Updated on 2026/03/07

写真a

 
HIRAOKA WAKAKO
 
Organization
Undergraduate School School of Science and Technology Professor
Title
Professor
External link

Degree

  • Doctor of Veterinary Science ( Hokkaido University )

Research Areas

  • Life sciences / Biophysics

Professional Memberships

Papers

  • Redox Potential of Single Octarepeat from Prion Protein with Divalent Metals

    Wakako Hiraoka, Osamu Inanami, Shuhei Murakami

    Free Radical Biology and Medicine   159   S23   2020.11

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

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

    Nucleosides, nucleotides & nucleic acids   39 ( 1-3 )   439 - 452   2020

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    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.

    DOI： 10.1080/15257770.2019.1670839

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

    Kumiko Yamamoto, Yoshinori Ikenaka, Takahiro Ichise, Tomoki Bo, Mayumi Ishizuka, Hironobu Yasui, Wakako Hiraoka, Tohru Yamamori, Osamu Inanami

    Free radical research   52 ( 6 )   648 - 660   2018.6

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    Language：English   Publishing type：Research paper (scientific journal)  

    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.

    DOI： 10.1080/10715762.2018.1460472

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  • Applications of the spin-trapping method in radiation biology Reviewed

    Mikinori Kuwabara, Wakako Hiraoka, Osamu Inanami

    Applications of EPR in Radiation Research   9783319092164   353 - 384   2014.6

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    Language：English   Publishing type：Part of collection (book)   Publisher：Springer International Publishing  

    DOI： 10.1007/978-3-319-09216-4_9

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  • Hyper-O-GlcNAcylation inhibits the induction of heat shock protein 70 (Hsp 70) by sodium arsenite in HeLa cells. Reviewed

    Yuri Miura, Takatoshi Sato, Yoko Sakurai, Ryo Sakai, Wakako Hiraoka, Tamao Endo

    Biological & pharmaceutical bulletin   37 ( 8 )   1308 - 14   2014

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    O-Linked β-N-acetylglucosamine-modification (O-GlcNAcylation) is a reversible, post-translational, and regulatory modification of nuclear, mitochondrial, and cytoplasmic proteins that is responsive to cellular stress. However, the role of O-GlcNAcylation in the induction of heat shock proteins (Hsps) by arsenite remains unclear. We used O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino N-phenyl carbamate (PUGNAc), an inhibitor of O-GlcNAcase, and glucosamine (GlcN), an enhancer of the hexosamine biosynthesis pathway, or O-GlcNAc transferase (OGT) short interfering RNA (siRNA) to enhance or suppress cellular O-GlcNAcylation levels, respectively, in HeLa cells. The exposure to arsenite increased O-GlcNAcylation and Hsp 70 levels in HeLa cells. However, the pre-treatment with PUGNAc or GlcN, which enhanced O-GlcNAcylation levels, decreased the arsenite-induced expression of Hsp 70. The pre-treatment with OGT siRNA, which suppressed O-GlcNAcylation levels, did not affect the induction of Hsp 70. We then examined the effects of O-GlcNAcylation on the nuclear translocation and phosphorylation of heat shock factor 1 (HSF1), and found that neither the nuclear translocation nor phosphorylation of HSF1 was regulated by O-GlcNAcylation. Finally, Hsp 70 mRNA expression was induced by arsenite, whereas the addition of PUGNAc slightly suppressed its induction. These results indicate that O-GlcNAcylation is related to arsenite-induced Hsp 70 expression, and demonstrated that hyper-O-GlcNAcylation inhibited the induction of Hsp 70 via transcriptional factors instead of HSF1.

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

    Yasuko Watanabe, Wakako Hiraoka, Manabu Igarashi, Kimihito Ito, Yuhei Shimoyama, Motohiro Horiuchi, Tohru Yamamori, Hironobu Yasui, Mikinori Kuwabara, Fuyuhiko Inagaki, Osamu Inanami

    Biochemical and biophysical research communications   394 ( 3 )   522 - 8   2010.4

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    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 nitroxide spin probe, suggesting that each interspin distance was within 20 A. The interspin distance ranges between Cu(II) and the spin probes of moPrP(Y148R1), moPrP(Y151R1), moPrP(Y156R1), and moPrP(T189R1) were estimated to be 12.1 A, 18.1 A, 10.7 A, and 8.4 A, 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).

