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誌 上 発 表 （ 原 著 論 文 ） 185 誌 上 発 表 （原著論文） Summaries of Papers Published in Other Journals (Original Papers) Namekata, I.*, Shimada, H.*, Kawanishi, T., Tanaka, H.* were in good agreement with those previously reported in and Shigenobu, K*: Reduction by SEA0400 of myo- voltage clamped cardiomyocytes. cardial ischemia-induced cytoplasmic and mito- Keywords: Na+-Ca2+ exchange, cardiomyocyte, fl uorescence chondrial Ca2+ overload. microscopy （ ） *1 Eur J Pharmacol ., 543, 108-115 2006 東邦大学薬学部 + The cardioprotective effects of SEA0400, a novel Na - *2 大妻大学家政学部 Ca2+ exchanger inhibitor, were examined in isolated guinea pig myocardial tissue and ventricular myocytes. In a coro- Tanaka, H.*1, Shimada, H.*1, Namekata, I.*1, Kawanishi, T., nary-perfused right ventricular tissue preparation, SEA0400 Iida-Tanaka, N.*2, Shigenobu, K.*1: Involvement of the had no cardiosuppressive effect during normoxia and exper- Na+/Ca2+ exchanger in ouabain-induced inotropy imental ischemia, but enhanced the recovery of contractile and arrhythmogenesis in guinea-pig myocardium force during reperfusion. SEA0400 had no effect on tissue as revealed by SEA0400. ATP content during normoxia, but attenuated its decrease J Pharmacol Sci. , 103, 241-246（ 2007） during ischemia. Treatment of ventricular myocytes with Involvement of the Na+/Ca2+ exchanger in ouabain- an ischemia mimetic solution（ high K+, glucose free, pH induced inotropy and arrhythmogenesis was examined with 6.0, gassed with N2） resulted in the depolarization of the a specific inhibitor, SEA0400. In right ventricular papil- mitochondrial membrane potential and an increase in cyto- lary muscle isolated from guinea-pig ventricle, 1 microM plasmic and mitochondrial Ca2+ concentration, which had a SEA0400, which specifi cally inhibits the Na+/Ca2+ exchang- similar time course. SEA0400 significantly delayed these er by 80%, reduced the ouabain（ 1 μM） -induced posi- changes. These results suggest that SEA0400 maintains mi- tive inotropy by 40%, but had no effect on the inotropy tochondrial function and tissue ATP content during ischemia induced by 100μM isobutyl methylxantine. SEA0400 sig- through the inhibition of cytoplasmic and mitochondrial nifi cantly inhibited the contracture induced by low Na+ so- Ca2+ overload. lution. In HEK293 cells expressing the Na+/Ca2+ exchanger, Keywords: Na+-Ca2+ exchange, Calcium, cardiomyocyte 1 microM ouabain induced an increase in intracellular Ca2+, *東邦大学薬学部 which was inhibited by SEA0400. The arrhythmic contrac- tions induced by 3 μM ouabain were signifi cantly reduced Namekata, I.*1, Kawanishi, T., Iida-Tanaka, N.*2, Tanaka, by SEA0400. These results provide pharmacological evi- H.*1 and Shigenobu, K.*1: Quantitative fluorescence dence that the Na+/Ca2+ exchanger is involved in ouabain- measurement of cardiac Na+/Ca2+ exchanger in- induced inotropy and arrhythmogenesis. hibition by kinetic analysis in stably transfected Keywords: Na+/Ca2+ exchange, cardiomyocyte, fl uorescence HEK293 cells. microscopy J Pharmacol Sci ., 101, 356-360（ 2006） *1 東邦大学薬学部 We developed a method to quantitatively evaluate the *2 大妻大学家政学部 potency of Na+/Ca2+ exchanger（ NCX） inhibitors with fl uo- rescence microscopy in NCX1-transfected HEK 293 cells. Izutsu, K., Yomota, C., and Aoyagi, N.: Inhibition of The reverse mode and forward mode NCX activities were mannitol crystallization in frozen solutions by so- measured as the ascending slope of the early phase increase dium phosphates and citrates. in cytoplasmic Ca2+ concentration after change to low Na+ Chem. Pharm. Bull., 55, 565-570（ 2007） extracellular solution and the descending rate（ inverse of Effects of co-solutes on the physical property of mannitol the exponential time constant） on return to normal solu- and sorbitol in frozen solutions and freeze-dried solids were tion, respectively. Both