Instituto de Biología Molecular y Celular de Rosario

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,

Escherichia coli is one of the organisms of choice for the production of recombinant proteins. Its use as a cell factory is well-established and it has become the most popular expression platform. For this reason, there are many molecular tools and protocols at hand for the high-level production of heterologous proteins, such as a vast catalog of expression plasmids, a great number of engineered strains and many cultivation strategies. We review the different approaches for the synthesis of recombinant proteins in E. coli and discuss recent progress in this ever-growing field.

Considerable progress has been made in identifying the targets of plant microRNAs, many of which regulate the stability or translation of mRNAs that encode transcription factors involved in development. In most cases, it is unknown, however, which immediate transcriptional targets mediate downstream effects of the microRNA-regulated transcription factors. We identified a new process controlled by the miR319-regulated clade of TCP (TEOSINTE BRANCHED/CYCLOIDEA/PCF) transcription factor genes. In contrast to other miRNA targets, several of which modulate hormone responses, TCPs control biosynthesis of the hormone jasmonic acid. Furthermore, we demonstrate a previously unrecognized effect of TCPs on leaf senescence, a process in which jasmonic acid has been proposed to be a critical regulator. We propose that miR319-controlled TCP transcription factors coordinate two sequential processes in leaf development: leaf growth, which they negatively regulate, and leaf senescence, which they positively regulate.

In idiopathic Parkinson's disease, intracytoplasmic neuronal inclusions (Lewy bodies) containing aggregates of the protein alpha-synuclein (alphaS) are deposited in the pigmented nuclei of the brainstem. The mechanisms underlying the structural transition of innocuous, presumably natively unfolded, alphaS to neurotoxic forms are largely unknown. Using paramagnetic relaxation enhancement and NMR dipolar couplings, we show that monomeric alphaS assumes conformations that are stabilized by long-range interactions and act to inhibit oligomerization and aggregation. The autoinhibitory conformations fluctuate in the range of nanoseconds to micro-seconds corresponding to the time scale of secondary structure formation during folding. Polyamine binding and/or temperature increase, conditions that induce aggregation in vitro, release this inherent tertiary structure, leading to a completely unfolded conformation that associates readily. Stabilization of the native, autoinhibitory structure of alphaS constitutes a potential strategy for reducing or inhibiting oligomerization and aggregation in Parkinson's disease.

Cell proliferation is an important determinant of plant form, but little is known about how developmental programs control cell division. Here, we describe the role of microRNA miR396 in the coordination of cell proliferation in Arabidopsis leaves. In leaf primordia, miR396 is expressed at low levels that steadily increase during organ development. We found that miR396 antagonizes the expression pattern of its targets, the GROWTH-REGULATING FACTOR (GRF) transcription factors. miR396 accumulates preferentially in the distal part of young developing leaves, restricting the expression of GRF2 to the proximal part of the organ. This, in turn, coincides with the activity of the cell proliferation marker CYCLINB1;1. We show that miR396 attenuates cell proliferation in developing leaves, through the repression of GRF activity and a decrease in the expression of cell cycle genes. We observed that the balance between miR396 and the GRFs controls the final number of cells in leaves. Furthermore, overexpression of miR396 in a mutant lacking GRF-INTERACTING FACTOR 1 severely compromises the shoot meristem. We found that miR396 is expressed at low levels throughout the meristem, overlapping with the expression of its target, GRF2. In addition, we show that miR396 can regulate cell proliferation and the size of the meristem. Arabidopsis plants with an increased activity of the transcription factor TCP4, which reduces cell proliferation in leaves, have higher miR396 and lower GRF levels. These results implicate miR396 as a significant module in the regulation of cell proliferation in plants.

Microorganisms have the capacity to utilize a variety of nutrients and adapt to continuously changing environmental conditions. Many microorganisms, including yeast and bacteria, accumulate carbon and energy reserves to cope with the starvation conditions temporarily present in the environment. Glycogen biosynthesis is a main strategy for such metabolic storage, and a variety of sensing and signaling mechanisms have evolved in evolutionarily distant species to ensure the production of this homopolysaccharide. At the most fundamental level, the processes of glycogen synthesis and degradation in yeast and bacteria share certain broad similarities. However, the regulation of these processes is sometimes quite distinct, indicating that they have evolved separately to respond optimally to the habitat conditions of each species. This review aims to highlight the mechanisms, both at the transcriptional and at the post-transcriptional level, that regulate glycogen metabolism in yeast and bacteria, focusing on selected areas where the greatest increase in knowledge has occurred during the last few years. In the yeast system, we focus particularly on the various signaling pathways that control the activity of the enzymes of glycogen storage. We also discuss our recent understanding of the important role played by the vacuole in glycogen metabolism. In the case of bacterial glycogen, special emphasis is placed on aspects related to the genetic regulation of glycogen metabolism and its connection with other biological processes.

The aggregation of alpha-synuclein (AS) is characteristic of Parkinson's disease and other neurodegenerative synucleinopathies. We demonstrate here that Cu(II) ions are effective in accelerating AS aggregation at physiologically relevant concentrations without altering the resultant fibrillar structures. By using numerous spectroscopic techniques (absorption, CD, EPR, and NMR), we have located the primary binding for Cu(II) to a specific site in the N terminus, involving His-50 as the anchoring residue and other nitrogen/oxygen donor atoms in a square planar or distorted tetragonal geometry. The carboxylate-rich C terminus, originally thought to drive copper binding, is able to coordinate a second Cu(II) equivalent, albeit with a 300-fold reduced affinity. The NMR analysis of AS-Cu(II) complexes reveals the existence of conformational restrictions in the native state of the protein. The metallobiology of Cu(II) in Parkinson's disease is discussed by a comparative analysis with other Cu(II)-binding proteins involved in neurodegenerative disorders.

