaseにより調節される食細胞NADPH oxidase細胞質因子p47^phoxリン酸化のシグナル伝達機構  [Not invited]

  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  2003/09
• 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  [Not invited]

  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  2003/03
• 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  [Not invited]

  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  2003/03
• 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  [Not invited]

  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  2003/03
• Attention to phagocyte functions at perinatal period for the elucidation of mastitis occurrence mechanism and its control.  [Not invited]

  稲波修

  動物衛生試験研究成績・計画概要集 平成14年度  2003
• 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  [Not invited]

  山盛徹, 稲波修, 住本英樹, 永幡肇, 桑原幹典

  磁気共鳴と医学  2002/09
• 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  [Not invited]

  脇研二, 稲波修, 山盛徹, 永幡肇, 桑原幹典

  磁気共鳴と医学  2002/09
• 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  [Not invited]

  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  2002/08
• 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  [Not invited]

  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  2002/08
• 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  [Not invited]

  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  2002/08
• 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  [Not invited]

  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  2002/08
• 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  [Not invited]

  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  2002/08
• 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  [Not invited]

  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  2002/08
• 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  [Not invited]

  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  2002/08
• 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  [Not invited]

  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  2002/08
• 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  [Not invited]

  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  2002/08
• BOLD法によるα‐phenyl‐N‐tert‐butylnitoroneの脳機能防護効果の評価  [Not invited]

  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  2002/08
• 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  [Not invited]

  稲波修

  KEK Proceedings  2002/05
• 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  [Not invited]

  稲波修

  日本獣医学会学術集会講演要旨集  2002/03
• 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  [Not invited]

  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  2002/03
• 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  [Not invited]

  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  2002/03
• 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  [Not invited]

  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  2002/01  (一社)日本生理学会
  
• Contribution of p38 MAPK and Ca2+ in hydrogen peroxide-induced increase of macromolecular permeability in vascular endothelial cells  [Not invited]

  K Niwa, O Inanami, T Ohta, S Ito, T Karino, M Kuwabara

  FREE RADICAL BIOLOGY AND MEDICINE  2002  PERGAMON-ELSEVIER SCIENCE LTD
  
• 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  [Not invited]

  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  2002/01
• Radioprotective synergistic action of cytokine on the clonal growth of X-irradiated human megakaryocytic progenitor cells.  [Not invited]

  Kashiwakura, I, M Murakami, Y Takagi, O Inanami, M Kuwabara, TA Takahashi

  BLOOD  2001/11  AMER SOC HEMATOLOGY
  
• 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  [Not invited]

  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  2001/09
• 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  [Not invited]

  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  2001/09
• 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  [Not invited]

  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  2001/09
• 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  [Not invited]

  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  2001/09
• 高磁場MRIを用いた中枢神経防護効果の観察  [Not invited]

  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  2001/09
• 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  [Not invited]

  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  2001/09
• 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  [Not invited]

  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  2001/09
• 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  [Not invited]

  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  2001/09
• 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  [Not invited]

  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  2001/09
• ¹H‐MRSを用いた中枢神経防護薬PBNの非侵襲的評価法  [Not invited]

  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  2001/09
• 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  [Not invited]

  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  2001/09
• 好中球NADPHオキシダーゼ活性化におけるERKの役割  [Not invited]

  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  2001/09
• 神経病理 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  [Not invited]

  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  2001/09  (公社)日本獣医学会
  
• 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  [Not invited]

  桑原幹典, 稲波修

  Clinical Neuroscience  2001/05
• ウシ好中球のどん食殺菌機構におけるp38 MARKとPKCの役割  [Not invited]

  山盛徹, 稲波修, 永幡肇, 桑原幹典

  磁気共鳴と医学  2001/03
• 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  [Not invited]

  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  2001/03
• 活性酸素・フリーラジカルの生成機構と疾病  [Not invited]

  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  2001/03
• 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  [Not invited]

  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  2001/03
• 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  [Not invited]

  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  2001/03
• 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  [Not invited]

  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  2001/01
• 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  [Not invited]

  桑原幹典, 稲波修

  三島海雲記念財団研究報告書  2000/12
• 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  [Not invited]

  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  2000/09
• 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  [Not invited]

  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  2000/09
• 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  [Not invited]

  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  2000/09
• 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  [Not invited]

  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  2000/09
• 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  [Not invited]

  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  2000/09
• 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  [Not invited]

  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  2000/09
• 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  [Not invited]

  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  2000/09
• 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  [Not invited]

  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  2000/08
• 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  [Not invited]

  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  2000/08
• 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  [Not invited]

  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  2000/08
• 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  [Not invited]

  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  2000/08
• Oxidative stress and intracellular signal transduction. Role of MAP kinase and calcium.  [Not invited]

  稲波修, 太田利男, 昆泰寛, 平岡和佳子, 桑原幹典

  放射線科学  2000/04
• Damage Induced in DNA as well as Double‐Stranded PolyA/PolyU by Direct and Quasi‐Direct Effects of Carbon Beams.  [Not invited]

  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)  2000/04
• ヒト末梢血由来巨核球前駆細胞の放射線感受性 (第3報)  [Not invited]

  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  2000/03
• 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  [Not invited]

  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  2000/03
• 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  [Not invited]

  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  2000/03
• Free radicals in cell‐biology. Apoptotic signal transduction and free radicals.  [Not invited]

  稲波修, 高橋賢次, 飯田義晴, 桑原幹典

  月刊細胞  2000/02
• 放射線誘発アポトーシスに対する抗酸化剤の影響  [Not invited]

  稲波修, 高橋賢次, 桑原幹典

  磁気共鳴と医学  2000
• 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  [Not invited]

  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  1999/09
• 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  [Not invited]

  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  1999/09
• 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  [Not invited]

  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  1999/09
• 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  [Not invited]

  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  1999/09
• 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  [Not invited]

  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  1999/08
• 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  [Not invited]

  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  1999/08
• 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  [Not invited]

  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  1999/08
• Detection and Identification of Precursor Radicals of DNA Strand Breaks Inducedby Direct and Quasi‐Direct Actions of Heavy Ions.  [Not invited]

  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)  1999/04
• 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  [Not invited]

  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  1999/03
• ラット実験的中枢神経変性のMRIによる評価法の確立  [Not invited]

  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  1999/03
• catechin経口投与によるLECラット肝炎の遅延効果  [Not invited]

  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  1999/03
• 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  [Not invited]

  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  1999/03
• 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  [Not invited]

  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  1999/03
• Effects of BAPTA-AM and forskolin on apoptosis and cytochrome C release in photosensitized Chinese hamster V79 cells  [Not invited]

  O Inanami, A Yoshito, K Takahashi, W Hiraoka, M Kuwabara

  FREE RADICAL BIOLOGY AND MEDICINE  1999  PERGAMON-ELSEVIER SCIENCE LTD
  
• Bone marrow transplantation in a Holstein heifer with bovine leucocyte adhesion deficiency (vol 156, pg 15, 1998)  [Not invited]

  H Nagahata, S Matsuki, H Higuchi, O Inanami, M Kuwabara, K Kobayashi

  VETERINARY JOURNAL  1999/01  BAILLIERE TINDALL
  
• 二重鎖PolyA/PolyUにおける放射線誘発ラジカルの解析  [Not invited]

  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
• 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  [Not invited]

  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
• 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  [Not invited]

  稲波修, 高橋賢次, 吉戸あすか, 桑原幹典

  磁気共鳴と医学  1999
• 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  [Not invited]

  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
• Magnetic resonance imaging of young and aged rat brains under a magnetic field of 7.05 T (vol 55, pg 933, 1994)  [Not invited]

  M Kuwabara, T Asanuma, O Inanami, T Jin, S Shimokawa, N Kasai, F Sato

  JOURNAL OF VETERINARY MEDICAL SCIENCE  1998/12  JAPAN SOC VET SCI
  
• Effects of postirradiation processing of lipid oxidation depressant Trolox on X-ray induced apoptosis.  [Not invited]

