Toxicologie, Pharmacologie et Signalisation Cellulaire

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,

A decrease in the abundance and biodiversity of intestinal bacteria within the dominant phylum Firmicutes has been observed repeatedly in Crohn disease (CD) patients. In this study, we determined the composition of the mucosa-associated microbiota of CD patients at the time of surgical resection and 6 months later using FISH analysis. We found that a reduction of a major member of Firmicutes, Faecalibacterium prausnitzii, is associated with a higher risk of postoperative recurrence of ileal CD. A lower proportion of F. prausnitzii on resected ileal Crohn mucosa also was associated with endoscopic recurrence at 6 months. To evaluate the immunomodulatory properties of F. prausnitzii we analyzed the anti-inflammatory effects of F. prausnitzii in both in vitro (cellular models) and in vivo [2,4,6-trinitrobenzenesulphonic acid (TNBS)-induced] colitis in mice. In Caco-2 cells transfected with a reporter gene for NF-kappaB activity, F. prausnitzii had no effect on IL-1beta-induced NF-kappaB activity, whereas the supernatant abolished it. In vitro peripheral blood mononuclear cell stimulation by F. prausnitzii led to significantly lower IL-12 and IFN-gamma production levels and higher secretion of IL-10. Oral administration of either live F. prausnitzii or its supernatant markedly reduced the severity of TNBS colitis and tended to correct the dysbiosis associated with TNBS colitis, as demonstrated by real-time quantitative PCR (qPCR) analysis. F. prausnitzii exhibits anti-inflammatory effects on cellular and TNBS colitis models, partly due to secreted metabolites able to block NF-kappaB activation and IL-8 production. These results suggest that counterbalancing dysbiosis using F. prausnitzii as a probiotic is a promising strategy in CD treatment.

We present TrackMate, an open source Fiji plugin for the automated, semi-automated, and manual tracking of single-particles. It offers a versatile and modular solution that works out of the box for end users, through a simple and intuitive user interface. It is also easily scriptable and adaptable, operating equally well on 1D over time, 2D over time, 3D over time, or other single and multi-channel image variants. TrackMate provides several visualization and analysis tools that aid in assessing the relevance of results. The utility of TrackMate is further enhanced through its ability to be readily customized to meet specific tracking problems. TrackMate is an extensible platform where developers can easily write their own detection, particle linking, visualization or analysis algorithms within the TrackMate environment. This evolving framework provides researchers with the opportunity to quickly develop and optimize new algorithms based on existing TrackMate modules without the need of having to write de novo user interfaces, including visualization, analysis and exporting tools. The current capabilities of TrackMate are presented in the context of three different biological problems. First, we perform Caenorhabditis-elegans lineage analysis to assess how light-induced damage during imaging impairs its early development. Our TrackMate-based lineage analysis indicates the lack of a cell-specific light-sensitive mechanism. Second, we investigate the recruitment of NEMO (NF-κB essential modulator) clusters in fibroblasts after stimulation by the cytokine IL-1 and show that photodamage can generate artifacts in the shape of TrackMate characterized movements that confuse motility analysis. Finally, we validate the use of TrackMate for quantitative lifetime analysis of clathrin-mediated endocytosis in plant cells.

In autoimmune arthritis, traditionally classified as a T helper (Th) type 1 disease, the activation of T cells results in bone destruction mediated by osteoclasts, but how T cells enhance osteoclastogenesis despite the anti-osteoclastogenic effect of interferon (IFN)-gamma remains to be elucidated. Here, we examine the effect of various Th cell subsets on osteoclastogenesis and identify Th17, a specialized inflammatory subset, as an osteoclastogenic Th cell subset that links T cell activation and bone resorption. The interleukin (IL)-23-IL-17 axis, rather than the IL-12-IFN-gamma axis, is critical not only for the onset phase, but also for the bone destruction phase of autoimmune arthritis. Thus, Th17 is a powerful therapeutic target for the bone destruction associated with T cell activation.

