Tottori University

AUTORES: Daniel J Klionsky1745,1749*, Kotb Abdelmohsen840, Akihisa Abe1237, Md Joynal Abedin1762, Hagai Abeliovich425,
\nAbraham Acevedo Arozena789, Hiroaki Adachi1800, Christopher M Adams1669, Peter D Adams57, Khosrow Adeli1981,
\nPeter J Adhihetty1625, Sharon G Adler700, Galila Agam67, Rajesh Agarwal1587, Manish K Aghi1537, Maria Agnello1826,
\nPatrizia Agostinis664, Patricia V Aguilar1960, Julio Aguirre-Ghiso784,786, Edoardo M Airoldi89,422, Slimane Ait-Si-Ali1376,
\nTakahiko Akematsu2010, Emmanuel T Akporiaye1097, Mohamed Al-Rubeai1394, Guillermo M Albaiceta1294,
\nChris Albanese363, Diego Albani561, Matthew L Albert517, Jesus Aldudo128, Hana Alg€ul1164, Mehrdad Alirezaei1198,
\nIraide Alloza642,888, Alexandru Almasan206, Maylin Almonte-Beceril524, Emad S Alnemri1212, Covadonga Alonso544,
\nNihal Altan-Bonnet848, Dario C Altieri1205, Silvia Alvarez1497, Lydia Alvarez-Erviti1395, Sandro Alves107,
\nGiuseppina Amadoro860, Atsuo Amano930, Consuelo Amantini1554, Santiago Ambrosio1458, Ivano Amelio756,
\nAmal O Amer918, Mohamed Amessou2089, Angelika Amon726, Zhenyi An1538, Frank A Anania291, Stig U Andersen6,
\nUsha P Andley2079, Catherine K Andreadi1690, Nathalie Andrieu-Abadie502, Alberto Anel2027, David K Ann58,
\nShailendra Anoopkumar-Dukie388, Manuela Antonioli832,858, Hiroshi Aoki1791, Nadezda Apostolova2007,
\nSaveria Aquila1500, Katia Aquilano1876, Koichi Araki292, Eli Arama2098, Agustin Aranda456, Jun Araya591,
\nAlexandre Arcaro1472, Esperanza Arias26, Hirokazu Arimoto1225, Aileen R Ariosa1749, Jane L Armstrong1930,
\nThierry Arnould1773, Ivica Arsov2120, Katsuhiko Asanuma675, Valerie Askanas1924, Eric Asselin1867, Ryuichiro Atarashi794,
\nSally S Atherton369, Julie D Atkin713, Laura D Attardi1131, Patrick Auberger1787, Georg Auburger379, Laure Aurelian1727,
\nRiccardo Autelli1992, Laura Avagliano1029,1755, Maria Laura Avantaggiati364, Limor Avrahami1166, Suresh Awale1986,
\nNeelam Azad404, Tiziana Bachetti568, Jonathan M Backer28, Dong-Hun Bae1933, Jae-sung Bae677, Ok-Nam Bae409,
\nSoo Han Bae2117, Eric H Baehrecke1729, Seung-Hoon Baek17, Stephen Baghdiguian1368,
\nAgnieszka Bagniewska-Zadworna2, Hua Bai90, Jie Bai667, Xue-Yuan Bai1133, Yannick Bailly884,
\nKithiganahalli Narayanaswamy Balaji473, Walter Balduini2002, Andrea Ballabio316, Rena Balzan1711, Rajkumar Banerjee239,
\nG abor B anhegyi1052, Haijun Bao2109, Benoit Barbeau1363, Maria D Barrachina2007, Esther Barreiro467, Bonnie Bartel997,
\nAlberto Bartolom e222, Diane C Bassham550, Maria Teresa Bassi1046, Robert C Bast Jr1273, Alakananda Basu1798,
\nMaria Teresa Batista1578, Henri Batoko1336, Maurizio Battino970, Kyle Bauckman2085, Bradley L Baumgarner1909,
\nK Ulrich Bayer1594, Rupert Beale1553, Jean-Fran¸cois Beaulieu1360, George R. Beck Jr48,294, Christoph Becker336,
\nJ David Beckham1595, Pierre-Andr e B edard749, Patrick J Bednarski301, Thomas J Begley1135, Christian Behl1419,
\nChristian Behrends757, Georg MN Behrens406, Kevin E Behrns1627, Eloy Bejarano26, Amine Belaid490,
\nFrancesca Belleudi1041, Giovanni B enard497, Guy Berchem706, Daniele Bergamaschi983, Matteo Bergami1401,
\nBen Berkhout1441, Laura Berliocchi714, Am elie Bernard1749, Monique Bernard1354, Francesca Bernassola1880,
\nAnne Bertolotti791, Amanda S Bess272, S ebastien Besteiro1351, Saverio Bettuzzi1828, Savita Bhalla913,
\nShalmoli Bhattacharyya973, Sujit K Bhutia838, Caroline Biagosch1159, Michele Wolfe Bianchi520,1378,1381,
\nMartine Biard-Piechaczyk210, Viktor Billes298, Claudia Bincoletto1314, Baris Bingol350, Sara W Bird1128, Marc Bitoun1112,
\nIvana Bjedov1258, Craig Blackstone843, Lionel Blanc1183, Guillermo A Blanco1496, Heidi Kiil Blomhoff1812,
\nEmilio Boada-Romero1297, Stefan B€ockler1464, Marianne Boes1423, Kathleen Boesze-Battaglia1835, Lawrence H Boise286,287,
\nAlessandra Bolino2063, Andrea Boman693, Paolo Bonaldo1823, Matteo Bordi897, J€urgen Bosch608, Luis M Botana1308,
\nJoelle Botti1375, German Bou1405, Marina Bouch e1038, Marion Bouchecareilh1331, Marie-Jos ee Boucher1901,
\nMichael E Boulton481, Sebastien G Bouret1926, Patricia Boya133, Micha€el Boyer-Guittaut1345, Peter V Bozhkov1141,
\nNathan Brady374, Vania MM Braga469, Claudio Brancolini1997, Gerhard H Braus353, Jos e M Bravo-San Pedro299,393,508,1374,
\nLisa A Brennan322, Emery H Bresnick2022, Patrick Brest490, Dave Bridges1939, Marie-Agn es Bringer124, Marisa Brini1822,
\nGlauber C Brito1311, Bertha Brodin631, Paul S Brookes1872, Eric J Brown352, Karen Brown1690, Hal E Broxmeyer480,
\nAlain Bruhat486,1339, Patricia Chakur Brum1893, John H Brumell446, Nicola Brunetti-Pierri315,1171,
\nRobert J Bryson-Richardson781, Shilpa Buch1777, Alastair M Buchan1819, Hikmet Budak1022, Dmitry V Bulavin118,505,1789,
\nScott J Bultman1792, Geert Bultynck665, Vladimir Bumbasirevic1470, Yan Burelle1356, Robert E Burke216,217,
\nMargit Burmeister1750, Peter B€utikofer1473, Laura Caberlotto1987, Ken Cadwell896, Monika Cahova112, Dongsheng Cai24,
\nJingjing Cai2099, Qian Cai1018, Sara Calatayud2007, Nadine Camougrand1343, Michelangelo Campanella1700,
\nGrant R Campbell1525, Matthew Campbell1249, Silvia Campello556,1876, Robin Candau1769, Isabella Caniggia1983,
\nLavinia Cantoni560, Lizhi Cao116, Allan B Caplan1656, Michele Caraglia1051, Claudio Cardinali1043, Sandra Morais Cardoso1579, Jennifer S Carew208, Laura A Carleton874, Cathleen R Carlin101, Silvia Carloni2002,
\nSven R Carlsson1267, Didac Carmona-Gutierrez1643, Leticia AM Carneiro312, Oliana Carnevali971, Serena Carra1318,
\nAlice Carrier120, Bernadette Carroll900, Caty Casas1324, Josefina Casas1116, Giuliana Cassinelli324, Perrine Castets1462,
\nSusana Castro-Obregon214, Gabriella Cavallini1841, Isabella Ceccherini568, Francesco Cecconi253,555,1884,
\nArthur I Cederbaum459, Valent ın Ce~na199,1281, Simone Cenci1323,2064, Claudia Cerella444, Davide Cervia1996,
\nSilvia Cetrullo1478, Hassan Chaachouay2028, Han-Jung Chae187, Andrei S Chagin634, Chee-Yin Chai626,628,
\nGopal Chakrabarti1502, Georgios Chamilos1601, Edmond YW Chan1142, Matthew TV Chan181, Dhyan Chandra1003,
\nPallavi Chandra548, Chih-Peng Chang818, Raymond Chuen-Chung Chang1653, Ta Yuan Chang345, John C Chatham1434,
\nSaurabh Chatterjee1910, Santosh Chauhan527, Yongsheng Che62, Michael E Cheetham1263, Rajkumar Cheluvappa1783,
\nChun-Jung Chen1153, Gang Chen598,1676, Guang-Chao Chen9, Guoqiang Chen1078, Hongzhuan Chen1077, Jeff W Chen1514,
\nJian-Kang Chen370,371, Min Chen249, Mingzhou Chen2104, Peiwen Chen1823, Qi Chen1674, Quan Chen172,
\nShang-Der Chen138, Si Chen325, Steve S-L Chen10, Wei Chen2125, Wei-Jung Chen829, Wen Qiang Chen979, Wenli Chen1113,
\nXiangmei Chen1133, Yau-Hung Chen1157, Ye-Guang Chen1250, Yin Chen1447, Yingyu Chen953,955, Yongshun Chen2135,
\nYu-Jen Chen712, Yue-Qin Chen1145, Yujie Chen1208, Zhen Chen339, Zhong Chen2123, Alan Cheng1702,
\nChristopher HK Cheng184, Hua Cheng1728, Heesun Cheong814, Sara Cherry1836, Jason Chesney1703,
\nChun Hei Antonio Cheung817, Eric Chevet1359, Hsiang Cheng Chi140, Sung-Gil Chi656, Fulvio Chiacchiera308,
\nHui-Ling Chiang958, Roberto Chiarelli1826, Mario Chiariello235,567,577, Marcello Chieppa835, Lih-Shen Chin290,
\nMario Chiong1285, Gigi NC Chiu878, Dong-Hyung Cho676, Ssang-Goo Cho650, William C Cho982, Yong-Yeon Cho105,
\nYoung-Seok Cho1064, Augustine MK Choi2095, Eui-Ju Choi656, Eun-Kyoung Choi387,400,685, Jayoung Choi1563,
\nMary E Choi2093, Seung-Il Choi2116, Tsui-Fen Chou412, Salem Chouaib395, Divaker Choubey1574, Vinay Choubey1936,
\nKuan-Chih Chow822, Kamal Chowdhury730, Charleen T Chu1856, Tsung-Hsien Chuang827, Taehoon Chun657,
\nHyewon Chung652, Taijoon Chung978, Yuen-Li Chung1194, Yong-Joon Chwae18, Valentina Cianfanelli254,
\nRoberto Ciarcia1775, Iwona A Ciechomska886, Maria Rosa Ciriolo1876, Mara Cirone1042, Sofie Claerhout1694,
\nMichael J Clague1698, Joan Cl aria1457, Peter GH Clarke1687, Robert Clarke361, Emilio Clementi1045,1398, C edric Cleyrat1781,
\nMiriam Cnop1366, Eliana M Coccia574, Tiziana Cocco1459, Patrice Codogno1375, J€orn Coers271, Ezra EW Cohen1533,
\nDavid Colecchia235,567,577, Luisa Coletto25, N uria S Coll123, Emma Colucci-Guyon516, Sergio Comincini1829,
\nMaria Condello578, Katherine L Cook2073, Graham H Coombs1929, Cynthia D Cooper2076, J Mark Cooper1395,
\nIsabelle Coppens601, Maria Tiziana Corasaniti1387, Marco Corazzari485,1884, Ramon Corbalan1566,
\nElisabeth Corcelle-Termeau251, Mario D Cordero1899, Cristina Corral-Ramos1289, Olga Corti507,1109, Andrea Cossarizza1767,
\nPaola Costelli1993, Safia Costes1518, Susan L Cotman721, Ana Coto-Montes946, Sandra Cottet566,1688, Eduardo Couve1301,
\nLori R Covey1015, L Ashley Cowart762, Jeffery S Cox1536, Fraser P Coxon1427, Carolyn B Coyne1846, Mark S Cragg1919,
\nRolf J Craven1679, Tiziana Crepaldi1995, Jose L Crespo1300, Alfredo Criollo1285, Valeria Crippa558, Maria Teresa Cruz1576,
\nAna Maria Cuervo26, Jose M Cuezva1277, Taixing Cui1907, Pedro R Cutillas987, Mark J Czaja27, Maria F Czyzyk-Krzeska1572,
\nRuben K Dagda2068, Uta Dahmen1404, Chunsun Dai800, Wenjie Dai1187, Yun Dai2059, Kevin N Dalby1940,
\nLuisa Dalla Valle1822, Guillaume Dalmasso1340, Marcello D’Amelio557, Markus Damme188, Arlette Darfeuille-Michaud1340,
\nCatherine Dargemont950, Victor M Darley-Usmar1433, Srinivasan Dasarathy205, Biplab Dasgupta202, Srikanta Dash1254,
\nCrispin R Dass242, Hazel Marie Davey8, Lester M Davids1560, David D avila227, Roger J Davis1731, Ted M Dawson604,
\nValina L Dawson606, Paula Daza1898, Jackie de Belleroche470, Paul de Figueiredo1180,1182,
\nRegina Celia Bressan Queiroz de Figueiredo135, Jos e de la Fuente1023, Luisa De Martino1775,
\nAntonella De Matteis1171, Guido RY De Meyer1443, Angelo De Milito631, Mauro De Santi2002,