  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  1998/11
• Gamma-ray induced piperidine sensation nastic base damage in the double strand oligonucleotide frozen water solution containing 8-oxoguanine.  [Not invited]

  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  1998/11
• Irradiation response and apoptosis in human lymphoblast derivative MOLT-4 cells.  [Not invited]

  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  1998/11
• Analysis of OH induced radicals in double stranded Poly A/ Ply U using the spin trapping method.  [Not invited]

  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  1998/11
• ESR analysis of radiation inducing radical in double chain polyA/polyU (DNA model).  [Not invited]

  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  1998/10
• Involvement of calcium in radiation induction apoptosis of stump cell derived from human leukemia.  [Not invited]

  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  1998/08
• Effect of integrin family on active oxygen formability in granulocyte.  [Not invited]

  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  1998/08
• Effect of 2 - chloro deoxyadenosine on apoptosis related signal transduction system in human leukemia derived cell.  [Not invited]

  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  1998/08
• Chronic granulomatous disease and NADPH oxidase.  [Not invited]

  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  1998/08
• Radiosensitivity of human umbilical cord blood megakaryocyte progenitor cell.  [Not invited]

  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  1998/03
• Nitrogen monoxide generation by INF‐γ/LPS in monocyte of cattle granulocyte adhesion aplasia (BLAD).  [Not invited]

  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  1998/03
• Head NMR micro imaging in anesthetized mouse.  [Not invited]

  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  1998/03
• Oxidation stress and antioxidant ability in birth stage of light breed horse and heavy breed horse.  [Not invited]

  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  1998/03
• Detection of free radical which arises in C₂C₁₂ myotube cell inhibited energy metabolism.  [Not invited]

  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  1998/03
• Effects of Aging on Nitric Oxide Production in LPS‐treated Wistar Rats.  [Not invited]

  稲波修, 浅沼武敏, 桑原幹典, KOTAKE Y

  磁気共鳴と医学  1998
• Roles of water molecules in the hydration layer of DNA in the formation of radiation‐induced DNA damage.  [Not invited]

  桑原幹典, 大島秀基, 飯田義晴, 稲波修

  磁気共鳴と医学  1998
• 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  [Not invited]

  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  1997/10
• 酸化ストレス誘発細胞死におけるSAPK/JNKの活性化機構  [Not invited]

  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  1997/10
• Analysis of cell damage by photo sensitizer pheophorbide-a in a Chinese hamster lung fibroblast.  [Not invited]

  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  1997/09
• Activation mechanism of SAPK/JNK in the cell death induced by oxidation stress.  [Not invited]

  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  1997/09
• Involvement of ICE protease and SAPK/JNK in the radiation induction apoptosis of a human leukemia lymphoblast originated strain culture cell.  [Not invited]

  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  1997/09
• Preparation of three-dimensional MR image based on multi- sliced two-dimensional MR image of hepatic tumor and lung tumor mouse and visualization of focal part.  [Not invited]

  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  1997/03
• LPS induced NO generation and aging change in rat. Evaluation method using electron spin resonance.  [Not invited]

  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  1997/03
• Analysis of signal transduction mechanisms leading to the reactive oxygen generation by employing neutrophils from a Holstein calf with bovine leukocyte adhesion deficiency(BLAD).  [Not invited]

  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
• Localization of O<span style=text-decoration:overline>^-₂</span> and OH radicals in polymorphonuclear leukocytes stimulated by opsonized zymosan.  [Not invited]

  稲波修, 桑原幹典

  家畜生化学  1996/12
• Phosphorylation-dependent activation of the respiratory burst oxidase  [Not invited]

  JL Johnson, JW Park, JE Benna, O Inanami, BM Babior

  BLOOD  1996/11  W B SAUNDERS CO
  
• Activity expression mechanism of cattle serum opsonin.  [Not invited]

  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  1996/08
• Diversity of sugar-connective high polymer protein in cattle serum.  [Not invited]

  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  1996/08
• Property of chitin-connective protein in horse serum.  [Not invited]

  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  1996/08
• CRP concentration of cattle serum rises by estrus.  [Not invited]

  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  1996/08
• Effect of .BETA.-catechin on cell death by cerebral ischemia - reperfusion.  [Not invited]

  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  1996/08
• Significance of phosphorylation of p47phox in NADPH oxidase activation.  [Not invited]

  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  1996/08
• Comparison of glycoproteins in 3 kinds of olfactory receptors of Xenopus.  [Not invited]

  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  1996/08
• Movement of serum protein to egg.  [Not invited]

  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  1996/08
• Active oxygen formation abnormal mechanism of cattle leucocyte adhesion aplasia (BLAD) blood neutrophil.  [Not invited]

  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  1996/03
• The superoxide production from opsonized‐zymosan‐ and PMA‐stimulated polymorphonuclear leukocyte in neonatal calf.  [Not invited]

  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
• The small molecular weight GTP‐binding proteins(Rho) regulate the superoxide production from polymorphonuclear leukocytes.  [Not invited]

  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
• Lipid peroxide levels and superoxide‐scavenging abilities of sera obtained from hotbred(Thoroughbred) horses.  [Not invited]

  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
• ヨシ血清中のキチン結合生蛋白質の性状  [Not invited]

  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  1995/09
• PBN(N‐tert‐butyl‐α‐phenylnitrone)による遅発性脳神経細胞死の抑制  [Not invited]

  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  1995/09
• 神経細胞内指向性分子の調製  [Not invited]

  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  1995/09
• 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  [Not invited]

  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  1995/09
• The function of C‐reactive protein on milk production and fertilization in the cow.  [Not invited]

  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  1995/03
• Isolation and Charatrerization of Chitin‐Binding Protein in Bovine Serum.  [Not invited]

  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  1995/03
• ボツリヌス菌C3の多形核白血球に対する作用  [Not invited]

  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  1995/03
• 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  [Not invited]

  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  1995/03
• 子牛多形核白血球のcytochrome b₅₅₈について  [Not invited]

  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  1995/03
• 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  [Not invited]

  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  1995/03
• 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  [Not invited]

  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  1995/03
• 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  [Not invited]

  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  1994/08
• ラット各組織における抗酸化物質の比較  [Not invited]

  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  1994/03
• Free radical. Nitric oxide and cerebral blood flow.  [Not invited]

  稲波修, 足立健彦, さき山陽一郎, 佐藤昭夫

  神経精神薬理  1994/03
• 子牛の好中球のスーパーオキシド生成機能低下の解明  [Not invited]

  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  1994/03
• 牛の好中球のスーパーオキシド生成機構について  [Not invited]

  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  1994/03
• Biological regulation system in which NO is concerned.In particular, cerebral circulation.  [Not invited]

  稲波修

  大気汚染学会講演要旨集  1994
• 放射線による染色体蛋白質損傷とその意義  [Not invited]

  稲波修, 桑原幹典

  放射線生物研究  1993/09
• 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  [Not invited]

  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  1993/08
• 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  [Not invited]

  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  1993/08
• 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  [Not invited]

  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  1993/08
• The spin trap-2-methyl-2-nitrosopropane enhances hydroxyl-radical-induced degradation in histone Hl and inhubits hydroxyl-radical-induced intermolecular cross-links in histone H2B.

  INANAMI O.