The aggregation of alpha-synuclein (AS) is characteristic of Parkinson's disease and other neurodegenerative synucleinopathies. Interactions with metal ions affect dramatically the kinetics of fibrillation of AS in vitro and are proposed to play a potential role in vivo. We recently showed that Cu(II) binds at the N-terminus of AS with high affinity (K(d) approximately 0.1 microM) and accelerates its fibrillation. In this work we investigated the binding features of the divalent metal ions Fe(II), Mn(II), Co(II), and Ni(II), and their effects on AS aggregation. By exploiting the different paramagnetic properties of these metal ions, NMR spectroscopy provides detailed information about the protein-metal interactions at the atomic level. The divalent metal ions bind preferentially and with low affinity (millimolar) to the C-terminus of AS, the primary binding site being the (119)DPDNEA(124) motif, in which Asp121 acts as the main anchoring residue. Combined with backbone residual dipolar coupling measurements, these results suggest that metal binding is not driven exclusively by electrostatic interactions but is mostly determined by the residual structure of the C-terminus of AS. A comparative analysis with Cu(II) revealed a hierarchal effect of AS-metal(II) interactions on AS aggregation kinetics, dictated by structural factors corresponding to different protein domains. These findings reveal a strong link between the specificity of AS-metal(II) interactions and the enhancement of aggregation of AS in vitro. The elucidation of the structural basis of AS metal binding specificity is then required to elucidate the mechanism and clarify the role of metal-protein interactions in the etiology of Parkinson's disease.

