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34 results on '"Génotoxicité '

1. Cytolethal Distending Toxin Promotes Replicative Stress Leading to Genetic Instability Transmitted to Daughter Cells

Author

William Tremblay, Florence Mompart, Elisa Lopez, Muriel Quaranta, Valérie Bergoglio, Saleha Hashim, Delphine Bonnet, Laurent Alric, Emmanuel Mas, Didier Trouche, Julien Vignard, Audrey Ferrand, Gladys Mirey, Anne Fernandez-Vidal, Génotoxicité & Signalisation (ToxAlim-GS), ToxAlim (ToxAlim), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut de Recherche en Santé Digestive (IRSD ), Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Unité de biologie moléculaire, cellulaire et du développement (MCD), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Contaminants & Stress Cellulaire (ToxAlim-COMICS), CHU Toulouse [Toulouse], La Ligue Contrele Cancer (Haute-Garonne’s committee), ANR-10-CESA-0011,GENOTOXTRACK,Biomarqueurs de génotoxicité ex vivo et in vivo.(2010), ANR-14-CE21-0008,GENOTRACE,Traceurs de génotoxicité et traceurs biologiques pour un essai micronoyau en cellules vivantes(2014), Mirey, Gladys, CONTAMINANTS, ECOSYSTEMES, SANTE - Biomarqueurs de génotoxicité ex vivo et in vivo. - - GENOTOXTRACK2010 - ANR-10-CESA-0011 - CES - VALID, Appel à projets générique - Traceurs de génotoxicité et traceurs biologiques pour un essai micronoyau en cellules vivantes - - GENOTRACE2014 - ANR-14-CE21-0008 - Appel à projets générique - VALID, Unité de biologie moléculaire, cellulaire et du développement - UMR5077 (MCD), Centre de Biologie Intégrative (CBI), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Centre Hospitalier Universitaire de Toulouse (CHU Toulouse), and La Ligue Contre le Cancer (Haute-Garonne's committee) (AF-V)

Subjects
Cell division, Cytolethal distending toxin, DNA damage, QH301-705.5, [SDV]Life Sciences [q-bio], Cell, replicative stress, [SDV.GEN] Life Sciences [q-bio]/Genetics, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, medicine.disease_cause, Cell and Developmental Biology, 03 medical and health sciences, medicine, Biology (General), Mitosis, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, Original Research, 030304 developmental biology, 0303 health sciences, humancolorectal organoid, [SDV.GEN]Life Sciences [q-bio]/Genetics, 030302 biochemistry & molecular biology, DNA bridge, DNA replication, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, genetic instability, Cell Biology, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, 3. Good health, Cell biology, [SDV] Life Sciences [q-bio], medicine.anatomical_structure, human colorectal organoid, Stem cell, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Carcinogenesis, cytolethal distending toxin, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology, Developmental Biology
Abstract

The cytolethal distending toxin (CDT) is produced by several Gram-negative pathogenic bacteria. In addition to inflammation, experimental evidences are in favor of a protumoral role of CDT-harboring bacteria such as Escherichia coli, Campylobacter jejuni, or Helicobacter hepaticus. CDT may contribute to cell transformation in vitro and carcinogenesis in mice models, through the genotoxic action of CdtB catalytic subunit. Here, we investigate the mechanism of action by which CDT leads to genetic instability in human cell lines and colorectal organoids from healthy patients’ biopsies. We demonstrate that CDT holotoxin induces a replicative stress dependent on CdtB. The slowing down of DNA replication occurs mainly in late S phase, resulting in the expression of fragile sites and important chromosomic aberrations. These DNA abnormalities induced after CDT treatment are responsible for anaphase bridge formation in mitosis and interphase DNA bridge between daughter cells in G1 phase. Moreover, CDT-genotoxic potential preferentially affects human cycling cells compared to quiescent cells. Finally, the toxin induces nuclear distension associated to DNA damage in proliferating cells of human colorectal organoids, resulting in decreased growth. Our findings thus identify CDT as a bacterial virulence factor targeting proliferating cells, such as human colorectal progenitors or stem cells, inducing replicative stress and genetic instability transmitted to daughter cells that may therefore contribute to carcinogenesis. As some CDT-carrying bacterial strains were detected in patients with colorectal cancer, targeting these bacteria could be a promising therapeutic strategy.

Published
2021

2. Functional Study of Haemophilus ducreyi Cytolethal Distending Toxin Subunit B

Author

Saleha Hashim, Nicolas Loiseau, Julien Vignard, Benoît J. Pons, Soraya Tadrist, Gladys Mirey, Génotoxicité & Signalisation (ToxAlim-GS), ToxAlim (ToxAlim), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Toxicologie Intégrative & Métabolisme (ToxAlim-TIM), Contaminants & Stress Cellulaire (ToxAlim-COMICS), ANR-10-CESA-0011,GENOTOXTRACK,Biomarqueurs de génotoxicité ex vivo et in vivo.(2010), and ANR-14-CE21-0008,GENOTRACE,Traceurs de génotoxicité et traceurs biologiques pour un essai micronoyau en cellules vivantes(2014)

Subjects
Cytolethal distending toxin, DNA damage, Health, Toxicology and Mutagenesis, Protein subunit, [SDV]Life Sciences [q-bio], Mutant, Bacterial Toxins, lcsh:Medicine, Toxicology, Article, Protein Structure, Secondary, phosphatase, Haemophilus ducreyi, 03 medical and health sciences, chemistry.chemical_compound, Jurkat Cells, 0302 clinical medicine, Humans, nuclease, 030304 developmental biology, 0303 health sciences, Nuclease, biology, lcsh:R, CdtB, Cell cycle, Molecular biology, Protein Subunits, chemistry, 030220 oncology & carcinogenesis, Mutation, biology.protein, cytotoxicity, cell cycle checkpoint, site-directed mutagenesis, Cytolethal Distending Toxin, DNA, Intracellular, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology, HeLa Cells
Abstract

International audience; The Cytolethal Distending Toxin (CDT) is produced by many Gram-negative pathogenic bacteria responsible for major foodborne diseases worldwide. CDT induces DNA damage and cell cycle arrest in host-cells, eventually leading to senescence or apoptosis. According to structural and sequence comparison, the catalytic subunit CdtB is suggested to possess both nuclease and phosphatase activities, carried by a single catalytic site. However, the impact of each activity on cell-host toxicity is yet to be characterized. Here, we analyze the consequences of cell exposure to different CDT mutated on key CdtB residues, focusing on cell viability, cell cycle defects, and DNA damage induction. A first class of mutant, devoid of any activity, targets putative catalytic (H160A), metal binding (D273R), and DNA binding residues (R117A-R144A-N201A). The second class of mutants (A163R, F156-T158, and the newly identified G114T), which gathers mutations on residues potentially involved in lipid substrate binding, has only partially lost its toxic effects. However, their defects are alleviated when CdtB is artificially introduced inside cells, except for the F156-T158 double mutant that is defective in nuclear addressing. Therefore, our data reveal that CDT toxicity is mainly correlated to CdtB nuclease activity, whereas phosphatase activity may probably be involved in CdtB intracellular trafficking.

Published
2020

3. Concurrent Nanopore Next-Generation Sequencing of Hepatitis B and Delta Virus Genomes Directly From Patient Plasma

Author

Anne Motte, Céline Boschi, Ludivine Brechard, Jessica Grace Bengone-Abogourin, Philippe Colson, Isabelle Allemand, Microbes évolution phylogénie et infections (MEPHI), Institut de Recherche pour le Développement (IRD)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Institut Hospitalier Universitaire Méditerranée Infection (IHU Marseille), Gamétogénèse Apoptose et Génotoxicité - LGAG (Equipe - U967 INSERM), Stabilité génétique, Cellules Souches et Radiations (SCSR (U_967)), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité)-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), ANR-10-IAHU-0003,Méditerranée Infection,I.H.U. Méditerranée Infection(2010), European Project, Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Aix Marseille Université (AMU), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP)-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP), COMBE, Isabelle, Instituts Hospitalo-Universitaires - I.H.U. Méditerranée Infection - - Méditerranée Infection2010 - ANR-10-IAHU-0003 - IAHU - VALID, and FEDER PRIMI - INCOMING

Subjects
Delta, Hepatitis B virus, Clinical Biochemistry, Computational biology, Genome, Viral, Biology, Genome, DNA sequencing, Virus, 03 medical and health sciences, Nanopores, [SDV.MHEP.CSC]Life Sciences [q-bio]/Human health and pathology/Cardiology and cardiovascular system, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, medicine, Humans, [SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, Letter to the Editor, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, [SDV.MP.VIR] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, [SDV.MHEP.ME] Life Sciences [q-bio]/Human health and pathology/Emerging diseases, 0303 health sciences, [SDV.MHEP.ME]Life Sciences [q-bio]/Human health and pathology/Emerging diseases, 030306 microbiology, Biochemistry (medical), High-Throughput Nucleotide Sequencing, General Medicine, Hepatitis B, medicine.disease, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, [SDV.MHEP.CSC] Life Sciences [q-bio]/Human health and pathology/Cardiology and cardiovascular system, 3. Good health, Nanopore, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, [SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Diagnostic Genetics, [SDV.MP.PAR] Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology
Abstract

International audience

Published
2021

4. CRISPR-Cas9 globin editing can induce megabase-scale copy-neutral losses of heterozygosity in hematopoietic cells

Author

Jean-Marc Blouin, F. Prat, J. Rosier, Jean-Philippe Merlio, J. Bouron, Emmanuel Richard, Cécile Ged, Julian Boutin, Isabelle Lamrissi-Garcia, Aurélie Bedel, Sandrine Dabernat, François Moreau-Gaudry, G. Cullot, J. Toutain, Caroline Rooryck, Perrine Pennamen, Véronique Guyonnet-Dupérat, D. Cappellen, Samuel Amintas, Biothérapies des maladies génétiques et cancers, Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire Maladies Rares: Génétique et Métabolisme (Bordeaux) (U1211 INSERM/MRGM), Université de Bordeaux (UB)-Groupe hospitalier Pellegrin-Institut National de la Santé et de la Recherche Médicale (INSERM), Bordeaux Research In Translational Oncology [Bordeaux] (BaRITOn), Université de Bordeaux (UB)-CHU Bordeaux [Bordeaux]-Institut National de la Santé et de la Recherche Médicale (INSERM), Association Française contre les Myopathies, ANR-21-CE18-0002,CRISPR-genotox,Génotoxicité induite par CRISPR-CAS9(2021), Admin, Oskar, and Génotoxicité induite par CRISPR-CAS9 - - CRISPR-genotox2021 - ANR-21-CE18-0002 - AAPG2021 - VALID