Six DNA regions were evaluated as potential DNA barcodes for Fungi, the second largest kingdom of eukaryotic life, by a multinational, multilaboratory consortium. The region of the mitochondrial cytochrome c oxidase subunit 1 used as the animal barcode was excluded as a potential marker, because it is difficult to amplify in fungi, often includes large introns, and can be insufficiently variable. Three subunits from the nuclear ribosomal RNA cistron were compared together with regions of three representative protein-coding genes (largest subunit of RNA polymerase II, second largest subunit of RNA polymerase II, and minichromosome maintenance protein). Although the protein-coding gene regions often had a higher percent of correct identification compared with ribosomal markers, low PCR amplification and sequencing success eliminated them as candidates for a universal fungal barcode. Among the regions of the ribosomal cistron, the internal transcribed spacer (ITS) region has the highest probability of successful identification for the broadest range of fungi, with the most clearly defined barcode gap between inter- and intraspecific variation. The nuclear ribosomal large subunit, a popular phylogenetic marker in certain groups, had superior species resolution in some taxonomic groups, such as the early diverging lineages and the ascomycete yeasts, but was otherwise slightly inferior to the ITS. The nuclear ribosomal small subunit has poor species-level resolution in fungi. ITS will be formally proposed for adoption as the primary fungal barcode marker to the Consortium for the Barcode of Life, with the possibility that supplementary barcodes may be developed for particular narrowly circumscribed taxonomic groups.

