131 results on '"Tsuru, D."'
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2. In-vessel components for initial operation of JT-60SA
- Author
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Takechi, M., Tsuru, D., Fukumoto, M., Sasajima, T., Matsunaga, G., Nakamura, S., Yamamoto, S., Itashiki, Y., Hayashi, T., and Isayama, A.
- Published
- 2021
- Full Text
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3. Search for reality of solid breeder blanket for DEMO
- Author
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Tobita, K., Utoh, H., Liu, C., Tanigawa, H., Tsuru, D., Enoeda, M., Yoshida, T., and Asakura, N.
- Published
- 2010
- Full Text
- View/download PDF
4. Thermo-hydraulic testing and integrity of ITER Test Blanket Module (TBM) First Wall mock-up in JAEA
- Author
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Ezato, K., Seki, Y., Tanigawa, H., Hirose, T., Tsuru, D., Nishi, H., Dairaku, M., Yokoyama, K., Suzuki, S., and Enoeda, M.
- Published
- 2010
- Full Text
- View/download PDF
5. Analysis of ELM stability with extended MHD models in JET, JT-60U and future JT-60SA tokamak plasmas
- Author
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Aiba N., Pamela S., Honda M., Urano H., Giroud C., Delabie E., Frassinetti L., Lupelli I., Hayashi N., Huijsmans G. T. A., Litaudon X., Abduallev S., Abhangi M., Abreu P., Afzal M., Aggarwal K. M., Ahlgren T., Ahn J. H., Aho-Mantila L., Airila M., Albanese R., Aldred V., Alegre D., Alessi E., Aleynikov P., Alfier A., Alkseev A., Allinson M., Alper B., Alves E., Ambrosino G., Ambrosino R., Amicucci L., Amosov V., Sunden E. A., Angelone M., Anghel M., Angioni C., Appel L., Appelbee C., Arena P., Ariola M., Arnichand H., Arshad S., Ash A., Ashikawa N., Aslanyan V., Asunta O., Auriemma F., Austin Y., Avotina L., Axton M. D., Ayres C., Bacharis M., Baciero A., Baiao D., Bailey S., Baker A., Balboa I., Balden M., Balshaw N., Bament R., Banks J. W., Baranov Y. F., Barnard M. A., Barnes D., Barnes M., Barnsley R., Wiechec A. B., Orte L. B., Baruzzo M., Basiuk V., Bassan M., Bastow R., Batista A., Batistoni P., Baughan R., Bauvir B., Baylor L., Bazylev B., Beal J., Beaumont P. S., Beckers M., Beckett B., Becoulet A., Bekris N., Beldishevski M., Bell K., Belli F., Bellinger M., Belonohy E., Ayed N. B., Benterman N. A., Bergsaker H., Bernardo J., Bernert M., Berry M., Bertalot L., Besliu C., Beurskens M., Bieg B., Bielecki J., Biewer T., Bigi M., Bilkova P., Binda F., Bisoffi A., Bizarro J. P. S., Bjorkas C., Blackburn J., Blackman K., Blackman T. R., Blanchard P., Blatchford P., Bobkov V., Boboc A., Bodnar G., Bogar O., Bolshakova I., Bolzonella T., Bonanomi N., Bonelli F., Boom J., Booth J., Borba D., Borodin D., Borodkina I., Botrugno A., Bottereau C., Boulting P., Bourdelle C., Bowden M., Bower C., Bowman C., Boyce T., Boyd C., Boyer H. J., Bradshaw J. M. A., Braic V., Bravanec R., Breizman B., Bremond S., Brennan P. D., Breton S., Brett A., Brezinsek S., Bright M. D. J., Brix M., Broeckx W., Brombin M., Broslawski A., Brown D. P. D., Brown M., Bruno E., Bucalossi J., Buch J., Buchanan J., Buckley M. 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K., Shibanuma K., Shimizu K., Shinohara K., Shirai H., Shiraishi J., Someya Y., Sukegawa A., Suzuki S., Takase H., Takechi M., Takenaga H., Tanigawa H., Tobita K., Toma M., Tsuchiya K., Tsuru D., Wakatsuki T., Yamoto S., Yagi M., Yoshida K., Yoshida M., Horiike H., Nobuta Y., Yamauchi Y., Idomura Y., Hatayama A., Hoshino K., Okano K., Masamune S., Sanpei A., Fukuyama A., Kado S., Kobayashi S., Konishi S., Kunugi T., Maekawa T., Minami T., Mizuuchi T., Murakami S., Nagasaki K., Shikama T., Watanabe F., Yamamoto S., Hanada K., Idei H., Katayama K., Nishikawa M., Inagaki S., Fujita T., Kajita S., Maeyama S., Ohno N., Yamazaki K., Watanabe T., Akiyama T., Isobe M., Kanno R., Kobayashi M., Masuzaki S., Miyazawa J., Morisaki T., Nakajima N., Nakamura Y., Nakata M., Narushima Y., Nishimura S., Ohdachi S., Oishi T., Osakabe M., Sagara A., Sakakibara S., Satake S., Suzuki Y., Takeiri Y., Tamura N., Tanaka K., Todo Y., Toi K., Yokoyama M., Watanabe K., Shibata Y., Fukuda T., Takizuka T., Ueda Y., Oya Y., Ejiri A., Inomoto M., Nishiura M., Ogawa Y., Takase Y., Kitajima S., Iio S., Matsuda S., Furukawa M., Ichimura M., Imai T., Nakashima Y., Sakamoto M., Sumida S., Barabaschi P., Cardella A., Clement-Lorenzo S., Coletti A., Davis S., Di Pietro E., Duglue D., Farthing J., Frello G., Hajnal N., Hurzlmeier H., Jokinen A., Kanapienyte D., Novello L., Peretti E., Phillips G., Rancsik P., Salpietro E., Scherber A., Spears B., Teuchner B., Tomarchio V., Verrecchia M., Wanner M., Zani L., Baulaigue O., Benoit F., Ciazynski D., Decool P., Dougnac H., Duchateau J. -L., Dumas N., Fejoz P., Geraud A., Gharafi S., Goncalves R., Gonde R., Gros G., Jestin F., Jiolat G., Lacroix B., Lamy S., Marechal J. -L., Nicollet S., Peluso B., Santagiustina A., Stephnie B., Torre A., Vagliani A., Vallet J. -C., Verger J. -M., Bonne F., Girard S., Hoa C., Lamaison V., Michel F., Poncet J. -M., Roussel P., Abdel Maksoud W., Ardellier F., Disset G., Donati A., Genini L., Mayri C., Molinie F., Nunio F., Ponsot P., Salanon B., Scola L., Vieillard L., Alonso J., Barrera G., Botija J., Cabrera Perez S., Fernandez P., Medrano M., Ramos F. J., Rincon E., Soleto A., Ferro A., Gaio E., Gasparini F., Maistrello A., Brolatti G., Coccoluto G., Corato V., Costa P., Cristofani C., Cucchiaro A., De Vellis A., Di Pace L., Frosi P., Ginoulhiac G., Lampasi A., Maffia G., Pizzuto A., Polli G. M., Rossi P., Starace F., Fiamozzi Zignani C., Zito P., Drotziger S., Fietz W., Heller R., Massimi A., Meyer I., Radloff D., Rita C., Urbach E., Collin B., Delrez C., Jamotton P., Massaut V., Sarkimaki K., Benkadda S., Artaud J. -F., Becoulet M., Falchetto G., Hoang T., Lotte P., Moreau P., Pegourie B., Travere J. -M., Bettini P., Canton A., Fassina A., Giudicotti L., Guo S. C., Marchiori G., Pigatto L., Vallar M., Xu X., Cismondi F., Barbato E., Mastrostefano S., de Baar M., Bruschi A., Farina D., Figini L., Granucci G., Moro A., Nowak S., Perelli-Cippo E., Platania P., Ricci D., Zuin M., Conway G., Dibon M., Fantz U., Happel T., Lauber P., Lackner K., Pautasso G., Schneider P., Kuhner G., Zocco A., Stankiewicz R., Stepniewski W., Sips G., Gleason Gonzalez C., Luo X., Scannapiego M., Bonifetto R., Decker J., Goodman T., Theiler C., Ayllon-Guerola J., Kovacsik A., Szepesi T., Kawashima H., Ogawa T., Sato M., Seki M., Universidad de Sevilla. Departamento de Física Atómica, Molecular y Nuclear, Universidad de Sevilla. RNM138: Física Nuclear Aplicada, Aiba, N, Pamela, S, Honda, M, Urano, H, Giroud, C, Delabie, E, Frassinetti, L, Lupelli, I, Hayashi, N, Huijsmans, G, Litaudon, X, Abduallev, S, Abhangi, M, Abreu, P, Afzal, M, Aggarwal, K, Ahlgren, T, Ahn, J, Aho-Mantila, L, Airila, M, Albanese, R, Aldred, V, Alegre, D, Alessi, E, Aleynikov, P, Alfier, A, Alkseev, A, Allinson, M, Alper, B, Alves, E, Ambrosino, G, Ambrosino, R, Amicucci, L, Amosov, V, Sunden, E, Angelone, M, Anghel, M, Angioni, C, Appel, L, Appelbee, C, Arena, P, Ariola, M, Arnichand, H, Arshad, S, Ash, A, Ashikawa, N, Aslanyan, V, Asunta, O, Auriemma, F, Austin, Y, Avotina, L, Axton, M, Ayres, C, Bacharis, M, Baciero, A, Baiao, D, Bailey, S, Baker, A, Balboa, I, Balden, M, Balshaw, N, Bament, R, Banks, J, Baranov, Y, Barnard, M, Barnes, D, Barnes, M, Barnsley, R, Wiechec, A, Orte, L, Baruzzo, M, Basiuk, V, Bassan, M, Bastow, R, Batista, A, Batistoni, P, Baughan, R, Bauvir, B, Baylor, L, Bazylev, B, Beal, J, Beaumont, P, Beckers, M, Beckett, B, Becoulet, A, Bekris, N, Beldishevski, M, Bell, K, Belli, F, Bellinger, M, Belonohy, E, Ayed, N, Benterman, N, Bergsaker, H, Bernardo, J, Bernert, M, Berry, M, Bertalot, L, Besliu, C, Beurskens, M, Bieg, B, Bielecki, J, Biewer, T, Bigi, M, Bilkova, P, Binda, F, Bisoffi, A, Bizarro, J, Bjorkas, C, Blackburn, J, Blackman, K, Blackman, T, Blanchard, P, Blatchford, P, Bobkov, V, Boboc, A, Bodnar, G, Bogar, O, Bolshakova, I, Bolzonella, T, Bonanomi, N, Bonelli, F, Boom, J, Booth, J, Borba, D, Borodin, D, Borodkina, I, Botrugno, A, Bottereau, C, Boulting, P, Bourdelle, C, Bowden, M, Bower, C, Bowman, C, Boyce, T, Boyd, C, Boyer, H, Bradshaw, J, Braic, V, Bravanec, R, Breizman, B, Bremond, S, Brennan, P, Breton, S, Brett, A, Brezinsek, S, Bright, M, Brix, M, Broeckx, W, Brombin, M, Broslawski, A, Brown, D, Brown, M, Bruno, E, Bucalossi, J, Buch, J, Buchanan, J, Buckley, M, Budny, R, Bufferand, H, Bulman, M, Bulmer, N, Bunting, P, Buratti, P, Burckhart, A, Buscarino, A, Busse, A, Butler, N, Bykov, I, Byrne, J, Cahyna, P, Calabro, G, Calvo, I, Camenen, Y, Camp, P, Campling, D, Cane, J, Cannas, B, Capel, A, Card, P, Cardinali, A, Carman, P, Carr, M, Carralero, D, Carraro, L, Carvalho, B, Carvalho, I, Carvalho, P, Casson, F, Castaldo, C, Catarino, N, Caumont, J, Causa, F, Cavazzana, R, Cave-Ayland, K, Cavinato, M, Cecconello, M, Ceccuzzi, S, Cecil, E, Cenedese, A, Cesario, R, Challis, C, Chandler, M, Chandra, D, Chang, C, Chankin, A, Chapman, I, Chapman, S, Chernyshova, M, Chitarin, G, Ciraolo, G, Ciric, D, Citrin, J, Clairet, F, Clark, E, Clark, M, Clarkson, R, Clatworthy, D, Clements, C, Cleverly, M, Coad, J, Coates, P, Cobalt, A, Coccorese, V, Cocilovo, V, Coda, S, Coelho, R, Coenen, J, Coffey, I, Colas, L, Collins, S, Conka, D, Conroy, S, Conway, N, Coombs, D, Cooper, D, Cooper, S, Corradino, C, Corre, Y, Corrigan, G, Cortes, S, Coster, D, Couchman, A, Cox, M, Craciunescu, T, Cramp, S, Craven, R, Crisanti, F, Croci, G, Croft, D, Crombe, K, Crowe, R, Cruz, N, Cseh, G, Cufar, A, Cullen, A, Curuia, M, Czarnecka, A, Dabirikhah, H, Dalgliesh, P, Dalley, S, Dankowski, J, Darrow, D, Davies, O, Davis, W, Day, C, Day, I, De Bock, M, de Castro, A, de la Cal, E, De La Luna, E, De Masi, G, de Pablos, J, De Temmerman, G, De Tommasi, G, De Vries, P, Deakin, K, Deane, J, Degli Agostini, F, Dejarnac, R, den Harder, N, Dendy, R, Denis, J, Denner, P, Devaux, S, Devynck, P, Di