Recent evidence indicates that reactive oxygen species (ROS) may function as intracellular messengers in receptor signaling pathways. The possible role of ROS in epidermal growth factor (EGF) signaling was therefore investigated. Stimulation of A431 human epidermoid carcinoma cells with EGF resulted in a transient increase in the intracellular concentration of ROS, measured with the oxidation-sensitive fluorescent probe 2′,7′-dichlorofluorescin diacetate and laser-scanning confocal microscopy. The predominant ROS produced appeared to be H2O2, because the EGF-induced increase in fluorescence was completely abolished by incorporation of catalase into the cells by electroporation. The elimination of H2O2 by catalase also inhibited the EGF-induced tyrosine phosphorylation of various cellular proteins including the EGF receptor and phospholipase C-γ1. The dependence of H2O2 production on the intrinsic tyrosine kinase activity of the EGF receptor and the autophosphorylation sites located in its COOH-terminal tail was investigated. EGF failed to induce H2O2 generation in cells expressing a kinase-inactive EGF receptor. However, normal H2O2 generation was observed in cells expressing a mutant receptor from which the 126 COOH-terminal amino acids had been deleted to remove four (out of the total of five) autophosphorylation sites. These results suggest that EGF-induced H2O2 formation requires the kinase activity but probably not the autophosphorylation sites of the EGF receptor and that inhibition of protein tyrosine phosphatase activity by H2O2 may be required for EGF-induced protein tyrosine phosphorylation to be manifested. Recent evidence indicates that reactive oxygen species (ROS) may function as intracellular messengers in receptor signaling pathways. The possible role of ROS in epidermal growth factor (EGF) signaling was therefore investigated. Stimulation of A431 human epidermoid carcinoma cells with EGF resulted in a transient increase in the intracellular concentration of ROS, measured with the oxidation-sensitive fluorescent probe 2′,7′-dichlorofluorescin diacetate and laser-scanning confocal microscopy. The predominant ROS produced appeared to be H2O2, because the EGF-induced increase in fluorescence was completely abolished by incorporation of catalase into the cells by electroporation. The elimination of H2O2 by catalase also inhibited the EGF-induced tyrosine phosphorylation of various cellular proteins including the EGF receptor and phospholipase C-γ1. The dependence of H2O2 production on the intrinsic tyrosine kinase activity of the EGF receptor and the autophosphorylation sites located in its COOH-terminal tail was investigated. EGF failed to induce H2O2 generation in cells expressing a kinase-inactive EGF receptor. However, normal H2O2 generation was observed in cells expressing a mutant receptor from which the 126 COOH-terminal amino acids had been deleted to remove four (out of the total of five) autophosphorylation sites. These results suggest that EGF-induced H2O2 formation requires the kinase activity but probably not the autophosphorylation sites of the EGF receptor and that inhibition of protein tyrosine phosphatase activity by H2O2 may be required for EGF-induced protein tyrosine phosphorylation to be manifested.

In the last two decades, the vast majority of microfluidic systems have been built in poly(dimethylsiloxane) (PDMS) by soft lithography, a technique based on PDMS micromolding. A long list of key PDMS properties have contributed to the success of soft lithography: PDMS is biocompatible, elastomeric, transparent, gas-permeable, water-impermeable, fairly inexpensive, copyright-free, and rapidly prototyped with high precision using simple procedures. However, the fabrication process typically involves substantial human labor, which tends to make PDMS devices difficult to disseminate outside of research labs, and the layered molding limits the 3D complexity of the devices that can be produced. 3D-printing has recently attracted attention as a way to fabricate microfluidic systems due to its automated, assembly-free 3D fabrication, rapidly decreasing costs, and fast-improving resolution and throughput. Resins with properties approaching those of PDMS are being developed. Here we review past and recent efforts in 3D-printing of microfluidic systems. We compare the salient features of PDMS molding with those of 3D-printing and we give an overview of the critical barriers that have prevented the adoption of 3D-printing by microfluidic developers, namely resolution, throughput, and resin biocompatibility. We also evaluate the various forces that are persuading researchers to abandon PDMS molding in favor of 3D-printing in growing numbers.