A very early step in the response of mammalian cells to DNA double-strand breaks is the phosphorylation of histone H2AX at serine 139 at the sites of DNA damage. Although the phosphatidylinositol 3-kinases, DNA-PK (DNA-dependent proteinkinase), ATM (ataxia telangiectasiamutated), and ATR (ATM andRad3-related), have all been implicated in H2AX phosphorylation, the specific kinase involved has not yet been identified. To definitively identify the specific kinase(s) that phosphorylates H2AX in vivo, we have utilized DNA-PKcs−/− and Atm−/− cell lines and mouse embryonic fibroblasts. We find that H2AX phosphorylation and nuclear focus formation are normal in DNA-PKcs−/− cells and severely compromised in Atm−/− cells. We also find that ATM can phosphorylate H2AX in vitro and that ectopic expression of ATM in Atm−/− fibroblasts restores H2AX phosphorylation in vivo. The minimal H2AX phosphorylation in Atm−/− fibroblasts can be abolished by low concentrations of wortmannin suggesting that DNA-PK, rather than ATR, is responsible for low levels of H2AX phosphorylation in the absence of ATM. Our results clearly establish ATM as the major kinase involved in the phosphorylation of H2AX and suggest that ATM is one of the earliest kinases to be activated in the cellular response to double-strand breaks. A very early step in the response of mammalian cells to DNA double-strand breaks is the phosphorylation of histone H2AX at serine 139 at the sites of DNA damage. Although the phosphatidylinositol 3-kinases, DNA-PK (DNA-dependent proteinkinase), ATM (ataxia telangiectasiamutated), and ATR (ATM andRad3-related), have all been implicated in H2AX phosphorylation, the specific kinase involved has not yet been identified. To definitively identify the specific kinase(s) that phosphorylates H2AX in vivo, we have utilized DNA-PKcs−/− and Atm−/− cell lines and mouse embryonic fibroblasts. We find that H2AX phosphorylation and nuclear focus formation are normal in DNA-PKcs−/− cells and severely compromised in Atm−/− cells. We also find that ATM can phosphorylate H2AX in vitro and that ectopic expression of ATM in Atm−/− fibroblasts restores H2AX phosphorylation in vivo. The minimal H2AX phosphorylation in Atm−/− fibroblasts can be abolished by low concentrations of wortmannin suggesting that DNA-PK, rather than ATR, is responsible for low levels of H2AX phosphorylation in the absence of ATM. Our results clearly establish ATM as the major kinase involved in the phosphorylation of H2AX and suggest that ATM is one of the earliest kinases to be activated in the cellular response to double-strand breaks. double-strand break ionizing radiation phosphatidylinositol mouse embryonic fibroblasts DNA double-strand breaks (DSBs)1 are probably the most dangerous of the many different types of DNA damage that occur within the cell. DSBs are generated by exogenous agents such as ionizing radiation (IR) or by endogenously generated reactive oxygen species and occur as intermediates during meiotic and V(D)J recombination (1Khanna K.K. Jackson S.P. Nat. Genet. 2001; 27: 247-254Crossref PubMed Scopus (1867) Google Scholar). A very early step in the cellular response to DSBs is the phosphorylation of a histone H2A variant, H2AX, at the sites of DNA damage (2Modesti M. Kanaar R. Curr. Biol. 2001; 11: R229-R232Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). H2AX is rapidly phosphorylated (within seconds) at serine 139 when DSBs are introduced into mammalian cells (3Rogakou E.P. Pilch D.R. Orr A.H. Ivanova V.S. Bonner W.M. J. Biol. Chem. 1998; 273: 5858-5868Abstract Full Text Full Text PDF PubMed Scopus (4049) Google Scholar) resulting in discrete γ-H2AX (phosphorylated-H2AX) foci at the DNA damage sites (4Rogakou E.P. Boon C. Redon C. Bonner W.M. J. Cell Biol. 1999; 146: 905-915Crossref PubMed Scopus (1931) Google Scholar). In experiments involving the use of “laser scissors” to introduce breaks into living cells, γ-H2AX foci localized specifically with the laser path through the cell nuclei clearly demonstrating that H2AX phosphorylation is specific to the sites of DNA damage (4Rogakou E.P. Boon C. Redon C. Bonner W.M. J. Cell Biol. 1999; 146: 905-915Crossref PubMed Scopus (1931) Google Scholar, 5Paull T.T. Rogakou E.P. Yamazaki V. Kirchgessner C.U. Gellert M. Bonner W.M. Curr. Biol. 2000; 10: 886-895Abstract Full Text Full Text PDF PubMed Scopus (1661) Google Scholar). H2AX phosphorylation also appears to be a general cellular response to processes involving DSB intermediates including V(D)J recombination in lymphoid cells (6Chen H.T. Bhandoola A. Difilippantonio M.J. Zhu J. Brown M.J. Tai X. Rogakou E.P. Brotz T.M. Bonner W.M. Ried T. Nussenzweig A. Science (Wash. D. C.). 2000; 290: 1962-1964Crossref PubMed Scopus (283) Google Scholar) and meiotic recombination in mice (7Mahadevaiah S.K. Turner J.M.A. Baudat F. Rogakou E.P. de Boer P. Blanco-Rodriguez J. Jasin M. Keeney S. Bonner W.M. Burgoyne P.S. Nat. Genet. 2001; 27: 271-276Crossref PubMed Scopus (685) Google Scholar). Phosphorylation of yeast H2A at serine 129 (homologous to serine 139 of mammalian H2AX) causes chromatin decondensation and is required for efficient DNA double-strand break repair (8Downs J.A. Lowndes N.F. Jackson S.P. Nature. 2000; 408: 1001-1004Crossref PubMed Scopus (529) Google Scholar). In mammals, phosphorylation of H2AX appears to play a critical role in the recruitment of repair or damage-signaling factors to the sites of DNA damage (5Paull T.T. Rogakou E.P. Yamazaki V. Kirchgessner C.U. Gellert M. Bonner W.M. Curr. Biol. 2000; 10: 886-895Abstract Full Text Full Text PDF PubMed Scopus (1661) Google Scholar, 9Rappold I. Iwabuchi K. Date T. Chen J. J. Cell Biol. 2001; 153: 613-620Crossref PubMed Scopus (396) Google Scholar). As H2AX phosphorylation plays a very early and important role in the cellular response to DNA double-strand breaks, it is important to specifically identify the kinase(s) involved in this event. Members of the PI 3-kinase family, including DNA-PK (DNA-dependent proteinkinase), ATM (ataxia telangiectasiamutated), and ATR (ATM andRad3-related), are involved in the responses of mammalian cells to DSBs (10Durocher D. Jackson S.P. Curr. Opin. Cell Biol. 2001; 13: 225-231Crossref PubMed Scopus (443) Google Scholar). γ-H2AX focus formation is inhibited by the PI 3-kinase inhibitor wortmannin, and H2AX phosphorylation is reduced in the DNA-PK-deficient human cell line M059J (5Paull T.T. Rogakou E.P. Yamazaki V. Kirchgessner C.U. Gellert M. Bonner W.M. Curr. Biol. 2000; 10: 886-895Abstract Full Text Full Text PDF PubMed Scopus (1661) Google Scholar). This led to the conclusion that DNA-PK and at least one other kinase, possibly ATM and/or ATR, can phosphorylate H2AX upon DNA damage (2Modesti M. Kanaar R. Curr. Biol. 2001; 11: R229-R232Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 5Paull T.T. Rogakou E.P. Yamazaki V. Kirchgessner C.U. Gellert M. Bonner W.M. Curr. Biol. 2000; 10: 886-895Abstract Full Text Full Text PDF PubMed Scopus (1661) Google Scholar, 10Durocher D. Jackson S.P. Curr. Opin. Cell Biol. 2001; 13: 225-231Crossref PubMed Scopus (443) Google Scholar, 11van Gent D.C. Hoeijmakers J.H. Kanaar R. Nat. Rev. Genet. 2001; 2: 196-206Crossref PubMed Scopus (938) Google Scholar). To unambiguously define the roles of ATM and DNA-PK in H2AX phosphorylation, we utilized cells derived from knockout mice for ATM or DNA-PKcs (the catalytic subunit of DNA-PK). We observed normal H2AX phosphorylation and γ-H2AX focus formation in irradiated fibroblasts derived from wild type or DNA-PKcs−/− mice. In contrast, H2AX phosphorylation and γ-H2AX focus formation were strikingly reduced to near background levels in fibroblasts from Atm−/− mice. Ectopic expression of ATM in Atm−/− cells restored H2AX phosphorylation. Moreover, we show that immunoprecipitated ATM can phosphorylate recombinant H2AX in vitro. These results indicate that ATM, not DNA-PK, is the major kinase responsible for modifying H2AX upon irradiation. The minimal H2AX phosphorylation in Atm−/− cells could be abolished by low concentrations of wortmannin suggesting that DNA-PK, rather than ATR, is responsible for low levels of γ-H2AX formation in the absence of ATM. Spontaneously immortalized mouse fibroblasts, derived from wild type, DNA-PKcs−/− (12Kurimasa A. Ouyang H. Dong L.J. Wang S. Li X. Cordon-Cardo C. Chen D.J. Li G.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1403-1408Crossref PubMed Scopus (158) Google Scholar), or Atm−/− mice (13Xu Y. Ashley T. Brainerd E.E. Bronson R.T. Meyn M.S. Baltimore D. Genes Dev. 1996; 10: 2411-2422Crossref PubMed Scopus (736) Google Scholar), were maintained in a humidified atmosphere with 5% CO2 in α-minimum Eagle’s medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Mouse embryonic fibroblasts (MEFs) were isolated from 13.5-day-old embryos and maintained in α-minimum Eagle’s medium supplemented with 15% fetal calf serum. Cells were grown to about 70% confluence and irradiated with x-rays (300-kV, 12-mA, 0.5-mm Cu) at the rate of 5.5 gray/min to achieve a cumulative dose of 10 gray for all experiments unless otherwise mentioned. Cells were UV-irradiated at the rate of 0.15 J/m2/s to achieve a cumulative dose of 10 J/m2. Cells were harvested after 30 min, except in the case of time courses where they were harvested at time points ranging from 5 min to 8 h. Drug treatment of cells was carried out by the addition of the following DNA-damaging agents to the culture media for 1 h at the indicated concentrations: neocarzinostatin (0.2 μg/ml), bleomycin (50 μg/ml), etoposide (30 μg/ml), methyl methanesulfonate (50 μg/ml), and hydroxyurea (1 mm). Anti-γ-H2AX antibody was generated against a synthetic peptide consisting of the last nine amino acids of H2AX with phospho-Ser-139 as described before (3Rogakou E.P. Pilch D.R. Orr A.H. Ivanova V.S. Bonner W.M. J. Biol. Chem. 1998; 273: 5858-5868Abstract Full Text Full Text PDF PubMed Scopus (4049) Google Scholar). SDS extracts for Western blotting were prepared from mock-irradiated or irradiated cells as described previously (14D'Anna J.A. Valdez J.G. Habbersett R.C. Crissman H.A. Radiat. Res. 1997; 148: 260-271Crossref PubMed Scopus (31) Google Scholar). The antibodies used for Western blotting are anti-γ-H2AX, anti-H2A (H-124; Santa Cruz Biotechnology Inc., Santa Cruz, CA), and anti-ATM monoclonal antibody MAT3–4G10/8 (15Andegeko Y. Moyal L. Mitelman L. Tsarfaty I. Shiloh Y. Rotman G. J. Biol. Chem. 2001; 276: 38224-38230Abstract Full Text Full Text PDF PubMed Google Scholar). Transient transfection of exponentially growing Atm−/− spontaneously immortalized fibroblasts with the ATM cDNA expression vector pMAT1 (16Zhang N. Chen P. Khanna K.K. Scott S. Gatei M. Kozlov S. Watters D. Spring K. Yen T. Lavin M.