  Mol. Biol. (Life Sci. Adv. )  1993
• Nitric oxide is involved in increased cerebral cortical blood flow following stimulation of the nucleus basalis of Meynert in anesthetized rats.  [Not invited]

  足立健彦, 稲波修, 佐藤昭夫

  基礎老化研究  1992/09
• Vasodilative responses in the hippocampus induced by electrical and chemical stimulation of septal complex.  [Not invited]

  CAO W‐H, 稲波修, 西園寺麗, 佐藤昭夫

  自律神経  1990/06
• ELECTRON-SPIN-RESONANCE OBSERVATION OF ACTIVATED OXYGENS BY PHOTO-EXCITED PHEOPHORBIDE-A  [Not invited]

  T YAMAMOTO, O INANAMI, M KUWABARA, F SATO

  JOURNAL OF RADIATION RESEARCH  1988/03  JAPAN RADIATION RESEARCH SOC
  
• TRH投与による麻酔ラット脳血流量の増加反応  [Not invited]

  稲波修, 佐藤昭夫

  生理学研究所年報  1988
• Mechanisms of OH‐induced damage formation in nucleic acids: Commonality and disparity in nucleoside, nucleotide and polynucleotide.  [Not invited]

  桑原幹典, 稲波修

  放射線化学  1987/09
• TRAPPING OF PRECURSORS OF OH-INDUCED CROSS-LINKAGES IN HISTONE H2B  [Not invited]

  O INANAMI, M KUWABARA, F SATO

  JOURNAL OF RADIATION RESEARCH  1987/03  JAPAN RADIATION RESEARCH SOC
  
• PROTECTIVE EFFECT OF HISTONE-H3 AGAINST THE ATTACK OF HYDRATED ELECTRON ON THE OTHER HISTONE SUBFRACTIONS  [Not invited]

  O INANAMI, M KUWABARA, F SATO

  JOURNAL OF RADIATION RESEARCH  1986/03  JAPAN RADIATION RESEARCH SOC
  
• OH-INDUCED FREE-RADICALS IN POLY-U AS STUDIED BY SPIN-TRAPPING COMBINED WITH ENZYMATIC DIGESTION  [Not invited]

  M KUWABARA, O INANAMI, D ENDOH, F SATO

  JOURNAL OF RADIATION RESEARCH  1986/03  JAPAN RADIATION RESEARCH SOC
  
• REACTIONS OF HYDRATED ELECTRON WITH ISOLATED HISTONE SUBFRACTIONS  [Not invited]

  O INANAMI, M HAYASHI, M KUWABARA, F SATO

  JOURNAL OF RADIATION RESEARCH  1985  JAPAN RADIATION RESEARCH SOC
  
• SPIN-TRAPPING OF OH-INDUCED FREE-RADICALS IN DNA  [Not invited]

  M KUWABARA, O INANAMI, D ENDOH, F SATO

  JOURNAL OF RADIATION RESEARCH  1985  JAPAN RADIATION RESEARCH SOC
  
• HYDRATED ELECTRON-INDUCED MAIN CHAIN SCISSION IN CHROMOSOMAL BASIC-PROTEINS AND THEIR RELATED-COMPOUNDS  [Not invited]

  O INANAMI, M HAYASHI, M KUWABARA, B SYUTO, G YOSHII

  JOURNAL OF RADIATION RESEARCH  1984  JAPAN RADIATION RESEARCH SOC
  

Teaching Experience

RadiologyRadiology Hokkaido University
Radiation BiologyRadiation Biology Hokkaido University

Association Memberships

• SOCIETY FOR FREE RADICAL RESEARCH JAPAN   JAPAN SOCIETY FOR BIOMEDICAL GERONTOLOGY   THE JAPANESE BIOCHEMICAL SOCIETY   THE SOCIETY OF ELECTRON SPIN SCIENCE AND TECHNOLOGY   日本放射線影響学会   日本獣医学会   

Research Projects

• Research for local control and high accuracy of cancer senolytic therapy by non-invasive imaging

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2023/06 -2026/03 

  Author : 安井 博宣, 稲波 修, 平田 健司, 久下 裕司
• Development of a precise oxygen mapping method for malignant tumors using electron paramagnetic resonance

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

  Date (from‐to) : 2021/07 -2025/03 

  Author : 平田 拓, 稲波 修

   

  本研究課題では、悪性腫瘍における酸素分圧を高精度に可視化する連続波電子スピン共鳴分光イメージンク法の理論と技術を構築することを目指している。具体的には、窒素同位体で標識した二つの分子プローブを用いるコンセプトにより、4 mmHg以下の計測精度を有する三次元酸素分圧計測の実現を目指す。初年度となる今年は、高精度酸素分圧計測を行うための標準試料となるラジカル水溶液の作製法について実験条件および測定法を確立した。具体的には、標準試料となるラジカル水溶液の酸素分圧を、クエンチングを利用した光ファイバセンサにより高精度で測定する手技を獲得した。また、ラジカル水溶液のラジカル濃度を正確に定量するため、Xバンド電子スピン分光計測およびスペクトルフィッティングを適用した。これにより、酸素分圧およびラジカル濃度を高精度で定量することが可能になり、今後の酸素分圧測定の校正データ取得の準備ができた。 酸素分圧測定の基礎となる緩和時間マッピングの精度を向上させるため、磁場勾配のヒステリシスの影響を確認する実験を進めた。磁場勾配のシーケンスによって緩和時間マッピングの精度が変化することが分かった。これは、マグネットのヒステリシス効果により磁場勾配のステップ状の変化を正確に実現できていないことが原因である。磁場勾配の線形性を改善するシーケンスの開発が必要である。 酸素分圧モニタOxyLiteを用いて、腫瘍および正常組織の酸素分圧測定を試みた。直径0.35mmの光ファイバ酸素分圧／温度プローブを用いて、マウスに作製した腫瘍の酸素分圧測定を試みた。実験手技を検討し、光ファイバプローブの腫瘍組織への刺入をファイバの損傷なく実施できるようになった。酸素分圧測定の時間応答が早くないことが明らかとなったが、十分に計測が安定してから酸素分圧を取得することで、応答時間に関する課題を解決することができた。
• Targetting of cancer-specific metabolic system for senolytic therapy

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

  Date (from‐to) : 2020/04 -2025/03 

  Author : 稲波 修, 平岡 和佳子, 岡松 優子, 安井 博宣, 滝口 満喜, 平田 拓

   

  本年度はインビトロの培養細胞の系の実験で、以下の四つの結果が明らかとなった。 1）グルタミン要求性の強いヒト肺がんA549細胞やH460細胞では、放射線照射により、これらの細胞でのグルタミンの取り込みの増大とグルタメートの増大、すなわちグルタミナーゼの活性化を起こす事を明らかにした。さらに、グルタミン代謝阻害剤添加あるいはグルタミン欠乏培地下で培養すると治療線量レベルの放射線照射によって多くの老化様細胞死とSASPの培養液中への分泌を増強させることが可能である事が明らかとなった。 2）グルタミノリシス阻害条件下で放射線による老化様細胞死の誘発機構には活性酸素種の生成量、グルタチオン量の変化DNA損傷の量、さらにはDNA損傷に伴う伴うp53やp21、p16の発現変化などの既知のメカニズムの関与は殆ど無く、未知の機構の関与が示唆されている。 3）更にグルタミノリシス阻害条件下で放射線誘発する老化様細胞死を起こす条件で、Bcl-XL阻害剤ABT-737によって、この増大した老化様細胞死の多くはアポトーシスに変換できること、いわゆるセノリシスを起こせることを明らかにした。 4）一方では、同じ肺がん由来細胞でも786-o細胞ではグルタミン代謝阻害が老化様細胞の増強を起こさず、A549やH460細胞と同様の現象は観察されず、全ての肺がん細胞に於いて同様の機構が働いていない事も明らかとなった。また、脂質代謝阻害剤は老化様細胞増強には関与しなかった。 以上の結果はグルタミン要求性の強い肺がん細胞を中心にその亜致死レベルのグルタミノリシス阻害条件下で治療線量レベルの放射線照射をすると、多量の老化様細胞死の増大が起こす事を示しており、さらに、アポトーシス抑制因子の阻害剤で効率よく老化様細胞死をアポトーシスに変換できることが明確になった。
• Spatio-temporal dynamics analysis of cell death induction due to disruption of biometal homeostasis and its application to radiotherapy

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2022/04 -2025/03 

  Author : 安井 博宣, 稲波 修, 平田 拓, 久下 裕司

   