NFATc1 and NFATc2 are functionally redundant in the immune system, but it was suggested that NFATc1 is required exclusively for differentiation of osteoclasts in the skeletal system. Here we provide genetic evidence that NFATc1 is essential for osteoclast differentiation in vivo by adoptive transfer of NFATc1(-/-) hematopoietic stem cells to osteoclast-deficient Fos(-/-) mice, and by Fos(-/-) blastocyst complementation, thus avoiding the embryonic lethality of NFATc1(-/-) mice. However, in vitro osteoclastogenesis in NFATc1-deficient cells was rescued by ectopic expression of NFATc2. The discrepancy between the in vivo essential role of NFATc1 and the in vitro effect of NFATc2 was attributed to selective autoregulation of the NFATc1 gene by NFAT through its promoter region. This suggested that an epigenetic mechanism contributes to the essential function of NFATc1 in cell lineage commitment. Thus, this study establishes that NFATc1 represents a potential therapeutic target for bone disease and reveals a mechanism that underlies the essential role of NFATc1 in bone homeostasis.

The Ras gene is frequently mutated in cancer, and mutant Ras drives tumorigenesis. Although Ras is a central oncogene, small molecules that bind to Ras in a well-defined manner and exert inhibitory effects have not been uncovered to date. Through an NMR-based fragment screen, we identified a group of small molecules that all bind to a common site on Ras. High-resolution cocrystal structures delineated a unique ligand-binding pocket on the Ras protein that is adjacent to the switch I/II regions and can be expanded upon compound binding. Structure analysis predicts that compound-binding interferes with the Ras/SOS interactions. Indeed, selected compounds inhibit SOS-mediated nucleotide exchange and prevent Ras activation by blocking the formation of intermediates of the exchange reaction. The discovery of a small-molecule binding pocket on Ras with functional significance provides a new direction in the search of therapeutically effective inhibitors of the Ras oncoprotein.

Zinc is an essential trace element required for enzymatic activity and for maintaining the conformation of many transcription factors; thus, zinc homeostasis is tightly regulated. Although zinc affects several signaling molecules and may act as a neurotransmitter, it remains unknown whether zinc acts as an intracellular second messenger capable of transducing extracellular stimuli into intracellular signaling events. In this study, we report that the cross-linking of the high affinity immunoglobin E receptor (Fcepsilon receptor I [FcepsilonRI]) induced a release of free zinc from the perinuclear area, including the endoplasmic reticulum in mast cells, a phenomenon we call the zinc wave. The zinc wave was dependent on calcium influx and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase activation. The results suggest that the zinc wave is involved in intracellular signaling events, at least in part by modulating the duration and strength of FcepsilonRI-mediated signaling. Collectively, our findings indicate that zinc is a novel intracellular second messenger.

Recognition of the melanoma Ag gp100 by tumor-infiltrating lymphocytes (TIL) in vitro has been correlated with tumor regression in patients with metastatic melanoma treated with the adoptive transfer of TIL plus IL-2. Three common gp100 epitopes have been identified that are recognized in the context of HLA-A2 by TIL from different patients: G9154 (KTWGQYWQV), G9209 (ITDQVPFSV), and G9280 (YLEPGPVTA). Upon stimulation with these peptides, melanoma-reactive CTL could be induced in vitro from PBL of some HLA-A2+ melanoma patients. However, numerous restimulations were required, and specific reactivity could not be generated in many patients. Therefore, to enhance the immunogenicity of gp100 peptides, amino acid substitutions were introduced into G9154, G9209, and G9280 at HLA-A*0201-binding anchor positions, but not at TCR contact residues, to increase peptide class I MHC-binding affinity. Several modified gp100 peptides bound with greater affinity to HLA-A*0201 than unmodified peptides and were recognized by TIL specific for the natural epitopes. These peptides were used to sensitize PBL from HLA-A2+ melanoma patients in vitro using peptide-pulsed autologous PBMC as stimulators. After five weekly restimulations with either the native G9209 or G9280 peptide, melanoma-reactive CTL could only be induced from two of seven patients. However, amino acid substitutions in these peptides enabled the induction of melanoma-reactive CTL from all seven patients. These results suggest that modified gp100 peptides may be more immunogenic than the native epitopes, and may be useful in immunotherapy protocols for patients with melanoma.