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8021-8026Crossref PubMed Scopus (97) Google Scholar) was carried out using Superfect transfection reagent (Qiagen Inc., Valencia, CA) as per the manufacturer’s protocols. Immediately after transfection, cells were induced for ATM expression with 5 μm CdCl2 for 16 h and then mock-irradiated or irradiated as described above. ATM immunoprecipitations were carried out as described (17Ziv Y. Banin S. Lim D.S. Canman C.E. Kastan M.B. Shiloh Y. Chan D.W. Gately D.P. Urban S. Galloway A.M. Lees-Miller S.P. Yen T. Allalunis-Turner J. Methods Mol. Biol. 2000; 99: 99-108PubMed Google Scholar). Approximately 1 × 107spontaneously immortalized mouse fibroblasts were grown to 70% confluence, mock-irradiated or irradiated, harvested after 30 min, and lysed in fresh cold lysis buffer containing protease and phosphatase inhibitors. The lysate was cleared by centrifugation, and the supernatant was incubated with 10 μg of anti-ATM monoclonal antibody MAT3–4G10/8 (15Andegeko Y. Moyal L. Mitelman L. Tsarfaty I. Shiloh Y. Rotman G. J. Biol. Chem. 2001; 276: 38224-38230Abstract Full Text Full Text PDF PubMed Google Scholar) for 2 h at 4 °C followed by incubation with protein A/G-Sepharose beads for an additional 2 h. The beads were washed repeatedly with lysis buffer, once with high salt buffer, and twice with kinase buffer. The beads were then incubated in a kinase mix (20 μl of kinase buffer, 500 ng of recombinant H2AX (purified from bacteria), 2 μl of 100 μm ATP, and 10 μCi of γ[32P]ATP) at 30 °C for 10 min. After SDS-polyacrylamide gel electrophoresis, the reaction products were visualized by autoradiography. Spontaneously immortalized fibroblasts were grown on chamber slides to about 70% confluence and then mock-irradiated or irradiated and incubated for 30 min. Cells were fixed in 4% paraformaldehyde for 10 min, permeabilized for 10 min in 0.2% Triton X-100, and blocked in 10% normal goat serum for 1 h at room temperature. The slides were incubated with anti-γ-H2AX antibody for 1 h, washed in phosphate-buffered saline, and incubated with Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (Molecular Probes, Eugene, OR) for 1 h at room temperature. Cells were washed in phosphate-buffered saline and mounted using Vectashield mounting medium with 4,6 diamidino-2-phenylindole (Vector Laboratory, Burlingame, CA). Fluorescence images were captured using an Olympus BH2 epifluorescent microscope equipped with a CCD camera and Cytovision software (Applied Imaging, Santa Clara, CA). To allow direct comparisons, all the cells were irradiated and processed simultaneously, and all the images were obtained using the same parameters (brightness, contrast, etc.). To examine H2AX phosphorylation in mouse cells, a rabbit polyclonal antibody (anti-γ-H2AX) was generated against a synthetic phosphorylated polypeptide consisting of the last nine amino acids of H2AX with phospho-Ser-139 (CKATQAS(PO4)QEY). The purified anti-γ-H2AX antibody reacted specifically with the immunizing polypeptide (phosphorylated at serine 139) but not with the unphosphorylated peptide (CKATQASQEY) (Fig. 1 a). Thus, the anti-γ-H2AX antibody is immunoreactive only with H2AX specifically phosphorylated at serine 139. Spontaneously immortalized wild type mouse fibroblasts were mock-irradiated or irradiated with x-rays and harvested after 30 min, and H2AX phosphorylation was analyzed by Western blotting of SDS extracts with anti-γ-H2AX antibody. We observed significant phosphorylation of histone H2AX in response to ionizing radiation (Fig. 1 b). The observed phosphorylation was specific to serine 139 as no signal was detected in irradiated samples when the immunizing polypeptide (phosphorylated at serine 139) was used as competitor in Western blotting (data not shown). Significant phosphorylation of H2AX was also observed after treatment of cells with the DSB-inducing agents neocarzinostatin, bleomycin, and etoposide. In contrast, there was no increase in γ-H2AX formation when these cells were irradiated with UV rays or treated with the DNA-alkylating agent methyl methanesulfonate confirming that H2AX is phosphorylated at serine 139 specifically in response to DNA double-strand breaks. Low levels of H2AX phosphorylation were also observed in cells treated with the DNA replication inhibitor hydroxyurea. This is probably because cells treated with hydroxyurea accumulate DSBs because of replication fork collapse (18Saintigny Y. Delacote F. Vares G. Petitot F. Lambert S. Averbeck D. Lopez B.S. EMBO J. 2001; 20: 3861-3870Crossref PubMed Scopus (259) Google Scholar, 19Skog S. Heiden T. Eriksson S. Wallström B. Tribukait B. Anticancer Drugs. 1992; PubMed Scopus Google Scholar). As the PI 3-kinases, DNA-PK, ATM, and ATR, have all been implicated in H2AX phosphorylation (2Modesti M. Kanaar R. Curr. Biol. 2001; 11: R229-R232Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 5Paull T.T. Rogakou E.P. Yamazaki V. Kirchgessner C.U. Gellert M. Bonner W.M. Curr. Biol. 2000; 10: 886-895Abstract Full Text Full Text PDF PubMed Scopus (1661) Google D. Jackson S.P. Curr. Opin. Cell Biol. 2001; 13: 225-231Crossref PubMed Scopus (443) Google Scholar, 11van Gent D.C. Hoeijmakers J.H. Kanaar R. Nat. Rev. Genet. 2001; 2: 196-206Crossref PubMed Scopus (938) Google Scholar), we to of these kinases a major role in the The PI 3-kinase inhibitor wortmannin the kinase of ATM and DNA-PK in cells with at concentrations of about 5 R.T. Res. 1998; Google Scholar). The kinase of ATR is to this with at concentrations than 100 Spontaneously immortalized wild type mouse fibroblasts were treated with concentrations of wortmannin for 30 min, irradiated with harvested after 30 min, and analyzed by Western We that H2AX phosphorylation was inhibited by low concentrations of wortmannin that ATM and/or DNA-PK, but not ATR, is involved in this (Fig. 1 Spontaneously immortalized fibroblasts from wild type, or Atm−/− mice were mock-irradiated or irradiated, harvested at time points ranging from 5 min to 8 h, and for H2AX phosphorylation by Western H2AX phosphorylation in wild type and DNA-PKcs−/− cells very rapidly (within 5 and for about 2 h, with levels of phosphorylation observed at 30 min (Fig. 2 a). In contrast, we observed minimal H2AX phosphorylation in irradiated Atm−/− cells. Although we observed H2AX phosphorylation in DNA-PKcs−/− cells at 30 min γ-H2AX formation in Atm−/− cells was reduced to about 5% of that in wild type cells (Fig. 2 that ATM is the major kinase responsible for H2AX phosphorylation upon DNA damage. Atm−/− fibroblasts were treated with concentrations of wortmannin for 30 min, irradiated with harvested after 30 min, and analyzed by Western We that the minimal H2AX phosphorylation in Atm−/− cells was abolished by low concentrations of wortmannin (Fig. 2 As ATR is inhibited by high concentrations of wortmannin R.T. Res. 1998; Google Scholar), results suggest that DNA-PK, rather than ATR, is responsible for low levels of γ-H2AX formation in the absence of ATM. is that other in the Atm−/− cell line used could also be responsible for the of H2AX phosphorylation in these cells. H2AX phosphorylation in a of early or or H2AX phosphorylation was observed in irradiated (Fig. In contrast, very low levels of γ-H2AX formation was observed in Atm−/− confirming that ATM is required for H2AX phosphorylation in response to significant in H2AX phosphorylation was observed irradiated and (Fig. To that ATM is required in for H2AX phosphorylation, the ATM cDNA expression vector pMAT1 (16Zhang N. Chen P. Khanna K.K. Scott S. Gatei M. Kozlov S. Watters D. Spring K. Yen T. Lavin M.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8021-8026Crossref PubMed Scopus (97) Google Scholar) was into Atm−/− spontaneously immortalized fibroblasts. The ectopic expression of ATM in the cells in of H2AX phosphorylation upon (Fig. 2 and the other cells with the vector no increase in γ-H2AX formation (Fig. The ATM expression and H2AX phosphorylation that ATM is required in for γ-H2AX formation in response to ionizing To ATM can phosphorylate H2AX in ATM was immunoprecipitated from spontaneously immortalized wild type fibroblasts using an anti-ATM monoclonal antibody against a peptide of ATM (15Andegeko Y. Moyal L. Mitelman L. Tsarfaty I. Shiloh Y. Rotman G. J. Biol. Chem. 2001; 276: 38224-38230Abstract Full Text Full Text PDF PubMed Google Scholar). The immunoprecipitated ATM phosphorylated recombinant H2AX in vitro (Fig. of cells in a significant increase in H2AX phosphorylation (Fig. no ATM protein or kinase was detected when was with normal mouse or from Atm−/− fibroblasts (Fig. The in vitro phosphorylation of H2AX by ATM that ATM could phosphorylate H2AX within the cell in response to DNA damage. H2AX phosphorylation in response to DNA damage results in the formation of discrete γ-H2AX foci at the sites of DNA double-strand breaks (4Rogakou E.P. Boon C. Redon C. Bonner W.M. J. Cell Biol. 1999; 146: 905-915Crossref PubMed Scopus (1931) Google Scholar). To the of γ-H2AX focus formation in wild type, and Atm−/− spontaneously immortalized fibroblasts, these cells were irradiated and to for 30 min before and with anti-γ-H2AX antibody. We observed γ-H2AX focus formation upon of wild type and DNA-PKcs−/− cells (Fig. In contrast, focus formation was very in Atm−/− cells confirming that ATM is required for γ-H2AX focus formation at the sites of H2AX is rapidly phosphorylated at serine 139 in response to DNA double-strand breaks (3Rogakou E.P. Pilch D.R. Orr A.H. Ivanova V.S. Bonner W.M. J. Biol. Chem. 1998; 273: 5858-5868Abstract Full Text Full Text PDF PubMed Scopus (4049) Google Scholar). The PI 3-kinases, DNA-PK, ATM, and ATR, have all been implicated in this (2Modesti M. Kanaar R. Curr. Biol. 2001; 11: R229-R232Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 5Paull T.T. Rogakou E.P. Yamazaki V. Kirchgessner C.U. Gellert M. Bonner W.M. Curr. Biol. 2000; 10: 886-895Abstract Full Text Full Text PDF PubMed Scopus (1661) Google Scholar, 10Durocher D. Jackson S.P. Curr. Opin. Cell Biol. 2001; 13: 225-231Crossref PubMed Scopus (443) Google Scholar, 11van Gent D.C. Hoeijmakers J.H. Kanaar R. Nat. Rev. Genet. 2001; 2: 196-206Crossref PubMed Scopus (938) Google Scholar). Although the of these kinases are in they have clearly in D. Jackson S.P. Curr. Opin. Cell Biol. 2001; 13: 225-231Crossref PubMed Scopus (443) Google Scholar). ATM phosphorylates and to cell DNA-PK is not required for of these processes S. A. G. Y. R. M. Crissman H.A. Ouyang H. Li G.C. Chen D.J. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, M.B. Lim D.S. Nat. Rev. Mol. Biol. 2000; PubMed Scopus Google Scholar). the other DNA-PK, ATM, be involved in the recruitment of and DNA to the sites of DSBs G.C. Jackson S.P. Genes Dev. 1999; 13: PubMed Scopus Google Scholar). is important to definitively the roles of these kinases in the phosphorylation of We that ATM can phosphorylate H2AX in vitro and that H2AX phosphorylation and γ-H2AX focus formation are severely compromised in Atm−/− cells. Ectopic expression of ATM this In contrast, these are normal in DNA-PKcs−/− cells. DNA-PK, but not ATR, be responsible for the minimal levels of H2AX phosphorylation in Atm−/− cells as this can be abolished by low concentrations of We also find that immunoprecipitated ATM can with recombinant H2AX in and J. and experiments be to examine formation H2AX and ATM in vivo. Our results establish that ATM is the major kinase responsible for histone H2AX phosphorylation in response to DNA double-strand breaks in fibroblasts. The reduced H2AX phosphorylation in M059J cells (5Paull T.T. Rogakou E.P. Yamazaki V. Kirchgessner C.U. Gellert M. Bonner W.M. Curr. Biol. 2000; 10: 886-895Abstract Full Text Full Text PDF PubMed Scopus (1661) Google Scholar) be because of the low levels of ATM in these cells B.S. Kirchgessner C.U. Radiat. Res. 2000; 153: PubMed Scopus Google Scholar, D.P. Chan Yen Mol. Biol. 1998; PubMed Scopus Google Scholar, D.W. Gately D.P. Urban S. Galloway A.M. Lees-Miller S.P. Yen T. Allalunis-Turner J. J. Radiat. Biol. 1998; PubMed Scopus Google Scholar) rather than because of the absence of ATM plays a role in the of in response to to repair of DNA of cell and cellular responses G. Shiloh Y. 1999; PubMed Scopus Google Scholar). As γ-H2AX focus formation is a very early within of DNA damage (3Rogakou E.P. Pilch D.R. Orr A.H. Ivanova V.S. Bonner W.M. J. Biol. Chem. 1998; 273: 5858-5868Abstract Full Text Full Text PDF PubMed Scopus (4049) Google Scholar), results indicate that ATM is one of the earliest kinases to be activated in the cellular response to DNA double-strand breaks. the important role of ATM in chromatin is a that ATM is also required for the of histone is to to chromatin decondensation Wang Y. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). Our results are with a that a of nuclear ATM with γ-H2AX at the sites of DSBs in response to DNA damage (15Andegeko Y. Moyal L. Mitelman L. Tsarfaty I. Shiloh Y. Rotman G. J. Biol. Chem. 2001; 276: 38224-38230Abstract Full Text Full Text PDF PubMed Google Scholar). A very was the of and of γ-H2AX foci and ATM This that DNA by ATM and H2AX phosphorylation occur very rapidly after DNA and at the same rate In the of this suggest that once ATM is activated at a it could phosphorylate histone H2AX at the of the break to the cell that a DSB has This very early could then the recruitment of DNA repair or damage-signaling factors to the break by chromatin and/or direct of these factors with We are to Bonner of for the anti-γ-H2AX antibody used in Shiloh for anti-ATM and Lavin of for We for and and for the