modes of NCX were inhibited by studied as a model of controlling component crystallinity SEA0400（ 2-[4-[（2,5-difl uorophenyl）methoxy]phenoxy]- in pharmaceutical formulations. A frozen mannitol solution 5-ethoxyaniline） and KB-R7943（ 2-[2-[4-（4-nitrobenzy- （500 mM） showed a eutectic crystallization exotherm at loxy）phenyl]ethyl]isothiourea methanesulfonate）, and the -22.8 degrees C, whereas sorbitol remained amorphous in concentration-inhibition relationships for both inhibitors the freeze-concentrated fraction in the thermal scan. Various 186 国 立 衛 研 報 第 125 号（2007） inorganic salts reduced the eutectic mannitol crystalliza- lowing derivatization with 2-nitrophenylhydrazine tion peak. Trisodium and tripotassium phosphates or cit- by high-performance liquid chromatography-UV rates prevented the mannitol crystallization at much lower detection-electrospray ionization mass spectrom- concentrations than other salts. They also raised transition etry. temperatures of the frozen mannitol and sorbitol solutions J.Chromatogr . A, 1142, 231-235（2007） （T（g）’: glass transition temperature of maximally freeze- Currently, biotin is typically determined in Japan using a concentrated amorphous phase）. Crystallization of some microbiological method. Such microbiological assays are sen- salts（ e.g., NaCl） induced crystallization of mannitol at sitive, but they are not always highly specific and are also above certain salt concentration ratios. Thermal and near- rather tedious and time-consuming. In the present study, RP- infrared analyses of cooled-melt amorphous sorbitol solids HPLC and LC-MS methods for the determination of biotin indicated increased intermolecular hydrogen-bonding in the have been developed by coupling the carboxyl group with presence of trisodium phosphate. The sodium phosphates 2-nitrophenylhydrazine hydrochloride. 2-Nitrophenylhydrazine and citrates should prevent crystallization of mannitol in is used for the derivatization of carboxylic acids, and these frozen solutions and freeze-dried solids by the intense derivatives are known to be applicable to LC-MS detection. hydrogen-bonding and reduced molecular mobility in the Good recovery rates of over 80% were obtained for the ad- amorphous phase. dition of 0.20~0.41μ mol of biotin per formulation. The Keywords: amorphous, crystallization, formulation, freeze- detection limit in HPLC at 400 nm was 5 ng per injection, drying, thermal analysis with good linearity being obtained over the concentration range 0.01-1.5 μ g per injection. Further, derivatives were Yonezawa, Y. *1, Izutsu, K., Tokunaga, H. *1, Maeda, determined by LC-MS with electrospray ionization, where H. *1, Arakawa, T. *2 and Tokunaga, M. *1: Dimeric the spectra indicated the molecular-related ions [M+H]+. The structure of nucleoside diphosphate kinase from detection limit was 0.2 ng per injection in SIMS analysis, moderately halophilic bacterium: contrast to te- and linearity was observed in the range of 5~50 ng per in- trameric Pseudomonas counterpart. jection. The proposed method could be used to specifically FEMS Microbiol Lett., 268, 52-58（ 2007） determine the presence of biotin for relatively clean samples Light scattering and chemical cross-linking analyses with almost pharmacologic amounts of biotin. of nucleoside diphosphate kinase（ NDK） from moderate Keywords: biotin, 2-nitrophenylhydrazine derivative, LC- halophile, Halomonas sp. 593（ HaNDK）, unambiguously MS, electrospray ionization demonstrated that this enzyme formed a dimeric structure, in contrast to the Pseudomonas NDK（ PaNDK）, a non- 四方田千佳子，保立仁美，伊豆津健一，青柳伸男：皮 halophilic counterpart, and other NDKs from Gram-negative 膚適用製剤の溶出試験に関する研究． bacteria, which all formed a tetrameric structure. Com- 医薬品研究, 38, 235-241（2007） parison of HaNDK and PaNDK showed that the HaNDK 皮膚適用製剤は，皮膚への作用を目的とした局所製剤 was less thermally stable than the PaNDK: the optimum の他，経口投与でバイオアベイラビリティーが低い医薬 temperature of PaNDK enzyme activity was 20 degrees C 品等の注射剤に変わる剤形としても広く検討されてい higher than that of HaNDK. However, the HaNDK readily る．