Subjects
CRISPR-Cas9 genome editing, Genetic enhancement, Science, General Physics and Astronomy, Gene Expression, Loss of Heterozygosity, Biology, Polymorphism, Single Nucleotide, General Biochemistry, Genetics and Molecular Biology, Article, Loss of heterozygosity, 03 medical and health sciences, 0302 clinical medicine, Genome editing, Insulin-Like Growth Factor II, medicine, CRISPR, Humans, Globin, Cells, Cultured, 030304 developmental biology, Sequence Deletion, Genetics, Gene Editing, 0303 health sciences, Multidisciplinary, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, Chromosomes, Human, Pair 11, Chromosome, General Chemistry, DNA Methylation, medicine.disease, Hematopoietic Stem Cells, Uniparental disomy, 3. Good health, Telomere, Globins, Targeted gene repair, HEK293 Cells, 030220 oncology & carcinogenesis, RNA, Long Noncoding, CRISPR-Cas Systems, Chromosome Deletion, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract

CRISPR-Cas9 is a promising technology for gene therapy. However, the ON-target genotoxicity of CRISPR-Cas9 nuclease due to DNA double-strand breaks has received little attention and is probably underestimated. Here we report that genome editing targeting globin genes induces megabase-scale losses of heterozygosity (LOH) from the globin CRISPR-Cas9 cut-site to the telomere (5.2 Mb). In established lines, CRISPR-Cas9 nuclease induces frequent terminal chromosome 11p truncations and rare copy-neutral LOH. In primary hematopoietic progenitor/stem cells, we detect 1.1% of clones (7/648) with acquired megabase LOH induced by CRISPR-Cas9. In-depth analysis by SNP-array reveals the presence of copy-neutral LOH. This leads to 11p15.5 partial uniparental disomy, comprising two Chr11p15.5 imprinting centers (H19/IGF2:IG-DMR/IC1 and KCNQ1OT1:TSS-DMR/IC2) and impacting H19 and IGF2 expression. While this genotoxicity is a safety concern for CRISPR clinical trials, it is also an opportunity to model copy-neutral-LOH for genetic diseases and cancers., CRISPR-Cas9 generates double-strand breaks, which pose a threat to genome integrity. Here the authors show loss of heterozygosity in edited hematopoietic progenitor cells.

Published
2021

5. In vitro micronucleus test in living cells associating biological tracers and high-content imaging

Author

Gladys Mirey, Vanessa Graillot, Odile Mondesert, Thibault Metenier, Julien Vignard, Valerie Lobjois, Emmanuelle Bazin, Valery Shevchenko, Christiane Guguen-Guillouzo, Christophe Chesne, Bernard Ducommun, Bernard Salles, Génotoxicité & Signalisation (ToxAlim-GS), ToxAlim (ToxAlim), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA), Institut des Technologies Avancées en sciences du Vivant (ITAV), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Eurosafe, and Bioprédic International

Subjects
0303 health sciences, 03 medical and health sciences, 0302 clinical medicine, [SDV]Life Sciences [q-bio], General Medicine, Toxicology, 030217 neurology & neurosurgery, ComputingMilieux_MISCELLANEOUS, 3. Good health, 030304 developmental biology
Abstract

International audience

Published
2017

6. Benzo[a]pyrene-induced DNA damage associated with mutagenesis in primary human activated T lymphocytes

Author

Lydie Sparfel, Marie Liamin, Emilien L. Jamin, Marc Audebert, Elisa Boutet-Robinet, Morgane Fernier, Julien Vignard, Eric Le Ferrec, Laure Khoury, Benjamin Kopp, Institut de recherche en santé, environnement et travail (Irset), Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique )-Institut National de la Santé et de la Recherche Médicale (INSERM)-École des Hautes Études en Santé Publique [EHESP] (EHESP)-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Université d'Angers (UA), Contaminants & Stress Cellulaire (ToxAlim-COMICS), ToxAlim (ToxAlim), Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, Analyse de Xénobiotiques, Identification, Métabolisme (E20 Metatoul-AXIOM), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-MetaToul-MetaboHUB, Génopole Toulouse Midi-Pyrénées [Auzeville] (GENOTOUL), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Génopole Toulouse Midi-Pyrénées [Auzeville] (GENOTOUL), Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Génotoxicité & Signalisation (ToxAlim-GS), Métabolisme et Xénobiotiques (ToxAlim-MeX), The French National Research Program for Environmental and Occupational Health of ANSES, Cancer TMOI of the French National Alliance for Life and Health Sciences (AVIESAN) [2014/1/052], ANSES, Université d'Angers (UA)-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-École des Hautes Études en Santé Publique [EHESP] (EHESP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA)-MetaToul-MetaboHUB, Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Ecole Nationale Vétérinaire de Toulouse (ENVT), Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Génopole Toulouse Midi-Pyrénées [Auzeville] (GENOTOUL), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université d'Angers (UA)-Université de Rennes (UR)-École des Hautes Études en Santé Publique [EHESP] (EHESP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Université de Toulouse (UT)-Université de Toulouse (UT)-Ecole Nationale Vétérinaire de Toulouse (ENVT), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Ecole d'Ingénieurs de Purpan (INP - PURPAN), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT), MetaToul AXIOM (E20), Université de Toulouse (UT)-Université de Toulouse (UT)-MetaboHUB-MetaToul, MetaboHUB-Génopole Toulouse Midi-Pyrénées [Auzeville] (GENOTOUL), Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-MetaboHUB-Génopole Toulouse Midi-Pyrénées [Auzeville] (GENOTOUL), Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Jonchère, Laurent, and Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-MetaboHUB-MetaToul

Subjects
0301 basic medicine, DNA damage, Stereochemistry, T-Lymphocytes, activated human t lymphocytes, Gene mutation, Toxicology, medicine.disease_cause, Biochemistry, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Benzo(a)pyrene, medicine, Humans, Cells, Cultured, Carcinogen, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Pharmacology, 0303 health sciences, [SDV.TOX.ECO] Life Sciences [q-bio]/Toxicology/Ecotoxicology, Primary (chemistry), Dose-Response Relationship, Drug, biology, Mutagenicity Tests, Chemistry, Mutagenesis, benzo[a]pyrene, General Medicine, T lymphocyte, Aryl hydrocarbon receptor, Molecular biology, 3. Good health, dna damage response, 030104 developmental biology, Leukocytes, Mononuclear, biology.protein, [SDV.TOX.ECO]Life Sciences [q-bio]/Toxicology/Ecotoxicology, 030217 neurology & neurosurgery, Genotoxicity, DNA Damage
Abstract

International audience; Polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene (B[a]P), are widely distributed environmental contaminants exerting toxic effects such as genotoxicity and carcinogenicity, mainly associated with aryl hydrocarbon receptor (AhR) activation and the subsequent induction of cytochromes P-450 (CYP) 1-metabolizing enzymes. We previously reported an up-regulation of AhR expression and activity in primary cultures of human T lymphocyte by a physiological activation. Despite the suggested link between exposure to PAHs and the risk of lymphoma, the potential of activated human T lymphocytes to metabolize AhR exogenous ligands such as B[a]P, and produce DNA damage has not been investigated. In the present study, we characterized the genotoxic response of primary activated T lymphocytes to B[a] P. We demonstrated that, following T lymphocyte activation, B[a]P treatment triggers a marked increase in CYP1 expression and activity generating, upon metabolic activation, DNA adducts and double-strand breaks (DSBs) after a 48-h treatment. At this time point, B[a]P also induces a DNA damage response with ataxia telangiectasia mutated kinase activation, thus producing a p53-dependent response and T lymphocyte survival. B[a]P activates DSB repair by mobilizing homologous recombination machinery but also induces gene mutations in activated human T lymphocytes which could consequently drive a cancer process. In conclusion, primary cultures of activated human T lymphocytes represent a good model for studying genotoxic effects of environmental contaminants such as PAHs, and predicting human health issues.

Published
2017

7. Cytolethal Distending Toxin Subunit B: A Review of Structure–Function Relationship

Author

Benoît J. Pons, Gladys Mirey, Julien Vignard, Génotoxicité & Signalisation (ToxAlim-GS), ToxAlim (ToxAlim), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA), and Mirey, Gladys

Subjects
Cytolethal distending toxin, DNA damage, Health, Toxicology and Mutagenesis, Bacterial Toxins, lcsh:Medicine, Context (language use), Review, Biology, Toxicology, medicine.disease_cause, Structure-Activity Relationship, 03 medical and health sciences, 0302 clinical medicine, cytolethal distending toxin, CdtB subunit, structure-function relationship, key residues, Catalytic Domain, medicine, Animals, Humans, Food and Nutrition, Amino Acid Sequence, Mode of action, Toxicologie, 030304 developmental biology, Genetics, 0303 health sciences, Mutation, lcsh:R, Pathogenic bacteria, Cell cycle, biology.organism_classification, 3. Good health, [SDV.TOX]Life Sciences [q-bio]/Toxicology, 030220 oncology & carcinogenesis, Alimentation et Nutrition, [SDV.AEN]Life Sciences [q-bio]/Food and Nutrition, Bacteria
Abstract

International audience; The Cytolethal Distending Toxin (CDT) is a bacterial virulence factor produced by several Gram-negative pathogenic bacteria. These bacteria, found in distinct niches, cause diverse infectious diseases and produce CDTs differing in sequence and structure. CDTs have been involved in the pathogenicity of the associated bacteria by promoting persistent infection. At the host-cell level, CDTs cause cell distension, cell cycle block and DNA damage, eventually leading to cell death. All these effects are attributable to the catalytic CdtB subunit, but its exact mode of action is only beginning to be unraveled. Sequence and 3D structure analyses revealed similarities with better characterized proteins, such as nucleases or phosphatases, and it has been hypothesized that CdtB exerts a biochemical activity close to those enzymes. Here, we review the relationships that have been established between CdtB structure and function, particularly by mutation experiments on predicted key residues in different experimental systems. We discuss the relevance of these approaches and underline the importance of further study in the molecular mechanisms of CDT toxicity, particularly in the context of different pathological conditions.