Maio, F, Di Siena, A, Di Troia, C, Dinca, P, D'Inca, R, Ding, B, Dittmar, T, Doerk, H, Doerner, R, Donne, T, Dorling, S, Dormido-Canto, S, Doswon, S, Douai, D, Doyle, P, Drenik, A, Drewelow, P, Drews, P, Duckworth, P, Dumont, R, Dumortier, P, Dunai, D, Dunne, M, Duran, I, Durodie, F, Dutta, P, Duval, B, Dux, R, Dylst, K, Dzysiuk, N, Edappala, P, Edmond, J, Edwards, A, Edwards, J, Eich, T, Ekedahl, A, El-Jorf, R, Elsmore, C, Enachescu, M, Ericsson, G, Eriksson, F, Eriksson, J, Eriksson, L, Esposito, B, Esquembri, S, Esser, H, Esteve, D, Evans, B, Evans, G, Evison, G, Ewart, G, Fagan, D, Faitsch, M, Falie, D, Fanni, A, Fasoli, A, Faustin, J, Fawlk, N, Fazendeiro, L, Fedorczak, N, Felton, R, Fenton, K, Fernades, A, Fernandes, H, Ferreira, J, Fessey, J, Fevrier, O, Ficker, O, Field, A, Fietz, S, Figueiredo, A, Figueiredo, J, Fil, A, Finburg, P, Firdaouss, M, Fischer, U, Fittill, L, Fitzgerald, M, Flammini, D, Flanagan, J, Fleming, C, Flinders, K, Fonnesu, N, Fontdecaba, J, Formisano, A, Forsythe, L, Fortuna, L, Fortuna-Zalesna, E, Fortune, M, Foster, S, Franke, T, Franklin, T, Frasca, M, Freisinger, M, Fresa, R, Frigione, D, Fuchs, V, Fuller, D, Futatani, S, Fyvie, J, Gal, K, Galassi, D, Galazka, K, Galdon-Quiroga, J, Gallagher, J, Gallart, D, Galvao, R, Gao, X, Gao, Y, Garcia, J, Garcia-Carrasco, A, Garcia-Munoz, M, Gardarein, J, Garzotti, L, Gaudio, P, Gauthier, E, Gear, D, Gee, S, Geiger, B, Gelfusa, M, Gerasimov, S, Gervasini, G, Gethins, M, Ghani, Z, Ghate, M, Gherendi, M, Giacalone, J, Giacomelli, L, Gibson, C, Giegerich, T, Gil, C, Gil, L, Gilligan, S, Gin, D, Giovannozzi, E, Girardo, J, Giruzzi, G, Gloggler, S, Godwin, J, Goff, J, Gohil, P, Goloborod'Ko, V, Gomes, R, Goncalves, B, Goniche, M, Goodliffe, M, Goodyear, A, Gorini, G, Gosk, M, Goulding, R, Goussarov, A, Gowland, R, Graham, B, Graham, M, Graves, J, Grazier, N, Grazier, P, Green, N, Greuner, H, Grierson, B, Griph, F, Grisolia, C, Grist, D, Groth, M, Grove, R, Grundy, C, Grzonka, J, Guard, D, Guerard, C, Guillemaut, C, Guirlet, R, Gurl, C, Utoh, H, Hackett, L, Hacquin, S, Hagar, A, Hager, R, Hakola, A, Halitovs, M, Hall, S, Cook, S, Hamlyn-Harris, C, Hammond, K, Harrington, C, Harrison, J, Harting, D, Hasenbeck, F, Hatano, Y, Hatch, D, Haupt, T, Hawes, J, Hawkes, N, Hawkins, J, Hawkins, P, Haydon, P, Hayter, N, Hazel, S, Heesterman, P, Heinola, K, Hellesen, C, Hellsten, T, Helou, W, Hemming, O, Hender, T, Henderson, M, Henderson, S, Henriques, R, Hepple, D, Hermon, G, Hertout, P, Hidalgo, C, Highcock, E, Hill, M, Hillairet, J, Hillesheim, J, Hillis, D, Hizanidis, K, Hjalmarsson, A, Hobirk, J, Hodille, E, Hogben, C, Hogeweij, G, Hollingsworth, A, Hollis, S, Homfray, D, Horacek, J, Hornung, G, Horton, A, Horton, L, Horvath, L, Hotchin, S, Hough, M, Howarth, P, Hubbard, A, Huber, A, Huber, V, Huddleston, T, Hughes, M, Hunter, C, Huynh, P, Hynes, A, Iglesias, D, Imazawa, N, Imbeaux, F, Imrisek, M, Incelli, M, Innocente, P, Irishkin, M, Stanik, I, Jachmich, S, Jacobsen, A, Jacquet, P, Jansons, J, Jardin, A, Jarvinen, A, Jaulmes, F, Jednorog, S, Jenkins, I, Jeong, C, Jepu, I, Joffrin, E, Johnson, R, Johnson, T, Johnston, J, Joita, L, Jones, G, Jones, T, Hoshino, K, Kallenbach, A, Kamiya, K, Kaniewski, J, Kantor, A, Kappatou, A, Karhunen, J, Karkinsky, D, Karnowska, I, Kaufman, M, Kaveney, G, Kazakov, Y, Kazantzidis, V, Keeling, D, Keenan, T, Keep, J, Kempenaars, M, Kennedy, C, Kenny, D, Kent, J, Kent, O, Khilkevich, E, Kim, H, Kinch, A, King, C, King, D, King, R, Kinna, D, Kiptily, V, Kirk, A, Kirov, K, Kirschner, A, Kizane, G, Klepper, C, Klix, A, Knight, P, Knipe, S, Knott, S, Kobuchi, T, Kochl, F, Kocsis, G, Kodeli, I, Kogan, L, Kogut, D, Koivuranta, S, Kominis, Y, Koppen, M, Kos, B, Koskela, T, Koslowski, H, Koubiti, M, Kovari, M, Kowalska-Strzeciwilk, E, Krasilnikov, A, Krasilnikov, V, Krawczyk, N, Kresina, M, Krieger, K, Krivska, A, Kruezi, U, Ksiazek, I, Kukushkin, A, Kundu, A, Kurki-Suonio, T, Kwak, S, Kwiatkowski, R, Kwon, O, Laguardia, L, Lahtinen, A, Laing, A, Lam, N, Lambertz, H, Lane, C, Lang, P, Lanthaler, S, Lapins, J, Lasa, A, Last, J, Laszynska, E, Lawless, R, Lawson, A, Lawson, K, Lazaros, A, Lazzaro, E, Leddy, J, Lee, S, Lefebvre, X, Leggate, H, Lehmann, J, Lehnen, M, Leichtle, D, Leichuer, P, Leipold, F, Lengar, I, Lennholm, M, Lerche, E, Lescinskis, A, Lesnoj, S, Letellier, E, Leyland, M, Leysen, W, Li, L, Liang, Y, Likonen, J, Linke, J, Linsmeier, C, Lipschultz, B, Liu, G, Liu, Y, Lo Schiavo, V, Loarer, T, Loarte, A, Lobel, R, Lomanowski, B, Lomas, P, Lonnroth, J, Lopez, J, Lopez-Razola, J, Lorenzini, R, Losada, U, Lovell, J, Loving, A, Lowry, C, Luce, T, Lucock, R, Lukin, A, Luna, C, Lungaroni, M, Lungu, C, Lungu, M, Lunniss, A, Lyssoivan, A, Macdonald, N, Macheta, P, Maczewa, K, Magesh, B, Maget, P, Maggi, C, Maier, H, Mailloux, J, Makkonen, T, Makwana, R, Malaquias, A, Malizia, A, Manas, P, Manning, A, Manso, M, Mantica, P, Mantsinen, M, Manzanares, A, Maquet, P, Marandet, Y, Marcenko, N, Marchetto, C, Marchuk, O, Marinelli, M, Marinucci, M, Markovic, T, Marocco, D, Marot, L, Marren, C, Marshal, R, Martin, A, Martin, Y, de Aguilera, A, Martinez, F, Martin-Solis, J, Martynova, Y, Maruyama, S, Masiello, A, Maslov, M, Matejcik, S, Mattei, M, Matthews, G, Maviglia, F, Mayer, M, Mayoral, M, May-Smith, T, Mazon, D, Mazzotta, C, Mcadams, R, Mccarthy, P, Mcclements, K, Mccormack, O, Mccullen, P, Mcdonald, D, Mcintosh, S, Mckean, R, Mckehon, J, Meadows, R, Meakins, A, Medina, F, Medland, M, Medley, S, Meigh, S, Meigs, A, Meisl, G, Meitner, S, Meneses, L, Menmuir, S, Mergia, K, Merrigan, I, Mertens, P, Meshchaninov, S, Messiaen, A, Meyer, H, Mianowski, S, Michling, R, Middleton-Gear, D, Miettunen, J, Militello, F, Militello-Asp, E, Miloshevsky, G, Mink, F, Minucci, S, Miyoshi, Y, Mlynar, J, Molina, D, Monakhov, I, Moneti, M, Mooney, R, Moradi, S, Mordijck, S, Moreira, L, Moreno, R, Moro, F, Morris, A, Morris, J, Moser, L, Mosher, S, Moulton, D, Murari, A, Muraro, A, Murphy, S, Asakura, N, Na, Y, Nabais, F, Naish, R, Nakano, T, Nardon, E, Naulin, V, Nave, M, Nedzelski, I, Nemtsev, G, Nespoli, F, Neto, A, Neu, R, Neverov, V, Newman, M, Nicholls, K, Nicolas, T, Nielsen, A, Nielsen, P, Nilsson, E, Nishijima, D, Noble, C, Nocente, M, Nodwell, D, Nordlund, K, Nordman, H, Nouailletas, R, Nunes, I, Oberkofler, M, Odupitan, T, Ogawa, M, O'Gorman, T, Okabayashi, M, Olney, R, Omolayo, O, O'Mullane, M, Ongena, J, Orsitto, F, Orszagh, J, Oswuigwe, B, Otin, R, Owen, A, Paccagnella, R, Pace, N, Pacella, D, Packer, L, Page, A, Pajuste, E, Palazzo, S, Panja, S, Papp, P, Paprok, R, Parail, V, Park, M, Diaz, F, Parsons, M, Pasqualotto, R, Patel, A, Pathak, S, Paton, D, Patten, H, Pau, A, Pawelec, E, Soldan, C, Peackoc, A, Pearson, I, Pehkonen, S, Peluso, E, Penot, C, Pereira, A, Pereira, R, Puglia, P, von Thun, C, Peruzzo, S, Peschanyi, S, Peterka, M, Petersson, P, Petravich, G, Petre, A, Petrella, N, Petrzilka, V, Peysson, Y, Pfefferle, D, Philipps, V, Pillon, M, Pintsuk, G, Piovesan, P, dos Reis, A, Piron, L, Pironti, A, Pisano, F, Pitts, R, Pizzo, F, Plyusnin, V, Pomaro, N, Pompilian, O, Pool, P, Popovichev, S, Porfiri, M, Porosnicu, C, Porton, M, Possnert, G, Potzel, S, Powell, T, Pozzi, J, Prajapati, V, Prakash, R, Prestopino, G, Price, D, Price, M, Price, R, Prior, P, Proudfoot, R, Pucella, G, Puiatti, M, Pulley, D, Purahoo, K, Putterich, T, Rachlew, E, Rack, M, Ragona, R, Rainford, M, Rakha, A, Ramogida, G, Ranjan, S, Rapson, C, Rasmussen, J, Rathod, K, Ratta, G, Ratynskaia, S, Ravera, G, Rayner, C, Rebai, M, Reece, D, Reed, A, Refy, D, Regan, B, Regana, J, Reich, M, Reid, N, Reimold, F, Reinhart, M, Reinke, M, Reiser, D, Rendell, D, Reux, C, Reynolds, S, Riccardo, V, Richardson, N, Riddle, K, Rigamonti, D, Rimini, F, Risner, J, Riva, M, Roach, C, Robins, R, Robinson, S, Robinson, T, Robson, D, Roccella, R, Rodionov, R, Rodrigues, P, Rodriguez, J, Rohde, V, Romanelli, F, Romanelli, M, Romanelli, S, Romazanov, J, Rowe, S, Rubel, M, Rubinacci, G, Rubino, G, Ruchko, L, Ruiz, M, Ruset, C, Rzadkiewicz, J, Saarelma, S, Sabot, R, Safi, E, Sagar, P, Saibene, G, Saint-Laurent, F, Salewski, M, Salmi, A, Salmon, R, Salzedas, F, Samaddar, D, Samm, U, Sandiford, D, Santa, P, Santala, M, Santos, B, Santucci, A, Sartori, F, Sartori, R, Sauter, O, Scannell, R, Schlummer, T, Schmid, K, Schmidt, V, Schmuck, S, Schneider, M, Schopf, K, Schworer, D, Scott, S, Sergienko, G, Sertoli, M, Shabbir, A, Sharapov, S, Shaw, A, Shaw, R, Sheikh, H, Shepherd, A, Shevelev, A, Shumack, A, Sias, G, Sibbald, M, Sieglin, B, Silburn, S, Silva, A, Silva, C, Simmons, P, Simpson, J, Simpson-Hutchinson, J, Sinha, A, Sipila, S, Sips, A, Siren, P, Sirinelli, A, Sjostrand, H, Skiba, M, Skilton, R, Slabkowska, K, Slade, B, Smith, N, Smith, P, Smith, R, Smith, T, Smithies, M, Snoj, L, Soare, S, Solano, E, Somers, A, Sommariva, C, Sonato, P, Sopplesa, A, Sousa, J, Sozzi, C, Spagnolo, S, Spelzini, T, Spineanu, F, Stables, G, Stamatelatos, I, Stamp, M, Staniec, P, Stankunas, G, Stan-Sion, C, Stead, M, Stefanikova, E, Stepanov, I, Stephen, A, Stephen, M, Stevens, A, Stevens, B, Strachan, J, Strand, P, Strauss, H, Strom, P, Stubbs, G, Studholme, W, Subba, F, Summers, H, Svensson, J, Swiderski, L, Szabolics, T, Szawlowski, M, Szepesi, G, Suzuki, T, Tal, B, Tala, T, Talbot, A, Talebzadeh, S, Taliercio, C, Tamain, P, Tame, C, Tang, W, Tardocchi, M, Taroni, L, Taylor, D, Taylor, K, Tegnered, D, Telesca, G, Teplova, N, Terranova, D, Testa, D, Tholerus, E, Thomas, J, Thomas, P, Thompson, A, Thompson, C, Thompson, V, Thorne, L, Thornton, A, Thrysoe, A, Tigwell, P, Tipton, N, Tiseanu, I, Tojo, H, Tokitani, M, Tolias, P, Tomes, M, Tonner, P, Towndrow, M, Trimble, P, Tripsky, M, Tsalas, M, Tsavalas, P, Jun, D, Turner, I, Turner, M, Turnyanskiy, M, Tvalashvili, G, Tyrrell, S, Uccello, A, Ul-Abidin, Z, Uljanovs, J, Ulyatt, D, Uytdenhouwen, I, Vadgama, A, Valcarcel, D, Valentinuzzi, M, Valisa, M, Olivares, P, Valovic, M, Van De Mortel, M, Van