α-Synuclein (α-syn) phosphorylation at serine 129 is characteristic of Parkinson disease (PD) and related α-synulceinopathies. However, whether phosphorylation promotes or inhibits α-syn aggregation and neurotoxicity in vivo remains unknown. This understanding is critical for elucidating the role of α-syn in the pathogenesis of PD and for development of therapeutic strategies for PD. To better understand the structural and molecular consequences of Ser-129 phosphorylation, we compared the biochemical, structural, and membrane binding properties of wild type α-syn to those of the phosphorylation mimics (S129E, S129D) as well as of in vitro phosphorylated α-syn using a battery of biophysical techniques. Our results demonstrate that phosphorylation at Ser-129 increases the conformational flexibility of α-syn and inhibits its fibrillogenesis in vitro but does not perturb its membrane-bound conformation. In addition, we show that the phosphorylation mimics (S129E/D) do not reproduce the effect of phosphorylation on the structural and aggregation properties of α-syn in vitro. Our findings have significant implications for current strategies to elucidate the role of phosphorylation in modulating protein structure and function in health and disease and provide novel insight into the underlying mechanisms that govern α-syn aggregation and toxicity in PD and related α-synulceinopathies. α-Synuclein (α-syn) phosphorylation at serine 129 is characteristic of Parkinson disease (PD) and related α-synulceinopathies. However, whether phosphorylation promotes or inhibits α-syn aggregation and neurotoxicity in vivo remains unknown. This understanding is critical for elucidating the role of α-syn in the pathogenesis of PD and for development of therapeutic strategies for PD. To better understand the structural and molecular consequences of Ser-129 phosphorylation, we compared the biochemical, structural, and membrane binding properties of wild type α-syn to those of the phosphorylation mimics (S129E, S129D) as well as of in vitro phosphorylated α-syn using a battery of biophysical techniques. Our results demonstrate that phosphorylation at Ser-129 increases the conformational flexibility of α-syn and inhibits its fibrillogenesis in vitro but does not perturb its membrane-bound conformation. In addition, we show that the phosphorylation mimics (S129E/D) do not reproduce the effect of phosphorylation on the structural and aggregation properties of α-syn in vitro. Our findings have significant implications for current strategies to elucidate the role of phosphorylation in modulating protein structure and function in health and disease and provide novel insight into the underlying mechanisms that govern α-syn aggregation and toxicity in PD and related α-synulceinopathies. Mounting evidence from pathologic, genetic, animal model, biochemical, and biophysical studies support the hypothesis that α-synuclein (α-syn) 3The abbreviations used are: α-syn, α-synuclein; PD, Parkinson disease; SEC, size exclusion chromatography; ThT, thioflavin T; TEM, transmission electron microscopy; MTSL, 1-oxy-2, 2, 5, 5-tetramethyl-d-pyrroline-3-methyl)-methanethiosulfonate; CK, casein kinase; POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt); MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; HSQC, heteronuclear single quantum coherence; NOE, nuclear Overhauser effect; LB, Lewy body; WT, wild type; HPLC, high performance liquid chromatography; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; PBS, phosphate-buffered saline. plays a central role in the pathogenesis of Parkinson disease (PD) and several other neurodegenerative diseases, including Alzheimer disease, multiple system atrophy, dementia with Lewy bodies, Down syndrome, and neurodegeneration with brain iron accumulation, collectively referred to as “synucleinopathies” (1Trojanowski J.Q. Lee V.M. Ann. N. Y. Acad. Sci. 2003; 991: 107-110Crossref PubMed Scopus (130) Google Scholar). Although the exact function of α-syn remains poorly understood, it is thought to play a role in regulating dopamine neurotransmission (2Abeliovich A. Schmitz Y. Farinas I. Choi-Lundberg D. Ho W.H. Castillo P.E. Shinsky N. Verdugo J.M. Armanini M. Ryan A. Hynes M. Phillips H. Sulzer D. Rosenthal A. Neuron. 2000; 25: 239-252Abstract Full Text Full Text PDF PubMed Scopus (1403) Google Scholar), vesicular trafficking (3Cooper A.A. Gitler A.D. Cashikar A. Haynes C.M. Hill K.J. Bhullar B. Liu K. Xu K. Strathearn K.E. Liu F. Cao S. Caldwell K.A. Caldwell G.A. Marsischky G. Kolodner R.D. Labaer J. Rochet J.C. Bonini N.M. Lindquist S. Science. 2006; 313: 324-328Crossref PubMed Scopus (1080) Google Scholar, 4Outeiro T.F. Lindquist S. Science. 2003; 302: 1772-1775Crossref PubMed Scopus (624) Google Scholar), and modulating synaptic function and plasticity (5Kahle P.J. Neumann M. Ozmen L. Haass C. Ann. N. Y. Acad. Sci. 2000; 920: 33-41Crossref PubMed Scopus (83) Google Scholar, 6George J.M. Jin H. Woods W.S. Clayton D.F. Neuron. 1995; 15: 361-372Abstract Full Text PDF PubMed Scopus (730) Google Scholar). Increasing evidence suggests that phosphorylation may be an important regulator of α-syn oligomerization, fibrillogenesis, Lewy body (LB) formation, and neurotoxicity in vivo (7Chen L. Feany M.B. Nat. Neurosci. 2005; 8: 657-663Crossref PubMed Scopus (525) Google Scholar). Immunohistochemical and biochemical studies suggest that the majority of α-syn within LBs from patients with PD and related synucleinopathies (8Fujiwara H. Hasegawa M. Dohmae N. Kawashima A. Masliah E. Goldberg M.S. Shen J. Takio K. Iwatsubo T. Nat. Cell Biol. 2002; 4: 160-164Crossref PubMed Scopus (159) Google Scholar, 9Anderson J.P. Walker D.E. Goldstein J.M. de Laat R. Banducci K. Caccavello R.J. Barbour R. Huang J. Kling K. Lee M. Diep L. Keim P.S. Shen X. Chataway T. Schlossmacher M.G. Seubert P. Schenk D. Sinha S. Gai W.P. Chilcote T.J. J. Biol. Chem. 2006; 281: 29739-29752Abstract Full Text Full Text PDF PubMed Scopus (912) Google Scholar, 10Kahle P.J. Neumann M. Ozmen L. Muller V. Jacobsen H. Spooren W. Fuss B. Mallon B. Macklin W.B. Fujiwara H. Hasegawa M. Iwatsubo T. Kretzschmar H.A. Haass C. EMBO Rep. 2002; 3: 583-588Crossref PubMed Scopus (264) Google Scholar, 11Takahashi M. Kanuka H. Fujiwara H. Koyama A. Hasegawa M. Miura M. Iwatsubo T. Neurosci. Lett. 2003; 336: 155-158Crossref PubMed Scopus (116) Google Scholar, 12Hasegawa M. Fujiwara H. Nonaka T. Wakabayashi K. Takahashi H. Lee V.M. Trojanowski J.Q. Mann D. Iwatsubo T. J. Biol. Chem. 2002; 277: 49071-49076Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar) is phosphorylated at Ser-129 (Ser(P)-129). Proteinaceous inclusions formed in cellular and animal models overexpressing WT or mutant α-syn can also be stained with an antibody against Ser(P)-129. A study by Fujiwara et al. (8Fujiwara H. Hasegawa M. Dohmae N. Kawashima A. Masliah E. Goldberg M.S. Shen J. Takio K. Iwatsubo T. Nat. Cell Biol. 2002; 4: 160-164Crossref PubMed Scopus (159) Google Scholar) reported that in vitro phosphorylated α-syn (at Ser-129, using casein kinase II (CK2)) forms fibrils more readily than unmodified α-syn. Phosphorylation at Ser-129 was also reported to promote the formation of cytoplasmic inclusions in some cell culture models of synucleinopathies (13Smith W.W. Margolis R.L. Li X. Troncoso J.C. Lee M.K. Dawson V.L. Dawson T.M. Iwatsubo T. Ross C.A. J. Neurosci. 2005; 25: 5544-5552Crossref PubMed Scopus (211) Google Scholar). Together, these findings suggested that phosphorylation at Ser-129 plays an important role in modulating α-syn aggregation, LB formation, and toxicity in vivo. However, in vivo studies by Feany and co-worker (7Chen L. Feany M.B. Nat. Neurosci. 