Published
2019

8. Use of a reliable metabolically competent human RGHep + hepatocyte model engineered with biological tracers for in vitro micronucleus test associating high-content imaging

Author

Emmanuelle Bazin, Valérie Lobjois, Sandrine Camus, Christophe Chesne, Odile Mondesert, Gladys Mirey, Thibault Metenier, Bernard Ducommun, Julien Vignard, Ruoya Li, Bernard Salles, Vanessa Graillot, Christiane Guguen-Guillouzo, ToxAlim (ToxAlim), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA), Institut des Technologies Avancées en sciences du Vivant (ITAV), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Génotoxicité & Signalisation (ToxAlim-GS), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Eurosafe, Bioprédic International, and Institut National de la Recherche Agronomique (INRA)

Subjects
Pharmacology, 0303 health sciences, Chemistry, Pharmaceutical Science, 030226 pharmacology & pharmacy, In vitro, 3. Good health, Cell biology, 03 medical and health sciences, 0302 clinical medicine, medicine.anatomical_structure, [SDV.TOX]Life Sciences [q-bio]/Toxicology, Hepatocyte, Micronucleus test, medicine, [SDV.SPEE]Life Sciences [q-bio]/Santé publique et épidémiologie, Pharmacology (medical), ComputingMilieux_MISCELLANEOUS, High content imaging, 030304 developmental biology
Abstract

International audience

Published
2018

9. PathogenicVibrio harveyi, in contrast to non-pathogenic strains, intervenes with the p38 MAPK pathway to avoid an abalone haemocyte immune response

Author

Florence Buzin, Bertrand Cougard, Marcel Koken, Ronan Le Bouffant, Carolyn S. Friedman, Marie-Agnès Travers, Christine Paillard, Sylvain Huchette, Laboratoire des Sciences de l'Environnement Marin (LEMAR) (LEMAR), Institut de Recherche pour le Développement (IRD)-Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER)-Université de Brest (UBO)-Institut Universitaire Européen de la Mer (IUEM), Institut de Recherche pour le Développement (IRD)-Institut national des sciences de l'Univers (INSU - CNRS)-Université de Brest (UBO)-Centre National de la Recherche Scientifique (CNRS)-Institut national des sciences de l'Univers (INSU - CNRS)-Université de Brest (UBO)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Scea France Haliotis, Gametogenèse et génotoxicité (UMR_S_566 ), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris Diderot - Paris 7 (UPD7)-IFR13-Institut National de la Santé et de la Recherche Médicale (INSERM), School of Aquatic and Fishery Sciences, University of Washington [Seattle], and Institut de Recherche pour le Développement (IRD)-Institut national des sciences de l'Univers (INSU - CNRS)-Université de Brest (UBO)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Institut national des sciences de l'Univers (INSU - CNRS)-Université de Brest (UBO)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Hemocytes, Abalone, MAP Kinase Signaling System, Phagocytosis, Gastropoda, Virulence, p38 Mitogen-Activated Protein Kinases, Biochemistry, Microbiology, 03 medical and health sciences, Immune system, Animals, Molecular Biology, Pathogen, Vibrio, 030304 developmental biology, 0303 health sciences, biology, Vibrio harveyi, immune escape, Aquatic animal, 04 agricultural and veterinary sciences, Cell Biology, biology.organism_classification, MAPK, 3. Good health, virulence, Vibrio Infections, [SDE]Environmental Sciences, 040102 fisheries, 0401 agriculture, forestry, and fisheries, Reactive Oxygen Species, Haliotis tuberculata, Bacteria
Abstract

International audience; Vibrio harveyi is a marine bacterial pathogen responsible for episodic abalone epidemics associated with massive mortalities in France, Japan, and Australia. The aim of this study was the understanding of a possible role of the p38 MAPK in abalone haemocyte responses towards this bacterium. First, the pathogenicity of different V. harveyi strains was compared in both immersion and injection trials, and clear differences were detected. The three strains, ORM4, 04/092, and 05/053, all isolated from moribund abalone, induced up to 80% mortalities in immersion or injection challenges (LD(50) (ORM4) = 2.5 x 10(2) CFU animal(-1)). The two strains, LMG 4044T and LMG 7890 were non-pathogenic towards abalone in immersion trials, and needed very high numbers for killing by intramuscular injections (LD(50) = 8.9 x 10(4) and 1.6 x 10(5) CFU animal(-1), respectively). To start unraveling the mechanism explaining these differences, the p38-MAPK, a keyplayer in antimicrobial immune response, was studied. The non-pathogenic strain, LMG 7890 can be eliminated by abalone haemocytes and induces haemocyte phagocytosis and high ROS production. With different concentrations of a p38-specific inhibitor, SB203580, p38 implication was shown. This inhibitor reduced phagocytosis and ROS induction leading to LMG 7890 proliferation. In the case of the pathogenic ORM4 which can not be eliminated by abalone haemocytes, no phagocytosis and ROS production was induced, and a retarded p38 activation was observed. Taken together, our results suggest that p38 MAPK modulation may be one of the ways of virulent V. harveyi to attack its host and escape abalone immune response.

Published
2009

10. In vivo genotoxic effects of dietary heme iron on rat colon mucosa and ex vivo effects on colon cells monitored by an optimized alkaline comet assay

Author

Océane Martin, Marianne Chevalier, Sylviane Taché, Nathalie Naud, Bernard Salles, Gladys Mirey, Pierre, Fabrice H. F., Elisa Boutet-Robinet, ToxAlim (ToxAlim), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA), Prévention et promotion de la cancérogénèse par les aliments (ToxAlim-PPCA), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Génotoxicité & Signalisation (ToxAlim-GS), and Contaminants & Stress Cellulaire (ToxAlim-COMICS)

Subjects
0303 health sciences, lcsh:QH426-470, Colon, [SDV]Life Sciences [q-bio], 030311 toxicology, Heme, 3. Good health, 03 medical and health sciences, lcsh:Genetics, Genetics, Molecular Medicine, Comet Assay, Genotoxicity, optimization, Genetics (clinical), ComputingMilieux_MISCELLANEOUS, 030304 developmental biology
Abstract

In vivo comet assay is increasingly used although this assay has serious limitations as standard conditions does not allow the use of frozen samples nor the processing -at the same time- of an important number of samples generated by in vivo studies. Therefore, the aim of our work was firstly to optimize samples preparation and alkaline comet assay protocol for frozen tissue and also to increase the number of samples processed in one experiment; secondly it was to use this optimized protocol to evaluate the genotoxic effects of dietary heme iron on colon mucosa and colon epithelial cells. To optimize samples preparation and include a step of samples freezing for further experiments, different experimental parameters were tested (i.e. buffer composition, temperature of freezing/defreezing… ). The lack of induced DNA strand breaks by the freezing process of colon rat samples was assessed by comparing the results obtained between frozen and fresh samples from the same rat. The extend of DNA damage was determined by alkaline comet assay and quantified by median % Tail DNA calculation on 100 cells from 2 experimental points. By modifying different experimental parameters, we determined the optimal conditions to prepare samples and perform alkaline comet assay on cryopreserved blood and colon mucosa without DNA strand breaks induction. In order to perform middle throughput comet assays, we replaced standard glass microscope slides by 20 wells Trevigen® comet slides. Quantification of DNA breaks by median % tail DNA was undertaken by using the Komet 6.0 software (Andor Technology). Once the protocol was suitable, we used it to study the effects of dietary heme iron. Epidemiological studies revealed that high red meat intake, rich in heme iron, is associated with an increased colorectal cancer risk. In rats, heme iron-induced colorectal carcinogenesis is associated with increased fecal lipoperoxidation. In our previous study (Bastide, Chenni et al. 2015), we showed that luminal lipid peroxidation induced by the hemoglobin diet is associated with a higher anaphase bridge index in the epithelium of intestine. The genotoxic effect of the hemoglobin diet in C57BL/6J mice was confirmed with immunolabelling of gammaH2AX, a Ser-139 phosphorylation of the histone variant H2A, a well-known marker of the occurrence of DNA double strand breaks and/or replication fork arrest. The aim of our present study was to check, with alkaline comet assay, if the heme-induced lipoperoxidation increase is associated with an increment of genotoxicity in the rat colon mucosa. For this purpose, four groups of rats received either control diet or hemin diet, added or not with calcium carbonate for 3 weeks. Calcium carbonate is used to increase the level of proof between heme-induced peroxidation and genotoxicity, since it annihilates the heme-induced increase of peroxidation via heme chelation. As expected, heme iron intake increased fecal lipoperoxidation and also genotoxicity. When calcium carbonate was added to the hemin diet, these parameters were normalized. These in vivo results were completed with ex vivo experiments to determine if aldehydes from heme-induced lipoperoxidation are responsible for an increase of genotoxicity. We used fecal water, the soluble part of colonic content, from control or hemin fed rats. Fecal waters were used before and after treatment with a polymer resin (4-Fmoc-hydrazinobenzoyl AM NovaGel™) for aldehyde trapping. With this trapping of aldehydes from fecal water, the weight of aldehydes in the genotoxic activity of fecal water can be determined. Fecal waters were used on murine colonic epithelial cells and genotoxicity was assayed. Similarly to the results obtained from in vivo study, fecal waters from hemin-fed rats induced genotoxicity. When fecal waters were depleted in aldehydes, the genotoxicity was normalized. Therefore, using the resin to specifically trap fecal aldehydes, we demonstrated that aldehydes alone are responsible -at least in great part- for fecal water genotoxicity. In conclusion, our results offer a suitable protocol to evaluate genotoxicity on in vivo cryopreserved colon mucosa and on in vitro murine colonic cells, with a middle throughput capacity. This protocol confirms the increase of genotoxicity in rat colon mucosa after an heme-iron diet. Moreover, this protocol enables the demonstration that aldehydes from heme-induced lipoperoxidation are responsible for this increase of genotoxicity.