Eester, D, Van Renterghem, W, van Rooij, G, Varje, J, Varoutis, S, Vartanian, S, Vasava, K, Vasilopoulou, T, Vega, J, Verdoolaege, G, Verhoeven, R, Verona, C, Rinati, G, Veshchev, E, Vianello, N, Vicente, J, Viezzer, E, Villari, S, Villone, F, Vincenzi, P, Vinyar, I, Viola, B, Vitins, A, Vizvary, Z, Vlad, M, Voitsekhovitch, I, Vondracek, P, Vora, N, Vu, T, de Sa, W, Wakeling, B, Waldon, C, Walkden, N, Walker, M, Walker, R, Walsh, M, Wang, E, Wang, N, Warder, S, Warren, R, Waterhouse, J, Watkins, N, Watts, C, Wauters, T, Weckmann, A, Weiland, J, Weisen, H, Weiszflog, M, Wellstood, C, West, A, Wheatley, M, Whetham, S, 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P., Peluso, E., Penot, C., Pereira, A., Pereira, R., Puglia, P. P. P., von Thun, C. P., Peruzzo, S., Peschanyi, S., Peterka, M., Petersson, P., Petravich, G., Petre, A., Petrella, N., Petrzilka, V., Peysson, Y., Pfefferle, D., Philipps, V., Pillon, M., Pintsuk, G., Piovesan, P., dos Reis, A. P., Piron, L., Pironti, A., Pisano, F., Pitts, R., Pizzo, F., Plyusnin, V., Pomaro, N., Pompilian, O. G., Pool, P. J., Popovichev, S., Porfiri, M. T., Porosnicu, C., Porton, M., Possnert, G., Potzel, S., Powell, T., Pozzi, J., Prajapati, V., Prakash, R., Prestopino, G., Price, D., Price, M., Price, R., Prior, P., Proudfoot, R., Pucella, G., Puglia, P., Puiatti, M. E., Pulley, D., Purahoo, K., Putterich, Th., Rachlew, E., Rack, M., Ragona, R., Rainford, M. S. J., Rakha, A., Ramogida, G., Ranjan, S., Rapson, C. J., Rasmussen, J. J., Rathod, K., Ratta, G., Ratynskaia, S., Ravera, G., Rayner, C., Rebai, M., Reece, D., Reed, A., Refy, D., Regan, B., Regana, J., Reich, M., Reid, N., Reimold, F., Reinhart, M., Reinke, M., Reiser, D., Rendell, D., Reux, C., Cortes, S. D. A. R., Reynolds, S., Riccardo, V., Richardson, N., Riddle, K., Rigamonti, D., Rimini, F. G., Risner, J., Riva, M., Roach, C., Robins, R. J., Robinson, S. A., Robinson, T., Robson, D. W., Roccella, R., Rodionov, R., Rodrigues, P., Rodriguez, J., Rohde, V., Romanelli, F., Romanelli, M., Romanelli, S., Romazanov, J., Rowe, S., Rubel, M., Rubinacci, G., Rubino, G., Ruchko, L., Ruiz, M., Ruset, C., Rzadkiewicz, J., Saarelma, S., Sabot, R., Safi, E., Sagar, P., Saibene, G., Saint-Laurent, F., Salewski, M., Salmi, A., Salmon, R., Salzedas, F., Samaddar, D., Samm, U., Sandiford, D., Santa, P., Santala, M. I. K., Santos, B., Santucci, A., Sartori, F., Sartori, R., Sauter, O., Scannell, R., Schlummer, T., Schmid, K., Schmidt, V., Schmuck, S., Schneider, M., Schopf, K., Schworer, D., Scott, S. D., Sergienko, G., Sertoli, M., Shabbir, A., Sharapov, S. E., Shaw, A., Shaw, R., Sheikh, H., Shepherd, A., Shevelev, A., Shumack, A., Sias, G., Sibbald, M., Sieglin, B., Silburn, S., Silva, A., Silva, C., Simmons, P. A., Simpson, J., Simpson-Hutchinson, J., Sinha, A., Sipila, S. K., Sips, A. C. C., Siren, P., Sirinelli, A., Sjostrand, H., Skiba, M., Skilton, R., Slabkowska, K., Slade, B., Smith, N., Smith, P. G., Smith, R., Smith, T. J., Smithies, M., Snoj, L., Soare, S., Solano, E. R., Somers, A., Sommariva, C., Sonato, P., Sopplesa, A., Sousa, J., Sozzi, C., Spagnolo, S., Spelzini, T., Spineanu, F., Stables, G., Stamatelatos, I., Stamp, M. F., Staniec, P., Stankunas, G., Stan-Sion, C., Stead, M. J., Stefanikova, E., Stepanov, I., Stephen, A. V., Stephen, M., Stevens, A., Stevens, B. D., Strachan, J., Strand, P., Strauss, H. R., Strom, P., Stubbs, G., Studholme, W., Subba, F., Summers, H. P., Svensson, J., Swiderski, L., Szabolics, T., Szawlowski, M., Szepesi, G., Suzuki, T. T., Tal, B., Tala, T., Talbot, A. R., Talebzadeh, S., Taliercio, C., Tamain, P., Tame, C., Tang, W., Tardocchi, M., Taroni, L., Taylor, D., Taylor, K. A., Tegnered, D., Telesca, G., Teplova, N., Terranova, D., Testa, D., Tholerus, E., Thomas, J., Thomas, J. D., Thomas, P., Thompson, A., Thompson, C. -A., Thompson, V. K., Thorne, L., Thornton, A., Thrysoe, A. S., Tigwell, P. A., Tipton, N., Tiseanu, I., Tojo, H., Tokitani, M., Tolias, P., Tomes, M., Tonner, P., Towndrow, M., Trimble, P., Tripsky, M., Tsalas, M., Tsavalas, P., Jun, D. T., Turner, I., Turner, M. M., Turnyanskiy, M., Tvalashvili, G., Tyrrell, S. G. J., Uccello, A., Ul-Abidin, Z., Uljanovs, J., Ulyatt, D., Uytdenhouwen, I., Vadgama, A. P., Valcarcel, D., Valentinuzzi, M., Valisa, M., Olivares, P. V., Valovic, M., Van De Mortel, M., Van Eester, D., Van Renterghem, W., van Rooij, G. J., Varje, J., Varoutis, S., Vartanian, S., Vasava, K., Vasilopoulou, T., Vega, J., Verdoolaege, G., Verhoeven, R., Verona, C., Rinati, G. V., Veshchev, E., Vianello, N., Vicente, J., Viezzer, E., Villari, S., Villone, F., Vincenzi, P., Vinyar, I., Viola, B., Vitins, A., Vizvary, Z., Vlad, M., Voitsekhovitch, I., Vondracek, P., Vora, N., Vu, T., de Sa, W. W. P., Wakeling, B., Waldon, C. W. F., Walkden, N., Walker, M., Walker, R., Walsh, M., Wang, E., Wang, N., Warder, S., Warren, R. J., Waterhouse, J., Watkins, N. W., Watts, C., Wauters, T., Weckmann, A., Weiland, J., Weisen, H., Weiszflog, M., Wellstood, C., West, A. T., Wheatley, M. R., Whetham, S., Whitehead, A. M., Whitehead, B. D., Widdowson, A. M., Wiesen, S., Wilkinson, J., Williams, J., Williams, M., Wilson, A. R., Wilson, D. J., Wilson, H. R., Wilson, J., Wischmeier, M., Withenshaw, G., Withycombe, A., Witts, D. M., Wood, D., Wood, R., Woodley, C., Wray, S., Wright, J., Wright, J. C., Wu, J., Wukitch, S., Wynn, A., Xu, T., Yadikin, D., Yanling, W., Yao, L., Yavorskij, V., Yoo, M. G., Young, C., Young, D., Young, I. D., Young, R., Zacks, J., Zagorski, R., Zaitsev, F. S., Zanino, R., Zarins, A., Zastrow, K. D., Zerbini, M., Zhang, W., Zhou, Y., Zilli, E., Zoita, V., Zoletnik, S., Zychor, I., Arakawa, H., Bando, T., Bierwage, A., Enoeda, M., Fukumoto, M., Hamamatsu, K., Hanada, M., Hatae, T., Hayashi, T., Higashijima, S., Hirota, M., Hiwatari, R., Ichikawa, M., Ide, S., Ikeda, Y., Imazawa, R., Inoue, S., Isayama, A., Ishida, S., Ishii, Y., Itami, K., Kamada, Y., Aiba, K. N., Kawano, Y., Kizu, K., Kawamura, Y., Kobayashi, T., Koide, Y., Kojima, A., Kubo, H., Kurihara, K., Kurita, G., Masaki, K., Matsukawa, M., Matsunaga, G., Matsuyama, A., Miki, K., Miya, N., Miyata, Y., Miyato, N., Mori, M., Moriyama, S., Murakami, H., Naito, O., Nakamura, M., Narita, E., Natsume, K., Oasa, K., Ohtani, Y., Ono, M., Oyama, N., Ozeki, T., Sakamoto, Y., Sakasai, A., Sakurai, S., Sano, R., Sasao, H., Shibama, Y. K., Shibanuma, K., Shimizu, K., Shinohara, K., Shirai, H., Shiraishi, J., Someya, Y., Sukegawa, A., Suzuki, S., Takase, H., Takechi, M., Takenaga, H., Tanigawa, H., Tobita, K., Toma, M., Tsuchiya, K., Tsuru, D., Wakatsuki, T., Yamoto, S., Yagi, M., Yoshida, K., Yoshida, M., Horiike, H., Nobuta, Y., Yamauchi, Y., Idomura, Y., Hatayama, A., Hoshino, K., Okano, K., Masamune, S., Sanpei, A., Fukuyama, A., Kado, S., Kobayashi, S., Konishi, S., Kunugi, T., Maekawa, T., Minami, T., Mizuuchi, T., Murakami, S., Nagasaki, K., Shikama, T., Watanabe, F., Yamamoto, S., Hanada, K., Idei, H., Katayama, K., Nishikawa, M., Inagaki, S., Fujita, T., Kajita, S., Maeyama, S., Ohno, N., Yamazaki, K., Watanabe, T., Akiyama, T., Isobe, M., Kanno, R., Kobayashi, M., Masuzaki, S., Miyazawa, J., Morisaki, T., Nakajima, N., Nakamura, Y., Nakata, M., Narushima, Y., Nishimura, S., Ohdachi, S., Oishi, T., Osakabe, M., Sagara, A., Sakakibara, S., Satake, S., Suzuki, Y., Takeiri, Y., Tamura, N., Tanaka, K., Todo, Y., Toi, K., Yokoyama, M., Watanabe, K., Shibata, Y., Fukuda, T., Takizuka, T., Ueda, Y., Oya, Y., Ejiri, A., Inomoto, M., Nishiura, M., Ogawa, Y., Takase, Y., Kitajima, S., Iio, S., Matsuda, S., Furukawa, M., Ichimura, M., Imai, T., Nakashima, Y., Sakamoto, M., Sumida, S., Barabaschi, P., Cardella, A., Clement-Lorenzo, S., Coletti, A., Davis, S., Di Pietro, E., Duglue, D., Farthing, J., Frello, G., Hajnal, N., Hurzlmeier, H., Jokinen, A., Kanapienyte, D., Novello, L., Peretti, E., Phillips, G., Rancsik, P., Salpietro, E., Scherber, A., Spears, B., Teuchner, B., Tomarchio, V., Verrecchia, M., Wanner, M., Zani, L., Baulaigue, O., Benoit, F., Ciazynski, D., Decool, P., Dougnac, H., Duchateau, J. -L., Dumas, N., Fejoz, P., Geraud, A., Gharafi, S., Goncalves, R., Gonde, R., Gros, G., Jestin, F., Jiolat, G., Lacroix, B., Lamy, S., Marechal, J. -L., Nicollet, S., Peluso, B., Santagiustina, A., Stephnie, B., Torre, A., Vagliani, A., Vallet, J. -C., Verger, J. -M., Bonne, F., Girard, S., Hoa, C., Lamaison, V., Michel, F., Poncet, J. -M., Roussel, P., Abdel Maksoud, W., Ardellier, F., Disset, G., Donati, A., Genini, L., Mayri, C., Molinie, F., Nunio, F., Ponsot, P., Salanon, B., Scola, L., Vieillard, L., Alonso, J., Barrera, G., Botija, J., Cabrera Perez, S., Fernandez, P., Medrano, M., Ramos, F. J., Rincon, E., Soleto, A., Ferro, A., Gaio, E., Gasparini, F., Maistrello, A., Brolatti, G., Coccoluto, G., Corato, V., Costa, P., Cristofani, C., Cucchiaro, A., De Vellis, A., Di Pace, L., Frosi, P., Ginoulhiac, G., Lampasi, A., Maffia, G., Pizzuto, A., Polli, G. M., Rossi, P., Starace, F., Fiamozzi Zignani, C., Zito, P., Drotziger, S., Fietz, W., Heller, R., Massimi, A., Meyer, I., Radloff, D., Rita, C., Urbach, E., Collin, B., Delrez, C., Jamotton, P., Massaut, V., Sarkimaki, K., Benkadda, S., Artaud, J. -F., Becoulet, M., Falchetto, G., Hoang, T., Lotte, P., Moreau, P., Pegourie, B., Travere, J. -M., Bettini, P., Canton, A., Fassina, A., Giudicotti, L., Guo, S. C., Marchiori, G., Pigatto, L., Vallar, M., Xu, X., Cismondi, F., Barbato, E., Mastrostefano, S., de Baar, M., Bruschi, A., Farina, D., Figini, L., Granucci, G., Moro, A., Nowak, S., Perelli-Cippo, E., Platania, P., Ricci, D., Zuin, M., Conway, G., Dibon, M., Fantz, U., Happel, T., Lauber, P., Lackner, K., Pautasso, G., Schneider, P., Kuhner, G., Zocco, A., Stankiewicz, R., Stepniewski, W., Sips, G., Gleason Gonzalez, C., Luo, X., Scannapiego, M., Bonifetto, R., Decker, J., Goodman, T., Theiler, C., Ayllon-Guerola, J., Kovacsik, A., Szepesi, T., Kawashima, H., Ogawa, T., Sato, M., and Seki, M.