  本研究では、「正常細胞とがん細胞におけるX線照射後の遷移金属や関連因子の動態がレドックス状態や細胞死にどのような影響を与えるのか？また、イメージング等検出技術によってそのメカニズムを解明することで、生体金属の恒常性破綻をがん放射線治療のアプローチとすることが可能か？」という問いに対し、初年度では(1)X線照射による遷移金属や関連因子の動態と細胞死誘導の評価と作用点を探索することおよび(2)増感標的となる分子の決定と実験的治療検討をin vitroで行うことを目的とした。研究成果として、昨年報告された銅によって引きおこされる新しい細胞死について放射線作用との関係を調べるために、銅イオノフォアであるElesclomolを用いて、HCT116細胞やMIA-Paca-2細胞においてピルビン酸デヒドロゲナーゼキナーゼ阻害剤ジクロロ酢酸（DCA）や乳酸脱水素酵素阻害剤FX11と併用した際のElesclomolの細胞毒性の増強作用の有無について調べた。その結果、既報ではあるがDCAのElesclomolの増強作用が確認できたことに加えて、FX11においてもElesclomolの細胞毒性の増強作用を有することがコロニー形成法によって明らかとなった。各種阻害剤を用いることで、この増感作用が、アポトーシス、ネクロプトーシス、フェロトーシスではなく、銅キレート剤や抗酸化剤によって抑制されたことから、銅に関連する酸化ストレスによる細胞死に依存していることが明らかとなった。以上の結果から、銅代謝に介入することが放射線感受性を引き上げることに繋がることが示唆された。
• Development of a novel ESR oximetry for tumor-therapy-effect prediction

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Fund for the Promotion of Joint International Research (Fostering Joint International Research (B))

  Date (from‐to) : 2020/10 -2024/03 

  Author : 稲波 修, 安井 博宣, 永根 大幹, 山下 匡

   

  今までオキシメトリーで広く用いられてきた5,9,14,18,23,27,32,36-octa-n-butoxy-2,3-naphthalocyanine (LiNc-BuO)プローブを合成のための原料であるブトキシナフタロシアニンの一般業者からの入手が難しくなってきており、初年度はまず最初にこれが研究遂行に必須である事から、1からのNcーBuO合成を試みる必要があった。これまでに北大の稲波・安井によって３ステップで数ｍｇレベルの合成が北大で可能となった。現在、実用レベルに必要な数十ミリグラムの合成を試みている。また、現在、実験動物施設内にESRイメージング装置がなく、動物施設から動物を装置設置の部屋に移動させて測定する必要がある。現在、実験動物管理は現在非常に厳しく、特に遺伝子組換えマウスやSPFマウスは容易に動物の輸送が許されない。そこで、稲波と共同研究を旧来から共同研究を行っている赤羽（阪大）と藤井（北海道医療大）が最近開発した移動型ESRイメージング装置を完成させており、共同研究としてえ動物実験施設へこの機器を持ち込んでPET-CTと組み合わせたがんを対象とした研究を計画している。そのための研究打合せと借用の交渉を開始し、共同研究に発展させるたいと考えている。現在、動物モデル、機器の性能調整等の打合せを行っており、実験計画を立てている段階である。分担者 永根と山下は制癌作用を持つニトロキシド付加クルクミン誘導体HO-3867のDNA損傷修復の遅延と放射線増感作用を明らかにした。今後、オキシチップを用いて、インビボで移植腫瘍モデルでのオキシメトリーを用いて、HO-3867の効果について検討する予定である。また、米国のクプサミー教授とZOOMで連絡を取り合い、臨床用ESRと移動可能ESR装置の麻布大への移設についても交渉中である
• Development of a method of 3D oxygen and pH mapping of tumors using electron spin resonance spectroscopy

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

  Date (from‐to) : 2019/04 -2022/03 

  Author : Hirata Hiroshi

   

  We aimed to develop quantitative imaging techniques for the partial pressure of oxygen and extracellular pH for malignant tumors. In this study, electron spin resonance (ESR) spectroscopy was applied to the three-dimensional mapping of the partial pressure of oxygen and pH in solution samples. Using a molecular probe that can concurrently detect the partial pressure of oxygen and pH, we showed the feasibility of concurrent mapping the partial pressure of oxygen and pH in solution samples.
• Development of a novel radiotherapy targeting iron-dependent cell death ferroptosis

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

  Date (from‐to) : 2019/04 -2022/03 

  Author : YASUI HIRONOBU

   

  In this study, (1) we searched for a point of action that can effectively induce ferrotosis with various candidate drugs, (2) we show evidence of the obtained mechanism of radiosensitizing action, and (3) therapeutic effect at the biological level. The ultimate goal was to develop an imaging method that could support this. Toward this goal, during this study, ferroptosis inducers were shown to increase radiosensitivity by reducing antioxidants in cancer cells, and intracellular iron metabolism in cancer cells by transferrin probes. Focusing on the mechanism, we succeeded in developing a method that makes it possible to predict the therapeutic effect of ferrotosis-induced cancer in advance.
• Elucidation of pathological pain via nociceptors and its application to pain relief

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

  Date (from‐to) : 2018/04 -2022/03 

  Author : OHTA Toshio

   

  This study clarified that the activation of TRPA1 expressed in sensory neurons is regulated by low-threshold voltage-gated Ca channels and that TRPA1 activity by oxidative stress substances produced during inflammation is modulated by endogenous sulfur compounds. Studies on species differences in TRP channel function revealed the importance of TRPA1 channels as a site of action in bird repellents and differences between mammals and birds in TRPV1 antagonists. A search for compounds that inhibit TRPV1 function revealed that some vanilloids showed inhibitory action on TRPV1.
• Development of a MS imaging-based diagnosis for novel cancer senotherapy

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

  Date (from‐to) : 2019/06 -2021/03 

  Author : Yasui Hironobu

   

  Permanently stopping the cell cycle, that is, senescence, is considered to be an effective therapeutic strategy for cancer cells having infinite proliferation ability. The purpose of this study was to develop a new therapeutic method targeting cellular senescence and to establish a method that can diagnose it. First, it was clarified that senescence can be efficiently induced by the combination with radiation and Id-1 inhibition, which is known as a senescence-inducing factor in human-derived cancer cells and mouse-derived cancer cells both in vitro and in vivo. Although mass spectrometry could not determine the degree of senescence induction in this model, it revealed that 18F-FLT PET imaging may be effective as a method for detecting senescence.
• Analysis of radioresistance factors of tumors in radiotherapy by using MS imaging and ESR imaging techniques

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Challenging Research (Exploratory)

  Date (from‐to) : 2018/06 -2021/03 

  Author : Inanami Osamu

   

  The aim of this study is to develop new metabolic analysis methods and imaging techniques for cancer metabolism and its novel radiation response, which will lead to the development of future therapies. In this study, we found cancer cells with dependence on mitochondrial electron transport chain (ETC), glutamine metabolism, and lipid metabolism, confirming the diversity of cancer cell metabolism. As a new cancer-specific metabolic evaluation system, ESR oximetry was established to evaluate mitochondrial ETC activity at the cellular level, and radiation response was clarified. In addition, we established a rapid ESR imaging method for tissue reduction state reflecting glutathione metabolism and an in vivo imaging method using pH-sensitive probes for extracellular pH changes reflecting glucose metabolism in an international collaboration.
• 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)

  Date (from‐to) : 2019 -2021 

  Author : 稲波 修
• Sono-mechanotherapy for neurite outgrowth

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Challenging Research (Exploratory)

  Date (from‐to) : 2018/06 -2020/03 

  Author : Mitsuyoshi Takiguchi

   