    DOI： 10.1016/j.bbrc.2010.03.003

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  • 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   48   S23   2008

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    Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

    DOI： 10.2142/biophys.48.S23_1

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

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

    Biochemical and biophysical research communications   350 ( 3 )   549 - 56   2006.11

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    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 (H1, codon 143-151), four amino acid residues of beta-sheet1 (S1, codon 127-130), and four amino acid residues of beta-sheet2 (S2, codon 160-163) were substituted for by cysteine residues. These recombinant mouse PrP(C) (moPrP(C)) mutants were reacted with a methane thiosulfonate sulfhydryl-specific spin labeling reagent (MTSSL). The 1/deltaH of the central (14N hyperfine) component (M(I) = 0) in the ESR spectrum of spin-labeled moPrP(C) 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 H1 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 S1 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 H1 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 PrP(C) to PrP(Sc) in acidic organelles such as the endosome.

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  • Comparison between sonodynamic effect and photodynamic effect with photosensitizers on free radical formation and cell killing. Reviewed International journal

    Wakako Hiraoka, Hidemi Honda, Loreto B Feril Jr, Nobuki Kudo, Takashi Kondo

    Ultrasonics sonochemistry   13 ( 6 )   535 - 42   2006.9

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    Although enhancement of ultrasound-induced cell killing by photodynamic reagents has been shown, the sonochemical mechanism in detail is still not clear. Here, comparison between sonodynamic effect and photodynamic effect with photosensitizers at a concentration of 10 microM on free radical formation and cell killing was made. When electron paramagnetic-resonance spectroscopy (EPR) was used to detect 2,2,6,6-tetramethyl-4-piperidone-N-oxyl (TAN) after photo-irradiation or sonication with 2,2,6,6-tetramethyl-4-piperidone (TMPD), the order of TAN formation in the photo-irradiated samples was as follows: rhodamine 6G (R6) > sulforhodamine B (SR) > hematoporphyrin (Hp) > rhodamine 123 (R123) > rose bengal (RB)>erythrosine B (Er) = 0; although there was time-dependent TAN formation when the samples were sonicated, no significant difference among these agents were observed. All these agents suppressed ultrasound-induced OH radical formation detected by EPR-spin trapping. Sensitizer-derived free radicals were markedly observed in SR, RB and Er, while trace level of radicals derived from R6 and R123 were observed. Enhancement of ultrasound-induced decrease of survival in human lymphoma U937 cells was observed at 1.5 W/cm(2) (less than inertial cavitation threshold) for R6, R123, SR and Er, and at 2.3 W/cm(2) for R6, R123, Er, RB and SR. On the other hand, photo-induced decrease of survival was observed for R6, Hp and RB at the same concentration (10 microM). These comparative results suggest that (1) (1)O(2) is not involved in the enhancement of ultrasound-induced loss of cell survival, (2) OH radicals and sensitizer-derived free radicals do not take part in the enhancement, and (3) the mechanism is mainly due to certain mechanical stress such as augmentation of physical disruption of cellular membrane by sensitizers in the close vicinity of cells and/or cavitation bubbles.