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Tokamak ,Rotation ,ELM ,extended MHD model ,H-mode ,rotation ,tokamaks ,01 natural sciences ,Stability (probability) ,010305 fluids & plasmas ,law.invention ,law ,Physics::Plasma Physics ,0103 physical sciences ,Extended MHD model ,010306 general physics ,tokamak ,Physics ,Jet (fluid) ,Plasma ,BOOTSTRAP CURRENT ,SIMULATION ,Condensed Matter Physics ,Computational physics ,Settore ING-IND/20 - Misure e Strumentazione Nucleari ,Nuclear Energy and Engineering ,Physics::Space Physics ,Magnetohydrodynamics ,Tokamaks - Abstract
The stability with respect to a peeling–ballooning mode (PBM) was investigated numerically with extended MHD simulation codes in JET, JT-60U and future JT-60SA plasmas. The MINERVA-DI code was used to analyze the linear stability, including the effects of rotation and ion diamagnetic drift ( *w i), in JET-ILW and JT-60SA plasmas, and the JOREK code was used to simulate nonlinear dynamics with rotation, viscosity and resistivity in JT-60U plasmas. It was validated quantitatively that the ELM trigger condition in JET-ILW plasmas can be reasonably explained by taking into account both the rotation and *w i effects in the numerical analysis. When deuterium poloidal rotation is evaluated based on neoclassical theory, an increase in the effective charge of plasma destabilizes the PBM because of an acceleration of rotation and a decrease in *w i. The difference in the amount of ELM energy loss in JT-60U plasmas rotating in opposite directions was reproduced qualitatively with JOREK. By comparing the ELM affected areas with linear eigenfunctions, it was confirmed that the difference in the linear stability property, due not to the rotation direction but to the plasma density profile, is thought to be responsible for changing the ELM energy loss just after the ELM crash. A predictive study to determine the pedestal profiles in JT-60SA was performed by updating the EPED1 model to include the rotation and *w i effects in the PBM stability analysis. It was shown that the plasma rotation predicted with the neoclassical toroidal viscosity degrades the pedestal performance by about 10% by destabilizing the PBM, but the pressure pedestal height will be high enough to achieve the target parameters required for the ITER-like shape inductive scenario in JT-60SA. JSPS KAKENHI 15K06656 EURATOM 633053
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- 2018
6. Immunoelectron-microscopic demonstration of histamine depletion in the gastric enterochromaffin-like cells of rats treated with α-fluoromethylhistidine
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Fujiwara, K., Karasuyama, M., Murata, I., Tanabe, T., Yabuuchi, M., Inoue, Y., and Tsuru, D.
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- 2001
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7. Consideration on blanket structure for fusion DEMO plant at JAERI
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Nishio, S., Ohmori, J., Kuroda, T., Tobita, K., Enoeda, M., Tsuru, D., Hirose, T., Sato, S., Kawamura, Y., Nakamura, H., and Sato, M.
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- 2006
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8. Design study of fusion DEMO plant at JAERI
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Tobita, K., Nishio, S., Enoeda, M., Sato, M., Isono, T., Sakurai, S., Nakamura, H., Sato, S., Suzuki, S., Ando, M., Ezato, K., Hayashi, T., Hirose, T., Inoue, T., Kawamura, Y., Koizumi, N., Kudo, Y., Kurihara, R., Kuroda, T., Matsukawa, M., Mouri, K., Nakamura, Y., Nishi, M., Nomoto, Y., Ohmori, J., Oyama, N., Sakamoto, K., Suzuki, T., Takechi, M., Tanigawa, H., Tsuchiya, K., and Tsuru, D.
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- 2006
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9. Adoption of a genetic algorithm (GA) for tomographic reconstruction of line-of-sight optical images
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Kihm, K. D., Okamoto, K., Tsuru, D., and Ko, H. S.
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- 1996
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10. Visualization of 3D gas density distribution using optical tomography
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Feng, J., Okamoto, K., Tsuru, D., Madarame, H., and Fumizawa, M.
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- 2002
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11. Safety activities in JAERI related to ITER
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O'hira, S., Tada, E., Hada, K., Neyatani, Y., Maruo, T., Hashimoto, M., Araki, T., Nomoto, K., Tsuru, D., Ishida, K., and Tsunematsu, T.
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- 2001
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12. STUDIES ON BACTERIAL PROTEASES.
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Tsuru, D., Yoshimoto, T., Yoshida, H., Kira, H., and Fukumoto, J.
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- 1970
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13. Crystallization and preliminary X-ray crystallographic studies of 7α-hydroxysteroid dehydrogenase from Escherichia coli.
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Tanaka, N., Nonaka, T., Yoshimoto, T., Tsuru, D., and Mitsui, Y.
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- 1996
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14. Mechanism of proline-specific proteinases: (I) substrate specificity of dipeptidyl peptidase IV from pig kidney and proline-specific endopeptidase from Flavobacterium meningosepticum
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Heins, J., Welker, P., Schönlein, Chr., Born, I., Hartrodt, B., Neubert, K., Tsuru, D., and Barth, A.
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- 1988
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15. Torus configuration and materials selection on a fusion DEMO reactor, SlimCS
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Tobita, K., Nishio, S., Tanigawa, H., Enoeda, M., Isono, T., Nakamura, H., Tsuru, D., Suzuki, S., Hayashi, T., Tsuchiya, K., and Nishitani, T.
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- *
FUSION reactors , *TORUS , *WATER cooled reactors , *BREEDER reactors , *LITHIUM compounds , *MATERIALS science , *STAINLESS steel - Abstract
Abstract: SlimCS is the conceptual design of a compact fusion DEMO plant assuming technologies foreseeable in 2020–2030s. For continuity of blanket technology from the Japanese ITER-TBM, the prime option of blanket is water-cooled solid breeder with Li2TiO3 (or Li4SiO4) and Be. A reduced-activation ferritic–martensitic steel (RAFM) and subcritical water are chosen as the structural material and coolant, respectively. The reactor has somewhat complex torus configuration with a sector-wide conducting plate slipped in between the replaceable (front) and permanent (back) blanket. In order to allow flexibility of maintenance in such a complex configuration, sector transport hot cell maintenance scheme is adopted. This paper describes characteristics of SlimCS with a focus on materials selection. [Copyright &y& Elsevier]
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- 2009
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16. The effects of transcranial static magnetic fields stimulation over the supplementary motor area on anticipatory postural adjustments.
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Tsuru D, Watanabe T, Chen X, Kubo N, Sunagawa T, Mima T, and Kirimoto H
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- Adult, Female, Humans, Male, Movement physiology, Muscle Contraction physiology, Muscle, Skeletal physiology, Psychomotor Performance physiology, Young Adult, Adaptation, Physiological physiology, Anticipation, Psychological physiology, Electromyography methods, Motor Cortex physiology, Postural Balance physiology, Transcranial Magnetic Stimulation methods
- Abstract
We investigated the influence of transcranial static magnetic field stimulation (tSMS) over the supplementary motor area (SMA) on anticipatory postural adjustments (APAs), in which the activation of the postural muscles of the legs and trunk that control standing posture precedes the activation of the prime mover muscles during rapid shoulder flexion movement. Eighteen subjects performed a self-paced rapid shoulder flexion task before, during, and after tSMS. Electromyogram (EMG) activity was recorded from the deltoid anterior (AD) as the prime mover muscle and the biceps femoris (BF) as the postural muscle during the task. The EMG latency difference (ΔEMG onset) between the two muscles was calculated by subtracting the EMG burst onset of the BF from that of the AD. The ΔEMG onset was significantly shortened, but center-of-pressure parameters were not affected after tSMS stimulation. These findings suggest that tSMS applied over the SMA could inhibitively modulate APAs function., (Copyright © 2020 Elsevier B.V. All rights reserved.)
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- 2020
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17. Flavoxobin, a serine protease from Trimeresurus flavoviridis (habu snake) venom, independently cleaves Arg726-Ser727 of human C3 and acts as a novel, heterologous C3 convertase.
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Yamamoto C, Tsuru D, Oda-Ueda N, Ohno M, Hattori S, and Kim ST
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- Amino Acid Sequence, Animals, Chromatography, Gel, Chromatography, Ion Exchange, Complement Activation drug effects, Complement C3 chemistry, Complement C3a metabolism, Complement Membrane Attack Complex metabolism, Crotalid Venoms isolation & purification, Humans, Serine Endopeptidases isolation & purification, Snakes, Thrombin immunology, Trypsin immunology, Complement C3 metabolism, Complement C3-C5 Convertases immunology, Crotalid Venoms immunology, Serine Endopeptidases immunology
- Abstract
We have recently shown that crude Trimeresurus flavoviridis (habu snake) venom has a strong capability for activating the human alternative complement system. To identify the active component, the crude venom was fractionated and purified by serial chromatography using Sephadex G-100, CM-cellulose C-52, diethylaminoethyl-Toyopearl 650M, and Butyl-Toyopearl, and the active fractions were evaluated by the C3a-releasing and soluble membrane attack complex-forming activities. Two peak fractions with the highest activities were detected after gel filtration and ion exchange chromatography, and the first fraction was purified to homogeneity. The homogeneous protein was examined for its N-terminal amino acid sequence by Edman degradation. The determined sequence of 25 amino acids completely coincided with that of a previously reported serine protease with coagulant activity, flavoxobin, purified from the same snake venom. To elucidate the molecular mechanism of the complement activation, the reactive products of the mixture of the purified human C3 and flavoxobin were examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The digesting pattern revealed that flavoxobin cleaves the alpha chain of the C3 molecule into two fragments. The N-terminal amino acid sequences for the remnant fragments of C3 disclosed that flavoxobin severs the human C3 at the Arg726-Ser727 site to form C3b and C3a the way C3bBb, the human alternative C3 convertase, does. In conclusion, flavoxobin acts as a novel, heterologous C3 convertase that independently cleaves human C3 and kick-starts the complement cascade.
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- 2002
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18. Trimeresurus flavoviridis (habu snake) venom induces human erythrocyte lysis through enzymatic lipolysis, complement activation and decreased membrane expression of CD55 and CD59.
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Yamamoto C, Tsuru D, Oda-Ueda N, Ohno M, Hattori S, and Kim ST
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- Animals, Complement Activation physiology, Dose-Response Relationship, Drug, Erythrocyte Membrane drug effects, Erythrocyte Membrane metabolism, Erythrocyte Membrane ultrastructure, Flow Cytometry, Fluorescent Antibody Technique, Hemolysis physiology, Humans, In Vitro Techniques, Lipolysis physiology, CD55 Antigens metabolism, CD59 Antigens metabolism, Complement Activation drug effects, Crotalid Venoms pharmacology, Hemolysis drug effects, Lipolysis drug effects, Trimeresurus
- Abstract
Trimeresurus flavoviridis (habu snake) bites can be fatal to man because of its virulent venom, which is clinicopathologically classified as haemorrhagic, necrotic, and haemolytic toxins. Trimeresurus flavoviridis venom causes lysis of human erythrocytes in conditions where plasma is present as well as in plasma-free conditions in a dose-dependent manner. The haemolytic process requires Ca2+ and Mg2+ ions in the solution. Additionally, the venom initiates activation of the human complement cascade as evidenced by C3a and C5a releases, complement consumption indicated by CH50 and formation of soluble membrane attack complex. The insertion of membrane attack complex into the erythrocyte membranes is morphologically identified by electronmicroscopy. Immunofluorescence analysis reveals that incubation of erythrocytes with the venom decreased cell-surface expression of CD55 (decay accelerating factor) and CD59 (protectin), which renders erythrocyte more vulnerable to adherent C3 and C5 convertases and to polymerization of C9 into membranes, and may enhance autologous complement-mediated haemolysis triggered by the venom. Our data demonstrate that Trimeresurus flavoviridis venom induces haemolysis in the presence of plasma by three distinct mechanisms, direct lipolysis through PLA2 activity, activation of the human complement system, and cleavages of CD55 and CD59 from erythrocyte membranes.
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- 2001
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19. A monoclonal antibody against the glutaraldehyde-conjugated polyamine, putrescine: application to immunocytochemistry.