現在，欧州薬局方（EP）や米国薬局方（USP）には， refolded and reassembled back to the active dimeric struc- 皮膚適用製剤の品質評価法として溶出試験のベッセル内 ture, upon heat denaturation at 0.2 M NaCl, as soon as the に製剤を固定する装置を設置する方法，パドル部分を改 temperature was lowered. On the contrary, the thermally 変した溶出試験法等が数種収載されている．しかし，日 more stable PaNDK was irreversibly denatured at its melt- 本薬局方（日局）では，皮膚適用製剤の品質評価法を収 ing temperature. 載しておらず，日局への適切な取り込みを目指すことと Keywords: protein structure, stability, halophilic, nucleoside した．それぞれの収載試験法を試みると共に，新たなメ diphosphate kinase ンブランフィルターを熱溶着する手法を開発し，有用な *1 Faculty of Agriculture, Kagoshima University 試験法であることを示した． *2 Alliance Protein Laboratories 簡便な試験法として，貼付剤の他，軟膏剤等への広い 応用が期待される． Yomota,C., Ohnishi,Y.: Determination of biotin fol- Keywords: 貼付剤，放出試験，溶出試験器，メンブラン 誌 上 発 表 （ 原 著 論 文 ） 187 フィルター brary. Our results clearly demonstrate the importance of the improved protocol for the library preparation of antibodies Kawamura, M.*1, Shibata, H., Kamada, H.*2, Oka- and the resulting isolation of antibodies for clinical and re- moto, T.*1, Mukai, Y.*1, Sugita, T.*1, Abe, Y.*1, Imai, S.*1, search applications. Nomura, T.*1, Nagano, K.*1, Mayumi, T.*3, Nakagawa, Keywords: non-immune antibody library, phage display S.*1, Tsutsumi, Y.*2, Tsunoda, S.*2 : A novel method system, single-chain Fv, high-throughput screening, vascular for constructing of gene fragment library to endothelial growth factor receptor 2 searching epitopes. *1 大阪大学大学院薬学研究科 Biochem. Biophys . Res. Commun ., 346 ,198-201（2006） *2 医薬基盤研 Identification of the epitope sequence or the functional domain of proteins is a laborious process but a neces- Mukai, Y.*1, Sugita, T.*1, Yamato, T.*1, Yamanada, N.*1 sary one for biochemical and immunological
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  • Genetically Modified Pest-Protected Plants Science and Regulation

    Genetically Modified Pest-Protected Plants Science and Regulation

    Copyright © National Academy of Sciences. All rights reserved. GENETICALLY MODIFIED PEST-PROTECTED PLANTS SCIENCE AND REGULATION Committee on Genetically Modified Pest-Protected Plants Board on Agriculture and Natural Resources National Research Council NATIONAL ACADEMY PRESS Washington, D.C. Copyright © National Academy of Sciences. All rights reserved. NATIONAL ACADEMY PRESS • 2101 Constitution Avenue, NW • Washington, DC 20418 NOTICE: The project that is the subject of this report was approved by the Governing Board of the National Research Council, whose members are drawn from the councils of the National Academy of Sciences, the National Academy of Engineering, and the Institute of Medicine. The members of the committee responsible for the report were chosen for their special competences and with regard for appropriate balance. Library of Congress Cataloging-in-Publication Data Genetically modified pest-protected plants : science and regulation / Committee on Genetically Modified Pest-Protected Plants, Board on Agriculture and Natural Resources, National Research Council. p. cm. Includes bibliographical references and index. ISBN 0-309-06930-0 (casebound) 1. Transgenic plants—Risk assessment. 2. Plants—Disease and pest resistance—Genetic aspects. I. National Research Council (U.S.). Committee on Genetically Modified Pest-Protected Plants. SB123.57 .G48 2000 631.5’233—dc21 00-009457 Genetically Modified Pest-Protected Plants: Science and Regulation is available from National Academy Press, 2101 Constitution Avenue, NW, Lockbox 285, Washington, DC 20055; (800) 624-6242 or (202) 334-3313 (in the Washington metropolitan area); Internet, http://www.nap.edu Copyright 2000 by the National Academy of Sciences. All rights reserved. Printed in the United States of America Copyright © National Academy of Sciences.