  Axon regeneration is crucial for the functional recovery after spinal cord injury. Myelin-related growth inhibitors, such as Nogo-A, inhibit neurites regrowth and elongation. Low-intensity pulsed ultrasound (LIPUS) is known to show neuroprotective effects in neurodegenerative diseases. In the present study, we evaluated the feasibility of LIPUS for enhancing axon regeneration in the presence of Nogo-A. Low-intensity pulsed ultrasound was exposed to rat cortical neurons in vitro after Nogo-A treatment. The total length of neurites per cell in LIPUS group was significantly larger than that of non-LIPUS group. There was no significant difference in the number of neurites. The results suggest that LIPUS may enhance the elongation of neurites in the presence of Nogo-A while may not affect on neurites sprouting and branching. This preliminary study implies the feasibility of LIPUS for axonal regeneration.
• Development of the optimal medication for individuals exposed to lethal radiation

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

  Date (from‐to) : 2016/04 -2020/03 

  Author : Kashiwakura Ikuo

   

  The aim of this study was to establish an effective emergency drug treatment protocol using biomarkers and approved drugs as indicators of disability prediction and treatment efficacy. Microarray results from mouse blood-derived RNAs after 24 h of 0.5 to 3 Gy total body irradiation showed a dose-dependent response of seven different mRNA expressions, suggesting their potential as exposure markers (patent application). Validation of several approved drugs in lethal-dose-irradiated mice revealed that Romiprostim, a drug for chronic idiopathic thrombocytopenic purpura, produced a 100% 30-day survival rate, and its mechanism of action included the reduction and recovery of multiple organ failure by various actions such as promoting DNA damage repair, suppressing cell death and inflammation (patented).
• 次世代放射線治療の最適化のための新規機能イメージング技術による基盤研究

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2014/04 -2017/03 

  Author : 稲波 修
• 腫瘍転移巣の術中迅速診断を目標とした新規近赤外発光イメージング技術の開発

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2014/04 -2015/03 

  Author : 稲波 修
• Role of functional change of microglia in neuropathogenesis of prion diseases

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2011/04 -2015/03 

  Author : HORIUCHI Motohiro, INANAMI Osamu, HASEBE Rie, SASSA Yukiko, OHSIMA Masanobu

   

  We established PrPSc-specific staining method and using this method we elucidated a part of neuropathophysiology in the early stage of prion infection; astrocytes recognize prion propagation directly or subtle changes in neurons by prion propagation and astrocyte activation precedes over microglial activation. Gene expression analysis of microglia in prion-infected mice showed that microglia play a neuroprotective role by secreting neurotrophic factors at the early stage of prion infection in the early stage of prion infection, but the microglial activation state shifts to pro-inflammatory or neurotoxic state along with the progression of prion disease. Furthermore, anti-inflammatory cytokines such as IL-10 interfere the propagation of prions in brain.
• Risk-communication of exposure to radioacive contaminants after the Great East Japan Earthquake disaster

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

  Date (from‐to) : 2012/04 -2014/03 

  Author : NAKAI Kunihiko, INANAMI Osamu

   

  The Fukushima Dai-ichi nuclear disaster raised concerns about the health consequences by exposure to and an intake of radionuclides even at the low-level contaminated areas. We performed surveys to detect the presence of radioactive substances in breast milk and placenta from breast-feeding mothers, and food using the duplicate diet method. The monitoring study suggests that there were no detectable contamination in breast milk and placenta, and a trace level of contamination in food. Then, a trial of risk communication was performed to the citizen using the monitoring information. To assess the effects of educational lecture, a questionnaire survey regarding the radiation health knowledge, risk perception, health issue, and trust was asked to the lecture attendees. The results suggest that to attend the lectures may effective to reduce the anxiety level.
• Characterization of malignant glioma and development of potent treatment strategy focusing on hypoxia dynamics

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2012 -2012 

  Author : INANAMI Osamu, YAMAMORI Tohru, YASUI Hironobu

   

  The purpose of this study is to clarify the hypothesis that the frequency of tumor hypoxia influences the prognosis of malignant glioma rather than an amount of tumor hypoxia. The results revealed that sequential PET analysis using 18F-FMISO could not visualize the transient hypoxia in glioma, because it took the relatively long time for the radioactivity attenuation. However, this study achieved the establishment of hypoxia imaging in transplanted murine glioma by using electron spin resonance technique. Furthermore, it was revealed that doranidazole is a potent drug to improve the radiation efficiency against hypoxic glioma cells.
• Cellular and Immune Responses of Dairy Cows Induced by Intramammary Infusion of Lactic Acid Bacteria and Therapeutic Trials to Quarters of Cows with Mastitis.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2010 -2012 

  Author : NAGAHATA Hajime, HIGUCHI Hidetoshi, TANJI Yasunori, INANAMI Osamu, SONOMOTO Kenji

   

  The aim of this study was to characterize the cellular and immune responses of mammary gland by intramammary infusion (IMI) of Lactic acid bacteria (LAB) in cows, and to evaluate the therapeutic effects of IMI of LAB to lactating cows withmastitis. Marked increase of SCC were found in infused quarter at 24-48 hr post infusion. Significantly increased concentrations of immunoglobulins, anti-bacterial proteins and mRNA expression of cytokines on milk leukocytes were found in cows. In therapeutic trials, IMI of LAB to quarters leads to a marked cellular and immune responses in mammary gland. The IMI of LAB may have a possible rolefor control of mastitis.(key words:lactic acid bacteria, mammary gland defense, immune-therapeutic trial, bovine mastitis control)
• Structural analysis of zoonosis-related high-molecular protein aggregation by double electron-electron resonance (DEER) technique

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2009 -2011 

  Author : INANAMI Osamu, INABA Mustumi, HORIUCHI Motohiro, KUWABARA Mikinori, YAMAMORI Tohru

   

  In this study, to obtain the structural information for this pathological fibrous form of PrPs', we measured the intermolecular distance in the pathological fibrous form with D178N mutation by the site-directed spin labeling (SDSL) with continuous wave (CW) ESR (X-band) and pulsed ESR (Q-band). After the formation of the fibrous structure by exposure to 1M of guanidine hydrochloride in pH4.0, the ESR spectra were measured by CW-ESR. The interspin-distances in pathological fibrous form were measured by continuous wave ESR (CW-ESR) and the four-pulse double electron-electron resonance (DEER) technique. The information of intermolecular distances will be useful to estimate the pathogenic fibrous structure of the familial prion disease, since it is difficult to analyze the macromolecules such as the fibrous protein by NMR or X-ray crystallography.
• 膜透過性ペプチド融合アポトーシス誘導タンパク質を用いた新規放射線がん治療

  文部科学省：科学研究費補助金(挑戦的萌芽研究)

  Date (from‐to) : 2009 -2010 

  Author : 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...
• From the ER to the plasma membrane : Vesicular transport of membrane skeleton units and the diseases

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(A))

  Date (from‐to) : 2007 -2009 

  Author : Mutsumi INABA, 山本 雅之, 高桑 雄一, Kota SATO, Takashi AGUI, Yayoi OTSUKA, Osamu INANAMI, Nobuhisa WATANABE, Masayuki YAMAMOTO, Yuichi TAKAKUWA, Hiroshi OHTA, Takashi UMEMURA, Yoshiaki HIKASA

   

  The long-range goal of this project is to investigate the mechanism for assembly and functional organization of the red cell membrane skeleton. Here, we analyzed the synthesis, quality control, interaction with an erythroid scaffolding protein ankyrin in the endoplasmic reticulum (ER), and the ER exit signal of anion exchanger 1 (AE1) and its mutants. The results obtained together indicate that the membrane skeletal network is assembled in the ER membrane as a small unit followed by vesicular transport to the plasma membrane.
• Elucidation of the mechanism on the formation of early pathological lesion of prion diseases

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(A))

  Date (from‐to) : 2006 -2009 

  Author : Motohiro HORIUCHI, 落合 謙爾, 瓜生 匡秀, 大島 正伸, Osamu INANAMI, Kazuhiro KIMURA, Rie HASEBE, Chang-hyun SON, Kenji OCHIAI, Masahide URYU, Masanobu OOSHIMA, Hidefumi FURUOKA

   