2005; 8: 657-663Crossref PubMed Scopus (525) Google Scholar) suggest a lack of correlation between phosphorylation at Ser-129 and the level of α-synfibrillation. Overexpression of the phosphomimic S129D or coexpression of WT α-syn and G protein-coupled receptor kinase 2 (Gprk2), which phosphorylates α-syn specifically at Ser-129, in the Drosophila model of PD results in increased α-syn toxicity without an increase in the number of α-syn inclusions (compared with overexpression of WT α-syn). Interestingly, overexpression of S129A results in a significant increase (4×) in the number of inclusions and suppression of dopaminergic neuronal loss produced by expression of WT human α-syn. We considered that a rigorous examination and comparison of the biochemical and biophysical properties of phosphorylation and and WT α-syn may the as well as the molecular mechanisms by which phosphorylation at Ser-129 may α-syn aggregation and toxicity in vivo. In to the phosphorylation we and the in vitro phosphorylated of α-syn. To better understand the role of the in modulating α-syn aggregation and membrane binding we compared the structural, oligomerization, and membrane binding properties of WT α-syn to those of the phosphorylation mimics (S129E, S129D) as well as the in vitro phosphorylated of α-syn using size exclusion thioflavin and transmission electron results demonstrate that phosphorylation at Ser-129 inhibits than promotes α-syn formation in vitro. important is that the phosphorylation mimics (S129E/D) do not reproduce the effect of phosphorylation at on α-syn structure and aggregation properties in vitro. findings have significant implications for in vivo and understanding of the role of α-syn in the pathogenesis of PD and related and of and α-syn using and by used in these studies as J.C. Rochet J.C. 2003; PubMed Scopus Google Scholar). the expression and of and α-syn as R. D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, W. T. D. G. T.M. V. J. Biol. 2002; PubMed Scopus Google Scholar). To of a a single was into α-syn at for with the mutant was 1-oxy-2, 2, 5, to with α-syn and the was as W. C. T.M. M. Acad. Sci. S. A. 2005; PubMed Scopus Google Scholar). In Phosphorylation of or mutant α-syn was phosphorylated by at a of was in the of with the and of of α-syn. phosphorylation was at for the and the was with of the was by and the effect of phosphorylation on the aggregation of α-syn, WT α-syn was phosphorylated for at and the was with the to at with for the to the but was not studies the in at a of and formation was by using an as H.A. J. M. R.J. T. J. Biol. 2002; PubMed Scopus Google Scholar). and of from of α-syn was by the of and α-syn in at the the aggregation at the from a α-syn at at for at to was from the and on an system with a and using an was also on and stained with to the of the protein and in and on stained with or to the the membrane and the membrane was with in for at membrane was with the antibody (211) at a of or at a of or α-syn Ser-129 at a of or α-syn at a of at for the membrane was with the antibody from at for was with and with and in a at a of WT or mutant α-syn on at a of with 2 of and stained with 2 of on a electron at with a the phosphorylation and of the phosphorylated of α-syn or was using on a with a and using an or or A was and the was at In the of the HPLC, a was of and and (sodium was in which was by and was with to a of To increase the of formation, of in and at by a membrane to the at and used within and of α-syn in or was with the of to a of of was for 2 at structure of α-syn was by using a of 2 for at in a of α-syn in or and from was from and and from and from of for the A was for the molecular a of in and a in A was on the using a with of was with of and of was on of the and to and with a laser by of the to the that on the of and was with the and to the of α-syn WT or mutant α-syn in at on and was to in the and using F. S. G. J. A. J. 1995; PubMed Scopus Google Scholar) and D. and D. G. of and J. 4: PubMed Scopus Google Scholar). to and using the of for the of mutant and phosphorylated by in the heteronuclear single quantum of the WT protein to the in by and for and to in WT α-syn, phosphorylated α-syn, and S129D α-syn using on WT and mutant α-syn in and as an and V. C.M. PubMed Scopus Google Scholar). as a function of the V. C.M. PubMed Scopus Google Scholar, C.M. J. Scopus Google Scholar). was at was increased from to in a with a of to the of the and to using of α-syn from the of α-syn and and the of C.M. J. Scopus Google Scholar). in from WT and phosphorylated α-syn, from the of between in the and of the for on α-syn on α-syn. or was in in with on a at a of and a of for of and the structural and between and suggest that type of a to phosphorylation at a at in the in a of in to the single by To whether a of Ser-129 by an is to the effect of phosphorylation, we in which Ser-129 was by or To provide insight into the in vivo studies with the phosphorylation the S129A was also and S129A within the human of by and the mutant in and to than as by and To whether of Ser-129 into a is to reproduce the effect of phosphorylation on the structural and aggregation properties of α-syn in we the α-syn using We that phosphorylates α-syn more than casein kinase II To phosphorylation at the mutant of α-syn was and phosphorylated in vitro to α-syn To that phosphorylation by at Ser-129, we of the antibody can be as as the of not and increases with and of elucidate the consequences of phosphorylation on the structure and of α-syn, we a of and high in of WT and α-syn, and at the and a of a high of in WT and α-syn in the in of the phosphorylation of WT α-syn by the of and Ser-129 at the in the of the the in the in which of phosphorylated and and the to and α-syn, phosphorylation at was that the of Ser-129 was at its and at its phosphorylated by phosphorylation that the of to by phosphorylation of Ser-129 in α-syn. In addition, for the in α-syn, in with studies H. E. Y. M. T. E. T. Iwatsubo T. Hasegawa M. K. PubMed Scopus Google Scholar). to the of the protein We in WT α-syn and phosphorylated α-syn. to and for WT and phosphorylated not that phosphorylation effect on the structure of α-syn. of WT, and α-syn and phosphorylated WT and α-syn and with a structure and Phosphorylation the of by of the of a the a can be that an of the of a WT α-syn, we a of the of it increased to α-syn, the and α-syn a a of be WT α-syn was its increased by to phosphorylation of α-syn at Ser-129 increased by to of to phosphorylated α-syn increased the to that phosphorylation the of by α-syn to its Phosphorylation the effect of phosphorylation on we of between a specifically and than of of an increase in D. J. Biol. PubMed Scopus Google Scholar). This effect an on the the of between the and the in H. S. 25: PubMed Scopus Google Scholar). the of α-syn was into a to provide an for the the of the the of the the or the of aggregation for α-syn W. C. T.M. M. Acad. Sci. S. A. 2005; PubMed Scopus Google Scholar). the for WT and phosphorylated α-syn. In WT α-syn, the of a effect to and with in with W. C. T.M. M. Acad. Sci. S. A. 2005; PubMed Scopus Google Scholar). In phosphorylated α-syn, the was to the of the for and for to To the of phosphorylation at Ser-129 on we in WT and α-syn. of the of WT and α-syn, heteronuclear R. D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). and as R. D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Phosphorylation at Ser-129 not in the that of the of α-syn that to of the on the to by phosphorylation not the of into a can the effect of phosphorylation on the of α-syn, we S129D and α-syn by of α-syn was to that for the WT to the of the and and we of and for S129D and α-syn to WT protein but than that for phosphorylated α-syn of Ser-129 into or does not the that WT α-syn. Phosphorylation at Ser-129 the of of Ser-129 phosphorylation on the structure of membrane-bound α-syn, of α-syn compared with of protein A and of the protein is thought to that of the membrane-bound of the protein D. E. R. G. J. Biol. PubMed Scopus Google Scholar). the of phosphorylation, that Ser-129 phosphorylation does not to structural of of the protein In the between the and the binding D. E. R. G. J. Biol. PubMed Scopus Google Scholar, R. D. J. Biol. 2003; PubMed Scopus (345) Google Scholar, A. R.L. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar), and of Ser-129 phosphorylation is some that membrane binding the of the of α-syn A. R.L. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, S. M.G. H. V. J.M. V. 2006; PubMed Scopus Google Scholar), and a of an effect by Ser-129 phosphorylation be on these is to a to binding to To the effect of phosphorylation at Ser-129 on we the structure of the in the and of by In we that phosphorylation does not perturb the of and its to binding to studies to the effect of phosphorylation on membrane binding by multiple in as the the effect of the on the aggregation properties of α-syn, we compared the aggregation of and S129A to that of the WT protein as a function of using the binding and In a the formed more fibrils than WT protein but these not and in other in formation between the and In we that the S129A more and forms more fibrils than the WT and the phosphorylation S129A and α-syn formed fibrils with to that of WT α-syn Phosphorylation the effect of in vitro phosphorylation on α-syn formation, WT α-syn was with in the at for which the to and formation was by and and compared with that to the in the of the We that phosphorylation inhibits α-syn formation, of as from the and studies To the and we also the of α-syn at using by the of the to the that the of α-syn in to phosphorylation remains of formation the of α-syn with with the and In we not of that can be by SEC, we the of in the Phosphorylation at Ser-129 to the of studies that phosphorylates α-syn at and Ser-129 M. J. Koyama A. S. M. Iwatsubo T. L. P.J. Haass C. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, A. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). To the of phosphorylation at Ser-129 to the of α-syn formation, we the effect of phosphorylation on the of mutant of α-syn. of with results in significant of α-syn to the forms of the of formed significant of the of α-syn 5, A and with the and the of the of the aggregation the of with To demonstrate that of α-syn fibrillogenesis is to phosphorylation at Ser-129, we the phosphorylated of and compared its aggregation properties to that of and WT α-syn. WT, and α-syn to aggregation at with and not fibrils and in a of the and WT α-syn high of as by the increased and the significant in of with and of by However, of in the formation of that from those formed by and WT α-syn the of in a that of α-syn is thought to be phosphorylated at Y. Iwatsubo T. Hasegawa M. Lett. PubMed Scopus Google Scholar), we to whether the forms and of α-syn and the consequences of on the aggregation of α-syn. In the of of we not significant in the of However, in the of we significant of α-syn as by TEM, ThT, and and aggregation of of α-syn was by and of aggregation, of α-syn to level as at findings suggest that of aggregation by is Phosphorylation of α-syn at Ser-129 in the pathogenesis of PD and related However, the exact mechanisms by which phosphorylation the and properties of α-syn in vivo unknown. To understand the structural underlying the effect of phosphorylation on the and properties of α-syn, we the effect of phosphorylation at Ser-129 on the conformational and properties of α-syn. Phosphorylation at Ser-129 in studies that α-syn that by and to and aggregation W. C. T.M. M. Acad. Sci. S. A. 2005; PubMed Scopus Google Scholar, K. J. M. C.M. J. Chem. 2005; PubMed Scopus Google Scholar). binding and that aggregation in structure W. C. T.M. M. Acad. Sci. S. A. 2005; PubMed Scopus Google Scholar). also that high of the of α-syn, to an we the effect of phosphorylation on the of α-syn. by that phosphorylation of α-syn at Ser-129 increased the of α-syn In addition, of and that phosphorylation at Ser-129 phosphorylation the of by α-syn in may its as well as function of α-syn. Ser-129 is the of α-syn that is to its with phosphorylation at these is not to α-syn membrane Phosphorylation at Ser-129 not the binding of α-syn to it with the formation of structure as by the the of the protein in its is also by Ser-129 phosphorylation, with to the of the phosphorylation within the and not into the binding This that of Ser-129 phosphorylation on the function of α-syn, which is to be with its membrane-bound is to be by the of the on the the of Phosphorylation at and used to the structural and consequences of protein phosphorylation, a comparison between the phosphorylation mimics and the phosphorylated of the protein is Our in vitro studies demonstrate that the and S129D do not reproduce the effect of phosphorylation on the structural and aggregation properties of α-syn in vitro. of Ser-129 into or not to an of α-syn and the and of to the of that of or to or the effect of phosphorylation on the structure and of α-syn and other Together, these findings the critical of the increased of the to the of and in the structural and consequences of studies have reported that by serine is by In some of these studies it was that the N. B. M. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), or conformational S. Acad. Sci. S. A. PubMed Scopus Google Scholar) by the than its is for the of can that phosphorylation of α-syn may an important and regulator of α-syn aggregation and with other have important implications for of to elucidate the role of phosphorylation in modulating in vivo and the of results using and for molecular understanding of the consequences of phosphorylation may insight into the of α-syn and the mechanisms by which it to neurodegeneration in PD and related to the effect of phosphorylation on α-syn aggregation and toxicity was by Feany and co-worker (7Chen L. Feany M.B. Nat. Neurosci. 2005; 8: 657-663Crossref PubMed Scopus (525) Google Scholar) in a Drosophila model of PD overexpressing WT, or Interestingly, the aggregation properties of the and and WT α-syn in vivo to we in with WT and the number of S129A forms more (4×) than WT and S129D mutant toxicity and of α-syn expression of the S129A mutant dopaminergic cell loss and increased the of α-syn formed to the WT or S129D for these is that may the formation and of of α-syn than of which may in be However, in vitro studies not with a we that Ser-129 phosphorylation inhibits formation and also the of α-syn to In to modulating the and fibrillogenesis of α-syn, phosphorylation may be in regulating its properties by modulating its with other neuronal synaptic and α-syn with the protein and phosphorylation H. M.S. P. J. R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and fibrillogenesis M. M. L. D. Lee J.M. C. P. B. 2005; PubMed Scopus Google Scholar) in vitro and in vivo. binding was to the of α-syn. Phosphorylation at Ser-129, but not was also reported to the of α-syn Hill J. A.A. S. V.L. J. Cell Sci. PubMed Scopus Google Scholar). Together, these findings suggest that phosphorylation within the or may be in regulating the with and other neuronal G. M. K. Lee E. Lee J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), A. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, R.J. Woods W.S. J.M. J. Biol. PubMed Scopus Google Scholar), N. L. M. N. P. J. B. J. Neurosci. PubMed Google Scholar), (3Cooper A.A. Gitler A.D. Cashikar A. Haynes C.M. Hill K.J. Bhullar B. Liu K. Xu K. Strathearn K.E. Liu F. Cao S. Caldwell K.A. Caldwell G.A. Marsischky G. Kolodner R.D. Labaer J. Rochet J.C. Bonini N.M. Lindquist S. Science. 2006; 313: 324-328Crossref PubMed Scopus (1080) Google Scholar), and Phosphorylation within the or or the of phosphorylation at these also membrane binding and of M. J. Koyama A. S. M. Iwatsubo T. L. P.J. Haass C. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, A. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, R.J. Woods W.S. J.M. J. Biol. PubMed Scopus Google Scholar), an in the of and vesicular Although results demonstrate that α-syn is of binding to in an (compared with the WT we have not the of phosphorylation on membrane which may be Phosphorylation of or LB findings that α-syn is of the of α-syn within of the fibrils formed by is from those formed by the protein the consequences of in structure or not studies to the molecular and cellular that the aggregation of phosphorylated α-syn and whether phosphorylation aggregation or and LB studies have that α-syn with several to α-syn at including K. Lee G. J. Sci. Full Text Full Text PDF PubMed Scopus Google Scholar) and S. M. S. H. M. Koyama S. H. H. T. K. K. M. M. T. S. K. H. C. E. S. T. T. Y. K. T. K. M. I. K. K. Iwatsubo T. M. H. T. J. Neurosci. 2006; PubMed Scopus Google Scholar) in findings with the that the Ser-129 remains in the forms and of α-syn support the hypothesis that phosphorylation of α-syn also within LBs and is not a for α-syn and LB formation in PD. Together, findings suggest that phosphorylation may an important regulator of α-syn aggregation and the of the and in regulating α-syn phosphorylation in for therapeutic strategies for PD and related have as therapeutic for neurodegenerative diseases, including Alzheimer disease M. J. J. 2006; PubMed Scopus Google Scholar), and to be the of of the of and on the development of kinase with