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  • Confirmation of enhanced expression of heme oxygenase-1 gene induced by ultrasound and its mechanism: analysis by cDNA microarray system, real-time quantitative PCR, and Western blotting. Reviewed

    Go Kagiya, Yoshiaki Tabuchi, Loreto B Feril Jr, Ryohei Ogawa, Qing-Li Zhao, Nobuki Kudo, Wakako Hiraoka, Katsuro Tachibana, Shin-Ichiro Umemura, Takashi Kondo

    Journal of medical ultrasonics (2001)   33 ( 1 )   3 - 10   2006.3

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    PURPOSE: The present study was undertaken to reconfirm heme oxygenase-1 (HO-1) induction by ultrasound, and elucidate the mechanism by which this occurs. METHODS: After exposure of human lymphoma U937 cells to 1 MHz continuous ultrasound (US), gene profiling by using cDNA microarray analysis, cell viability by using the trypan blue dye exclusion test, mRNA expression by using real-time quantitative polymerase chain reaction, and protein expression by using Western blotting were examined. As an indicator of cavitation, hydroxyl radical formation was studied by using electron paramagnetic resonance-spin trapping. RESULTS: The cDNA microarray analysis reconfirmed HO-1 induction in human lymphoma U937 cells after exposure to US, and further identified one upregulated and two downregulated genes. When U937 cells were exposed to US for 1 min, HO-1 induction, as examined by real-time quantitative polymerase chain reaction and Western blotting, was observed at intensities higher than the cavitational threshold. When a potent antioxidant, N-acetyl-L-cysteine, was added to the culture medium before or after sonication, the induction was attenuated, indicating that reactive oxygen species are involved in HO-1 induction. A decrease in mitochondrial membrane potential and generation of superoxide anion radicals were also observed in the cells exposed to US. CONCLUSION: We used a cDNA microarray system to confirm upregulation of the HO-1 gene and to discover new genes that respond to ultrasonic cavitation. Increased intracellular oxidative stress secondary to the sonomechanical effects arising from ultrasonic cavitation is suggested to be the mechanism of enhancement of HO-1 expression.

    DOI： 10.1007/s10396-005-0066-7

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  • Conformational change in full-length mouse prion: a site-directed spin-labeling study. Reviewed International journal

    Osamu Inanami, Shukichi Hashida, Daisuke Iizuka, Motohiro Horiuchi, Wakako Hiraoka, Yuhei Shimoyama, Hideo Nakamura, Fuyuhiko Inagaki, Mikinori Kuwabara

    Biochemical and biophysical research communications   335 ( 3 )   785 - 92   2005.9

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    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, Wüthricha, 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 Cu(2+) binding region, Helix1 and Helix2, respectively, three recombinant moPrP mutations (N96C, D143C, and T189C) 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 4.8ns were evaluated from the X-band ESR spectra at pH 7.4 and 20 degrees C for N96R1, D143R1, and T189R1, respectively. tau reflects the fact that the Cu(2+) 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 4.8ns. 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 Cu(2+) binding region and D143 in Helix1 were conserved.

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  • 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. Reviewed International journal

    Maiko Ogata, Osamu Inanami, Mihoko Nakajima, Takayuki Nakajima, Wakako Hiraoka, Mikinori Kuwabara

    Photochemistry and photobiology   78 ( 3 )   241 - 7   2003.9

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    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-tetrabromorhodamine 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 (1O2) and superoxide anion (O2-). Apoptosis induction by TBR was inhibited by pyrrolidine dithiocarbamate, an O2- scavenger, but not by NaN3, a quencher of 1O2. 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 O2- was responsible for TBR-induced apoptosis, and Ca(2+)-dependent and caspase-3-independent nuclease such as DNase gamma played an important role in apoptotic signaling triggered by Golgi dysfunction.

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

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

    PHOTOCHEMISTRY AND PHOTOBIOLOGY   70 ( 4 )   650 - 655   1999.10

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  • Reactions and Rate Constants Between Hydrated Electrons and the Monomer and Dimer of 2-Methyl-2-Nitrosopropane Determined by the Pulse Radiolysis Method Reviewed

    Mikinori Kuwabara, Wakako Hiraoka, Sadashi Sawamura, Meiseki Katayama

    Journal of the American Chemical Society   113 ( 10 )   3995 - 3997   1991.5

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    DOI： 10.1021/ja00010a054

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  • Free-radical reactions induced by OH-radical attack on cytosine-related compounds: A study by a method combining esr, spin trapping and HPLC Reviewed