Animals and humans are exposed each day to a multitude of chemicals in the air, water and food. They have developed a battery of enzymes and transporters that facilitate the biotransformation and elimination of these compounds. Moreover, a majority of these enzymes and transporters are inducible due to the activation of xenobiotic receptors which act as transcription factors for the regulation of their target genes (such as xenobiotic metabolizing enzymes, see below §4 for the AhR). These receptors include several members of the nuclear/steroid receptor family (CAR for Constitutive Androstane Receptor, PXR for Pregnane X Receptor) but also the Aryl hydrocarbon Receptor or AhR, a member of the bHLH-PAS family (basic Helix-Loop-Helix - Period/ARNT/Single minded). In addition to the regulation of xenobiotic metabolism, numerous alternative functions have been characterized for the AhR since its discovery. These alternative functions will be described in this review along with its endogenous functions as revealed by experiments performed on knock-out animals.

The RNA genome of the human T-cell leukemia virus type 1 (HTLV-1) codes for proteins involved in infectivity, replication, and transformation. We report in this study the characterization of a novel viral protein encoded by the complementary strand of the HTLV-1 RNA genome. This protein, designated HBZ (for HTLV-1 bZIP factor), contains a N-terminal transcriptional activation domain and a leucine zipper motif in its C terminus. We show here that HBZ is able to interact with the bZIP transcription factor CREB-2 (also called ATF-4), known to activate the HTLV-1 transcription by recruiting the viral trans-activator Tax on the Tax-responsive elements (TxREs). However, we demonstrate that the HBZ/CREB-2 heterodimers are no more able to bind to the TxRE and cyclic AMP response element sites. Taking these findings together, the functional inactivation of CREB-2 by HBZ is suggested to contribute to regulation of the HTLV-1 transcription. Moreover, the characterization of a minus-strand gene protein encoded by HTLV-1 has never been reported until now.

STAT (signal transducers and activators of transcription) proteins are transcription factors which are activated by phosphorylation on tyrosine residues upon stimulation by cytokines. Seven members of the STAT family are known, including the closely related STAT5A and STAT5B, which are activated by various cytokines. Except for prolactin-dependent beta-casein production in mammary gland cells, the biological consequences of STAT5 activation in various systems are not clear. We applied PCR-driven random mutagenesis and a retrovirus-mediated expression screening system to identify constitutively active forms of STAT5. By this strategy, we have identified a constitutively active STAT5 mutant which has two amino acid substitutions; one is located upstream of the putative DNA binding domain (H299R), and the other is located in the transactivation domain (S711F). The mutant STAT5 was constitutively phosphorylated on tyrosine residues, localized in the nucleus, and was transcriptionally active. Expression of the mutant STAT5 partially dispenses with interleukin 3 (IL-3) as a growth stimulant of IL-3-dependent cell lines. Further analyses of the mutant STAT5 have demonstrated that both of the mutations are required for nuclear localization, efficient transcriptional activation, and induction of IL-3-independent growth of an IL-3-dependent cell line, Ba/F3, and have indicated that a molecular basis for the constitutive activation is the stability of the phosphorylated form of the mutant STAT5.

Solute carrier family 11, member 2 (SLC11A2) is the only transmembrane iron transporter known to be involved in cellular iron uptake. It is widely expressed and has been postulated to play important roles in intestinal iron absorption, erythroid iron utilization, hepatic iron accumulation, placental iron transfer, and other processes. Previous studies have suggested that other transporters might exist, but their physiological significance remained uncertain. To define the activities of Slc11a2 in vivo, we inactivated the murine gene that encodes it globally and selectively. We found that fetal Slc11a2 is not needed for materno-fetal iron transfer but that Slc11a2 activity is essential for intestinal non-heme iron absorption after birth. Slc11a2 is also required for normal hemoglobin production during the development of erythroid precursors. However, hepatocytes and most other cells must have an alternative, as-yet-unknown, iron uptake mechanism. We previously showed that Slc11a2 serves as the primary portal for intestinal iron entry in hemochromatosis. However, inactivation of murine Hfe ameliorates the phenotype of animals lacking Slc11a2.