The dried flower heads of Matricaria recutita L. (Asteraceae) are used in folk medicine to prepare a spasmolytic and sedative tea. Our fractionation of the aqueous extract of this plant led to the detection of several fractions with significant affinity for the central benzodiazepine receptor and to the isolation and identification of 5,7,4'-trihydroxyflavone (apigenin) in one of them. Apigenin competitively inhibited the binding of flunitrazepam with a Ki of 4 microM and had no effect on muscarinic receptors, alpha 1-adrenoceptors, and on the binding of muscimol to GABAA receptors. Apigenin had a clear anxiolytic activity in mice in the elevated plusmaze without evidencing sedation or muscle relaxant effects at doses similar to those used for classical benzodiazepines and no anticonvulsant action was detected. However, a 10-fold increase in dosage produced a mild sedative effect since a 26% reduction in ambulatory locomotor activity and a 35% decrement in hole-board parameters were evident. The results reported in this paper demonstrate that apigenin is a ligand for the central benzodiazepine receptors exerting anxiolytic and slight sedative effects but not being anticonvulsant or myorelaxant.

The production of proteins in sufficient amounts is key for their study or use as biotherapeutic agents. Escherichia coli is the host of choice for recombinant protein production given its fast growth, easy manipulation, and cost-effectiveness. As such, its protein production capabilities are continuously being improved. Also, the associated tools (such as plasmids and cultivation conditions) are subject of ongoing research to optimize product yield. In this work, we review the latest advances in recombinant protein production in E. coli.