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Fujiwara K, Tanabe T, Yabuuchi M, Ueoka R, and Tsuru D
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- Animals, Antibodies, Monoclonal biosynthesis, Antibody Formation, Antibody Specificity, Chromatography, High Pressure Liquid methods, Enzyme-Linked Immunosorbent Assay methods, HeLa Cells, Humans, Immunoenzyme Techniques, Mice, Polyamines, Tumor Cells, Cultured, Antibodies, Monoclonal immunology, Glutaral, Putrescine immunology
- Abstract
We developed a mouse monoclonal antibody (mAb; APUT-32, IgG1 subisotype mAb) against putrescine (Put) conjugated to bovine serum albumin using a glutaraldehyde (GA)-sodium borohydride procedure, for applications in immunocytochemistry (ICC). The antibody specificity was evaluated by an ELISA binding test, simulating the ICC of tissue sections. APUT-32 mAb was highly specific to Put, and distinguished alterations in the chemical structure of other polyamine (PA) analogs, showing 3.8% crossreaction with cadaverine, 3.3% with spermidine (Spd), and 2.3% with 1,3-diaminopropane. Comparable results in immunoreactivity of APUT-32 mAb were obtained with the ELISA inhibition test. By the indirect immunoperoxidase method using the APUT-32 mAb, Put-like immunoreactivities were observed in the cytoplasm of HeLa and MCF-7 cell lines fixed with GA in combination with NaBH4 reduction, but almost no immunoreaction was seen in the cytoplasm of the human melanoma BD cell line. On the other hand, the same method but using a previously prepared ASPM-29 mAb, specific for spermine (Spm) and Spd, produced intense immunostaining in the cytoplasm of all the three cell types. The Put-like immunoreaction was completely abolished by absorption of the APUT-32 mAb with 10 microg/ml Put-human serum albumin conjugate prepared using GA and NaBH4. HPLC analysis was also performed for the levels of each of the PAs in the three types of cell, showing that the levels of Put detected were much lower than those of Spm and Spd, and were strikingly different in the three cell lines among which the human melanoma BD cell line contained the lowest levels of Put. These results strongly suggest that APUT-32 mAb reacts specifically with Put in the tumor cells and therefore has the potential as a new tool for elucidating the biological roles of Put in cells and tissues.
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- 2001
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20. Expression and secretion of scytalidopepsin B, an acid protease from Scytalidium lignicolum, in yeast.
- Author
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Shimuta K, Oda-Ueda N, Washio M, Oyama H, Oda K, and Tsuru D
- Subjects
- Amino Acid Sequence, Base Sequence, DNA, Fungal, Electrophoresis, Polyacrylamide Gel methods, Mitosporic Fungi genetics, Molecular Sequence Data, Saccharomyces cerevisiae metabolism, Aspartic Acid Endopeptidases genetics, Enzyme Precursors genetics, Gene Expression, Mitosporic Fungi enzymology
- Abstract
An expression and secretion system for scytalidopepsin B, an acid protease from Scytalidium lignicolum, was constructed in yeast. Saccharomyces cerevisiae AH22 was transformed with an yeast-E. coli shuttle vector, pAM82, in which an yeast invertase signal segment and the cDNA encoding the pro- and mature enzyme regions were inserted. The transformant was found to secret a pepstatin-insensitive acid protease, when cultured aerobically in a low phosphate (Pi) medium. Amino terminal amino acid sequencing analysis indicated that the recombinant acid protease was accurately processed and secreted as a mature form.
- Published
- 2000
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21. Glutaraldehyde (GA)-hapten adducts, but without a carrier protein, for use in a specificity study on an antibody against a GA-conjugated hapten compound: histamine monoclonal antibody (AHA-2) as a model.
- Author
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Fujiwara K, Murata I, Yagisawa S, Tanabe T, Yabuuchi M, Sakakibara R, and Tsuru D
- Subjects
- Animals, Antibody Specificity, Cross Reactions, Enzyme-Linked Immunosorbent Assay, Rats, Antibodies, Monoclonal immunology, Glutaral immunology, Haptens immunology, Histamine immunology
- Abstract
In our recent study on monoclonal antibodies (mAbs AHA-1-5) against glutaraldehyde (GA)-conjugated histamine (HA), we identified one mAb (AHA-2) which can detect neuronal HA in the rat brain with an immunocytochemistry method (ICC) [Fujiwara et al. (1999) J. Biochem. 126, 503-509]. In the present study the specificity of AHA-2 mAb for use for ICC has been examined by means of competitive experiments involving HA and analogs, all of which had been allowed to react with GA followed by sodium borohydride, but not allowed to couple with the carrier protein. It was demonstrated that the antibody distinguished alterations in the chemical structure of the molecule, showing decreased immunoreactivity with all the GA-adducts of (R)-(-)-alpha-methylhistamine, 1- and 3-methylhistamine, L-histidine, and 1- and 3-methyl-L-histidine. On the other hand, AHA-1 mAb only reacted with GA-adducts of 3-MeHA (3-MeHA-GA) and HA (HA-GA), to almost the same degree, in relatively high concentration ranges. AHA-3, 4, and 5 mAbs reacted about 10-times more strongly with 1-MeHA-GA than with HA-GA, but reacted very little or not at all with the other analogs. These results may suggest that AHA-2 mAb recognized both the non-substituted imidazole and alpha-methine groups of a HA molecule in addition to the conjugation site of GA including the part(s) reduced with NaBH(4), and especially the imidazole group more strictly than the other mAbs. This may partly explain why AHA-2, among the five AHA mAbs, can detect neuronal HA with an ICC method. The present ELISA method for GA-hapten adducts should be applicable to other antibodies against GA-conjugated biologically active amines or amino acids, thus allowing the study of antibody specificity for ICC more easily and accurately than was previously possible with hapten-protein conjugates as antigens.
- Published
- 1999
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22. Histamine monoclonal antibody for brain immunocytochemistry.
- Author
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Fujiwara K, Masuyama Y, Yagisawa S, Tanabe T, Yabuuchi M, and Tsuru D
- Subjects
- Animals, Antibody Specificity, Enzyme-Linked Immunosorbent Assay, Evaluation Studies as Topic, Hypothalamus immunology, Immune Sera, Immunohistochemistry, Male, Rats, Rats, Wistar, Antibodies, Monoclonal immunology, Histamine immunology, Hypothalamus metabolism
- Abstract
Among five monoclonal antibodies (AHA-1 to 5 mAbs) prepared against glutaraldehyde (GA)-conjugated histamine (HA) in our previous study, only mAb AHA-2 was found to detect HA specifically in rat brain neurons by an immunocytochemistry method (ICC) using GA as a tissue fixative. All the other mAbs, except for AHA-5, reacted with HA in the enterochromaffin-like cells (ECL cells) of rat stomach [Fujiwara et al. (1997) Histochem. Cell Biol. 107, 39-45]. Enzyme-linked immunosorbent assay (ELISA) binding and inhibition tests demonstrated that AHA-2 is specific for HA, with almost no detectable cross-reaction with any other established or putative amino acid neurotransmitters, LH-RH, TRH, or peptides with N-terminal histidines. ELISA assays also suggested that the AHA-2 mAb recognizes a HA epitope structure different from the one recognized by the AHA-1 mAb. The immunostaining patterns with AHA-2 mAb, as seen in the five subgroups of the tuberomammillary nuclei in the rat posterior hypothalamus, were very similar to those described by Inagaki et al. [(1988) Brain Res. 439, 402-405; (1990) Exp. Brain Res. 80, 374-380] and Panula et al. [(1984) Proc. Natl. Acad. Sci. USA 81, 2572-2576; (1988) J. Histochem. Cytochem. 36, 259-269] using polyclonal anti-HA serum. However, it was also noted that moderate numbers of immunoreactive nerve fibers projected into the median eminence. The present HA ICC method using AHA-2 mAb allows highly sensitive HA detection in brain, and thus might permit detailed studies of HA localization hitherto impossible using previously available anti-HA polyclonal antibodies produced against carbodiimide-conjugated HA.
- Published
- 1999
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23. Immunocytochemical localization of histamine in enterochromaffin-like (ECL) cells in rat oxyntic mucosa: a transmission electron microscopy study using monoclonal antibodies and conventional glutaraldehyde-based fixation.
- Author
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Fujiwara K, Bai G, Tamura C, and Tsuru D
- Subjects
- Animals, Antibodies, Monoclonal, Enterochromaffin Cells drug effects, Fixatives pharmacology, Gastric Mucosa drug effects, Glutaral pharmacology, Histamine immunology, Male, Microscopy, Electron, Parietal Cells, Gastric drug effects, Parietal Cells, Gastric metabolism, Rats, Rats, Wistar, Enterochromaffin Cells metabolism, Gastric Mucosa metabolism, Histamine metabolism
- Abstract
Histamine (HA), contained in the enterochromaffin-like (ECL) cells of the gastric mucosa in animals, plays an important role in gastric acid secretion, although methods for its exact morphological localization are still lacking. We used a pre-embedding indirect immunoperoxidase approach to define the fine structural localization of HA in rat oxyntic mucosa that was fixed with a glutaraldehyde-based fixative and HA monoclonal antibodies (MAbs AHA-1 and 2). Transmission electron microscopy showed that the peroxidase endproduct not only was concentrated in the cores of cytoplasmic granules but also was distributed to a high degree in the cytoplasm peripheral to the granules of the ECL cells. These results suggest that in ECL cells HA is enzymatically synthesized in the cytoplasm, then is transported and stored in the cores of the granules before its release from the basal lamina. The present HA immunoelectron microscopic method with MAbs would be applicable more generally to the ultrastructural identification of HA-containing cells.
- Published
- 1999
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24. The crystal structure of pyroglutamyl peptidase I from Bacillus amyloliquefaciens reveals a new structure for a cysteine protease.
- Author
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Odagaki Y, Hayashi A, Okada K, Hirotsu K, Kabashima T, Ito K, Yoshimoto T, Tsuru D, Sato M, and Clardy J
- Subjects
- Amino Acid Sequence, Binding Sites, Biopolymers, Catalytic Domain, Crystallography, X-Ray, Cysteine Endopeptidases chemistry, Models, Molecular, Molecular Sequence Data, Sequence Alignment, Sequence Homology, Amino Acid, Structure-Activity Relationship, Bacillus enzymology, Bacterial Proteins chemistry, Protein Conformation, Pyroglutamyl-Peptidase I chemistry
- Abstract
Background: The N-terminal pyroglutamyl (pGlu) residue of peptide hormones, such as thyrotropin-releasing hormone (TRH) and luteinizing hormone releasing hormone (LH-RH), confers resistance to proteolysis by conventional aminopeptidases. Specialized pyroglutamyl peptidases (PGPs) are able to cleave an N-terminal pyroglutamyl residue and thus control hormonal signals. Until now, no direct or homology-based three-dimensional structure was available for any PGP., Results: The crystal structure of pyroglutamyl peptidase I (PGP-I) from Bacillus amyloliquefaciens has been determined to 1.6 A resolution. The crystallographic asymmetric unit of PGP-I is a tetramer of four identical monomers related by noncrystallographic 222 symmetry. The protein folds into an alpha/beta globular domain with a hydrophobic core consisting of a twisted beta sheet surrounded by five alpha helices. The structure allows the function of most of the conserved residues in the PGP-I family to be identified. The catalytic triad comprises Cys144, His168 and Glu81., Conclusions: The catalytic site does not have a conventional oxyanion hole, although Cys144, the sidechain of Arg91 and the dipole of an alpha helix could all stabilize a negative charge. The catalytic site has an S1 pocket lined with conserved hydrophobic residues to accommodate the pyroglutamyl residue. Aside from the S1 pocket, there is no clearly defined mainchain substrate-binding region, consistent with the lack of substrate specificity. Although the overall structure of PGP-I resembles some other alpha/beta twisted open-sheet structures, such as purine nucleoside phosphorylase and cutinase, there are important differences in the location and organization of the active-site residues. Thus, PGP-I belongs to a new family of cysteine proteases.
- Published
- 1999
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25. Immunoelectron microscopic study for polyamines.
- Author
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Fujiwara K, Bai G, Kitagawa T, and Tsuru D
- Subjects
- Animals, Endoplasmic Reticulum, Rough chemistry, Male, Medulla Oblongata chemistry, Medulla Oblongata ultrastructure, Neurons chemistry, Neurons ultrastructure, Rats, Rats, Wistar, Ribosomes chemistry, Microscopy, Immunoelectron, Polyamines analysis
- Abstract
The polyamines (PAs) are ubiquitous polycationic metabolites in eukaryotic and prokaryotic cells and are believed to be intimately involved in the regulation of DNA, RNA, and protein biosynthesis, the exact function of which remains unclear, mainly because of a lack of knowledge of PA subcellular localization. In this study, using immunoelectron microscopy, we have demonstrated that PAs are predominantly located on free and attached ribosomes of the rough endoplasmic reticulum in the neurons of the lateral reticular nucleus of rat medulla oblongata. The nuclei, axons, and nerve endings were devoid of PA. This suggests that PAs are one of the components of biologically active ribosomes, being closely involved in the translation processes of protein biosynthesis.
- Published
- 1998
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26. Nucleotide sequence of the gene encoding the precursor protein of pepstatin insensitive acid protease B, scytalidopepsin B, from Scytalidium lignicolum.
- Author
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Oda N, Gotoh Y, Oyama H, Murao S, Oda K, and Tsuru D
- Subjects
- Amino Acid Sequence, Aspartic Acid Endopeptidases chemistry, Base Sequence, DNA Primers chemistry, Enzyme Precursors chemistry, Mitosporic Fungi enzymology, Molecular Sequence Data, Pepstatins chemistry, Polymerase Chain Reaction, Protease Inhibitors chemistry, Protein Precursors chemistry, Sequence Analysis, DNA, Sequence Homology, Amino Acid, Aspartic Acid Endopeptidases genetics, Enzyme Precursors genetics, Mitosporic Fungi genetics, Protein Precursors genetics
- Abstract
A chromosomal DNA of Scytalidium lignicolum was digested with Sau3AI. The digest was self-ligated and amplified by inverse PCR procedure using primers designed based on the nucleotide sequences of up- and down-stream regions of an intron present in the scytalidopepsin B gene. Analysis of the nucleotide sequence of PCR product (700 bp) showed that the enzyme is synthesized as a precursor protein consisting of the prepro- and mature enzyme regions. The deduced amino acid sequence was highly similar to those of aspergillopepsin A and recently reported endothiapepsins B and C, but quite different from those of pepstatin-insensitive bacterial acid proteases and the pepstatin-sensitive aspartic protease family.