Analysis of transgenic mice expressing familial amyotrophic lateral sclerosis (ALS)-linked mutations in the enzyme superoxide dismutase (SOD1) have shown that motor neuron death arises from a mutant-mediated toxic property or properties. In testing the disease mechanism, both elimination and elevation of wild-type SOD1 were found to have no effect on mutant-mediated disease, which demonstrates that the use of SOD mimetics is unlikely to be an effective therapy and raises the question of whether toxicity arises from superoxide-mediated oxidative stress. Aggregates containing SOD1 were common to disease caused by different mutants, implying that coaggregation of an unidentified essential component or components or aberrant catalysis by misfolded mutants underlies a portion of mutant-mediated toxicity.

A 16S rRNA-based polymerase chain reaction (PCR) detection method was used to determine the prevalence of Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Campylobacter rectus, Eikenella corrodens, Porphyromonas gingivalis, Prevotella intermedia. Prevotella nigrescens and Treponema denticola in subgingival specimens of 50 advanced periodontitis, 50 adult gingivitis and 50 pediatric gingivitis subjects. The optimal PCR conditions were determined for each study species. Agarose gel electrophoresis of PCR products from each study species revealed a single band of the predicted size. Restriction enzyme digestion of amplicons confirmed the specificity of the amplification. PCR detection limits were in the range of 25-100 cells. No cross-reactivity with other oral micro-organisms or nonspecific amplification was observed. The prevalence by PCR in advanced periodontitis, adult gingivitis and pediatric gingivitis subjects was 30%, 14% and 14% for A. actinomycetemcomitans, 86%, 18% and 8% for B. forsythus, 74%, 52% and 78% for C. rectus, 80%, 70% and 66% for E. corrodens, 70%, 10% and 14% for P. gingivalis, 58%, 12% and 18% for P. intermedia, 52%, 20% and 22% for P. nigrescens, and 54%, 16% and 16% for T. denticola, respectively. The prevalence was higher in the advanced periodontitis group than in both adult gingivitis and pediatric gingivitis for A. actinomycetemcomitans, B. forsythus, P. gingivalis, P. intermedia, P. nigrescens and T. denticola at P < 0.01, and for E. corrodens at P < 0.05. The prevalence of C. rectus was significantly higher in the advanced periodontitis group than in the adult gingivitis group at P < 0.01. Matching results between PCR and culture occurred in 28% (B. forsythus) to 71% (A. actinomycetemcomitans) of the samples; the major discrepancy occurred in the PCR-positive/culture-negative category. Matching results between PCR and DNA probe methods were found in 84% of the subjects (B. forsythus) and 70% (P. gingivalis). Odds ratio analysis revealed statistically significant positive associations between 17 of the 28 possible combinations (P < 0.01). This study demonstrated the utility of a 16S rRNA-based PCR detection method for identifying important subgingival microorganisms. The results indicated a strong association between the study species and periodontitis. Several previously unreported symbiotic relationships were found between the 8 species tested.