Published
2015

11. DNA damage in B and T lymphocytes of farmers during onepesticide spraying season

Author

Elisa Boutet-Robinet, Yannick Lecluse, Gladys Mirey, Pierre Lebailly, Fabrice Herin, Bernard Salles, Cancers et préventions, Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Normandie Université (NU), Centre Régional de Lutte contre le Cancer François Baclesse [Caen] (UNICANCER/CRLC), UNICANCER-Tumorothèque de Caen Basse-Normandie (TCBN)-Normandie Université (NU), Génotoxicité & Signalisation (ToxAlim-GS), ToxAlim (ToxAlim), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA), Epidémiologie et analyses en santé publique : risques, maladies chroniques et handicaps (LEASP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM), CHU Toulouse [Toulouse], Contaminants & Stress Cellulaire (ToxAlim-COMICS), Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre Régional de Lutte contre le Cancer François Baclesse (CRLC François Baclesse ), Normandie Université (NU)-Tumorothèque de Caen Basse-Normandie (TCBN), and Boutet, Elisa

Subjects
Adult, Male, DNA damage, T-Lymphocytes, [SDV]Life Sciences [q-bio], Biology, Toxicology, 03 medical and health sciences, 0302 clinical medicine, Crop production, Humans, Longitudinal Studies, Lymphocytes, Pesticides, Comet assay, 030304 developmental biology, 2. Zero hunger, 0303 health sciences, B-Lymphocytes, Farmers, Public Health, Environmental and Occupational Health, food and beverages, Pesticide, Middle Aged, Occupational exposure, Crop Production, respiratory tract diseases, 13. Climate action, 030220 oncology & carcinogenesis, Comet assay · DNA damage · Lymphocytes · Occupational exposure · Pesticides, Original Article, France, Seasons
Abstract

Purpose The effect of one pesticide spraying season on DNA damage was measured on B and T lymphocytes among open-field farmers and controls. Methods At least two peripheral blood samples were collected from each individual: one in a period without any pesticide application, several weeks after the last use (January, at period P0), and another in the intensive pesticide spraying period (May or June, at period P4). DNA damage was studied by alkaline comet assay on isolated B or T lymphocytes. Results Longitudinal comparison of DNA damage observed at both P0 and P4 periods revealed a statistically significant genotoxic effect of the pesticide spraying season in both B (P = 0.02) and T lymphocytes (P = 0.02) in exposed farmers. In contrast, non-farmers did not show any significant modifications. DNA damage levels in B and T lymphocytes were significantly higher in farmers than in non-farmers during the P4 period (P = 0.003 and P = 0.001 for B and T lymphocytes, respectively) but not during the P0 period. The seasonal effect observed among farmers was not correlated with either total farm area, farm area devoted to crops or recent solar exposure. On average, farmers used pesticides for 21 days between P0 and P4. Between the two time points studied, there was a tendency for a potential effect of the number of days of fungicide treatments (r2 = 0.43; P = 0.11) on T lymphocyte DNA damage. Conclusions A genotoxic effect was found in lymphocytes of farmers exposed to pesticides, suggesting in particular the possible implication of fungicides. Electronic supplementary material The online version of this article (doi:10.1007/s00420-015-1024-3) contains supplementary material, which is available to authorized users.

Published
2015

12. Structure of the GTPase-binding Domain of Sec5 and Elucidation of its Ral Binding Site

Author

Darerca Owen, Helen R. Mott, Louise J. Hopkins, Daniel Nietlispach, Gladys Mirey, Jacques Camonis, Mott, Helen R, Department of Biochemistry, University of Cambridge [UK] (CAM), Génotoxicité & Signalisation (ToxAlim-GS), ToxAlim (ToxAlim), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA), and Institut Curie [Paris]

Subjects
[SDV.OT]Life Sciences [q-bio]/Other [q-bio.OT], Protein Conformation, Molecular Sequence Data, 030303 biophysics, Vesicular Transport Proteins, Small G Protein, Exocyst, Plasma protein binding, Biology, Biochemistry, Exocytosis, GTP Phosphohydrolases, Mice, 03 medical and health sciences, Escherichia coli, Animals, Amino Acid Sequence, Binding site, Molecular Biology, 030304 developmental biology, 0303 health sciences, Binding Sites, Membrane Proteins, Cell Biology, Protein Structure, Tertiary, Cell biology, Ral GTP-Binding Proteins, Immunoglobulin superfamily, ral GTP-Binding Proteins, Sequence Alignment, Autre (Sciences du Vivant), Protein Binding, GTPase binding
Abstract

International audience; The exocyst complex is involved in the final stages of exocytosis, when vesicles are targeted to the plasma membrane and dock. The regulation of exocytosis is vital for a number of processes, for example, cell polarity, embryogenesis, and neuronal growth formation. Regulation of the exocyst complex in mammals was recently shown to be dependent upon binding of the small G protein, Ral, to Sec5, a central component of the exocyst. This interaction is thought to be necessary for anchoring the exocyst to secretory vesicles. We have determined the structure of the Ral-binding domain of Sec5 and shown that it adopts a fold that has not been observed in a G protein effector before. This fold belongs to the immunoglobulin superfamily in a subclass known as IPT domains. We have mapped the Ral binding site on this domain and found that it overlaps with protein-protein interaction sites on other IPT domains but that it is completely different from the G protein-geranyl-geranyl interaction face of the Ig-like domain of the Rho guanine nucleotide dissociation inhibitor. This mapping, along with available site-directed mutagenesis data, allows us to predict how Ral and Sec5 may interact.

Published
2003

13. RECQ helicase RECQL4 participates in non-homologous end joining and interacts with the Ku complex

Author

Gladys Mirey, Dharmendra Kumar Singh, Huiming Lu, Vilhelm A. Bohr, Guido Keijzers, Bernard Salles, Raghavendra A. Shamanna, Deborah L. Croteau, National Institute on Aging, Génotoxicité & Signalisation (ToxAlim-GS), ToxAlim (ToxAlim), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA), University of Copenhagen = Københavns Universitet (KU), and Intramural Program of the National Institute on Aging, National Institutes of Health, USA ZIA AG000726

Subjects
Cancer Research, Ku80, unwinding activities, DNA End-Joining Repair, DNA repair, RecQ helicase, homologous recombination, werner protein, [SDV.CAN]Life Sciences [q-bio]/Cancer, Original Manuscript, DNA-Activated Protein Kinase, Biology, in-vitro, Radiation Tolerance, functional interaction, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Humans, DNA Breaks, Double-Stranded, Ku Autoantigen, 030304 developmental biology, 0303 health sciences, Ku70, double-strand breaks, RecQ Helicases, DNA-repair, Intracellular Signaling Peptides and Proteins, Rothmund-Thomson Syndrome, Antigens, Nuclear, General Medicine, DNA repair protein XRCC4, genomic stability, Molecular biology, Non-homologous end joining, DNA-Binding Proteins, enzymes and coenzymes (carbohydrates), chemistry, Gamma Rays, 030220 oncology & carcinogenesis, rothmund-thomson-syndrome, Gene Knockdown Techniques, roles, Tumor Suppressor p53-Binding Protein 1, DNA, HeLa Cells
Abstract

International audience; RECQL4, a member of the RecQ helicase family, is a multifunctional participant in DNA metabolism. RECQL4 protein participates in several functions both in the nucleus and in the cytoplasm of the cell, and mutations in human RECQL4 are associated with three genetic disorders: Rothmund-Thomson, RAPADILINO and Baller-Cerold syndromes. We previously reported that RECQL4 is recruited to laser-induced DNA double-strand breaks (DSB). Here, we have characterized the functional roles of RECQL4 in the non-homologous end joining (NHEJ) pathway of DSB repair. In an in vitro NHEJ assay that depends on the activity of DNA-dependent protein kinase (DNA-PK), extracts from RECQL4 knockdown cells display reduced end-joining activity on DNA substrates with cohesive and non-cohesive ends. Depletion of RECQL4 also reduced the end joining activity on a CFP reporter plasmid in vivo. Knockdown of RECQL4 increased the sensitivity of cells to gamma-irradiation and resulted in accumulation of 53BP1 foci after irradiation, indicating defects in the processing of DSB. We find that RECQL4 interacts with the Ku70/Ku80 heterodimer, part of the DNA-PK complex, via its N-terminal domain. Further, RECQL4 stimulates higher order DNA binding of Ku70/Ku80 to a blunt end DNA substrate. Taken together, these results implicate that RECQL4 participates in the NHEJ pathway of DSB repair via a functional interaction with the Ku70/Ku80 complex. This is the first study to provide both in vitro and in vivo evidence for a role of a RecQ helicase in NHEJ.

Published
2014

14. Increased expression of prostaglandin reductase 1 in hepatocellular carcinomas from clinical cases and experimental tumors in rats

Author

Patrick Rouimi, Monica De-la-Luz-Cruz, Luis Del-Pozo-Yauner, Ricardo Sánchez-Rodríguez, Saúl Villa-Treviño, Luis Landero-López, Julia Esperanza Torres-Mena, Rebeca García-Román, Gloria Alejandra Bernal-Ramos, Jorge Meléndez-Zajgla, Victoria Chagoya-Hazas, Julio Isael Pérez-Carreón, Instituto Nacional de Medicina Genomica, Instituto Politécnico Nacional, Partenaires INRAE, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México (UNAM), Centro de Especialidades Medicas, Universidad Veracruzana, Génotoxicité & Signalisation (ToxAlim-GS), ToxAlim (ToxAlim), Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, CONACYT 421197, Fondo Sectorial de Investigacion en Salud y Seguridad Social SSA/IMSS/ISSSTE-CONACYT SALUD-2009-01-115431, Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Toulouse III - Paul Sabatier (UT3), and Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)

Subjects
acylfulvene, Cirrhosis, Carcinoma, Hepatocellular, alkenal/one oxidoreductase, [SDV]Life Sciences [q-bio], enzymes, [SDV.CAN]Life Sciences [q-bio]/Cancer, Biology, Biochemistry, 03 medical and health sciences, GSTP1, 0302 clinical medicine, Liver Neoplasms, Experimental, medicine, chemoprevention, Animals, Humans, Tumor marker, Diethylnitrosamine, ComputingMilieux_MISCELLANEOUS, Carcinogen, 030304 developmental biology, mechanisms, 0303 health sciences, [SDV.BA]Life Sciences [q-bio]/Animal biology, hepatocarcinogenesis, Liver Neoplasms, 13-reductase, Cell Biology, medicine.disease, Molecular biology, 3. Good health, Neoplasm Proteins, Rats, 4-Hidroxynonenal, Blot, Gene Expression Regulation, Neoplastic, Alcohol Oxidoreductases, Real-time polymerase chain reaction, inflammation, 030220 oncology & carcinogenesis, Hepatocellular carcinoma, Carcinogen detoxification enzymes, identification, Immunohistochemistry, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology, b-4 12-hydroxydehydrogenase
Abstract

International audience; To identify novel tumor-associated proteins, we analyzed the protein expression patterns from experimental hepatocellular carcinoma (HCC) that were induced using hepatocarcinogenesis models in rats. Rats were subjected to two previously described protocols of hepatocarcinogenesis using diethylnitrosamine as a carcinogen: the alternative Solt-Farber (aS&F) protocol, which induces HCC within 9 months, and Schiffer's model, which induces cirrhosis and multifocal HCC within 18 weeks. The patterns of protein expression from tumors and normal liver tissue were examined by SDS-PAGE and the bands identified at 33-34 kDa were analyzed by mass spectrometry. The prostaglandin reductase 1 (PTGR1) showed the highest number of peptides, with a confidence of level >99%. The increased expression of PTGR1 in tumors was confirmed in these two models by Western blotting and by increase in alkenal/one oxidoreductase activity (25-fold higher than normal liver). In addition, the gene expression level of Ptgr1, as measured by gRT-PCR, was increased during cancer development in a time-dependent manner (200-fold higher than normal liver). Furthermore, PTGR1 was detected in the cytoplasm of neoplastic cells in rat tumors and in 12 human HCC cases by immunohistochemistry. These analyses were performed by comparing the expression of PTGR1 to that of two well-known markers of hepatocarcinoma, Glutathione S-transferase pi 1 (GSTP1) in rats and glypican-3 in humans. The increased expression and activity of PTGR1 in liver carcinogenesis encourage further research aimed at understanding the metabolic role of PTGR1 in HCC and its potential application for human cancer diagnosis and treatment.