The growth-regulating factors (GRFs) are plant-specific transcription factors. They form complexes with GRF-interacting factors (GIFs), a small family of transcriptional co-activators. In Arabidopsis thaliana, seven out of the nine GRFs are controlled by microRNA miR396. Analysis of Arabidopsis plants carrying a GRF3 allele insensitive to miR396 revealed a strong boost in the number of cells in leaves, which was further enhanced synergistically by an additional increase of GIF1 levels. Genetic experiments revealed that GRF3 can still increase cell number in gif1 mutants, albeit to a much lesser extent. Genome-wide transcript profiling indicated that the simultaneous increase of GRF3 and GIF1 levels causes additional effects in gene expression compared to either of the transgenes alone. We observed that GIF1 interacts in vivo with GRF3, as well as with chromatin-remodeling complexes, providing a mechanistic explanation for the synergistic activities of a GRF3-GIF1 complex. Interestingly, we found that, in addition to the leaf size, the GRF system also affects the organ longevity. Genetic and molecular analysis revealed that the functions of GRFs in leaf growth and senescence can be uncoupled, demonstrating that the miR396-GRF-GIF network impinges on different stages of leaf development. Our results integrate the post-transcriptional control of the GRF transcription factors with the progression of leaf development.

Sexually reproducing animals require an orchestrated communication between spermatozoa and the egg to generate a new individual. Capacitation, a maturational complex phenomenon that occurs in the female reproductive tract, renders spermatozoa capable of binding and fusing with the oocyte, and it is a requirement for mammalian fertilization. Capacitation encompasses plasma membrane reorganization, ion permeability regulation, cholesterol loss and changes in the phosphorylation state of many proteins. Novel tools to study sperm ion channels, image intracellular ionic changes and proteins with better spatial and temporal resolution, are unraveling how modifications in sperm ion transport and phosphorylation states lead to capacitation. Recent evidence indicates that two parallel pathways regulate phosphorylation events leading to capacitation, one of them requiring activation of protein kinase A and the second one involving inactivation of ser/thr phosphatases. This review examines the involvement of ion transporters and phosphorylation signaling processes needed for spermatozoa to achieve capacitation. Understanding the molecular mechanisms leading to fertilization is central for societies to deal with rising male infertility rates, to develop safe male gamete-based contraceptives and to preserve biodiversity through better assisted fertilization strategies.

Increasing evidence suggests that phosphorylation may play an important role in the oligomerization, fibrillogenesis, Lewy body (LB) formation, and neurotoxicity of alpha-synuclein (alpha-syn) in Parkinson disease. Herein we demonstrate that alpha-syn is phosphorylated at S87 in vivo and within LBs. The levels of S87-P are increased in brains of transgenic (TG) models of synucleinopathies and human brains from Alzheimer disease (AD), LB disease (LBD), and multiple system atrophy (MSA) patients. Using antibodies against phosphorylated alpha-syn (S129-P and S87-P), a significant amount of immunoreactivity was detected in the membrane in the LBD, MSA, and AD cases but not in normal controls. In brain homogenates from diseased human brains and TG animals, the majority of S87-P alpha-syn was detected in the membrane fractions. A battery of biophysical methods were used to dissect the effect of S87 phosphorylation on the structure, aggregation, and membrane-binding properties of monomeric alpha-syn. These studies demonstrated that phosphorylation at S87 expands the structure of alpha-syn, increases its conformational flexibility, and blocks its fibrillization in vitro. Furthermore, phosphorylation at S87, but not S129, results in significant reduction of alpha-syn binding to membranes. Together, our findings provide novel mechanistic insight into the role of phosphorylation at S87 and S129 in the pathogenesis of synucleinopathies and potential roles of phosphorylation in alpha-syn normal biology.

Bacteria can encounter a wide range of environments and must adapt to new conditions in order to survive. As the selective barrier between living cells and their environment, the plasma membrane plays a key role in cell viability. The barrier function of the cytoplasmic membrane is known to depend

BACKGROUND: Development of eukaryotic organisms is controlled by transcription factors that trigger specific and global changes in gene expression programs. In plants, MADS-domain transcription factors act as master regulators of developmental switches and organ specification. However, the mechanisms by which these factors dynamically regulate the expression of their target genes at different developmental stages are still poorly understood. RESULTS: We characterized the relationship of chromatin accessibility, gene expression, and DNA binding of two MADS-domain proteins at different stages of Arabidopsis flower development. Dynamic changes in APETALA1 and SEPALLATA3 DNA binding correlated with changes in gene expression, and many of the target genes could be associated with the developmental stage in which they are transcriptionally controlled. We also observe dynamic changes in chromatin accessibility during flower development. Remarkably, DNA binding of APETALA1 and SEPALLATA3 is largely independent of the accessibility status of their binding regions and it can precede increases in DNA accessibility. These results suggest that APETALA1 and SEPALLATA3 may modulate chromatin accessibility, thereby facilitating access of other transcriptional regulators to their target genes. CONCLUSIONS: Our findings indicate that different homeotic factors regulate partly overlapping, yet also distinctive sets of target genes in a partly stage-specific fashion. By combining the information from DNA-binding and gene expression data, we are able to propose models of stage-specific regulatory interactions, thereby addressing dynamics of regulatory networks throughout flower development. Furthermore, MADS-domain TFs may regulate gene expression by alternative strategies, one of which is modulation of chromatin accessibility.