Abstract In this study, we performed a greenhouse experiment to investigate the effect of cow manure biochar on maize yield, nutrient uptake and physico‐chemical properties of a dryland sandy soil. Biochar was derived from dry cow manure pyrolysed at 500 °C. Cow manure biochar was mixed with a sandy soil at the rate equivalent to 0, 10, 15 and 20 t biochar per hectare. Maize was used as a test crop. Results of the study indicated that cow manure biochar contains some important plant nutrients which significantly affected the maize crop growth. Maize yield and nutrient uptake were significantly improved with increasing the biochar mixing rate. Application of biochar at 15 and 20 t/ha mixing rates significantly increased maize grain yield by 150 and 98% as compared with the control, respectively. Maize net water use efficiency (WUE) increased by 6, 139 and 91% as compared with the control, with the 10, 15 and 20 t/ha mixing rate, respectively. Nutrient uptake by maize grain was significantly increased with higher biochar applications. Application of cow manure biochar improved the field‐saturated hydraulic conductivity of the sandy soil, as a result net WUE also increased. Results of the soil analysis after the harvesting indicated significant increase in the pH, total C, total N, Oslen‐P, exchangeable cations and cation exchange capacity. The results of this study indicated that application of cow manure biochar to sandy soil is not only beneficial for crop growth but it also significantly improved the physico‐chemical properties of the coarse soil.