Published
2014

15. Mouse Testis Development and Function Are Differently Regulated by Follicle-Stimulating Hormone Receptors Signaling During Fetal and Prepubertal Life

Author

Evelyne Moreau, Pirjo Pakarinen, Stéphanie Migrenne, Jorge Merlet, René Habert, Andrée Dierich, Chrystèle Racine, Gametogenèse et génotoxicité (UMR_S_566 ), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris Diderot - Paris 7 (UPD7)-IFR13-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
Male, Anatomy and Physiology, Mouse, Cellular differentiation, Follicle-stimulating hormone, Mice, 0302 clinical medicine, Endocrinology, Reproductive Physiology, Testis, Morphogenesis, Testosterone, Receptor, Mice, Knockout, 0303 health sciences, Multidisciplinary, Sexual Differentiation, Leydig Cells, Animal Models, Sertoli cell, medicine.anatomical_structure, Receptors, FSH, Medicine, hormones, hormone substitutes, and hormone antagonists, Signal Transduction, Research Article, medicine.medical_specialty, Cell Physiology, endocrine system, Science, 030209 endocrinology & metabolism, Endocrine System, Biology, Paracrine Mechanisms, ta3111, 03 medical and health sciences, Paracrine signalling, Model Organisms, Internal medicine, medicine, Animals, Reproductive Endocrinology, Desert hedgehog, 030304 developmental biology, Sertoli Cells, Endocrine Physiology, Reproductive System, [SDV.BDLR]Life Sciences [q-bio]/Reproductive Biology, Follicle Stimulating Hormone, Hormone, Developmental Biology
Abstract

It is currently admitted that Follicle-Stimulating Hormone (FSH) is physiologically involved in the development and function of fetal/neonatal Sertoli cells in the rat but not the mouse. However, FSH is produced by both species from late fetal life onwards. We thus reinvestigated the role of FSH in mouse testis development at day 0 (birth) 6, 8 and 10 post-partum (dpp) by using mice that lack functional FSH receptors (FSH-R(-/-)). At birth, the number and proliferative index of Sertoli cells were significantly lower in FSH-R(-/-) mice than in wild type neonates. Claudin 11 mRNA expression also was significantly reduced in FSH-R(-/-) testes at 0 and 8 dpp, whereas the mRNA levels of other Sertoli cell markers (Transferrin and Desert hedgehog) were comparable in FSH-R(-/-) and wild type testes. Conversely, AMH mRNA and protein levels were higher at birth, comparable at 6 dpp and then significantly lower in FSH-R(-/-) testes at 8-10 dpp in FSH-R(-/-) mice than in controls. Although the plasma concentration of LH and the number of Leydig cells were similar in FSH-R(-/-) and control (wild type), testosterone concentration and P450c17 mRNA expression were significantly increased in FSH-R(-/-) testes at birth. Conversely, at 10 dpp when adult Leydig cells appear, expression of the steroidogenic genes P450scc, P450c17 and StAR was lower in FSH-R(-/-) testes than in controls. In conclusion, our results show that 1) like in the rat, signaling via FSH-R controls Sertoli cell development and function during late fetal life in the mouse as well; 2) paracrine factors produced by Sertoli cells are involved in the FSH-R-dependent regulation of the functions of fetal Leydig cells in late fetal life; and 3) the role of FSH-R signaling changes during the prepubertal period.

Published
2012

16. DNA-PK, a Pharmacological Target in Cancer Chemotherapy and Radiotherapy?

Author

Gladys Mirey, Bernard Salles, Patrick Calsou, Génotoxicité & Signalisation (ToxAlim-GS), ToxAlim (ToxAlim), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA), Institut de pharmacologie et de biologie structurale (IPBS), Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), and Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées

Subjects
Genome instability, 0303 health sciences, Chemotherapy, DNA repair, [SDV]Life Sciences [q-bio], medicine.medical_treatment, DNA Repair Inhibition, Cancer, Biology, medicine.disease, Bioinformatics, 3. Good health, Non-homologous end joining, Radiation therapy, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, chemistry, 030220 oncology & carcinogenesis, medicine, Cancer research, ComputingMilieux_MISCELLANEOUS, DNA, 030304 developmental biology
Abstract

In the search for ways of sensitizing tumor cells to chemotherapy or radiotherapy, the inhibition of DNA repair has recently been proposed as a target of clinical interest. Ionizing radiations, as well as several antitumor drugs, induce the formation of DNA double-strand breaks (DSBs), that are highly damaging to the DNA, leading to cell death and genomic instability. DSBs are mainly repaired by the Nonhomologous End joining (NHEJ) process, in which DNA dependent protein kinase (DNA-PK) is the key complex. Consequently, specific DNA-PK inhibitors have been selected and evaluated for sensitizing cells to chemotherapy or radiotherapy. The choice of DNA-PK as a pharmacological target of interest in cancer treatment is discussed.

Published
2012

17. A new in vitro micronucleus test in living cells associating biological tracers and high-content imaging

Author

Julien Vignard, Christophe Chesne, Bernard Salles, Bernard Ducommun, O. Mondesert, E. Bazin, V. Graillot, T. Méténier, C. Guillouzo, Gladys Mirey, Valérie Lobjois, V. Shevchenko, ToxAlim (ToxAlim), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA), Institut des Technologies Avancées en sciences du Vivant (ITAV), Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, Génotoxicité & Signalisation (ToxAlim-GS), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Eurosafe, Biopredic, Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), and ANR

Subjects
0303 health sciences, [SDV]Life Sciences [q-bio], education, Mutagenesis (molecular biology technique), General Medicine, Biology, Toxicology, In vitro, 03 medical and health sciences, 0302 clinical medicine, Biochemistry, Micronucleus test, health care economics and organizations, 030217 neurology & neurosurgery, High content imaging, 030304 developmental biology
Abstract

Poster presented at the 52nd Congress of the European-Societies-of-Toxicology (EUROTOX); A new in vitro micronucleus test in living cells associating biological tracers and high-content imaging

Published
2016

18. Evaluation of three simple direct or indirect carbonyl detection methods for characterization of oxidative modifications of proteins

Author

Saúl Villa-Treviño, Françoise Guéraud, Georg Waeg, Verónica R. Vásquez-Garzón, Neven Zarkovic, Patrick Rouimi, Isabelle Jouanin, Centro de Investigacion y Estudios Avanzados del I.P.N, Génotoxicité & Signalisation (ToxAlim-GS), ToxAlim (ToxAlim), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA), Analyse de Xénobiotiques, Identification, Métabolisme (E20 Metatoul-AXIOM), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA)-MetaToul-MetaboHUB, Génopole Toulouse Midi-Pyrénées [Auzeville] (GENOTOUL), Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Génopole Toulouse Midi-Pyrénées [Auzeville] (GENOTOUL), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Karl-Franzens-Universität [Graz, Autriche], Rudjer Boskovic Institute [Zagreb], Prévention et promotion de la cancérogénèse par les aliments (ToxAlim-PPCA), Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-MetaToul-MetaboHUB, Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Ecole Nationale Vétérinaire de Toulouse (ENVT), Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Génopole Toulouse Midi-Pyrénées [Auzeville] (GENOTOUL), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Institut National de la Santé et de la Recherche Médicale (INSERM), ProdInra, Archive Ouverte, Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-MetaboHUB-MetaToul, Karl-Franzens-Universität Graz, Dept Biol Celular, Mexico City 07738, Ctr Invest & Estudios Avanzados ( IPN ), Toxicologie Alimentaire ( UTA ), Institut National de la Recherche Agronomique ( INRA ) -Université de Bourgogne ( UB ) -AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement, Inst Mol Biosci, University of Graz, Div Mol Med, Ruder Boskovic Institute, Ctr Invest & Estudios Avanzados, Dept Biol Celular, Instituto Politecnico Nacional [Mexico] ( IPN ), Ctr Invest & Estudios Avanzados (IPN), Toxicologie Alimentaire (UTA), Institut National de la Recherche Agronomique (INRA)-Université de Bourgogne (UB)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement, Instituto Politecnico Nacional [Mexico] (IPN), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Rouimi, Patrick, Jouanin, Isabelle, Waeg, Georg, Zarkovic, Neven, Villa-Trevino, Saul, and Guéraud, Françoise

Subjects
Male, STRESS, Health, Toxicology and Mutagenesis, [SDV]Life Sciences [q-bio], Toxicology, medicine.disease_cause, Protein Carbonylation, Lipid peroxidation, chemistry.chemical_compound, 0302 clinical medicine, CHEMISTRY, ADDUCTION, CELLULAR DYSFUNCTION, biotin-hydrazide, ComputingMilieux_MISCELLANEOUS, 0303 health sciences, Chemistry, lipid peroxidation, HUMAN-DISEASE, 3. Good health, [SDV] Life Sciences [q-bio], Hydrazines, Biochemistry, 030220 oncology & carcinogenesis, oxidative stress, carbonylation, hydroxynonenal, Oxidation-Reduction, Biotin, Biotin hydrazide, Oxidative phosphorylation, COVALENT MODIFICATION, Antibodies, 4-Hydroxynonenal, 03 medical and health sciences, Lipid oxidation, TARGETS, medicine, Animals, Rats, Wistar, LIPID-PEROXIDATION PRODUCTS, 030304 developmental biology, Aldehydes, [ SDV ] Life Sciences [q-bio], Protein carbonyls, 4-hydroxynonenal, dinitrophenylhydrazine, MASS-SPECTROMETRY, IN-VITRO, Proteins, Rats, Oxidative Stress, Cumene hydroperoxide, Streptavidin, Carbonylation, Oxidative stress
Abstract