  To identify host genes whose expression is up-regulated in the early stage of prion-infection, DNA microarray analysis was carried out and involvement of the genes in pathobiology of prion diseases was analyzed. The results showed that Cd14 molecule, known as receptor for LPS, influences the progression of the disease, whereas, Cxcl10, known as chemokine with neuroprotective effect, plays a role in antagonizing the disease progression. In addition, microglial activation was suggested to have a protective effect on the disease progression.
• Regulatory mechanisms of vanilloid receptor on pain signaling

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 2006 -2009 

  Author : Toshio OHTA, 今川 敏明, 稲波 修, Toshiaki IMAGAWA, Osamu INANAMI

   

  We cloned cDNA encording vanilloid receptor (TRPV1) from porcine and human spinal cord cDNA library. We established in vitro assay system for monitoring vanilloid receptor activities. Heterologous expressed TRPV1 receptors are activated by not only capsaicin, heat and protons, but also regulated by extracellular sodium ions. Hyperalgesia induced by inflammatory mediators, such as serotonin and histamine is due to a hyper-activation of TRPV1 using heterologous expression system and cultured sensory neurons. We found that mustard oil and methylsalicylate act as TRPV1 agonist and the latter pr...
• Molecular imaging of prion protein by atomic force microscop yand a novel electro-spin-resonance techniques

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 2007 -2008 

  Author : NANAML I, 平岡 和佳子, 下山 雄平, Mutsumi INABA, Motohiro HORIUTCHI, Mikinori KUWABARA, Katetoshi ASANUMA, Wakako HIRAOKA, Yuhei SHINOYAMA

   

  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。
• Placental/Umbilical cord blood-derived mesenchymal stem cell-likestroma cells support the hematopoietic recovery of X-irradiated human CD34+ cells

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 2006 -2008 

  Author : Ikuo KASHIWAKURA, 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。
• プリオンと銅イオン相互作用の新しいパルス電子スピン共鳴法を用いたアプローチ

  文部科学省：科学研究費補助金(萌芽研究)

  Date (from‐to) : 2006 -2007 

  Author : 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...
• Immunological and molecular analysis of responses of mammary gland against infection and modulation of the immunological functions in dairy cows

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(C))

  Date (from‐to) : 2005 -2007 

  Author : Hajime NAGAHATA, 稲波 修, 桐沢 力雄, 林 正信, 桑原 幹典

   

  This study was conducted to evaluate the adhesion molecules, functional responses and cytokine expressions on blood and milk leukocytes from dairy cows with mammary infection, and to investigate the effects of possible substances on the leukocyte responses in lactating dairy cows. Major results of this study were summarized as follows :L-selectin expression on neutrophils in milk from cows with S. aureus mastitis was significantly decreased compared to that in blood; however, significantly (P<0.05) higher than that of normal controls. CD18 expression on neutrophils in milk from cows with S....
• Structural analysis of prion proteins and mechanism of PrPsc transition by using a novel dynamic molecular structure analysis

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 2005 -2006 

  Author : Osamu INANAMI, 堀内 基広, 苅和 宏明, 稲葉 睦, 桑原 幹典

   

  We examined the influence of D177N (178N in humans) mutation on the conformational stability of the S2 region of moPrPc with varying pHs by using the SDSL-ESR technique. We prepared moPrPc mutants that reacted with methane thiosulfonate spin-probes (Y161R1 and Y161R1/D177N). 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 from 7.5 to 4.0.These results suggested that the disappearance of a salt bridge (D177-R163) induced the increase in the instability of S2 region. The value...
• 実験動物頭部のファンクショナルMRIによる痛覚のビジュアリゼーション

  文部科学省：科学研究費補助金(萌芽研究)

  Date (from‐to) : 2005 -2006 

  Author : 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...
• Molecular pathology for down-regulation of erythroid-specific genes in prion diseases

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(A))

  Date (from‐to) : 2004 -2006 

  Author : Mutsumi INABA, 山本 雅之, 陰山 聡一, 堀内 基広, 稲波 修, 梅村 孝司

   

  AHSP is an erythroid-specific molecular chaperone that stabilizes newly synthesised a-globin. Previous studies demonstrated that mRNA levels of AHSP were specifically reduced in hematopoietic tissues of prion-infected animals. The purpose of the present study is to clarify the mechanism for down-regulation of AHSP transcription. A series of truncation and mutation analyses on 2.5-kb 5' upstream region of the AHSP gene in MELhide8 cells and electrophoretic mobility shift assay showed that the minimal 5'-promoter region located at-328-286 to the translation initiation site including a GATA bi...
• Hierarchical Correlation Dynamics among Cellular Rhythms: Coordination and Dissociation of Non-linear Biological Oscillators.

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 2004 -2006 

  Author : Koichi KAWAHARA, 米山 満, 稲波 修, 内貴 猛, 山内 芳子

   

  There are many rhythmic phenomena in the biological system. A variety of oscillatory phenomena such as a cyclic change in the concentration of intracellular Ca2+ are also observed in the cellular level. The present study aims at elucidating the mechanisms responsible for interactions among cellular oscillatory rhythms, and for the coordination and dissociation of such cellular rhythms. The results are summarized as follows:1. In cultured cardiac myocytes at 4 DIV, the cyclic changes in the concentration of intracellular free Ca2+ (Ca2+ oscillation) in association with spontaneous rhythmic c...
• Does the bovine lactoferrin improve chronic inflammation in the immuno-suppressive condition induced by FIV infection?

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(C))

  Date (from‐to) : 2004 -2006 

  Author : Reeko SATO, 寺口 進, 内藤 善久, 安田 準, 稲波 修

   

  1)The effects of orally-administered bovine lactoferrin (bLF) on lymphocyte functions in FIV-infected catsThe effects of bLF on ConA-induced proliferation of peripheral lymphocyte in healthy and FIV-infected cats were investigated. Orally-administered bLF showed the inhibition of lymphocyte proliferation both in healthy and FIV- infected cats. Co-incubation of bLF and peripheral lymphocytes also showed the inhibition of lymphocyte proliferation both in healthy and FIV-infected cats.2)The effects of bLF and ConA on cytokine mRNA expression in feline peripheral lymphocyteAfter the co-incubati...
• 神経因性疼痛発生の分子機構の解明と痛覚評価系の開発に関する研究

  文部科学省：科学研究費補助金(萌芽研究)

  Date (from‐to) : 2004 -2006 

  Author : 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。
• Role of store-operated Ca entry mechanism in signal transduction in gastrointestinal intrinsic neurons

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 2003 -2005 

  Author : Toshio OHTA, 稲波 修

   

  The aim of this project was to identify the regulatory mechanisms of capacitative Ca entry in gastrointestinal intrinsic neurons. For these purposes, we analyzed the properties of capacitative Ca entry, its regulatory mechanisms with intracellular messengers and putative channel molecules. 1) We established experimental system for visualization of Ca signaling in single cultured gastrointestinal intrinsic neurons. 2) ATP elicited Ca increases through release of Ca from intracellular stores, the activation of capacitative Ca entry and P2X2 channels in rat cultured gastrointestinal intrinsic ...
• Dysfunction of neutrophil during parturition and proteome analysis of placenta and serum in cow

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 2003 -2004 

  Author : Osamu INANAMI, 佐藤 基佳, 稲葉 睦, 桑原 幹典, 浅沼 武敏, 太田 利男

   

  The purpose of this project is to identify and characterize internal factors to reduce neutrophil functions after parturion in cows, since bovine mastitis sometimes occurs during parturition. In the experiments, the analysis of signal transduction pathways for activation of bovine neutrophils was performed. As important molecules for activation of NADPH oxidase in bovine neutrophils, cPKC, p38MAPK, ERK, PI3K and nPKC(pkcδ) were identified, but these factors did not changes during parturition in the cow. Furthermore, I tried to identify causative factors to reduce immune functions of cow aft...
• Characterization of host defence capability of dairy cows during peripartum period and modulation of the immunological functions.