An integrated human-mouse positional candidate approach was used to identify the gene responsible for the phenotypes observed in a mouse model of Niemann-Pick type C (NP-C) disease. The predicted murine NPC1 protein has sequence homology to the putative transmembrane domains of the Hedgehog signaling molecule Patched, to the cholesterol-sensing regions of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and SREBP cleavage-activating protein (SCAP), and to the NPC1 orthologs identified in human, the nematode Caenorhabditis elegans, and the yeast Saccharomyces cerevisiae. The mouse model may provide an important resource for studying the role of NPC1 in cholesterol homeostasis and neurodegeneration and for assessing the efficacy of new drugs for NP-C disease.

BACKGROUND: A retrospective review of treatment results comparing women with clear cell carcinoma of the ovary (CCC) with a group with serous adenocarcinoma of the ovary (SAC) was conducted. METHODS: Between 1988-1998, 662 patients with epithelial ovarian carcinoma were identified through the medical records department and the tumor registry at 4 institutions. After the central pathologic review, 101 patients with pure or dominant (>/= 90%) CCC (15.3%) were entered into the current study. Two hundred thirty five patients with pure SAC were selected as a group for comparison. All patients underwent staging laparotomy followed by platinum-based chemotherapy. Distribution of the International Federation of Gynecology and Obstetrics (FIGO) disease stage, response to chemotherapy, and prognosis for patients with CCC were compared with the same values in patients with SAC. RESULTS: Patients with CCC were significantly more likely to have FIGO Stage I disease than were patients with SAC (48.5% vs. 16.6%). A high recurrence rate was noted in those patients with Stage IC CCC (37%). In those patients with Stage IC disease, the survival rates for patients with CCC were lower than those for patients with SAC. The 3-year and 5-year survival rates for Stage III CCC patients were significantly lower compared with Stage III SAC patients. The response rate to platinum-based chemotherapy in patients with CCC was significantly lower than that in patients with SAC. CONCLUSIONS: CCC is an intriguing histologic type of epithelial ovarian cancer that demonstrates a clinical behavior distinctly different from that of SAC.

Some sintered oxides based on were found to exhibit appreciable proton conduction under hydrogen‐containing atmosphere at high temperature. The verification of proton conduction was made by studying the EMF of various gas cells using the specimen ceramics as the solid electrolyte. The protonic conductivity in the doped was higher than that in the proton conductor found previously by us. These materials could be applied to the solid electrolyte for a hydrogen fuel cell, a hydrogen pump, and a steam electrolyzer to produce hydrogen.

To gain a better understanding of the global application of soil erosion prediction models, we comprehensively reviewed relevant peer-reviewed research literature on soil-erosion modelling published between 1994 and 2017. We aimed to identify (i) the processes and models most frequently addressed in the literature, (ii) the regions within which models are primarily applied, (iii) the regions which remain unaddressed and why, and (iv) how frequently studies are conducted to validate/evaluate model outcomes relative to measured data. To perform this task, we combined the collective knowledge of 67 soil-erosion scientists from 25 countries. The resulting database, named 'Global Applications of Soil Erosion Modelling Tracker (GASEMT)', includes 3030 individual modelling records from 126 countries, encompassing all continents (except Antarctica). Out of the 8471 articles identified as potentially relevant, we reviewed 1697 appropriate articles and systematically evaluated and transferred 42 relevant attributes into the database. This GASEMT database provides comprehensive insights into the state-of-the-art of soil- erosion models and model applications worldwide. This database intends to support the upcoming country-based United Nations global soil-erosion assessment in addition to helping to inform soil erosion research priorities by building a foundation for future targeted, in-depth analyses. GASEMT is an open-source database available to the entire user-community to develop research, rectify errors, and make future expansions.

BACKGROUND: Up-regulation of hepatic delta-aminolevulinic acid synthase 1 (ALAS1), with resultant accumulation of delta-aminolevulinic acid (ALA) and porphobilinogen, is central to the pathogenesis of acute attacks and chronic symptoms in acute hepatic porphyria. Givosiran, an RNA interference therapy, inhibits ALAS1 expression. METHODS: In this double-blind, placebo-controlled, phase 3 trial, we randomly assigned symptomatic patients with acute hepatic porphyria to receive either subcutaneous givosiran (2.5 mg per kilogram of body weight) or placebo monthly for 6 months. The primary end point was the annualized rate of composite porphyria attacks among patients with acute intermittent porphyria, the most common subtype of acute hepatic porphyria. (Composite porphyria attacks resulted in hospitalization, an urgent health care visit, or intravenous administration of hemin at home.) Key secondary end points were levels of ALA and porphobilinogen and the annualized attack rate among patients with acute hepatic porphyria, along with hemin use and daily worst pain scores in patients with acute intermittent porphyria. RESULTS: A total of 94 patients underwent randomization (48 in the givosiran group and 46 in the placebo group). Among the 89 patients with acute intermittent porphyria, the mean annualized attack rate was 3.2 in the givosiran group and 12.5 in the placebo group, representing a 74% lower rate in the givosiran group (P<0.001); the results were similar among the 94 patients with acute hepatic porphyria. Among the patients with acute intermittent porphyria, givosiran led to lower levels of urinary ALA and porphobilinogen, fewer days of hemin use, and better daily scores for pain than placebo. Key adverse events that were observed more frequently in the givosiran group were elevations in serum aminotransferase levels, changes in serum creatinine levels and the estimated glomerular filtration rate, and injection-site reactions. CONCLUSIONS: Among patients with acute intermittent porphyria, those who received givosiran had a significantly lower rate of porphyria attacks and better results for multiple other disease manifestations than those who received placebo. The increased efficacy was accompanied by a higher frequency of hepatic and renal adverse events. (Funded by Alnylam Pharmaceuticals; ENVISION ClinicalTrials.gov number, NCT03338816.).

Nursing competency includes core abilities that are required for fulfilling one's role as a nurse. Therefore, it is important to clearly define nursing competency to establish a foundation for nursing education curriculum. However, while the concepts surrounding nursing competency are important for improving nursing quality, they are still not yet completely developed. Thus, challenges remain in establishing definitions and structures for nursing competency, competency levels necessary for nursing professionals, training methods and so on. In the present study, we reviewed the research on definitions and attributes of nursing competency in Japan as well as competency structure, its elements and evaluation. Furthermore, we investigated training methods to teach nursing competency.

The article contains sections titled: 1. Introduction 2. Molecular Structure and Conformation 2.1. Chitin 2.2. Chitosan 3. Raw Materials and Production 3.1. Isolation of Chitin from Crab and Shrimp Shells 3.2. Preparation of Chitosan from Chitin 4. Metabolism and Biosynthesis 5. Chemical Properties 5.1. Reactions on the Amino Group 5.1.1. N-Acylation 5.1.2. Formation of N-Alkylidene and N-Arylidene Derivatives 5.1.3. N-Alkylation and N-Arylation 5.2. Reactions at the Hydroxyl Group 5.3. Reactions at C-6 5.4. Graft Polymerization on Chitin and Chitosan 6. Application Forms and Formulations 7. Uses 8. Economic Aspects 9. Toxicology and Environmental Aspects

The heroin analogue 1-methyl-4-phenylpyridinium, MPP(+), both in vitro and in vivo, produces death of dopaminergic substantia nigral cells by inhibiting the mitochondrial NADH dehydrogenase multienzyme complex, producing a syndrome indistinguishable from Parkinson's disease. Similarly, a fragment of amyloid protein, Abeta(1-42), is lethal to hippocampal cells, producing recent memory deficits characteristic of Alzheimer's disease. Here we show that addition of 4 mM d-beta-hydroxybutyrate protected cultured mesencephalic neurons from MPP(+) toxicity and hippocampal neurons from Abeta(1-42) toxicity. Our previous work in heart showed that ketone bodies, normal metabolites, can correct defects in mitochondrial energy generation. The ability of ketone bodies to protect neurons in culture suggests that defects in mitochondrial energy generation contribute to the pathophysiology of both brain diseases. These findings further suggest that ketone bodies may play a therapeutic role in these most common forms of human neurodegeneration.