Among disruptions induced by oxidative stress, modifications of proteins, particularly irreversible carbonylation, are associated with the development of several diseases, including cardiovascular diseases, neurodegenerative diseases, and cancer. Carbonylation of proteins can occur directly or indirectly through the adduction of lipid oxidation products. In this study, three classical and easy-to-perform techniques to detect direct or indirect carbonylation of proteins were compared. A model protein apomyoglobin and a complex mixture of rat liver cytosolic proteins were exposed to cumene hydroperoxide oxidation or adduction to the lipid peroxidation product 4-hydroxynonenal in order to test direct or indirect carbonylation, respectively. The technique using a specific anti-4-hydroxynonenal-histidine adduct antibody was effective to detect in vitro modification of model apomyoglobin and cytosolic proteins by 4-hydroxynonenal but not by direct carbonylation which was achieved by techniques using biotin-coupled hydrazide or dinitrophenylhydrazine derivatization of carbonyls. Sequential use of these methods enabled the detection of both direct and indirect carbonyl modification in proteins, although constitutively biotinylated proteins were detected by biotin-hydrazide. Although rather classical and efficient, methods for carbonyl detection on proteins in oxidative stress studies may be biased by some artifactual detections and complicated by proteins multimerizations. The use of more and more specific available antibodies is recommended to complete detection of lipid peroxidation product adducts on proteins.

Published
2012

19. Impacts of low doses of pesticide mixtures on liver cell defence systems

Author

Georges de Sousa, Andrej Razpotnik, Gwendoline Dupont, Patrick Rouimi, Edwin Fouché, Nathalie Zucchini-Pascal, Roger Rahmani, Génotoxicité & Signalisation (ToxAlim-GS), ToxAlim (ToxAlim), Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, Institut Sophia Agrobiotech (ISA), Centre National de la Recherche Scientifique (CNRS)-Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Recherche Agronomique (INRA), Prévention et promotion de la cancérogénèse par les aliments (ToxAlim-PPCA), and Rouimi, Patrick

Subjects
Male, [SDV]Life Sciences [q-bio], Apoptosis, 010501 environmental sciences, Pharmacology, Toxicology, 01 natural sciences, AGRICULTURAL HEALTH, chemistry.chemical_compound, Mice, PARKINSONS-DISEASE, Pesticides mixture, Atrazine, Liver, Hepatocytes, HepaRG, Metabolic pathways, DRUG-METABOLIZING-ENZYMES, CONSTITUTIVE ANDROSTANE RECEPTOR, PREGNANE-X-RECEPTOR, IN-VITRO, NUCLEAR RECEPTORS, CANCER INCIDENCE, OCCUPATIONAL-EXPOSURE, HERBICIDE ATRAZINE, Cells, Cultured, chemistry.chemical_classification, 0303 health sciences, Liver cell, General Medicine, 3. Good health, Chlorpyrifos, [SDE]Environmental Sciences, Female, Endosulfan, Biology, Cell Line, 03 medical and health sciences, Detoxification, Animals, Humans, Pesticides, 030304 developmental biology, 0105 earth and related environmental sciences, Gene Expression Profiling, Pesticide, Mice, Inbred C57BL, Metabolic pathway, Enzyme, chemistry
Abstract

International audience; Low amounts of residual pesticides are present in the environment, often as mixtures of chemicals which contaminate drinking water and food, being a source of chronic exposure for humans and a growing matter of concern in public health policy. Despite of the needs and growing investigation, little is known about the impact of low doses and mixtures of these chemicals on human health. The purpose of this study was to enlighten if modifications of liver cell metabolic- and/or defence-related capacities could occur under such exposures. In vitro perturbations of several metabolic, stress and survival pathways in human and mice cultured hepatocytes and liver cells were evaluated under exposure to low doses of single molecules or equimolecular combinations of the three pesticides, atrazine, chlorpyrifos and endosulfan. Mainly phases land II enzymes of detoxification were found modulated, together with apoptotic process deregulation. Hence, CYP3A4 and CYP3A11 were upregulated in primary cultured human and mouse hepatocytes, respectively. These inductions were correlated to an anti-apoptotic process (increased Bcl-xL/Bax ratio, inhibition of the PARP protein cleavage). Such disturbances in pathways involved in cell protection may possibly account for initiation of pathologies or decrease in drugs efficiency in humans exposed to multiple environmental contaminants.

Published
2012

20. A diagnostic tool to assess genotoxic activity ex vivo

Author

B. Ducommun, Gilles Favre, Bernard Salles, Y. Fedor, Aurélien Olichon, Gladys Mirey, ToxAlim (ToxAlim), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA), Institut National de la Santé et de la Recherche Médicale (INSERM), Institut des Technologies Avancées en sciences du Vivant (ITAV), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Génotoxicité & Signalisation (ToxAlim-GS), and Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3)

Subjects
0303 health sciences, business.industry, [SDV]Life Sciences [q-bio], General Medicine, Toxicology, 03 medical and health sciences, 0302 clinical medicine, Cancer research, Medicine, business, 030217 neurology & neurosurgery, Ex vivo, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology
Abstract

National audience

Published
2011

21. SHOC1 and PTD form an XPF-ERCC1-like complexthat is required for formation of class I crossovers

Author

Raphael Mercier, Nicolas Macaisne, Julien Vignard, Institut Jean-Pierre Bourgin (IJPB), Institut National de la Recherche Agronomique (INRA)-AgroParisTech, Génotoxicité & Signalisation (ToxAlim-GS), ToxAlim (ToxAlim), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA), Macaisne, Nicolas, Vignard, Julien, Mercier, Raphaël, Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), and Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées

Subjects
0106 biological sciences, XPF, [SDV.SA]Life Sciences [q-bio]/Agricultural sciences, Saccharomyces cerevisiae, Arabidopsis, MLH3, 01 natural sciences, Evolution, Molecular, 03 medical and health sciences, Endonuclease, Meiosis, Recombination, ERCC1, Crossover, Humans, Crossing Over, Genetic, Cation Transport Proteins, Phylogeny, 030304 developmental biology, Genetics, 0303 health sciences, Endodeoxyribonucleases, biology, Arabidopsis Proteins, Proteins, Epistasis, Genetic, Cell Biology, Endonucleases, biology.organism_classification, Cell biology, DNA-Binding Proteins, MSH5, MSH4, Mutation, biology.protein, C9orf84, Carrier Proteins, Dimerization, Protein Binding, 010606 plant biology & botany
Abstract

Two distinct pathways for meiotic crossover formation coexist in most eukaryotes. The Arabidopsis SHOC1 protein is required for class I crossovers and shows sequence similarity with the XPF endonuclease family. Active XPF endonucleases form a heterodimer with ERCC1 proteins. Here, we show that PTD, an ERCC1-like protein, is required for class-I-interfering crossovers along with SHOC1, MSH4, MSH5, MER3 and MLH3. SHOC1 interacts with PTD in a two-hybrid assay, through its XPF-like nuclease–(HhH)2 domain. We propose that a XPF–ERCC1-like heterodimer, represented by SHOC1 and PTD in Arabidopsis, involving Zip2 in Saccharomyces cerevisiae and C9orf84 in human, is required for formation of class I crossovers.

Published
2011

22. Partners apart: Smc6-independent DNA binding activity of Smc5 on single-strand DNA

Author

Cyril Charbonnel, Jean-Yves Masson, Julien Vignard, Génotoxicité & Signalisation (ToxAlim-GS), ToxAlim (ToxAlim), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA), Canc Res Ctr, and University of Liverpool

Subjects
0303 health sciences, COMPLEX, [SDV]Life Sciences [q-bio], RECOMBINATION, Cell Biology, Biology, Single strand dna, DNA binding site, 03 medical and health sciences, 0302 clinical medicine, Biochemistry, SMC5-SMC6, Molecular Biology, BREAK REPAIR, 030217 neurology & neurosurgery, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Developmental Biology, Dna binding activity, COHESIN
Abstract

International audience

Published
2011

23. Mouse differentiating spermatogonia can generate germinal stem cells in vivo

Author

Isabelle Allemand, Lydia Riou, Florence Le Page, Jacques Testart, Pierre Fouchet, Vilma Barroca, Mathieu Coureuil, J.-P. Louis, Bruno Lassalle, Gametogenèse et génotoxicité (UMR_S_566 ), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris Diderot - Paris 7 (UPD7)-IFR13-Institut National de la Santé et de la Recherche Médicale (INSERM), Immunologie et Embryologie Moléculaires (IEM), and Université d'Orléans (UO)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Male, Transgene, Green Fluorescent Proteins, Mice, Transgenic, Biology, Mice, 03 medical and health sciences, 0302 clinical medicine, Testis, Glial cell line-derived neurotrophic factor, Animals, Regeneration, Cell Lineage, Glial Cell Line-Derived Neurotrophic Factor, Progenitor cell, Cell Proliferation, 030304 developmental biology, Stem cell transplantation for articular cartilage repair, 0303 health sciences, Stem Cells, fungi, food and beverages, Cell Differentiation, Cell Biology, Cell Dedifferentiation, Spermatogonia, Cell biology, Mice, Inbred C57BL, Endothelial stem cell, Haematopoiesis, Germ Cells, biology.protein, [SDV.IMM]Life Sciences [q-bio]/Immunology, Fibroblast Growth Factor 2, Stem cell, 030217 neurology & neurosurgery, Stem Cell Transplantation, Adult stem cell
Abstract

International audience; In adults, stem cells are responsible for the maintenance of many actively renewing tissues, such as haematopoietic, skin, gut and germinal tissues. These stem cells can self-renew or be committed to becoming progenitors. Stem-cell commitment is thought to be irreversible but in male and female Drosophila melanogaster, it was shown recently that differentiating germ cells can revert to functional stem cells that can restore germinal lineage. Whether progenitors are also able to generate stem cells in mammals remains unknown. Here we show that purified mouse spermatogonial progenitors committed to differentiation can generate functional germinal stem cells that can repopulate germ-cell-depleted testes when transplanted into adult mice. We found that GDNF, a key regulator of the stem-cell niche, and FGF2 are able to reprogram in vitro spermatogonial progenitors for reverse differentiation. This study supports the emerging concept that the stem-cell identity is not restricted in adults to a definite pool of cells that self-renew, but that stemness could be acquired by differentiating progenitors after tissue injury and throughout life.