ABSTRACT Based upon knowledge of the hydrolytic profile of major β-lactamases found in Gram-negative bacteria, we tested the efficacy of the combination of ceftazidime-avibactam (CAZ-AVI) with aztreonam (ATM) against carbapenem-resistant enteric bacteria possessing metallo-β-lactamases (MBLs). Disk diffusion and agar-based antimicrobial susceptibility testing were initially performed to determine the in vitro efficacy of a unique combination of CAZ-AVI and ATM against 21 representative Enterobacteriaceae isolates with a complex molecular background that included bla IMP , bla NDM , bla OXA-48 , bla CTX-M , bla AmpC , and combinations thereof. Time-kill assays were conducted, and the in vivo efficacy of this combination was assessed in a murine neutropenic thigh infection model. By disk diffusion assay, all 21 isolates were resistant to CAZ-AVI alone, and 19/21 were resistant to ATM. The in vitro activity of CAZ-AVI in combination with ATM against diverse Enterobacteriaceae possessing MBLs was demonstrated in 17/21 isolates, where the zone of inhibition was ≥21 mm. All isolates demonstrated a reduction in CAZ-AVI agar dilution MICs with the addition of ATM. At 2 h, time-kill assays demonstrated a ≥4-log 10 -CFU decrease for all groups that had CAZ-AVI with ATM (8 μg/ml) added, compared to the group treated with CAZ-AVI alone. In the murine neutropenic thigh infection model, an almost 4-log 10 -CFU reduction was noted at 24 h for CAZ-AVI (32 mg/kg every 8 h [q8h]) plus ATM (32 mg/kg q8h) versus CAZ-AVI (32 mg/kg q8h) alone. The data presented herein require us to carefully consider this new therapeutic combination to treat infections caused by MBL-producing Enterobacteriaceae .

The outer membrane proteins responsible for the influx of carbapenem beta-lactam antibiotics in the nonfermentative gram-negative pathogen Acinetobacter baumannii are still poorly characterized. Resistance to both imipenem and meropenem in multidrug-resistant clinical strains of A. baumannii is associated with the loss of a heat-modifiable 29-kDa outer membrane protein, designated CarO. The chromosomal locus containing the carO gene was cloned and characterized from different clinical isolates. Only one carO copy, present in a single transcriptional unit, was found in the A. baumannii genome. The carO gene encodes a polypeptide of 247 amino acid residues with a typical N-terminal signal sequence and a predicted transmembrane beta-barrel topology. Its absence from different carbapenem-resistant clinical isolates of A. baumannii resulted from the disruption of carO by distinct insertion elements. The overall data thus support the notion that CarO participates in the influx of carbapenem antibiotics in A. baumannii. Moreover, database searches identified the presence of carO homologs only in species of the genera Acinetobacter, Moraxella, and Psychrobacter, disclosing the existence of a novel family of outer membrane proteins restricted to the family Moraxellaceae of the class gamma-Proteobacteria.

Ferredoxin (flavodoxin)-NADP(H) reductases (FNR) are ubiquitous flavoenzymes that deliver NADPH or low potential one-electron donors (ferredoxin, flavodoxin) to redox-based metabolisms in plastids, mitochondria and bacteria. The plant-type reductase is also the basic prototype for one of the major families of flavin-containing electron transferases that display common functional and structural properties. Many aspects of FNR biochemistry have been extensively characterized in recent years using a combination of site-directed mutagenesis, steady-state and transient kinetic experiments, spectroscopy and X-ray crystallography. Despite these considerable advances, various key features in the enzymology of these important reductases remain yet to be explained in molecular terms. This article reviews the current status of these open questions. Measurements of electron transfer rates and binding equilibria indicate that NADP(H) and ferredoxin interactions with FNR result in a reciprocal decrease of affinity, and that this induced-fit step is a mandatory requisite for catalytic turnover. However, the expected conformational movements are not apparent in the reported atomic structures of these flavoenzymes in the free state or in complex with their substrates. The overall reaction catalysed by FNR is freely reversible, but the pathways leading to NADP+ or ferredoxin reduction proceed through entirely different kinetic mechanisms. Also, the reductases isolated from various sources undergo inactivating denaturation on exposure to NADPH and other electron donors that reduce the FAD prosthetic group, a phenomenon that might have profound consequences for FNR function in vivo. The mechanisms underlying this reductive inhibition are so far unknown. Finally, we provide here a rationale to interpret FNR evolution in terms of catalytic efficiency. Using the formalism of the Albery-Knowles theory, we identified which parameter(s) have to be modified to make these reductases even more proficient under a variety of conditions, natural or artificial. Flavoenzymes with FNR activity catalyse a number of reactions with potential importance for biotechnological processes, so that modification of their catalytic competence is relevant on both scientific and technical grounds.

MicroRNAs (miRNAs) are ∼21 nt small RNAs that regulate gene expression in animals and plants. They can be grouped into families comprising different genes encoding similar or identical mature miRNAs. Several miRNA families are deeply conserved in plant lineages and regulate key aspects of plant development, hormone signaling, and stress response. The ancient miRNA miR396 regulates conserved targets belonging to the GROWTH-REGULATING FACTOR (GRF) family of transcription factors, which are known to control cell proliferation in Arabidopsis leaves. In this work, we characterized the regulation of an additional target for miR396, the transcription factor bHLH74, that is necessary for Arabidopsis normal development. bHLH74 homologs with a miR396 target site could only be detected in the sister families Brassicaceae and Cleomaceae. Still, bHLH74 repression by miR396 is required for margin and vein pattern formation of Arabidopsis leaves. MiR396 contributes to the spatio-temporal regulation of GRF and bHLH74 expression during leaf development. Furthermore, a survey of miR396 sequences in different species showed variations in the 5' portion of the miRNA, a region known to be important for miRNA activity. Analysis of different miR396 variants in Arabidopsis thaliana revealed that they have an enhanced activity toward GRF transcription factors. The interaction between the GRF target site and miR396 has a bulge between positions 7 and 8 of the miRNA. Our data indicate that such bulge modulates the strength of the miR396-mediated repression and that this modulation is essential to shape the precise spatio-temporal pattern of GRF2 expression. The results show that ancient miRNAs can regulate conserved targets with varied efficiency in different species, and we further propose that they could acquire new targets whose control might also be biologically relevant.