BACKGROUND: Varicella zoster virus (VZV) vasculopathy produces stroke secondary to viral infection of cerebral arteries. Not all patients have rash before cerebral ischemia or stroke. Furthermore, other vasculitides produce similar clinical features and comparable imaging, angiographic, and CSF abnormalities. METHODS: We review our 23 published cases and 7 unpublished cases of VZV vasculopathy. All CSFs were tested for VZV DNA by PCR and anti-VZV IgG antibody and were positive for either or both. RESULTS: Among 30 patients, rash occurred in 19 (63%), CSF pleocytosis in 20 (67%), and imaging abnormalities in 29 (97%). Angiography in 23 patients revealed abnormalities in 16 (70%). Large and small arteries were involved in 15 (50%), small arteries in 11 (37%), and large arteries in only 4 (13%) of 30 patients. Average time from rash to neurologic symptoms and signs was 4.1 months, and from neurologic symptoms and signs to CSF virologic analysis was 4.2 months. CSF of 9 (30%) patients contained VZV DNA while 28 (93%) had anti-VZV IgG antibody in CSF; in each of these patients, reduced serum/CSF ratio of VZV IgG confirmed intrathecal synthesis. CONCLUSIONS: Rash or CSF pleocytosis is not required to diagnose varicella zoster virus (VZV) vasculopathy, whereas MRI/CT abnormalities are seen in almost all patients. Most patients had mixed large and small artery involvement. Detection of anti-VZV IgG antibody in CSF was a more sensitive indicator of VZV vasculopathy than detection of VZV DNA (p < 0.001). Determination of optimal antiviral treatment and benefit of concurrent steroid therapy awaits studies with larger case numbers.

NF-kappaB is activated by various stimuli including inflammatory cytokines and stresses. A key step in the activation of NF-kappaB is the phosphorylation of its inhibitors, IkappaBs, by an IkappaB kinase (IKK) complex. Recently, two closely related kinases, designated IKKalpha and IKKbeta, have been identified to be the components of the IKK complex that phosphorylate critical serine residues of IkappaBs for degradation. A previously identified NF-kappaB-inducing kinase (NIK), which mediates NF-kappaB activation by TNFalpha and IL-1, has been demonstrated to activate IKKalpha. Previous studies showed that mitogen-activated protein kinase/ERK kinase kinase-1 (MEKK1), which constitutes the c-Jun N-terminal kinase/stress-activated protein kinase pathway, also activates NF-kappaB by an undefined mechanism. Here, we show that overexpression of MEKK1 preferentially stimulates the kinase activity of IKKbeta, which resulted in phosphorylation of IkappaBs. Moreover, a catalytically inactive mutant of IKKbeta blocked the MEKK1-induced NF-kappaB activation. By contrast, overexpression of NIK stimulates kinase activities of both IKKalpha and IKKbeta comparably, suggesting a qualitative difference between NIK- and MEKK1-mediated NF-kappaB activation pathways. Collectively, these results indicate that NIK and MEKK1 independently activate the IKK complex and that the kinase activities of IKKalpha and IKKbeta are differentially regulated by two upstream kinases, NIK and MEKK1, which are responsive to distinct stimuli.

The distribution of sialic acid (SA) species varies among animal species, but the biological role of this variation is largely unknown. Influenza viruses differ in their ability to recognize SA-galactose (Gal) linkages, depending on the animal hosts from which they are isolated. For example, human viruses preferentially recognize SA linked to Gal by the alpha2,6(SAalpha2,6Gal) linkage, while equine viruses favor SAalpha2,3Gal. However, whether a difference in relative abundance of specific SA species (N-acetylneuraminic acid [NeuAc] and N-glycolylneuraminic acid [NeuGc]) among different animals affects the replicative potential of influenza viruses is uncertain. We therefore examined the requirement for the hemagglutinin (HA) for support of viral replication in horses, using viruses whose HAs differ in receptor specificity. A virus with an HA recognizing NeuAcalpha2,6Gal but not NeuAcalpha2,3Gal or NeuGcalpha2,3Gal failed to replicate in horses, while one with an HA recognizing the NeuGcalpha2,3Gal moiety replicated in horses. Furthermore, biochemical and immunohistochemical analyses and a lectin-binding assay demonstrated the abundance of the NeuGcalpha2,3Gal moiety in epithelial cells of horse trachea, indicating that recognition of this moiety is critical for viral replication in horses. Thus, these results provide evidence of a biological effect of different SA species in different animals.

The effects of silicon application on the drought tolerance of sorghum ( Sorghum bicolor (L.) Moench) were investigated for two cultivars differing in drought susceptibility. Silicon application ameliorated the decrease in dry weight under drought stress conditions, but had no effect on dry matter production under wet conditions. Under dry conditions, silicon‐applied sorghum had a lower shoot to root (S/R) ratio, indicating the facilitation of root growth and the maintenance of the photosynthetic rate and stomatal conductance at a higher level compared with plants grown without silicon application. The diurnal determination of the transpiration rate indicated that the silicon‐applied sorghum could extract a larger amount of water from drier soil and maintain a higher stomatal conductance. Very similar effects of silicon application were observed for both cultivars regardless of their drought susceptibility. These results suggest that silicon application may be useful to improve the drought tolerance of sorghum via the enhancement of water uptake ability.

The usual dietary sources of vitamin B(12) are animal foods, meat, milk, egg, fish, and shellfish. As the intrinsic factor-mediated intestinal absorption system is estimated to be saturated at about 1.5-2.0 microg per meal under physiologic conditions, vitamin B(12) bioavailability significantly decreases with increasing intake of vitamin B(12) per meal. The bioavailability of vitamin B(12) in healthy humans from fish meat, sheep meat, and chicken meat averaged 42%, 56%-89%, and 61%-66%, respectively. Vitamin B(12) in eggs seems to be poorly absorbed (< 9%) relative to other animal food products. In the Dietary Reference Intakes in the United States and Japan, it is assumed that 50% of dietary vitamin B(12) is absorbed by healthy adults with normal gastro-intestinal function. Some plant foods, dried green and purple lavers (nori) contain substantial amounts of vitamin B(12), although other edible algae contained none or only traces of vitamin B(12). Most of the edible blue-green algae (cyanobacteria) used for human supplements predominantly contain pseudovitamin B(12), which is inactive in humans. The edible cyanobacteria are not suitable for use as vitamin B(12) sources, especially in vegans. Fortified breakfast cereals are a particularly valuable source of vitamin B(12) for vegans and elderly people. Production of some vitamin B(12)-enriched vegetables is also being devised.

), the total soil loss from the basin could be reduced by ca. 52%. Our methodological framework identified the potential risk for soil erosion in large-scale zones, and with a more sophisticated model and input data of higher spatial and temporal resolution, results could be specified locally within these risk zones. Accurate assessment of soil erosion in the UBNR basin would support sustainable use of the basin's land resources and possibly open up prospects for cooperation in the Eastern Nile region.