BACKGROUND: Adipose tissue (AT) is involved in several physiological functions, including metabolic regulation, energy storage, and endocrine functions. OBJECTIVES: In this review we examined the evidence that an additional function of AT is to modulate persistent organic pollutant (POP) toxicity through several mechanisms. METHODS: We reviewed the literature on the interaction of AT with POPs to provide a comprehensive model for this additional function of AT. DISCUSSION: As a storage compartment for lipophilic POPs, AT plays a critical role in the toxicokinetics of a variety of drugs and pollutants, in particular, POPs. By sequestering POPs, AT can protect other organs and tissues from POPs overload. However, this protective function could prove to be a threat in the long run. The accumulation of lipophilic POPs will increase total body burden. These accumulated POPs are slowly released into the bloodstream, and more so during weight loss. Thus, AT constitutes a continual source of internal exposure to POPs. In addition to its buffering function, AT is also a target of POPs and may mediate part of their metabolic effects. This is particularly relevant because many POPs induce obesogenic effects that may lead to quantitative and qualitative alterations of AT. Some POPs also induce a proinflammatory state in AT, which may lead to detrimental metabolic effects. CONCLUSION: AT appears to play diverse functions both as a modulator and as a target of POPs toxicity.

Experiments performed in normal animals suggest that the basal ganglia (BG) are crucial in motor program selection. BG are also involved in movement disorders. In particular, BG neuronal activity in parkinsonian animals and patients is more oscillatory and more synchronous than in normal individuals. We propose a new model for the function and dysfunction of the motor part of BG. We hypothesize that the striatum, the subthalamic nucleus, the internal pallidum (GPi), the thalamus, and the cortex are involved in closed feedback loops. The direct (cortex-striatum-GPi-thalamus-cortex) and the hyperdirect loops (cortex-subthalamic nucleus-GPi-thalamus-cortex), which have different polarities, play a key role in the model. We show that the competition between these two loops provides the BG-cortex system with the ability to perform motor program selection. Under the assumption that dopamine potentiates corticostriatal synaptic transmission, we demonstrate that, in our model, moderate dopamine depletion leads to a complete loss of action selection ability. High depletion can lead to synchronous oscillations. These modifications of the network dynamical state stem from an imbalance between the feedback in the direct and hyperdirect loops when dopamine is depleted. Our model predicts that the loss of selection ability occurs before the appearance of oscillations, suggesting that Parkinson's disease motor impairments are not directly related to abnormal oscillatory activity. Another major prediction of our model is that synchronous oscillations driven by the hyperdirect loop appear in BG after inactivation of the striatum.

High frequency stimulation (HFS) of the subthalamic nucleus (STN) is a well-established therapeutic approach for the treatment of late-stage Parkinson's disease. Although the underlying cause of this illness remains a mystery, changes in firing rate and synchronized activity in different basal ganglia nuclei have been related to its symptoms. Here we investigated the impact of STN-HFS on firing rate as well as correlated and oscillatory activity in the STN network in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned non-human primates by using simultaneous extracellular single-unit recordings. STN-HFS reduced (i) the firing rate of STN neurons, (ii) the oscillatory activity at an individual STN neuron level as well as (iii) the correlated and oscillatory activity between pairs of STN neurons, while contralateral rigidity was improved. A detailed analysis showed that the decrease of mean firing rate resulted from the resetting of firing probability to virtually zero by the stimulus pulse. Subsequently, STN neurons resumed their activity after a mean duration of 2.9 +/- 0.1 ms and their firing probability returned to baseline values approximately 7 ms after the onset of the stimulus pulse, the recovery of the firing probability being represented by a sigmoid function. Thus, the overall decrease of the mean firing rate resulted from the repetition of this dynamical process with a frequency of 130 Hz (interstimulus interval approximately 7.7 ms), allowing the neuron to fire with its baseline firing rate only for a very short period. Although the mechanisms underlying the desynchronization of neuronal activity in the STN network remain unclear, the resetting of STN neuron firing probability by the electrical stimulus would rather be expected to increase oscillatory activity at an individual neuron level as well as correlated and oscillatory activity between pairs of STN neurons. However, assuming the resetting of firing rate to be the consequence of a transient GABAergic inhibition through excitation of presynaptic GABAergic axon terminals, different recovery periods of STN neurons might delay the appearance of synchronized oscillations, particularly if they are not generated locally. In conclusion, our study provides new evidence that STN-HFS decreases oscillatory activity in the STN network. Although the exact relation between oscillatory activity and Parkinson's disease symptoms remains to be determined, the present results suggest that STN-HFS might at least partially exert its beneficial effects through the reduction of oscillatory activity in the STN network and consequently in the entire cortex-basal ganglia-cortex network.