Published
2009

24. Characterization of Spo11-dependent and independent phospho-H2AX foci during meiotic prophase I in the male mouse

Author

Bernard Dutrillaux, Jacqueline Bernardino-Sgherri, Alexandra Chicheportiche, Bernard de Massy, Gametogenèse et génotoxicité (UMR_S_566 ), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris Diderot - Paris 7 (UPD7)-IFR13-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de génétique humaine (IGH), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Origine, structure et évolution de la biodiversité (OSEB), Muséum national d'Histoire naturelle (MNHN)-Centre National de la Recherche Scientifique (CNRS), and Larose, Catherine

Subjects
Male, Spo11, DNA Repair, DNA repair, cells, Guinea Pigs, [SDV.GEN] Life Sciences [q-bio]/Genetics, Histones, 03 medical and health sciences, Meiotic Prophase I, Mice, 0302 clinical medicine, Meiosis, Chinchilla, Spermatocytes, Testis, Animals, DNA Breaks, Double-Stranded, ComputingMilieux_MISCELLANEOUS, Cells, Cultured, 030304 developmental biology, Mice, Knockout, 0303 health sciences, [SDV.GEN]Life Sciences [q-bio]/Genetics, Endodeoxyribonucleases, biology, fungi, Histone H2AX, Esterases, Chromosome, Cell Biology, Molecular biology, 3. Good health, Chromatin, Mice, Inbred C57BL, enzymes and coenzymes (carbohydrates), Histone, 030220 oncology & carcinogenesis, biology.protein, Pachytene Stage, biological phenomena, cell phenomena, and immunity
Abstract

Meiotic DNA double strand breaks (DSBs) are indicated at leptotene by the phosphorylated form of histone H2AX (gamma-H2AX). In contrast to previous studies, we identified on both zygotene and pachytene chromosomes two distinct types of gamma-H2AX foci: multiple small (S) foci located along autosomal synaptonemal complexes (SCs) and larger signals on chromatin loops (L-foci). The S-foci number gradually declined throughout pachytene, in parallel with the repair of DSBs monitored by repair proteins suggesting that S-foci mark DSB repair events. We validated this interpretation by showing the absence of S-foci in Spo11(-/-) spermatocytes. By contrast, the L-foci number was very low through pachytene. Based on the analysis of gamma-H2AX labeling after irradiation of spermatocytes, the formation of DSBs clearly induced L-foci formation. Upon DSB repair, these foci appear to be processed and lead to the above mentioned S-foci. The presence of L-foci in wild-type pachytene and diplotene could therefore reflect delayed or unregulated DSB repair events. Interestingly, their distribution was different in Spo11(+/-) spermatocytes compared with Spo11(+/+) spermatocytes, where DSB repair might be differently regulated as a response to homeostatic control of crossing-over. The presence of these L-foci in Spo11(-/-) spermatocytes raises the interesting possibility of yet uncharacterized alterations in DNA or chromosome structure in Spo11(-/-) cells.

Published
2007

25. Effect of various doses of deoxynivalenol on liver xenobiotic metabolizing enzymes in mice

Author

Patrick Rouimi, Isabelle P. Oswald, Marie-Estelle Gouze, Joëlle Laffitte, Pierre Galtier, Nicolas Loiseau, Unité de recherche Pharmacologie-Toxicologie (UPT), Institut National de la Recherche Agronomique (INRA), Laboratoire de Pharmacologie et Toxicologie, Génotoxicité & Signalisation (ToxAlim-GS), ToxAlim (ToxAlim), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Biocomplexité des écosystèmes coralliens de l'Indo-Pacifique (CoReUS2), IFP Energies nouvelles (IFPEN), Xénobiotiques, Ecole Nationale Vétérinaire de Toulouse (ENVT), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA), Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire de Toulouse (ENVT), and Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées

Subjects
DEOXYNIVALENOL, Male, LIVER, [SDV]Life Sciences [q-bio], Administration, Oral, Food Contamination, 010501 environmental sciences, Pharmacology, Toxicology, 01 natural sciences, Isozyme, Xenobiotics, 03 medical and health sciences, chemistry.chemical_compound, Mice, Vomitoxin, Cytochrome P-450 Enzyme System, Animals, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0105 earth and related environmental sciences, GLUTATHIONE S-TRANSFERASE, 0303 health sciences, Mice, Inbred BALB C, biology, Dose-Response Relationship, Drug, Cytochrome P450, General Medicine, Glutathione, 3. Good health, Immunoglobulin A, Dose–response relationship, Glutathione S-transferase, CYTOCHROME P450, chemistry, Biochemistry, biology.protein, Microsomes, Liver, Alkaline phosphatase, Xenobiotic, Edible Grain, Trichothecenes, Food Science
Abstract

International audience; DON is one of the major mycotoxic contaminant of cereal grains throughout the world. The purpose of this investigation was to characterize the effects of a range of environmentally relevant doses of DON in mice exposed through a subchronic toxicological assay. Animals received 3 days per week for 4 weeks, 0.014, 0.071, 0.355 or 1.774 mg of toxin/kg b.w. All doses, except 0.014 mg/kg, provoked increases in plasma immunoglobulin A whereas there was no change in plasma biochemical parameters such as alkaline phosphatase, electrolytes or other immunoglobulins. Administration of 0.071 or 0.355 mg/kg doses led to increased liver microsomal pentoxyresorufin depentylase and cytosolic glutathione transferase activities. Examining protein modulation, western blot analyses liver fractions from mice receiving these doses revealed increased levels in both P450 2b, GST a and p isoenzymes without any change in P450 1a expression. A significant competitive inhibition of deoxynivalenol on CDNB conjugation in vitro suggests that the mycotoxin is a putative substrate for glutathione S-transferases. These changes in liver xenobiotic metabolizing enzymes are discussed by considering the structural nature of deoxynivalenol and previous reports on similar effects exerted by other trichothecenes. These results suggest that a subchronic exposure to low doses of deoxynivalenol causes changes in the normal liver metabolism of xenobiotics.

Published
2005

26. Mechanism of sulforaphane-induced cell cycle arrest and apoptosis in human colon cancer cells

Author

Lydie Combaret, Georges Cassar, Pengfei Li, Laurence Gamet-Payrastre, Patrick Rouimi, Géraldine Parnaud, Jacques Tulliez, Xénobiotiques, Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, Ecole Nationale Vétérinaire de Toulouse (ENVT), C. Eugene Bennett Department of Chemistry, West Virginia University [Morgantown], Génotoxicité & Signalisation (ToxAlim-GS), ToxAlim (ToxAlim), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Inconnu, Unité de nutrition et métabolisme protéique, Institut National de la Recherche Agronomique (INRA), ProdInra, Migration, and Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA)

Subjects
G2 Phase, Cancer Research, medicine.medical_specialty, Cell cycle checkpoint, [SDV]Life Sciences [q-bio], Medicine (miscellaneous), Mitosis, Apoptosis, Biology, 03 medical and health sciences, HT29 Cells, chemistry.chemical_compound, 0302 clinical medicine, Isothiocyanates, Internal medicine, MG132, CDC2 Protein Kinase, medicine, Roscovitine, Anticarcinogenic Agents, Humans, Phosphorylation, Cyclin B1, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, Cyclin-dependent kinase 1, Nutrition and Dietetics, Kinase, Cell Cycle, Cell cycle, Flow Cytometry, CANCER, 3. Good health, [SDV] Life Sciences [q-bio], Endocrinology, Oncology, chemistry, Purines, 030220 oncology & carcinogenesis, Sulfoxides, Cancer research, RAT, Thiocyanates
Abstract

Sulforaphane (SFN) is a natural micronutrient found in cruciferous vegetables that has been shown to possess antitumoral properties in carcinogen-treated rats. In vitro, SFN regulates phase II enzymes, cell cycle, and apoptosis. In the present study, we investigated the relationship between SFN induction of apoptosis and cell cycle arrest in HT29 human colon carcinoma cells. In previously published data, a significant increase in the G2/M phase of the cell cycle has been observed in SFN-treated cells that was associated with increased cyclin B1 protein levels. In the present study, our results show that SFN induced p21 expression. Moreover, preincubation of HT29 cells with roscovitine, a specific cdc2 kinase inhibitor, blocked the G2/M phase accumulation of HT29 cells treated with SFN and abolished its apoptotic effect (22.2 +/- 4 of floating cells in SFN-treated cells vs. 6.55 +/- 2 in cells treated with both SFN and roscovitine). These results suggest that the cdc2 kinase could be a key target for SFN in the regulation of G2/M block and apoptosis. Moreover, in SFN-treated cells the retinoblastoma tumor suppressor protein (Rb) is highly phosphorylated. Inhibition of the cdc2 kinase by roscovitine did not change the phosphorylation status of Rb in SFN-treated cells, suggesting that this cyclin-dependent kinase may not be involved. In our study, we did not observe any significant change in the proteasomal activity between control and SFN-treated cells. Moreover, inhibition of proteasomal activity through the use of MG132 diminished SFN-induced HT29 cell death, suggesting that the apoptotic effect of SFN requires a functional proteasome-dependent degradation system. In summary, we have elucidated part of the mechanism of action of SFN in the concomitant regulation of intestinal cell growth and death.