- Published
- 1998
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27. Preparation of monoclonal antibodies against N-(gamma-maleimidobutyryloxy)succinimide (GMBS)-conjugated acetylspermine, and development of an enzyme-linked immunosorbent assay (ELISA) for N1,N12-diacetylspermine.
- Author
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Fujiwara K, Kaminishi Y, Kitagawa T, Tsuru D, Yabuuchi M, Kanetake H, and Nomata K
- Subjects
- Animals, Humans, Mice, Mice, Inbred BALB C, Sensitivity and Specificity, Spermine immunology, Spermine urine, Antibodies, Monoclonal immunology, Enzyme-Linked Immunosorbent Assay methods, Spermine analogs & derivatives, Succinimides immunology
- Abstract
We have developed three mouse monoclonal antibodies (mAb) of types IgG1 and IgG2b, i.e. anti-acetylspermine (Ac-Spm)-1 and 2 (ACSPM-1 and 2), and anti-acetylspermine (Ac-Spm)-3 (ACSPM-3), respectively, against Ac-Spm conjugated to bovine serum albumin via a heterobifunctional cross-linker, N-(gamma-maleimidobutyryloxy)succinimide (GMBS). Among these mAbs, ACSPM-2 was the most useful for the development of an enzyme-linked immunosorbent assay (ELISA) for acetylpolyamines (Ac-PAs) with glutaraldehyde (GA)-conjugated N1,N12-diacetylspermine (2Ac-Spm) or acetylspermine (Ac-Spm) as the solid phase antigen. However, GMBS-conjugated Ac-Spm did not behave as a solid phase antigen in the competitive ELISA. The ELISA is based on the principle of competition between an analyte and the conjugated antigen for the mAb, followed by immunoreaction with biotinylated anti-mouse immunoglobulin and horseradish peroxidase-streptavidin. The ACSPM-2 mAb reacted with 2Ac-Spm to the highest degree, followed by Ac-Spm, N1-acetylspermidine (N1-Ac-Spd), N8,N8-diacetylspermidine (2Ac-Spd), and spermine (Spm), the EC50 values being 0.06, 0.25, 7.0, 10, and 60 microM, respectively, but exhibited almost no cross-reaction with other polyamine-related compounds or amino acids. The method was used to determine the urinary Ac-PA levels in healthy subjects, the average value of 0.36 microg of 2Ac-Spm/g creatinine (n = 16) being obtained. The ACSPM-2 ELISA for 2Ac-Spm, which was the PA most relevant to the analysis of human urine among the five PA analogs mentioned above, might have potential for elucidation of the correlation of urinary 2Ac-Spm levels in cancers.
- Published
- 1998
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28. Glutathione-independent formaldehyde dehydrogenase from Pseudomons putida: survey of functional groups with special regard for cysteine residues.
- Author
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Tsuru D, Oda N, Matsuo Y, Ishikawa S, Ito K, and Yoshimoto T
- Subjects
- Aldehyde Oxidoreductases biosynthesis, Aldehyde Oxidoreductases genetics, Amino Acid Sequence, Animals, Cysteine analysis, Horses, Kinetics, Models, Structural, Molecular Sequence Data, Mutagenesis, Site-Directed, Pseudomonas putida genetics, Recombinant Proteins biosynthesis, Aldehyde Oxidoreductases physiology, Cysteine metabolism, Glutathione physiology, Pseudomonas putida enzymology
- Abstract
The role of cysteine residues for structure and function of formaldehyde dehydrogenase from Pseudomonas putida was analysed by amino acid sequence comparison, homology-based structure modeling, site-directed mutagenesis, and chemical modification. Five out of seven cysteine residues found in the enzyme were concluded to coordinate with an active site zinc (Cys-46) and structural zinc atoms (Cys-97, -100, -103, and -111) from the sequence comparison with other Zn-containing medium-chain alcohol dehydrogenase homologues. The three-dimensional structure model based on the known structure of the horse liver E-type alcohol dehydrogenase (ADH) indicated that Cys-257 is located very far from the active site Zn and NAD+ binding region, suggesting that Cys-257 does not participate in the enzyme reaction. The structure also suggested that Cys-166 does not coordinate to active site Zn, but Asp-169 functions as a Zn-ligand, instead.
- Published
- 1997
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29. Nucleotide sequence of the gene encoding pepstatin-insensitive acid protease B, scytalidopepsin B, of Scytalidium lignicolum.
- Author
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Kakimori T, Yoshimoto T, Oyama H, Oda N, Gotoh Y, Oda K, Murao S, and Tsuru D
- Subjects
- Amino Acid Sequence, Aspartic Acid Endopeptidases biosynthesis, Base Sequence, Blotting, Southern, DNA, Fungal analysis, DNA, Fungal isolation & purification, Molecular Sequence Data, Molecular Weight, Polymerase Chain Reaction, Restriction Mapping, Aspartic Acid Endopeptidases genetics, Mitosporic Fungi genetics, Mitosporic Fungi metabolism, Pepstatins pharmacology, Protease Inhibitors pharmacology
- Abstract
A chromosomal DNA fragment of Scytalidium lignicolum that encodes the mature enzyme region of acid protease B (Scytalidopepsin B), was cloned and its nucleotides sequenced. The fragment contained a 76-bp intron at the middle of the mature enzyme-coding region. The mature enzyme was composed of 206 amino acid residues with a molecular weight of 21,550. There were some discrepancies between the amino acid sequence deduced from these results and that previously established by protein sequencing.
- Published
- 1996
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30. Crystal structures of the binary and ternary complexes of 7 alpha-hydroxysteroid dehydrogenase from Escherichia coli.
- Author
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Tanaka N, Nonaka T, Tanabe T, Yoshimoto T, Tsuru D, and Mitsui Y
- Subjects
- Amino Acid Sequence, Animals, Binding Sites, Computer Simulation, Crystallization, Crystallography, X-Ray, Drosophila, Glycochenodeoxycholic Acid analogs & derivatives, Glycochenodeoxycholic Acid metabolism, Hydroxysteroid Dehydrogenases isolation & purification, Hydroxysteroid Dehydrogenases metabolism, Macromolecular Substances, Models, Molecular, Models, Structural, Molecular Sequence Data, NAD metabolism, Rats, Sequence Homology, Amino Acid, Software, Streptomyces enzymology, Substrate Specificity, Thermodynamics, Escherichia coli enzymology, Hydroxysteroid Dehydrogenases chemistry, Protein Conformation, Protein Structure, Secondary
- Abstract
7 alpha-Hydroxysteroid dehydrogenase (7 alpha-HSDH;1 EC 1.1.1.159) is an NAD+-dependent oxidoreductase belonging to the short-chain dehydrogenase/reductase (SDR) 1 family. It catalyzes the dehydrogenation of a hydroxyl group at position 7 of the steroid skeleton of bile acids. The crystal structure of the binary (complexed with NAD+) complex of 7 alpha-HSDH has been solved at 2.3 A resolution by the multiple isomorphous replacement method. The structure of the ternary complex [the enzyme complexed with NADH, 7-oxoglycochenodeoxycholic acid (as a reaction product), and possibly partially glycochenodeoxycholic acid (as a substrate)] has been determined by a difference Fourier method at 1.8 A resolution. The enzyme 7 alpha-HSDH is an alpha/beta doubly wound protein having a Rossmann-fold domain for NAD (H) binding. Upon substrate binding, large conformation changes occur at the substrate binding loop (between the beta F strand and alpha G helix) and the C-terminal segment (residues 250-255). The variable amino acid sequences of the substrate-binding loop appear to be responsible for the wide variety of substrate specificities observed among the enzymes of the SDR family. The crystal structure of the ternary complex of 7 alpha-HSDH, which is the only structure available as the ternary complex among the enzymes of the SDR family, indicates that the highly conserved Tyr159 and Ser146 residues most probably directly interact with the hydroxyl group of the substrates although this observation cannot be definite due to an insufficiently characterized nature of the ternary complex. The strictly conserved Lys163 is hydrogen-bonded to both the 2'- and 3'-hydroxyl groups of the nicotinamide ribose of NAD(H). We propose a new catalytic mechanism possibly common to all the enzymes belonging to the SDR family in which a tyrosine residue (Tyr159) acts as a catalytic base and a serine residue (Ser146) plays a subsidiary role of stabilizing substrate binding.
- Published
- 1996
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31. Prolidase from Xanthomonas maltophilia: purification and characterization of the enzyme.
- Author
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Suga K, Kabashima T, Ito K, Tsuru D, Okamura H, Kataoka J, and Yoshimoto T
- Subjects
- Chromatography, Ion Exchange, Dipeptidases antagonists & inhibitors, Dipeptidases metabolism, Hydrogen-Ion Concentration, Indicators and Reagents, Metals pharmacology, Substrate Specificity, Temperature, Dipeptidases isolation & purification, Xanthomonas enzymology
- Abstract
Prolidase (iminodipeptidase, EC 3.4.13.9) was purified from an extract of Xanthomonas maltophilia, by ammonium sulfate fractionation and sequential chromatographies on DEAE-Toyopearl, Toyopearl HW65C, FPLC-Hiload Superdex 200 pg, and FPLC-Hitrap Q columns, which an activity recovery of 2.3%. The enzyme was the most active at pH 7.5 with Leu-Pro as substrate. It was stable between pH 6.0 and 8.5 for 60 min at 37 degrees C and retained half of activity after 60 min at 37 degrees C. The isoelectric point of the enzyme was 3.7. Its molecular weight was estimated to be 100,000 by gel filtration on FPLC-Hiload Superdex 200 and 51,000 by SDS-PAGE, suggesting that it is a dimer. It hydrolyzed dipeptides only if proline is located at the carboxyl terminal position. The enzyme was inhibited by PCMB and o-phenanthroline, and was activated by Mn2+.
- Published
- 1995
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32. Partial purification and characterization of an esterase acting on the anticancer pro-drugs, 7-ethylcamptothecin derivatives.
- Author
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Fujita Y, Yaegashi T, Sawada S, Oyama H, Yoshimoto T, and Tsuru D
- Subjects
- Animals, Camptothecin metabolism, Chromatography, High Pressure Liquid, Esterases antagonists & inhibitors, Esterases metabolism, Irinotecan, Liver enzymology, Male, Pancreas enzymology, Rats, Rats, Wistar, Antineoplastic Agents, Phytogenic metabolism, Camptothecin analogs & derivatives, Esterases isolation & purification, Prodrugs metabolism
- Abstract
A hydrolytic enzyme which catalyzes hydrolysis of the ester-linkage of a series of 17-O-acyl derivatives of 7-ethylcamptothecin-21-(2-dimethylamino)ethylamide [acyl derivatives of 22E] was purified from rat liver and its properties were characterized. It hydrolyzed the ester-linkage of all 22E derivatives tested as well as p-nitrophenyl acetate at pH 8-9 but had no effect on 7-ethyl-10-[4-(piperidino)-1-piperidino] carbonyloxycamptothecin (CPT-11: irinotecan), unlike CPT-11 converting carboxylesterase, which was previously purified from rat serum [Tsuji T. et al., J. Pharmacobio-Dyn., 14, 341 (1991)]. The enzyme had no effect on either acetyl choline or butyrylcholine. It was inhibited by several organophosphorous compounds such as diisopropyl fluorophosphate (DFP), bis-(p-nitrophenyl)phosphate and paraoxon, but was insensitive to inhibitors specific for choline esterases. These results indicate that this liver esterase is clearly distinct from choline esterase and serum CPT-11 converting enzyme and is able to convert pro-drugs, O-acyl derivatives of 22E, to an antitumor agent.
- Published
- 1995
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33. Prolylcarboxypeptidase (angiotensinase C): purification and characterization of the enzyme from Xanthomanas maltophilia.
- Author
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Suga K, Ito K, Tsuru D, and Yoshimoto T
- Subjects
- Amino Acid Sequence, Ammonium Sulfate chemistry, Carboxypeptidases antagonists & inhibitors, Carboxypeptidases chemistry, Carboxypeptidases metabolism, Chelating Agents pharmacology, Chemical Fractionation, Chloromercuribenzoates pharmacology, Chromatography, High Pressure Liquid, Electrophoresis, Polyacrylamide Gel, Hydrogen-Ion Concentration, Hydrolysis, Iodoacetamide pharmacology, Isoelectric Focusing, Isoflurophate pharmacology, Molecular Sequence Data, Molecular Weight, Phenylmethylsulfonyl Fluoride pharmacology, Proline metabolism, p-Chloromercuribenzoic Acid, Carboxypeptidases isolation & purification, Xanthomonas enzymology
- Abstract
Prolylcarboxypeptidase (Angiotensinase C, EC 3.4.16.2) was purified to homogeneity from cell free extracts of Xanthomonas maltophilia by ammonium sulfate fractionation and sequential chromatographies on DEAE-Toyopearl, Sephadex G-150, FPLC-Hiload Superdex 200 pg, and FPLC-Hitrap SP columns, with an activity recovery of 15%. The molecular weight of the enzyme was found to be 330,000 by gel filtration and 83,000 by SDS-PAGE, suggesting a tetrameric form for the native enzyme. It had an optimum pH of 8.5 and stability between pH 8.0 and 11.0. The isoelectric point of the enzyme was 6.6. The enzyme hydrolyzed Pro-X bonds when proline was in the penultimate position from the carboxyl terminal. The enzyme was strongly inhibited by diisopropylfluorophosphate (DFP), while phenylmethylsulfonyl fluoride (PMSF), p-chloromercuribenzoic acid (PCMB), iodoacetamide, and metal chelators had no effect.