Antibodies to transferrin receptor (TfR) have potential use for therapeutic entry into the brain. We have shown that bispecific antibodies against TfR and β-secretase (BACE1 [β-amyloid cleaving enzyme-1]) traverse the blood-brain barrier (BBB) and effectively reduce brain amyloid β levels. We found that optimizing anti-TfR affinity improves brain exposure and BACE1 inhibition. Here we probe the cellular basis of this improvement and explore whether TfR antibody affinity alters the intracellular trafficking of TfR. Comparing high- and low-affinity TfR bispecific antibodies in vivo, we found that high-affinity binding to TfR caused a dose-dependent reduction of brain TfR levels. In vitro live imaging and colocalization experiments revealed that high-affinity TfR bispecific antibodies facilitated the trafficking of TfR to lysosomes and thus induced the degradation of TfR, an observation which was further confirmed in vivo. Importantly, high-affinity anti-TfR dosing induced reductions in brain TfR levels, which significantly decreased brain exposure to a second dose of low-affinity anti-TfR bispecific. Thus, high-affinity anti-TfR alters TfR trafficking, which dramatically impacts the capacity for TfR to mediate BBB transcytosis.

The estrogen receptor alpha (ER), a member of the steroid receptor superfamily, contains an N-terminal hormone-independent transcriptional activation function (AF-1) and a C-terminal hormone-dependent transcriptional activation function (AF-2). Here, we used in-gel kinase assays to determine that pp90rsk1 activated by either epidermal growth factor (EGF) or phorbol myristate acetate specifically phosphorylates Ser-167 within AF-1. In vitro kinase assays demonstrated that pp90rsk1 phosphorylates the N terminus of the wild-type ER but not of a mutant ER in which Ser-167 was replaced by Ala. In vivo, EGF stimulated phosphorylation of Ser-167 as well as Ser-118. Ectopic expression of active pp90rsk1 increased the level of phosphorylation of Ser-167 compared to that of either a mutant pp90rsk1, which is catalytically inactive in the N-terminal kinase domain, or to that of vector control. The ER formed a stable complex with the mutant pp90rsk1 in vivo. Transfection of the mutant pp90rsk1 depressed ER-dependent transcription of both a wild-type ER and a mutant ER that had a defective AF-2 domain (ER TAF-1). Furthermore, replacing either Ser-118 or Ser-167 with Ala in ER TAF-1 showed similar decreases in transcription levels. A double mutant in which both Ser-118 and Ser-167 were replaced with Ala demonstrated a further decrease in transcription compared to either of the single mutations. Taken together, our results strongly suggest that pp90rsk1 phosphorylates Ser-167 of the human ER in vivo and that Ser-167 aids in regulating the transcriptional activity of AF-1 in the ER.

RNAi is proving to be a powerful experimental tool for the functional annotation of mammalian genomes. The full potential of this technology will be realized through development of approaches permitting regulated manipulation of endogenous gene expression with coordinated reexpression of exogenous transgenes. We describe the development of a lentiviral vector platform, pSLIK (single lentivector for inducible knockdown), which permits tetracycline-regulated expression of microRNA-like short hairpin RNAs from a single viral infection of any naïve cell system. In mouse embryonic fibroblasts, the pSLIK platform was used to conditionally deplete the expression of the heterotrimeric G proteins Galpha12 and Galpha13 both singly and in combination, demonstrating the Galpha13 dependence of serum response element-mediated transcription. In RAW264.7 macrophages, regulated knockdown of Gbeta2 correlated with a reduced Ca(2+) response to C5a. Insertion of a GFP transgene upstream of the Gbeta2 microRNA-like short hairpin RNA allowed concomitant reexpression of a heterologous mRNA during tetracycline-dependent target gene knockdown, significantly enhancing the experimental applicability of the pSLIK system.