    Wakako Hiraoka, Mikinori Kuwabara, Fumiaki Sato, Akira Matsuda, Tohru Ueda

    Nucleic Acids Research   18 ( 5 )   1217 - 1223   1990.3

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    DOI： 10.1093/nar/18.5.1217

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  • Effects of 3’-Deoxyadenosine (Cordycepin) on the Repair of X-Ray-Induced DNA Single- and Double-Strand Breaks in Chinese Hamster V79 Cells Reviewed

    Wakako Hiraoka, Mikinori Kuwabara, Fumiaki Sato

    Journal of Radiation Research   31 ( 2 )   156 - 161   1990

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    DOI： 10.1269/jrr.31.156

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  • Induction of DNA double-strand breaks in Chinese hamster V79 cells by 2-chlorodeoxyadenosine Reviewed

    Kiyoshi Tanabe, Wakako Hiraoka, Mikinori Kuwabara, Fumiaki Sato, Akira Matsuda, Tohru Ueda

    Chemico-Biological Interactions   71 ( 2-3 )   167 - 175   1989

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    Language：English   Publishing type：Research paper (scientific journal)  

    DOI： 10.1016/0009-2797(89)90032-X

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  • Evidence for the Formation of Strand-Break Precursors in Hydroxy-Attacked Thymidine 5'-Monophosphate by the Spin Trapping Method Reviewed

    Mikinori Kuwabara, Wakako Hiraoka, Fumiaki Sato

    Biochemistry   28 ( 25 )   9625 - 9632   1989

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    Language：English   Publishing type：Research paper (scientific journal)  

    DOI： 10.1021/bi00451a013

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  • Modification of the Repair of Potentially Lethal Damage in Plateau-Phase Chinese Hamster Cells by 2-Chlorodeoxyadenosine Reviewed

    Kiyoshi Tanabe, Wakako Hiraoka, Mikinori Kuwabara, Fumiaki Sato, Akira Matsuda, Tohru Ueda

    Journal of Radiation Research   29 ( 3 )   172 - 181   1988

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    Language：English   Publishing type：Research paper (scientific journal)  

    DOI： 10.1269/jrr.29.172

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  • Sensitization of X-Irradiated Chinese Hamster V79 Cells by Derivatives of Pyrimidine Nucleosides Reviewed

    Wakako Hiraoka, Kiyoshi Tanabe, Mikinori Kuwabara, Fumiaki Sato, Akira Matsuda, Tohru Ueda

    Journal of Radiation Research   29 ( 4 )   246 - 254   1988

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    DOI： 10.1269/jrr.29.246

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MISC

  • Radiation-induced response of mitochondrial energy metabolism in tumor cells

    Osamu Inanami, Kumiko Yamamoto, Tomoki Bo, Tohru Yamamori, Hironobu Yasui, Wakako Hiraoka

    FREE RADICAL BIOLOGY AND MEDICINE   120   S137 - S137   2018.5

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    Language：English   Publishing type：Research paper, summary (international conference)  

    DOI： 10.1016/j.freeradbiomed.2018.04.452

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  • The analysis of the ESR spectra derived from mitochondria in cancer cells

    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   56th   2017

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  • 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

    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)   60th   2017

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  • 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

    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   55th   156‐157   2016.11

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  • 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

    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   148th   199   2009

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  • 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Cu<sup>2+</sup>結合構造の同定

    稲波修, 渡邊康子, 平岡和佳子, 下山雄平, 稲垣冬彦, 桑原幹典, 桑原幹典

    生化学   1P-0090   2007

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  • プリオンタンパク質C末端領域の新たなCu<sup>2+</sup>結合構造の同定

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

    日本獣医学会学術集会講演要旨集   144th   2007

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  • Site‐directed spin label(SDSL)法によるp47<sup>phox</sup>リン酸化の解析

    鈴木友子, 稲波修, 稲波修, 桑原幹典, 下山雄平, 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   44th   262 - 263   2005.10

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  • 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―

    稲波修, 稲波修, 橋田修吉, 橋田修吉, 渡邊康子, 渡邊康子, 平岡和佳子, 下山雄平, 下山雄平, 中村秀夫, 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   44th   268 - 269   2005.10

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

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

    Biophysics   45 ( 1 )   S38   2005.10

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    Language：Japanese   Publisher：The Biophysical Society of Japan  

    DOI： 10.2142/biophys.45.S38_3

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  • プリオンタンパク質の銅イオンの新たな結合様式

    稲波修, 稲波修, 堀内基広, 堀内基広, 平岡和佳子, 稲垣冬彦, 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   140th   208   2005.8

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    Language：Japanese  

    J-GLOBAL

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  • 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

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Presentations

  • 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

    山本久美子、酒井友里、房知輝、平岡和佳子、山盛徹、稲波修

    第160回日本獣医学会学術集会  2017.9 

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    Presentation type：Oral presentation (general)  

    Venue：鹿児島  

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Awards

  • 日本ソノケミストリー学会論文賞

    2007  

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Research Projects

  • Targetting of cancer-specific metabolic system for senolytic therapy

    Grant number：20H00443  2020.4 - 2025.3

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

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    Grant amount：\45110000 （ Direct Cost: \34700000 、 Indirect Cost：\10410000 ）

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  • Redox reguration of self-organized peptides

    Grant number：20K12655  2020.4 - 2023.3

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

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

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  • Development of the peptide array which can distinguish metals, and its medical application

    Grant number：23500546  2011 - 2013

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

    HIRAOKA Wakako

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    Grant amount：\5200000 （ Direct Cost: \4000000 、 Indirect Cost：\1200000 ）

    Clarifying the mechanism of binding of peptides to various metals leads to development of self-organization and medical application. Binding of protein to metal is important as a function of active center of enzyme reactions and also may result in the misfolding of neurodegenerative protein. To elucidate the mechanism of metal recognition of peptide toward the development of new peptide array, we investigated the short peptides of human prion protein (PrP) that bind to divalent metal ions and the stability of these bound structure. UV, CD and ESR spectra indicated two kinds of coordination modes for the binding of peptide-Cu. By potentiometric titration, competitive binding of other divalent metal ions, such as Co, Ni, Zn, and Mn, to peptide-Cu revealed that these metal ions were substitute for Cu. Variation of amino-acid sequence of peptide affected the quantity of each metal substituted.

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  • Development of multi-probe sono-irradiation system on microscopy

    Grant number：20500428  2008 - 2010

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

    HIRAOKA Wakako, SAI Hiroshi

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

    Development of multi-probe sono-irradiation system on microscopy is a part of the research that aims at the development of new treatment and the diagnosis technique in sonomedicine. It is a diagnosis and treatment method development device that has both detection and the diagnostic system of biological function by sono-irradiation in providing with the multi probe. It succeeded in developing of a new drug delivery system and developing of a micro-scaling diagnostic system in tissues with this device.

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  • Molecular imaging of prion protein by atomic force microscop yand a novel electro-spin-resonance techniques

    Grant number：19380172  2007 - 2008

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

    I NANAML Osamu, INABA Mutsumi, HORIUTCHI Motohiro, KUWABARA Mikinori, ASANUMA Katetoshi, HIRAOKA Wakako, SHINOYAMA Yuhei

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    Grant amount：\20410000 （ Direct Cost: \15700000 、 Indirect Cost：\4710000 ）

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  • Development of ultrasound spot-irradiation system with real-time optical monitoring for biological applications

    Grant number：18500352  2006 - 2007

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

    HIRAOKA Wakako, CHOI Pak-kon, KONDO Takashi

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    Grant amount：\3870000 （ Direct Cost: \3600000 、 Indirect Cost：\270000 ）

    We have tried to develop an ultrasound spot-irradiation system with real-time optical monitoring for biological applications. Ultrasound has been used for various physical and chemical purposes for long time. In addition, therapeutic application of ultrasound has been a focus of constant attention recently. In this study, we made an ultrasound spot-irradiation system in which irradiated cells or tissues were monitored under microscopic stage. This system also equipped with micromanipulation and injection for microsurgery of cells and tissues. As a apparatus of ultrasound, we applied several types of piezzoceramic focusing of transducer for the range from 1 MHz to 7 MHz. These apparatus was designed for both continuous and burst wave. The power of ultrasound was determined by calorimetry.
    For the development of new application with this system, we surveyed several kinds of photo-activable caged reagents for sonodynamic therapy. As caged reagents, we employed 5-carboxymethoxy-2-nitrobenzyl(CMNB)-caged fluorescein, Dextran,((5-carboxyraethoxy-2-nitrobenzyl) oxy) carbonyl-caged-5-(and-6)-carboxy-Q-rhodamine, and CMNB-caged carboxy fluorescein. To access the cleavage of caged crosslinkage, the increase of fluorescence was determined after sonolysis or photolysis of drugs. After the sonolysis of the reagents at a concentration of 1 μM in PBS, the intensity of fluorescence of solution increased in time dependence . Activation of drugs within the cultured cells was assayed with flow cytometer(Cytomks FC 500, Beckman-Coulter USA). PLB-985 cells(human myelomonoblastic leukemia cells derived from B-cell) were used for this assay. The cells were incubated with 10 PA CMNB-caged fluorescein in PPS for 10 minutes at 37℃. Cytometric analysis of sonicated cells showed a partial activation of CMNB-caged fluorescein within the cells. These results suggest the possibility of the device of new caged compounds using ultrasound, which are also expected as tracers and therapeutic reagents. Our system will open the possibility of new application of sonotherapy.

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  • Fundamental research of the visualization of neuropathic pain in veterinary medical science by using functional MRI method

    Grant number：17580275  2005 - 2006

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

    NIWA Koichi, HIRAOKA Wakako, ASANUMA Taketoshi, KITAMURA Naoki

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    Grant amount：\3500000 （ Direct Cost: \3500000 ）

    In this study, the possibility that functional MRI (fMRI) method can non-invasively visualize a response of pain was examined in rat cerebral cortex. In order to demonstrate that blood oxygen level dependent (BOLD) signal changes actually reflect the pain in the cerebral cortex, we compared BOLD-signals with morphine with those without morphine.
    Experiment was performed using a 7.05 Tesla superconducting MRI system and a one-turned surface coil centered over the primary somatosensory cortex in cerebral cortex of rat under the mechanical ventilation. A set of multi-slice gradient echo images was acquired and analyzed using commercially available software for imaging analysis (MEDx). Isoflurane provided the stable anesthesia level and the favorable results concerning the fMRI in rat cerebral cortex. Following the subcutaneous injection of 50 μl of formalin or capsaicin into the left forepaw, a regional increase in the signal intensity in the MR images was observed in all rats.
    Formalin stimulation to the left forepaw of rat displayed that the signal increase was observed in contralateral somatosensory cortex and both anterior cingulated cortexes following its injection, it was maintained for 4 minutes. Furthermore, the continuous but weak signal increase in signal was observed for 20 minutes after stimulation. Pre-treatment with morphine vanished these responses. Capsaicin stimulation in left forepaw displayed that the signal increase was observed immediately after stimulation, but the signal disappeared within one minute. Pre-treatment with morphine vanished these responses in a similar fashion to the formalin stimulation.
    This BOLD-fMRI technique in anesthetized animal brain is a useful way to study the mechanism of pain and assess new analgesics.

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  • 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

    Grant number：17658126  2005 - 2006

    日本学術振興会  科学研究費助成事業  萌芽研究

    桑原 幹典, 稲波 修, 平岡 和佳子, 浅沼 武敏

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

    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分から再度信号は上昇した。第一体性感覚野領域でのBOLD信号は刺激後直ぐにピークを迎え、BOLD信号に観察された24分間に大きな変化がないことから、ラット脳CG領域は痛みの表現型そのものと考えることができる。第一体性感覚野などの体性感覚野領域は痛みの場所を特定し、ACCやCG領域では痛みに起因する感情的評価(不快感や嫌悪感)を表現していると考えることができる。今回実験を行った群において、CG領域で特異な反応が観察された。神経活動の上昇とBOLD-fMRI信号の上昇は等しいと考えられているが、現在までのところ、必ずしも等しいとは考えられていない。神経活動の増加により、局所的な酸素消費量の増加がおこり、デオキシヘモグロビン量がオキシヘモグロビン量を凌駕する。炎症性疼痛の後期や疼痛などの慢性痛における痛みはCG領域で強く起こっていることを示唆していることが可視化された。

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  • Study of reactions under ultrasonic cavitation using time-resolved vibrational spectroscopy

    Grant number：16560044  2004 - 2006

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

    CHOI Pak-kon, WAKAKO Hiraoka, SHIGEMI Saito

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    Grant amount：\3100000 （ Direct Cost: \3100000 ）

    We investigated the process of bubble collapse in ultrasonic cavitation through sonoluminescence study and vibratiional spectroscopic study. High-intensity ultrasound irradiated in liquids such as water causes cavitation, that is, the formation, growth and implosive collapse of bubbles. The energy stored in bubbles is released when bubble violently collapses in the compression phase of the ultrasound as acoustic noise, shock waves, chemical reactions, and the emission of light. The violent collapse is predicted to generate a hot spot of thousands of Kelvin with in bubbles. Water molecules in bubbles are dissociated to make H and OH radicals. To monitor the process of bubble collapse, we measured vibrational spectrum of OH-stretching mode using time-resolved Raman scattering. We observed a slight difference in the spectrum at 3500 cm-1 under cavitation and no-ultrasound. However, we obtained no clear evidence that the difference is due to the OH radicals produced in cavitation. Therefore, we changed the plan to study the sonoluminescence from water, ethanol solutions and NaCl solutions.
    Spectra of multi-bubble sonoluminescence (MBSL) were measured in argon-saturated water and ethanol solutions in the concentration range 2-100 mM at the frequencies of 116 kHz and 1.0 MHz. The spectral peaks from OH-radical emission near 310 nm were observed at relatively low ultrasonic power at both frequencies. The sub-peaks at 290 and 340 nm were also observed, which are attributed to OH-radical vibronic transition. The MBSL spectra from ethanol solutions indicated that the quenching of OH-radical emission was more efficient than that of underlying continuous spectrum. The continuous spectrum suggested the decrease in temperature with increasing ethanol concentration. The concentration dependence of spectrum quenching showed no frequency dependence. A new peak was observed at 385 nm only at small ethanol concentrations and at the frequency of 1.0 MHz, which attributed to CH or CN molecule emission.
    We have measured the spectra of multi-bubble sonoluminescence from Ar-saturated NaCl solutions at the frequencies of 108 kHz and 1.0 MHz. The intensity of sodium-atom emission observed takes a maximum at the concentration of 2 M, which corresponds to the formation of OH radicals measured using KI colorimetric method. High-resolution spectroscopic study was made on sodium D lines for various ultrasonic powers. The effects of ethanol on the sodium D line-width were also investigated. The line-width showed asymmetric broadening toward red region, and depended on the ultrasonic power. The addition of ethanol in the range of 0.5-2 mM caused sudden quenching and broadening of sodium D lines. The results indicate that collisions between excited sodium atoms and foreign atoms, that is, argon gas and molecules decomposed from ethanol are responsible for the observed line broadening. This clearly demonstrates that the sodium-atom emission occurs within bubbles. The electronic exciting process of sodium atom is discussed in terms of chemical excitation by OH radicals or H radicals.

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  • Development of the sonotherapy for skin homeostasis

    Grant number：16500324  2004 - 2005

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

    HIRAOKA Wakako, CHOI Pak-Kon, KONDO Takashi

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    Grant amount：\3100000 （ Direct Cost: \3100000 ）

    Homeostasis of skin physiology is maintained by the subcutaneous tissue structure and the normal turnover of hypodermically division cell. Change of skin environment disturb these skin physiology, and give a damage to subcutaneous tissue. This research aimed to develop sonotherapy system for maintenance of skin homeostasis.
    1.Ultrasonic Generator : PZT element attached to the bottom of irradiation container was connected by an amplifier and generator (IWATSU, SG-4105 and NF HSA4101). This system produced 1-7 MHz ultrasound.
    2.Influence to Cell growth : In order to estimate the safety of the created equipment, the influence of ultrasound to cell growth was investigated. U937 cell, human lymphoma cell, was irradiated with 1-7 MHz ultrasound. Dependencies of sound power and irradiation time were analyzed.
    3.Inactivation of mushroom tyrosinase. : When the enzyme solution was irradiated with 1 MHz ultrasound at intensity 0.47 W/cm^2 or less, tyrosinase activity was reduced during irradiation and then recovered immediately after irradiation. On the other hand, the enzyme was irradiated at intensity greater than 0.47 W/cm^2, the reduction of tyrosinase activity was continued after irradiation. Experiment of ESR-spin trapping with dimethyl-1-pyrroline N-oxide showed the production of hydroxyl radical in US-irradiated buffer solution at intensity rather than 0.47 W/cm^2. Although 0.25 M mannitol scavenged hydroxyl radical in US-irradiated buffer solution, the effect of mannitol on the US-induced inactivation of tyrosinase was not observed. While neither catalase nor SOD showed the effect on enzyme activity after US-irradiation, in the presence of Cu^<2+> (10^<-6>M〜10^<-4>M), ultrasound did not induce the inactivation of tyrosinase. These results suggest that the mechanism of US-induced inactivation of tyrosinase is based on Cu^<2+> release from reactive center of the enzyme rather than US-induced ROIs.

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  • 家畜におけるエンドトキシンショック時の血中No代謝物の測定法の確立とその臨床方法

    Grant number：07660416  1995

    日本学術振興会  科学研究費助成事業  一般研究(C)

    平岡 和佳子, 首藤 文栄

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

    基礎的実験として、単離マクロファージを用いて、各種サイトカインを用いた刺激により発生するNOの細胞毒性や細胞分化における役割を解析した。実験に用いたClostridium botulium exoenzyme C3(C3)はsG蛋白質(Small molecular weight GTP-binding proteins)のひとつであるRhoをADP-リボシル化することによって、不活化することが知られている。このC3がインターフェロンガンマによるNOの生成に関与するかどうかを観察した結果、Rhoがインターフエロンガンマによって誘発される、iNOSの遺伝子発現に関わっていることがあきらかとなった。
    これらの系におけるNOの検出系としては、NOの代謝物であるところのNO^<2->+NO^<3->の定量測定法として、グリース反応が用いられているが、この方法は、感度が低く、実際に血清中のNOを測定することは困難であると思われる。我々は、グリース反応よりも、検出感度が高いと報告されている2,3-ジアミノナフタレン(DAN)を用いての、蛍光光度法によるNOの検出と、N-メチル-D-グルカミンジチオカルバメート鉄錯体を用いたESR-スピントラッピング法を比較検討した。DANによるNOの検出限界は、ほぼ、グリース反応の50倍、スピントラッピング法では、10倍の検出感度が得られた。
    また、実際のニトロシルチオールの特異抗体の作成については、抗原として、内因性の物質として牛血清アルブミン、さらにはほ乳動物体内には存在しないヘモシアニンを用いそれぞれ、NO飽和水に溶解してニトロ化を行った。現在、これらの物質を用いて兎に免疫し、ニトロ化タンパク質の特異抗体の精製をおこなっており、今後ニトロ化タンパク特異抗体を用いて、感染を受けた家畜の血清中のニトロ化タンパク質の定量をおこなっていきたい。

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  • 透過投影X線顕微鏡による培養細胞観察への生体染色法の導入

    Grant number：04780280  1992

    日本学術振興会  科学研究費助成事業  奨励研究(A)

    平岡 和佳子

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

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  • 活性酸素が関わる情報伝達

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    Grant type：Competitive

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