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 2002 -2004 

  Author : Hajime NAGAHATA, 林 正信, 桐沢 力雄, 稲波 修, 桑原 幹典

   

  The above-entitled study was conducted to characterize the leukocyte functions of dairy cows during peripartum period, and to investigate the modulation of the host defence capability. Major results of this study were summarized as follows:Significantly decreased luminal-dependent chemiluminescent response was found in neutrophils from cows around parturition, compared to those of control cows. Similar changes were found in neutrophils stimulated with aggregated bovine IgG. These results were in consistent with previous findings that functions of neutrophils are impaired in peripartum perio...
• 抗酸化物質を用いた新たな神経突起・神経細胞分化誘導法の開発

  文部科学省：科学研究費補助金(萌芽研究)

  Date (from‐to) : 2002 -2003 

  Author : 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...
• Molecular mechanisms for the assembly of the red cell membrane skeleton during erythroid cell development

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 2002 -2003 

  Author : Mutsumi INABA, 滝口 満喜, 稲波 修, 前出 吉光, 山本 雅之, 高桑 雄一, 斉藤 昌之

   

  The "unit-assembly hypothesis" for red cell membrane skeleton during erythroid development was investigated.Stable transfectants of major constituents of red cell transmembrane proteins were developed in K562 and HEK293 cells using retroviral vectors. Band 3 and glycophorinCweretransportedtotheplasmamembranevia the ER/Golgi pathway. Band 3-binding domain of ankyrin (AnkN90) and protein 4.1 showed membranous localization in the cytoplasm with co-localization with markers for the ER and the Golgi apparatus in band 3-transfeted cells despite of the difference in erythroid differentiation. Endo...
• Analysis for putative relationship between causative genes for hereditary disorder and quantitative traits loci in cattle

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 2001 -2002 

  Author : Mutsumi INABA, 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 putative relationship between phenotypes for band 3 deficiency with the R664X mutation and contents of saturated/unsaturated fatty acids, which predominantly affect flavor and taste of beef, was evaluated. There was no direct linkage between phenotype/genotype and beef quality although carriers for band 3 deficiency appeared to have unsaturated fatty acids such as oleic acid at levels higher than normal animals. Moreover, we could not obtain direct evidence for remarkable advantage of homozygous/heterozygous states for claudin-16 deficiency. Present study, however, demonstrated a genetic ...
• ネコ免疫不全FIVの抗酸化剤治療の基礎的検討と臨床応用

  文部科学省：科学研究費補助金(萌芽的研究, 萌芽研究)

  Date (from‐to) : 2001 -2002 

  Author : 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...
• Hormonal system for regulating local defense

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 2000 -2002 

  Author : Yasuhiro KON, 岩永 敏彦, 稲波 修, 遠藤 大二

   

  It has been known that hormones regulate several functions as a physiological ligand. Renin-angiotensin system (RAS)plays functions for regulating blood pressure and water-mineral balance. Renin produced mainly from kidney is thought to be also synthesized by other tissues, however, its real functions are under discussion. In the present study, the relationship between RAS and local immune defense were investigated by morphological and biochemical techniques. The morphological analysis of renin knockout mice suggested the following results. The kidneys of null mutant mice showing more or le...
• NADPHオキシダーゼコンポーネントp47^のSH3Aドメインの構造解析 ―部位特異的スピンラベル法と飽和移動法による研究―

  文部科学省：科学研究費補助金(特定領域研究(A))

  Date (from‐to) : 2001 -2001 

  Author : 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...
• 新規ヌクレオシド系制ガン剤と放射線照射併用によるアポトーシス細胞死増感作用

  文部科学省：科学研究費補助金(特定領域研究(C))

  Date (from‐to) : 2001 -2001 

  Author : 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...
• Development of antitumor drugs to enhance apoptotic cell death and clinical application

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(C))

  Date (from‐to) : 2000 -2001 

  Author : Osamu INANAMI, 滝口 満喜, 松田 彰, 桑原 幹典

   

  Clinically, the combination treatment of solid tumor cells with an anticancer drug and radiation was widely used to enhance cell killing. 1-(3-C-Ethynyl-β-D-rijbo-pentofuranosyl) cytosine (ECyd) was newly developed as an anticancer drug to induce cell death by inhibiting RNA synthesis. In this study, we examined whether the exposure of human gastric adenocarcinoma MKN45 cells to X rays in the presence of ECyd at the limited concentration ranges with no apoptosis induced apoptotic cell death. MKN45 cells were treated with 0.1μM ECyd for 1 h before irradiation. After irradiation with 20 Gy, a...
• Development of new antitumor drugs and apoptotic signal transudation - Application to feline leukemia and canine lymphoma -

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 2000 -2001 

  Author : Mikinori KUWABARA, 太田 利男, 松田 彰, 稲波 修, 岡田 幸助, 佐藤 れえ子

   

  Clinically, the combination treatment of solid tumor cells with an anticancer drug and radiation was widely used to enhance cell killing. 1-(3-C-Ethynyl-β-D-ribo-pentofuranosyl) cytosine (ECyd) and 2-chrolo-deoxyadenosine (Cl-Ade) was newly developed as anticancer drugs to induce cell death by inhibiting RNA synthesis. In this study, we examined whether the exposure of these anticancer drugs or /and ionizing radiation to human gastric adenocarcinoma MKN45 cells, human lekemia cell line MOLT-4 cells induce apoptosis and canine lymphoma cell line CL-1. In MOLT-4 cells, Fas expression and casp...
• Molecular mechanism for the assembly of red cell membrane skeletons based on pathobiology of congenital hemolytic anemia in cattle

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 2000 -2001 

  Author : Mutsumi INABA, 辻本 元, 稲波 修, 高桑 雄一, 山本 雅之, 松木 直章, 小野 憲一郎

   

  Stable transfectants of normal bovine band 3 (bebWT) or the mutant band 3 with R664X mutation (bebRX) were established in K562 cells and HEK293 cells using retoriviral vectors. Transfected cells expressing EGFP-bebWT and EGFP-bebRX, and N-terminal domain of ankyrin (AnkN90) in combination with band 3 proteins were also prepared.The bebWT and EGFP-bebWT showed stable expression on the plasma membrane of the transfected cells, whereas the mutant proteins, bebRX and EGFP-bebRX were degraded by Ub-proteasome system soon after synthesis on the ER or after retrograde transported from the Golgi ap...
• Action of optimum cytokine for the recovery of X-irradiated human hematopoietic stem and progenitor cells

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(C))

  Date (from‐to) : 2000 -2001 

  Author : Ikuo KASHIWAKURA, 村上 美穂, 稲波 修, 桑原 幹典, 高橋 恒夫, 高木 良成

   

  We obtained the results from the project titled "Action of optimum cytokine for the recovery of X-irradiated human hematopoietic stem and progenitor cells" for the period from 2000 to 2001. The following points were clarified.(1) Human placental/umbilical cord blood (CB) CD34^+ CFU-Meg (megakaryocyte colony-forming units) is more radiosensitive than the other myeloid hematopoietic progenitor cells, including erythroid burst-forming units, granulocyte-macrophage colony-forming units and granulocyte-erythroid-macrophage-megakaryocyte colony-forming units.(2) The proportion of large megakaryoc...
• Characterization of CD18-Deficient Neutrophil Functions and Some Modulations of Bovine Neutrophils from Cattle with Bovine Leukocyte Adhesion Deficiency

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 1999 -2001 

  Author : Hajime NAGAHATA, 辻 正義, 林 正信, 稲波 修, 桑原 幹典, 樋口 豪紀

   