- Published
- 1995
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34. Isolation and characterization of the prolyl aminopeptidase gene (pap) from Aeromonas sobria: comparison with the Bacillus coagulans enzyme.
- Author
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Kitazono A, Kitano A, Tsuru D, and Yoshimoto T
- Subjects
- Aeromonas classification, Amino Acid Sequence, Aminopeptidases drug effects, Bacterial Proteins drug effects, Base Sequence, Cloning, Molecular, Gene Expression drug effects, Molecular Sequence Data, Sequence Homology, Amino Acid, Substrate Specificity, Aeromonas enzymology, Aeromonas genetics, Aminopeptidases genetics, Bacillus enzymology, Bacillus genetics, Bacterial Proteins genetics, Genes, Bacterial
- Abstract
The Aeromonas sobria pap gene encoding prolyl aminopeptidase (PAP) was cloned. It consists of 425 codons and encodes a homotetrameric enzyme of 205 kDa. The purified enzyme showed an almost absolute specificity for amino-terminal proline. Proline and hydroxyproline residues from many peptide and amide substrates could be easily removed, while no activity was detected for substrates having other amino terminals. The enzyme was very similar to that from Bacillus coagulans in many aspects, such as the strong inhibition caused by PCMB and the weak or no inhibition caused by DFP and chelators, respectively. However, these enzymes show only 15% identity in their amino acid sequences. Differences were also observed in their molecular weight, stability and activity toward some peptide substrates. When aligning the deduced amino acid sequence with known sequences from other microorganisms, conserved sequences were found at the amino-terminal region; the significance of these conserved regions is discussed. Based on the results of this work, and on the studies available to date, the occurrence of at least two types of PAPs is postulated. One group would be formed by the Bacillus, Neisseria, and Lactobacillus enzymes, and the other by enzymes such as the Aeromonas PAP.
- Published
- 1994
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35. Cloning and high-level expression of the glutathione-independent formaldehyde dehydrogenase gene from Pseudomonas putida.
- Author
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Ito K, Takahashi M, Yoshimoto T, and Tsuru D
- Subjects
- Alcohol Dehydrogenase genetics, Aldehyde Oxidoreductases metabolism, Amino Acid Sequence, Base Sequence, Cloning, Molecular, DNA Primers, Escherichia coli genetics, Formaldehyde metabolism, Metalloproteins metabolism, Molecular Sequence Data, Pseudomonas putida enzymology, Recombinant Proteins metabolism, Sequence Analysis, Sequence Homology, Amino Acid, Zinc, Aldehyde Oxidoreductases genetics, Genes, Bacterial genetics, Metalloproteins genetics, Pseudomonas putida genetics
- Abstract
A DNA fragment of 485 bp was specifically amplified by PCR with primers based on the N-terminal sequence of the purified formaldehyde dehydrogenase (EC 1.2.1.46) from Pseudomonas putida and on that of a cyanogen bromide-derived peptide. With this product as a probe, a gene coding for formaldehyde dehydrogenase (fdhA) in P. putida chromosomal DNA was cloned in Escherichia coli DH5 alpha. Sequencing analysis revealed that the fdhA gene contained 1,197-bp open reading frame, encoding a protein composed of 399 amino acid residues whose calculated molecular weight was 42,082. The transformant of E. coli DH5 alpha harboring the hybrid plasmid, pFDHK3DN71, showed about 50-fold-higher formaldehyde dehydrogenase activity than P. putida. The predicted amino acid sequence contained several features characteristic of the zinc-containing medium-chain alcohol dehydrogenase (ADH) family. Most of the glycine residues strictly conserved within the family, including a Gly-Xaa-Gly-Xaa-Xaa-Gly pattern in the coenzyme binding domain, were well conserved in this enzyme. Regions around both the catalytic and the structural zinc atoms were also conserved. Analyses of structural and enzymatic characteristics indicated that P. putida FDH belongs to the medium-chain ADH family, with mixed properties of mammalian class I and III ADHs.
- Published
- 1994
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36. Oligopeptidase B: protease II from Escherichia coli.
- Author
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Tsuru D and Yoshimoto T
- Subjects
- Amino Acid Sequence, Base Sequence, Escherichia coli enzymology, Molecular Sequence Data, Sequence Homology, Amino Acid, Serine Endopeptidases chemistry, Serine Endopeptidases metabolism, Serine Proteinase Inhibitors pharmacology, Substrate Specificity, Serine Endopeptidases isolation & purification
- Published
- 1994
- Full Text
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37. Location of the 7 alpha-hydroxysteroid dehydrogenase gene (hdhA) on the physical map of the Escherichia coli chromosome.
- Author
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Yoshimoto T, Nagai H, Ito K, and Tsuru D
- Subjects
- Base Sequence, Chromosomes, Bacterial, Escherichia coli enzymology, Genes, Bacterial, Molecular Sequence Data, Escherichia coli genetics, Hydroxysteroid Dehydrogenases genetics, Restriction Mapping
- Published
- 1993
- Full Text
- View/download PDF
38. Prolyl endopeptidase from Aeromonas hydrophila: cloning, sequencing, and expression of the enzyme gene, and characterization of the expressed enzyme.
- Author
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Kanatani A, Yoshimoto T, Kitazono A, Kokubo T, and Tsuru D
- Subjects
- Aeromonas hydrophila classification, Amino Acid Sequence, Base Sequence, Binding Sites, Cloning, Molecular, DNA, Bacterial genetics, Flavobacterium enzymology, Flavobacterium genetics, Gene Expression, Molecular Sequence Data, Prolyl Oligopeptidases, Restriction Mapping, Sequence Homology, Amino Acid, Serine Endopeptidases chemistry, Species Specificity, Aeromonas hydrophila enzymology, Aeromonas hydrophila genetics, Genes, Bacterial, Serine Endopeptidases genetics
- Abstract
A strain of Aeromonas hydrophila was found to show prolyl endopeptidase activity. The enzyme gene was cloned and expressed in Escherichia coli JM83. A 12 kbp EcoRI fragment containing the enzyme gene was subcloned at the HincII site of pUC19 to construct plasmid pAPEP-3 with a 3.5 kbp insert. E. coli JM83 transformed with this plasmid showed about 100-fold higher activity than the parent Aeromonas. Analysis of the nucleotide sequence of the insert revealed that the mature enzyme-encoding sequence starts just after the ATG initiation codon of the open reading frame. The enzyme was a single polypeptide composed of 689 amino acid residues with a molecular weight of 76,383. It showed properties very similar to those of Flavobacterium prolyl endopeptidase, except that the isoelectric point was 5.5. The amino acid sequence was 56 and 41% homologous to those of Flavobacterium and porcine brain prolyl endopeptidases, respectively. From a survey of sequence homology with other members of the prolyl endopeptidase family, the amino acid residues involved in the catalytic triad were deduced to be Ser-537, His-656, and Asp-512 (or Asp-621).
- Published
- 1993
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39. Pyroglutamyl peptidase gene from Bacillus amyloliquefaciens: cloning, sequencing, expression, and crystallization of the expressed enzyme.
- Author
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Yoshimoto T, Shimoda T, Kitazono A, Kabashima T, Ito K, and Tsuru D
- Subjects
- Amino Acid Sequence, Amino Acids analysis, Bacillus enzymology, Base Sequence, Cloning, Molecular, Cysteine chemistry, DNA, Bacterial, Electrophoresis, Polyacrylamide Gel, Escherichia coli genetics, Molecular Sequence Data, Pyroglutamyl-Peptidase I chemistry, Pyroglutamyl-Peptidase I metabolism, Restriction Mapping, Bacillus genetics, Genes, Bacterial, Pyroglutamyl-Peptidase I genetics
- Abstract
The pyroglutamyl peptidase [EC 3.4.11.8] gene from Bacillus amyloliquefaciens was cloned and expressed in Escherichia coli DH1. The transformant of E. coli DH1 harboring plasmid pBPG 1 with a 2.1 kb chromosomal DNA fragment showed 80-fold higher activity than B. amyloliquefaciens. The nucleotide sequence of a 0.9 kb fragment that contains the promoter and the mature protein coding region was determined by the dideoxy chain-termination method. An open reading frame of 648 bp starting with an ATG methionine codon was found, which encodes a protein of 215 amino acid residues with a deduced molecular weight of 23,286. The enzyme has two cysteine residues (Cys68 and Cys144) per subunit molecule. Substitution of Cys144 with Ser by site-directed mutagenesis resulted in a complete loss of the activity, while that of Cys68 with Ser did not affect the activity at all. This result and titration with DTNB suggest that Cys144 is concerned in the catalytic action and Cys68 is located inside the enzyme. The expressed enzyme was purified to homogeneity by hydrophobic chromatography on a Toyopearl HW-65C column and crystallization, with an activity recovery of 42.7%. The enzyme was most active at pH 6.5 and stable at pH 7.0-9.0. Its molecular weight was estimated to be 51,000 by gel filtration, suggesting it to be a dimer. Big crystals of the wild and PCMB-modified enzymes were obtained by the hanging drop method.
- Published
- 1993
- Full Text
- View/download PDF
40. Zinc protease of Bacillus subtilis var. amylosacchariticus: construction of a three-dimensional model and comparison with thermolysin.
- Author
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Tsuru D, Imajo S, Morikawa S, Yoshimoto T, and Ishiguro M
- Subjects
- Amino Acid Sequence, Metalloendopeptidases metabolism, Metalloendopeptidases ultrastructure, Models, Molecular, Molecular Sequence Data, Protein Conformation, Protein Folding, Sequence Alignment, Thermolysin metabolism, Thermolysin ultrastructure, Zinc, Bacillus subtilis enzymology, Metalloendopeptidases chemistry, Thermolysin chemistry
- Abstract
The active site structure of the Zn-containing neutral protease from Bacillus subtilis var. amylosacchariticus (BANP) was predicted by computer-aided modeling on the basis of the three-dimensional structure of thermolysin (TLN). As expected from the high homology in amino acid sequence of the two enzymes, the overall folding of BANP was very similar to that of TLN. Glu144, Tyr158, and His228 of BANP were located near the active site Zn ion, to which three amino acid residues, His143, His147, and Glu167, were coordinated. This model is supported by the previous results that chemical modifications of Tyr158 and photooxidation of His228 of BANP markedly affect the proteolytic activity of the enzyme. Interestingly, BANP was found to be significantly less sensitive to metalloprotease inhibitors such as phosphoramidon and talopeptin. From a comparison of the enzyme-inhibitor complex models between BANP and thermolysin, it is suggested that replacement of Thr129 in TLN by Phe130 in BANP is related to difference in inhibitor sensitivity between BANP and TLN.
- Published
- 1993
- Full Text
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41. Location of the protease II gene (ptrB) on the physical map of the Escherichia coli chromosome.
- Author
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Kanatani A, Yoshimoto T, Nagai H, Ito K, and Tsuru D
- Subjects
- Chromosome Mapping, Molecular Sequence Data, Escherichia coli genetics, Genes, Bacterial genetics, Metalloendopeptidases genetics
- Published
- 1992
- Full Text
- View/download PDF
42. Cloning, sequencing, and high expression of the proline iminopeptidase gene from Bacillus coagulans.
- Author
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Kitazono A, Yoshimoto T, and Tsuru D
- Subjects
- Amino Acid Sequence, Aminopeptidases chemistry, Aminopeptidases metabolism, Bacillus enzymology, Base Sequence, Cloning, Molecular, Crystallization, DNA, Bacterial, Electrophoresis, Polyacrylamide Gel, Enzyme Stability, Escherichia coli, Molecular Sequence Data, Oxidation-Reduction, Restriction Mapping, Substrate Specificity, Aminopeptidases genetics, Bacillus genetics
- Abstract
The gene coding for proline iminopeptidase in Bacillus coagulans was cloned and expressed in Escherichia coli. Nucleotide sequencing revealed an 861-bp open reading frame with an unusual TTG initiation codon, encoding a 287-amino-acid protein. The calculated molecular weight of the product was 32,415. The amino acid sequences of the amino-terminal region and those of some peptide fragments obtained by endoproteinase Asp-N digestion of the purified enzyme completely coincided with those deduced from the nucleotide sequence. The rare TTG initiation codon that normally codes for leucine was translated as a formal initiation codon; a methionine residue was found at the amino terminus of the enzyme. By using a vector bearing the strong tac promoter, an expression level as high as 200-fold that of the first clone was achieved. The replacement of the TTG initiation codon with ATG and a simultaneous reduction of the distance to the tac promoter resulted in a further increase of 2.5-fold. The expressed enzyme was easily purified to homogeneity by hydrophobic chromatography on a Toyopearl HW-65C column and crystallization, with a recovery of activity of 36%. The molecular weight was found to be 33,000 by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration on a Hi-Load 16/60 Superdex 200 fast protein liquid chromatography column. The expressed enzyme showed the same catalytic and physicochemical properties as those of the wild type, specifically cleaving the N-terminal proline from small substrates.
- Published
- 1992
- Full Text
- View/download PDF
43. Degradation of streptomyces metalloprotease inhibitor (SMPI) by neutral protease from Bacillus subtilis var. amylosacchariticus.