Published
2004

27. Ral GTPases regulate exocyst assembly through dual subunit interactions

Author

Serge Moskalenko, Carine Rossé, Jacques Camonis, Chao Tong, Gladys Mirey, Laurent Daviet, Michael A. White, Etienne Formstecher, University of Texas Southwestern Medical Center, Hybrigenics, Génotoxicité & Signalisation (ToxAlim-GS), ToxAlim (ToxAlim), Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, Institut Curie [Paris], and White, Michael A

Subjects
[SDV.OT]Life Sciences [q-bio]/Other [q-bio.OT], animal structures, Macromolecular Substances, Vesicular Transport Proteins, Exocyst, GTPase, Biology, Transfection, Biochemistry, Exocytosis, Cell Line, 03 medical and health sciences, 0302 clinical medicine, Humans, Molecular Biology, 030304 developmental biology, 0303 health sciences, RALB, Effector, Secretory Vesicles, Membrane Proteins, Cell Biology, Exocyst assembly, RALA, Cell biology, Protein Subunits, Ral GTP-Binding Proteins, 030220 oncology & carcinogenesis, ral GTP-Binding Proteins, Carrier Proteins, Autre (Sciences du Vivant), Protein Binding
Abstract

International audience; Ral GTPases have been implicated in the regulation of a variety of dynamic cellular processes including proliferation, oncogenic transformation, actin-cytoskeletal dynamics, endocytosis, and exocytosis. Recently the Sec6/8 complex, or exocyst, a multisubunit complex facilitating post-Golgi targeting of distinct subclasses of secretory vesicles, has been identified as a bona fide Ral effector complex. Ral GTPases regulate exocyst-dependent vesicle trafficking and are required for exocyst complex assembly. Sec5, a membrane-associated exocyst subunit, has been identified as a direct target of activated Ral; however, the mechanism by which Ral can modulate exocyst assembly is unknown. Here we report that an additional component of the exocyst, Exo84, is a direct target of activated Ral. We provide evidence that mammalian exocyst components are present as distinct subcomplexes on vesicles and the plasma membrane and that Ral GTPases regulate the assembly interface of a full octameric exocyst complex through interaction with Sec5 and Exo84.

Published
2003

28. A Ral guanine exchange factor-Ral pathway is conserved in Drosophila melanogaster and sheds new light on the connectivity of the Ral, Ras, and Rap pathways

Author

Gladys Mirey, Jacques Camonis, Maria Balakireva, Stéphanie Voegeling, Carine Rossé, Sébastien L'Hoste, Camonis, Jacques, Génotoxicité & Signalisation (ToxAlim-GS), ToxAlim (ToxAlim), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA), Transduction du signal et oncogénèse, and Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
[SDV.OT]Life Sciences [q-bio]/Other [q-bio.OT], animal structures, Rap GTP-binding protein, Genes, Insect, GTPase, Biology, Eye, Animals, Genetically Modified, 03 medical and health sciences, 0302 clinical medicine, Species Specificity, Two-Hybrid System Techniques, ral Guanine Nucleotide Exchange Factor, Animals, Drosophila Proteins, Humans, Ral Guanine Nucleotide Exchange Factor, Cell Growth and Development, Molecular Biology, Alleles, 030304 developmental biology, Genetics, 0303 health sciences, Effector, fungi, Gene Expression Regulation, Developmental, Cell Biology, biology.organism_classification, Cell biology, Drosophila melanogaster, rap GTP-Binding Proteins, Microscopy, Electron, Scanning, ras Proteins, Guanine nucleotide exchange factor, Signal transduction, 030217 neurology & neurosurgery, Drosophila Protein, Autre (Sciences du Vivant)
Abstract

International audience; Ras GTPases are central to many physiological and pathological signaling pathways and act via a combination of effectors. In mammals, at least three Ral exchange factors (RalGEFs) contain a Ras association domain and constitute a discrete subgroup of Ras effectors. Despite their ability to bind activated Rap as well as activated Ras, they seem to act downstream of Ras but not downstream of Rap. We have revisited the Ras/Rap-Ral connections in Drosophila melanogaster by using iterative two-hybrid screens with these three GTPases as primary baits and a subsequent genetic approach. We show that (i) the Ral-centered protein network appears to be extremely conserved in human and flies, (ii) in this network, RGL is a functional Drosophila orthologue of RalGEFs, and (iii) the RGL-Ral pathway functionally interacts with both the Ras and Rap pathways. Our data do not support the paradigmatic model where Ral is in the effector pathway of Ras. They reveal a signaling circuitry where Ral is functionally downstream of the Rap GTPase, at odds with the pathways described for mammalian cell lines. Thus, in vivo data show variations in the connectivity of pathways described for cell lines which might display only a subset of the biological possibilities.

Published
2003

29. RLIP76, an effector of the GTPase Ral, interacts with the AP2 complex: involvement of the Ral pathway in receptor endocytosis

Author

Jacques Camonis, Yannick Mahé, B. Meunier-Bisceuil, A. Sorkin, Corinne Leprince, Gladys Mirey, V. Jullien-Flores, Transduction du signal et oncogénèse, Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Génotoxicité & Signalisation (ToxAlim-GS), ToxAlim (ToxAlim), Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, University of Colorado, University of Colorado [Boulder], and ProdInra, Migration

Subjects
GTPase-activating protein, GTP Phosphohydrolases, 0302 clinical medicine, Epidermal growth factor, Genes, Reporter, RLIP76, 0303 health sciences, RALB, Ras signalling, GTPase-Activating Proteins, Transferrin, Signal transducing adaptor protein, RALA, Endocytosis, Cell biology, Adaptor Protein Complex mu Subunits, ErbB Receptors, 030220 oncology & carcinogenesis, Signal transduction, Protein Binding, Signal Transduction, [SDV.OT]Life Sciences [q-bio]/Other [q-bio.OT], animal structures, Adaptor Protein Complex 3, Adaptor Protein Complex 1, Green Fluorescent Proteins, Adaptor Protein Complex 2, Biology, Transfection, Evolution, Molecular, 03 medical and health sciences, Adaptor Protein Complex alpha Subunits, Two-Hybrid System Techniques, Receptors, Transferrin, Humans, 030304 developmental biology, adaptin, Epidermal Growth Factor, [SDV.OT] Life Sciences [q-bio]/Other [q-bio.OT], Membrane Proteins, Epithelial Cells, Cell Biology, Ral, Adaptor Proteins, Vesicular Transport, Luminescent Proteins, Ral GTP-Binding Proteins, Mutagenesis, ras Proteins, ATP-Binding Cassette Transporters, Indicators and Reagents, ral GTP-Binding Proteins, Carrier Proteins, Gene Deletion, HeLa Cells
Abstract

International audience; RLIP76 is a modular protein that was identified as a putative effector of Ral, a GTPase activated during Ras signaling. To explore further the contribution of the Ral-RLIP76 pathway to Ras signaling, we have looked for partners of RLIP76. Mu2, the medium chain of the AP2 complex is shown to interact with RLIP76. We show also that in vivo endogenous AP2 and RLIP76 form a complex and that this in vivo interaction is independent of cells being stimulated by a growth factor. Furthermore, RLIP76 differentiates AP2 from AP1 in vivo as RLIP76 differentiates mu2 from mu1 in vitro and in two hybrid assays. We show that activated Ral interferes with both tranferrin receptor endocytosis and epidermal growth factor (EGF) receptor endocytosis in HeLa cells. We propose a model where the Ral-RLIP76 pathway connects signal transduction and endocytosis through interaction on one hand between the Ras-Ral pathway and RLIP, on the other hand between RLIP and proteins belonging to the endocytotic machinery.

Published
2000

30. Interaction of the Grb7 adapter protein with Rnd1, a new member of the Rho family

Author

Jacques Camonis, Béatrice Vayssière, Pierre Chardin, Gérard Zalcman, K. Michael Weidner, Gladys Mirey, Yannick Mahé, Tanja Ligensa, Génétique et expression des oncogènes, Institut National de la Santé et de la Recherche Médicale (INSERM), Génotoxicité & Signalisation (ToxAlim-GS), ToxAlim (ToxAlim), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA), Pharma Research, and Centre National de la Recherche Scientifique (CNRS)

Subjects
rho GTP-Binding Proteins, [SDV.OT]Life Sciences [q-bio]/Other [q-bio.OT], GTP', yeast two-hybrid, Recombinant Fusion Proteins, Two-hybrid screening, Biophysics, GTPase, Plasma protein binding, Rnd1, Transfection, SH2 domain, Biochemistry, Antibodies, src Homology Domains, 03 medical and health sciences, 0302 clinical medicine, Structural Biology, Two-Hybrid System Techniques, Escherichia coli, Tumor Cells, Cultured, Genetics, Humans, Grb7, Phosphorylation, Phosphotyrosine, Molecular Biology, Sequence Deletion, 030304 developmental biology, 0303 health sciences, Binding Sites, Sequence Homology, Amino Acid, biology, GRB7, Proteins, Signal transducing adaptor protein, Cell Biology, Precipitin Tests, Cell biology, Molecular Weight, SH2, GRB7 Adaptor Protein, 030220 oncology & carcinogenesis, biology.protein, Signal transduction, HeLa Cells, Protein Binding
Abstract

International audience; Grb7 is a member of a family of molecular adapters which are able to contribute positively but also negatively to signal transduction and whose precise roles remain obscure. Rnd1 is a member of the Rho family, but, as opposed to usual GTPases, it is constitutively bound to GTP. We show here that Rnd1 and Grb7 interact, in two-hybrid assays, in vitro, and in pull-down experiments performed with SK-BR3, a breast cancer cell line that overexpresses Grb7. This interaction involves switch II loop of Rnd1, a region crucial for guanine nucleotide exchange in all GTPases, and a Grb7 SH2 domain, a region crucial for Grb7 interaction with several activated receptors. The contribution of the interaction between Rnd1 and Grb7 to their respective functions and properties is discussed.

Published
2000

31. Effector recognition by the small GTP-binding proteins Ras and Ral

Author

Ingrid R. Vetter, Alfred Wittinghofer, Robbert H. Cool, Jacques Camonis, Gladys Mirey, Juan Antonio García-Ranea, Alfonso Valencia, B. Bauer, Max Planck Institute of Molecular Plant Physiology (MPI-MP), Max-Planck-Gesellschaft, Génotoxicité & Signalisation (ToxAlim-GS), ToxAlim (ToxAlim), Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, Génétique et expression des oncogènes, Institut National de la Santé et de la Recherche Médicale (INSERM), Consejo Superior de Investigaciones Científicas [Madrid] (CSIC), and Max Planck Institute of Molecular Physiology

Subjects
Models, Molecular, [SDV.OT]Life Sciences [q-bio]/Other [q-bio.OT], animal structures, GTPase-activating protein, Sequence alignment, GTPase, Biology, Biochemistry, Conserved sequence, 03 medical and health sciences, GTP-binding protein regulators, GTP-Binding Proteins, Animals, Humans, Molecular Biology, Conserved Sequence, 030304 developmental biology, Genetics, Guanylyl Imidodiphosphate, 0303 health sciences, Effector, GTPase-Activating Proteins, 030302 biochemistry & molecular biology, Haplorhini, Cell Biology, Guanine Nucleotides, Recombinant Proteins, Cell biology, Ral GTP-Binding Proteins, