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Sato, Katsutoshi*; Nishikino, Masaharu; Kawachi, Tetsuya; Shimokawa, Takashi*; Imai, Takashi*; Teshima, Teruki*; Nishimura, Hiroaki*; Kando, Masaki
Journal of Radiation Research, 56(4), p.633 - 638, 2015/07
Times Cited Count：1 Percentile：6.94(Biology)While X-ray laser is expected to be widely applied to biomedical studies, this has not been achieved to date and its biological effects such as DNA damage have not been evaluated. As a first step for its biological application, we developed a culture cell irradiation system using laser-plasma soft X-ray laser and investigated whether the soft X-ray laser is able to induce the DNA double strand breaks (DSBs) in living cells or not. The human adenocarcimona cell line A549 was irradiated with the soft X-ray laser at a photon energy of 89 eV and then the repair focus formation of the DSBs was assessed by immunofluorescence staining with anti-phosphorylated DNA-PKcs antibody. As a result, the phosphorylated DNA-PKcs foci were clearly identified even with just a single shot of the soft X-ray laser. In this study, we successfully demonstrated for the first time that soft X-ray laser at 89 eV induced the DNA double strand breaks in living cells.
Tamada, Taro; Kinoshita, Takayoshi*; Kurihara, Kazuo; Adachi, Motoyasu; Ohara, Takashi; Imai, Keisuke*; Kuroki, Ryota; Tada, Toshiji*
Journal of the American Chemical Society, 131(31), p.11033 - 11040, 2009/07
Times Cited Count：58 Percentile：79.01(Chemistry, Multidisciplinary)To help resolve long-standing questions regarding the catalytic activity of the serine proteases the structure of porcine pancreatic elastase has been analyzed by high-resolution neutron and X-ray crystallography. In order to mimic the tetrahedral transition intermediate a peptidic inhibitor was used. A single large crystal was used to collect room-temperature neutron data to 1.65 
resolution and X-ray data to 1.20 resolution. Another crystal provided a low-temperature X-ray data set to 0.94 resolution. The neutron data are to higher resolution than previously reported for a serine protease and the X-ray data are comparable with other studies. The neutron and X-ray data show that the hydrogen bond between His57 and Asp102 (chymotrypsin numbering) is 2.60 in length and that the hydrogen-bonding hydrogen is 0.80-0.96 from the histidine nitrogen. This is not consistent with a low-barrier hydrogen which is predicted to have the hydrogen midway between the donor and acceptor atom. The observed interaction between His57 and Asp102 is essentially a short but conventional hydrogen bond, sometimes described as a short ionic hydrogen bond. The neutron analysis also shows that the oxygen of the oxopropyl group of the inhibitor is present as an oxygen anion rather than a hydroxyl group, supporting the role of the "oxyanion hole" in stabilizing the tetrahedral intermediate in catalysis.
Hamada, Nobuyuki*; Iwakawa, Mayumi*; Imadome, Kaori*; Funayama, Tomoo; Sakashita, Tetsuya; Sora, Sakura*; Ni, M.*; Imai, Takashi*; Kobayashi, Yasuhiko
Journal of Radiation Research, 50(Suppl.A), P. A118, 2009/05
Hamada, Nobuyuki*; Iwakawa, Mayumi*; Imai, Takashi*; Kobayashi, Yasuhiko
Hoshasen Kagaku, 51(8), p.31 - 41, 2008/08
no abstracts in English
Iwakawa, Mayumi*; Hamada, Nobuyuki*; Imadome, Kaori*; Funayama, Tomoo; Sakashita, Tetsuya; Kobayashi, Yasuhiko; Imai, Takashi*
Mutation Research; Fundamental and Molecular Mechanisms of Mutagenesis, 642(1-2), p.57 - 67, 2008/07
Times Cited Count：34 Percentile：66.2(Biotechnology & Applied Microbiology)Kinoshita, Takayoshi*; Tamada, Taro; Imai, Keisuke*; Kurihara, Kazuo; Ohara, Takashi; Kuroki, Ryota
Acta Crystallographica Section F, 63(4), p.315 - 317, 2007/04
Times Cited Count：8 Percentile：66.54(Biochemical Research Methods)Porcine pancreatic elastase (PPE) resembles the attractive drug target leukocyte elastase, which has been implicated in a number of inflammatory disorders. In order to investigate the structural characteristics of the covalent inhibitor bound to PPE, including hydrogen and hydration of water, a single crystal of PPE for neutron diffraction study was grown in D
O containing 0.2 M sodium sulfate (pD=5.0) using the sitting-drop vapor diffusion method. The crystal was grown to a size of 1.6 mm
by repeated macro-seeding. The neutron diffraction data were collected at room temperature using a BIX-3 diffractometer at the JRR-3 research reactor of the Japan Atomic Energy Agency (JAEA). The data set was integrated and scaled to 2.3 resolution with space group P212121 and cell dimensions of a=51.2, b=57.8 and c=75.6.
Hamada, Nobuyuki*; Ni, M.; Iwakawa, Mayumi*; Imadome, Kaori*; Funayama, Tomoo; Sakashita, Tetsuya; Sora, Sakura*; Imai, Takashi*; Kobayashi, Yasuhiko
no journal, ,
Hamada, Nobuyuki*; Ni, M.; Kanasugi, Yuichi*; Iwakawa, Mayumi*; Imadome, Kaori*; Funayama, Tomoo; Sakashita, Tetsuya; Sora, Sakura*; Imai, Takashi*; Takakura, Kaoru*; et al.
no journal, ,
Hamada, Nobuyuki*; Iwakawa, Mayumi*; Imadome, Kaori*; Kanasugi, Yuichi*; Funayama, Tomoo; Sakashita, Tetsuya; Takakura, Kaoru*; Imai, Takashi*; Kobayashi, Yasuhiko
no journal, ,
no abstracts in English
Hamada, Nobuyuki*; Iwakawa, Mayumi*; Kanasugi, Yuichi*; Imadome, Kaori*; Funayama, Tomoo; Sakashita, Tetsuya; Takakura, Kaoru*; Imai, Takashi*; Kobayashi, Yasuhiko
no journal, ,
no abstracts in English
Tamada, Taro; Kinoshita, Takayoshi*; Ohara, Takashi; Kurihara, Kazuo; Imai, Keisuke*; Kuroki, Ryota; Tada, Toshiji*
no journal, ,
Porcine pancreatic elastase (PPE) is a serine protease classified in the chymotrypsin family. We determined two crystal structures of PPE in complex with peptidic inhibitor FR130180, which mimics the tetrahedral transition intermediate. One is the structure determined using 1.65 resolution neutron diffraction in the combination with 1.20 resolution X-ray diffraction data obtained using the same crystal. The other is the sub-angstrom X-ray structure determined to 0.94 resolution. The structural features obtained from neutron diffraction and X-ray diffraction were compared to understand the detailed scheme of interaction between PPE and its inhibitor. From the observation of hydrogen atom located between the active site His57 and Asp102 by neutron and high resolution X-ray diffraction experiment, it is revealed that the interaction is not a low barrier hydrogen bond, but a short ionic hydrogen bond. Moreover, using neutron diffraction data we show that the hydroxyl group of inhibitor FR13080 bound within the "oxy-anion hole" exhibits an oxy-anion-like tetrahedral intermediate.