  The above-entitled study was performed and the results were summarized as follows :Clinical and pathobiological findings of the bone marrow (BM) -transplanted heifer with leukocyte adhesion deficiency were evaluated for 1.6 years, from 27 months after BM transplantation until deceased. Serum cytokine levels and their expression of mRNA on neutrophils from a bone marrow (BM) transplanted heifer with leukocyte adhesion deficiency were evaluated. the clinical condition of the affected heifer was relatively stable after BM-transplantation. Persistent hyper Ei - globulinemia and increased serum ...
• 部位特異的スピンラベル法を用いたオキシダーゼ活性化と蛋白構造変化に関する研究-p47phoxのリン酸化による構造変化とその細胞膜への移動との関連性-

  文部科学省：科学研究費補助金(特定領域研究(A))

  Date (from‐to) : 2000 -2000 

  Author : 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...
• Clonic granulomaous disease (CGD) and its deficients in bactricidal activity-Basic research for gene therapy for CGD-

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(C))

  Date (from‐to) : 1997 -1998 

  Author : Osamu INANAMI, 永幡 肇, 桑原 幹典

   

  This project was performed to clarify the signal transduction mechanisms for NADPH oxidase activation and phagocytotis by using normal neutrophil, human clonic granulomaous disease (CGD) and bovine leukocyte adhesion deficiency (BLAD) which were genetic deficient in p47phox and beta2-integrin CR3 corresponding to the receptor of complement iC3b, respectively The various reagents to inhibit NADPH oxidase-related signal transduction were used for this purpose. We found that inhibitors for protein kinase C (PKC), phosphatidyl inositol 3 kinase (P1 3-kinase) and p38 mitogen-activated protein ki...
• The defect of biodefense mechanisms in bovine leukocyte adhesion deficiency syndrome (BLAD) as observed from signal transduction pathway

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 1997 -1998 

  Author : Mikinori KUWABARA, 田島 誉士, 木村 和弘, 稲波 修, 永幡 肇, 首籐 文栄

   

  This project was performed to clarify the signal transduction mechanisms for NADPH oxidase activation and phagocytotis by using normal neutrophils and those with bovine leukocyte adhesion deficiency (BLAD) which was genetic deficient in beta2-integrin CR3 corresponding to the receptor of complement iC3b. Various reagents to inhibit NADPH oxidase-related signal transduction were used for this purpose. We found that inhibitors of protein kinase C (PKC), phosphatidyl inositol 3-kinase (PI 3-kinase) and p38 mitogen-activated protein kinase (p38 MAPK) were dose-dependently inhibited superoxide g...
• Steric visualization of diseased organs of laboratory small animals by three-dimensionally reconstructing MRI method

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 1997 -1998 

  Author : Mikinori KUWABARA, 入口 紀男, 下川 繁三, 昆 泰寛, 有川 二郎, 稲浪 修, 山本 強

   

  Using multislice MT images of thoracic and abdominal organs in laboratory small animals, three-dimensional (3-d) images were constructed by superimposing them on the computer with the volume ray tracing software. Positive contrast agent was orally administrated into stomach and duodenum cavities just before MR.imaging. Two-dimensional transversal multislices of MR images from thoracic to abdominal regions were taken by an SIS300/183 MRI system with a magnetic field of 7.05 T under the proton-weighting condition (TR=2000ms, TE=20ms). The slice thickness was I mm and the multislice spin-echo ...
• Pathobiology of leukocyte functions from cattle with leukocyte adhesion deficiency(BLAD)and trials of bone marrow transplantation to BLAD-cattle.

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(C))

  Date (from‐to) : 1997 -1998 

  Author : Hajime NAGAHATA, 桑原 幹典, 稲波 修, 寺岡 宏樹

   

  The above-entitled study was performed and the results were summarized as follows :Fc receptor for immunoglobulin G-mediated phagocytosis, superoxide production and intracellular calcium ([Ca^2+]^i) signaling of complement receptor type 3-(CR3) deficient neutrophils from a heifer with leukocyte adhesion deficiency were compared to those of control heifers. The mean phagocytic activity of IgG-coated yeasts and aggregated bovine IgG (Agg-IgG)-induced superoxide production of CR3-deficient neutrophils were 10% and 77.9%, respectively, of those of control neutrophils. The [Ca^2+]^i signals in C...
• Fundamental studies on the molecular therapy for botulisms

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 1996 -1997 

  Author : Bunei SYUTO, 稲波 修

   

  Botulisms is a very serious disease with high mortality. In many cases antitoxin therapy is applied, but it is not always effective after intoxication, because toxin molecules invade into inside of nerve terminals, where antitoxin molecules are inaccessible. To neutralize the toxin invaded into inside of the cell, it is necessary to develope the novel antitoxin molecules penetrable into inside of the cell like toxin molecules.Botulinum type C toxin (strain Stockholm) molecule consists of a heavy chain and a light chain with a disulfide linkage between them. The C-terminal region of the heav...
• 好中球NADPHオキシダーゼの解析と活性酸素生成機構-慢性肉芽腫症の遺伝子治療の基礎的解析-

  文部科学省：科学研究費補助金(基盤研究(C))

  Date (from‐to) : 1996 -1996 

  Author : 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...
• Spin-trapping detection of nitric oxide (NO) production from macrophage during matuation

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(一般研究(C), 基盤研究(C))

  Date (from‐to) : 1995 -1996 

  Author : Mikinori KUWABARA, 稲波 修

   

  In order to investigate nitric oxide (NO) production from macrophage during maturation, peneatrial macrophage was isolated from thioglycolate-stimulated mouse and the production of NO after stimulation by interferon-gamma (INF-gamma), lipopolysaccharide (LPS) and lactoferrin (LF) was measured by the method combining electron-spin-resonance spectroscopy (ESR) and spin-trapping. The complex (MGD-Fe) of Fe^<2+> and N- (Dithiocarbonyl) -N-methylglucamine was used as spin-trapping reagent. Triplet ESR signal due to MGD-Fe-NO,which was produced by the reaction of NO and MGD-Fe, was time-dependent...
• 嗅球におけるNO合成酵素およびMAPキナーゼ発現の分子制御機構-免疫組織化学、in situ PCR hybridizationならびにノーザン・ブロッティング法による解析

  文部科学省：科学研究費補助金(一般研究(C))

  Date (from‐to) : 1995 -1995 

  Author : 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...
• ボツリヌス中毒症の分子治療法に関する基礎的研究

  文部科学省：科学研究費補助金(一般研究(C))

  Date (from‐to) : 1995 -1995 

  Author : 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...
• 成牛ならび新生子牛の多核白血球の活性酸素産生機構解明に関する研究

  文部科学省：科学研究費補助金(奨励研究(A))

  Date (from‐to) : 1994 -1994 

  Author : 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...
• 嗅覚系におけるプログラム細胞死の発現動態とその分子制御機構-遺伝子組織化学,免疫組織化学ならびにレクチン組織化学的による解析

  文部科学省：科学研究費補助金(一般研究(C))

  Date (from‐to) : 1994 -1994 

  Author : 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...
• Regulation of cerebral blood flows by cholinergic fibers originating in the nucleus basalis of Meynert.

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(一般研究(B))

  Date (from‐to) : 1988 -1989 

  Author : 佐藤 昭夫, 佐藤, 昭夫, Akio SATO, 稲波 修, 佐藤 優子

   

  Intracerebral cholinergic fibers originating in the nucleus basalis of Meynert (NBM) and septum, and projecting to the cortex and hippocampus, respectively, have been reported to degenerate in Alzheimer's disease as well as in aged people. However, physiological function of these cholinergic fibers have remained obscure. The present experiment was performed to examine whether these fibers can act as vasodilators in the cortex and hippocampus using anesthetized rats. Regional cerebral blood flow was measured by either laser Doppler flowmetry or ^<14>C iodoantipirine technique. The unilateral...

Industrial Property Rights

• 特許第7440725号:化合物の分解方法及び化合物  

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  稲波 修, 白土 博樹, 松田 彰, 山盛 徹, 安井 博宣, 前田 憲一郎  国立大学法人北海道大学  201703015092486570
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  中野 昌俊, 桑原 幹典, 稲波 修, 原 征彦  三井農林株式会社, 中野 昌俊  200903086189344527