- Author
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Tsuru D, Fujita Y, Morikawa S, Ito K, and Yoshimoto T
- Subjects
- Amino Acid Sequence, Chromatography, High Pressure Liquid, Molecular Sequence Data, Substrate Specificity, Bacillus subtilis enzymology, Bacterial Proteins metabolism, Metalloendopeptidases antagonists & inhibitors, Streptomyces enzymology, Thermolysin antagonists & inhibitors
- Abstract
The zinc-containing neutral endopeptidase (neutral protease: BANP) from Bacillus subtilis var. amylosacchariticus was inhibited by the proteinaceous metalloprotease inhibitor isolated from Streptomyces nigrescens (SMPI). The degree of inhibition was, however, significantly less than that for thermolysin (TLN). During incubation of BANP with SMPI, the inhibitor was proteolytically degraded and inactivated. Analysis of the digestion products suggested that a minor diversity in their substrate specificities between TLN and BANP affects the sensitivity to the proteinaceous metalloprotease inhibitor, SMPI.
- Published
- 1992
- Full Text
- View/download PDF
44. Prolyl endopeptidase from Flavobacterium meningosepticum: cloning and sequencing of the enzyme gene.
- Author
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Yoshimoto T, Kanatani A, Shimoda T, Inaoka T, Kokubo T, and Tsuru D
- Subjects
- Amino Acid Sequence, Base Sequence, Binding Sites, Cloning, Molecular, DNA, Bacterial genetics, Endopeptidases chemistry, Flavobacterium genetics, Molecular Sequence Data, Prolyl Oligopeptidases, Sequence Homology, Nucleic Acid, Endopeptidases genetics, Flavobacterium enzymology, Serine Endopeptidases
- Abstract
The prolyl endopeptidase [EC 3.4.21.26] gene of Flavobacterium meningosepticum was cloned in Escherichia coli with the aid of an oligonucleotide probe which was prepared based on the amino acid sequence. The hybrid plasmid, pFPEP1, with a 3.5 kbp insert at the HincII site of pUC19 containing the enzyme gene, was subcloned into pUC19 to construct plasmid pFPEP3. The whole nucleotide sequence of an inserted HincII-BamHI fragment of plasmid pFPEP3 was determined by the dideoxy chain-terminating method. The purified prolyl endopeptidase was labeled with tritium DFP, and the sequence surrounding the reactive serine residue was found to be Ala (551)-Leu-Ser-Gly-Arg-*Ser-Asn(557). Ser-556 was identified as a reactive serine residue. The enzyme consists of 705 amino acid residues as deduced from the nucleotide sequence and has a molecular weight of 78,705, which coincides well with the value estimated by ultra centrifugal analysis. The amino acid sequence was 38.2% homologous to that of the porcine brain prolyl endopeptidase [Rennex et al. (1991) Biochemistry 30, 2195-2203] and 24.5% homologous to E. coli protease II, which has substrate specificity for basic amino acids [Kanatani et al. (1991) J. Biochem. 110, 315-320].
- Published
- 1991
- Full Text
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45. Acetyl-CoA carboxylase from Escherichia coli: gene organization and nucleotide sequence of the biotin carboxylase subunit.
- Author
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Kondo H, Shiratsuchi K, Yoshimoto T, Masuda T, Kitazono A, Tsuru D, Anai M, Sekiguchi M, and Tanabe T
- Subjects
- Amino Acid Sequence, Base Sequence, Cloning, Molecular, DNA, Bacterial genetics, Molecular Sequence Data, Restriction Mapping, Sequence Alignment, Acetyl-CoA Carboxylase genetics, Carbon-Nitrogen Ligases, Escherichia coli genetics, Genes, Bacterial, Ligases genetics
- Abstract
Biotin carboxylase [biotin-carboxyl-carrier-protein:carbon-dioxide ligase (ADP-forming), EC 6.3.4.14] is the enzyme mediating the first step of the acetyl-CoA carboxylase [acetyl-CoA:carbon-dioxide ligase (ADP-forming), EC 6.4.1.2] reaction. We screened an Escherichia coli DNA library and a DNA fragment carrying the biotin carboxylase gene fabG, and its flanking regions were cloned. The gene for biotin carboxyl carrier protein was found 13 base pairs upstream of the fabG gene. Nucleotide sequencing of the recombinant plasmids revealed that the fabG codes for a 449-amino acid residue protein with a calculated molecular weight of 49,320, a value in good agreement with that of 51,000 determined by SDS/polyacrylamide gel electrophoresis of the purified enzyme. The deduced amino acid sequence of biotin carboxylase is also consistent with the partial amino acid sequence determined by Edman degradation. The primary structure of this enzyme exhibits a high homology with those of other biotin-dependent enzymes and carbamoyl-phosphate synthetase [carbon-dioxide:L-glutamine amino-ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.5.5]; therefore, all these enzymes probably function through the same mechanism of reaction.
- Published
- 1991
- Full Text
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46. Chemical modification of neutral protease from Bacillus subtilis var. amylosacchariticus: assignment of tyrosyl residues iodinated.
- Author
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Morikawa S, Kanatani A, Kobayashi R, Yoshimoto T, and Tsuru D
- Subjects
- Amino Acid Sequence, Bacterial Proteins metabolism, Caseins metabolism, Iodine chemistry, Metalloendopeptidases metabolism, Molecular Sequence Data, Tyrosine chemistry, Bacillus subtilis enzymology, Bacterial Proteins chemistry, Metalloendopeptidases chemistry, Tyrosine analysis
- Abstract
The neutral protease of Bacillus subtilis var. amylosacchariticus (B. amylosacchariticus) was iodinated with a 25-fold molar excess of iodine at pH 9.4 for 3 min at 0 degree C, by which treatment the proteolytic activity toward casein was markedly reduced, while the hydrolytic activity toward an N-blocked peptide substrate was rather increased. The modified enzyme was digested with Staphylococcus aureus V8 protease at pH 8.0 and the amino acid sequences of resultant peptides were compared with those obtained from the native enzyme. One of the peptides was found to have an amino acid sequence of Thr-Ala-Asn-Leu-Ile-Tyr-Glu, which corresponds to residue Nos. 153-159 of the enzyme, where Tyr-158 was identified to be mono-iodotyrosine. The other two peptides were those containing Tyr-21 which was mono- and di-iodinated, respectively. Referring to nitration experiments on the neutral protease and the active site structure of thermolysin, it was concluded that the iodination of Tyr-158 is mainly responsible for the activity changes of B. amylosacchariticus neutral protease.
- Published
- 1991
47. Protease II from Escherichia coli: sequencing and expression of the enzyme gene and characterization of the expressed enzyme.
- Author
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Kanatani A, Masuda T, Shimoda T, Misoka F, Lin XS, Yoshimoto T, and Tsuru D
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Brain enzymology, Chromatography, High Pressure Liquid, Chromosomes, Bacterial, Cloning, Molecular, DNA, Bacterial genetics, DNA, Bacterial isolation & purification, Endopeptidases genetics, Escherichia coli genetics, Kinetics, Molecular Sequence Data, Peptide Fragments isolation & purification, Plasmids, Prolyl Oligopeptidases, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Restriction Mapping, Sequence Homology, Nucleic Acid, Serine Endopeptidases isolation & purification, Serine Endopeptidases metabolism, Substrate Specificity, Swine, Escherichia coli enzymology, Genes, Bacterial, Serine Endopeptidases genetics
- Abstract
Protease II gene of Escherichia coli HB101 was cloned and expressed in E. coli JM83. The transformant harboring a hybrid plasmid, pPROII-12, with a 2.4 kbp fragment showed 90-fold higher enzyme activity than the host. The whole nucleotide sequence of the inserted fragment of plasmid pPROII-12 was clarified by the dideoxy chain-terminating method. The sequence that encoded the mature enzyme protein was found to start at an ATG codon, as judged by comparison with amino terminal protein sequencing. The molecular weight of the enzyme was estimated to be 81,858 from the nucleotide sequence. The reactive serine residue of protease II was identified as Ser-532 with tritium DFP. The sequence around the serine residue is coincident with the common sequence of Gly-X-Ser-X-Gly, which has been found in the active site of serine proteases. Except for this region, protease II showed no significant sequence homology with E. coli serine proteases, protease IV and protease La (lon gene), or other known families of serine proteases. However, 25.3% homology was observed between protease II and prolyl endopeptidase from porcine brain. Although the substrate specificities of these two enzymes are quite different, it seems possible to classify protease II as a member of the prolyl endopeptidase family from the structural point of view.
- Published
- 1991
- Full Text
- View/download PDF
48. Dye-sensitized photooxidation of neutral protease from Bacillus subtilis var. amylosacchariticus: assignment of histidine residue oxidized.
- Author
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Morikawa S, Kanatani A, Yoshimoto T, and Tsuru D
- Subjects
- Amino Acid Sequence, Chromatography, High Pressure Liquid, Endopeptidases metabolism, Metalloendopeptidases metabolism, Molecular Sequence Data, Oxidation-Reduction, Peptides chemistry, Peptides isolation & purification, Photochemistry, Bacillus subtilis enzymology, Bacterial Proteins, Histidine chemistry, Metalloendopeptidases chemistry
- Abstract
The neutral protease of Bacillus subtilis var. amylosacchariticus was photooxidized in the presence of methylene blue, by which treatment the enzyme was rapidly inactivated. The inactive enzyme was digested with endoproteinase Asp-N, the resultant peptides were separated by HPLC, and their amino acid sequences were compared with those obtained from the unmodified enzyme. Of four peptides that contained histidine residues, only the recovery of one peptide was found to be decreased by the photooxidation with the appearance of a new peptide. Comparisons of amino acid compositions and sequences between these two peptides showed that the latter peptide lacked His228 of the former one, indicating that His228 was photooxidized. This result suggests that His228 is involved in the catalytic reaction of the neutral protease or interaction with substrates.
- Published
- 1991
49. CPT-11 converting enzyme from rat serum: purification and some properties.
- Author
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Tsuji T, Kaneda N, Kado K, Yokokura T, Yoshimoto T, and Tsuru D
- Subjects
- Animals, Antineoplastic Agents, Phytogenic metabolism, Camptothecin blood, Camptothecin metabolism, Carboxylic Ester Hydrolases chemistry, Carboxylic Ester Hydrolases isolation & purification, Hydrogen-Ion Concentration, Hydrolysis, Irinotecan, Isoelectric Point, Kinetics, Male, Molecular Weight, Rats, Rats, Inbred Strains, Camptothecin analogs & derivatives, Carboxylic Ester Hydrolases blood, Prodrugs metabolism
- Abstract
A rat serum enzyme that catalyzes the conversion of a pro-drug, 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin (CPT-11), to an anticancer drug, 7-ethyl-10-hydroxycamptothecin (SN-38), was purified and its properties were characterized. The enzyme was purified by column chromatography on diethylaminoethyl Toyopearl 650M, QAE-Sephadex, Sephadex G-150, Con A-Sepharose and high performance liquid chromatography with an ion-exchanger column. It was most active at pH 7.5 and was stable at pH 4-9 for 1 h at 30 degrees C. The molecular weight was estimated to be 60 and 57 kDa by gel filtration and sodium dodecylsulfate-polyacrylamide gel electrophoresis methods, respectively, and the isoelectric point was 4.6, as determined by isoelectric focusing. The Km value for CPT-11 was 0.28 microM. This enzyme was inhibited by diisopropyl phosphorofluoridate (DFP) and phenylmethanesulfonyl fluoride (PMSF) but insensitive to eserine, p-chloromercuribenzoate (PCMB) and ethylenediaminetetraacetate (EDTA). The enzyme also hydrolyzed p-nitrophenylacetate (p-NPA), a commonly used substrate for esterases, but was not active toward acetylcholine, suggesting that the enzyme is a carboxylesterase[EC 3.1.1.1]. During the hydrolyses of CPT-11 and p-NPA, an initial burst phenomenon similar to that found in the alpha-chymotrypsin-catalyzed hydrolysis of p-NPA was observed. Kinetic analysis revealed that the deacylation of the enzyme is the rate-limiting step in substrate hydrolysis. This enzyme was found to also split other ester derivatives of SN-38 besides CPT-11.
- Published
- 1991
- Full Text
- View/download PDF
50. Cloning and sequencing of the 7 alpha-hydroxysteroid dehydrogenase gene from Escherichia coli HB101 and characterization of the expressed enzyme.
- Author
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Yoshimoto T, Higashi H, Kanatani A, Lin XS, Nagai H, Oyama H, Kurazono K, and Tsuru D
- Subjects
- Amino Acid Sequence, Base Sequence, Cloning, Molecular, Crystallography, Hydroxysteroid Dehydrogenases antagonists & inhibitors, Hydroxysteroid Dehydrogenases chemistry, Hydroxysteroid Dehydrogenases metabolism, Molecular Sequence Data, Molecular Weight, Restriction Mapping, Substrate Specificity, Escherichia coli genetics, Genes, Bacterial, Hydroxysteroid Dehydrogenases genetics
- Abstract
The 7 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.159) gene from Escherichia coli HB101 was cloned and expressed in E. coli DH1. The hybrid plasmid pSD1, with a 2.8-kbp insert of chromosomal DNA at the BamHI site of pBR322, was subcloned into pUC19 to construct plasmid pSD3. The entire nucleotide sequence of an inserted PstI-BamHI fragment of plasmid pSD3 was determined by the dideoxy chain-termination method. Within this sequence, the mature enzyme protein-encoding sequence was found to start at a GTG initiation codon and to comprise 765 bp, as judged by comparison with the protein sequence. The deduced amino acid sequence of the enzyme indicated that the molecular weight is 26,778. The transformant of E. coli DH1 harboring pSD3 with a 1.8-kbp fragment showed about 200-fold-higher enzyme activity than the host. The enzyme was purified by a single chromatography step on DEAE-Toyopearl and obtained as crystals, with an activity yield of 39%. The purified enzyme was homogeneous, as judged by sodium dodecyl sulfate gel electrophoresis. The enzyme was most active at pH 8.5 and stable between pH 8 and 9. The enzyme was NAD+ dependent and had a pI of 4.3. The molecular mass was estimated to be 120 kDa by the gel filtration method and 28 kDa by electrophoresis, indicating that the enzyme exists in a tetrameric form.
- Published
- 1991
- Full Text
- View/download PDF
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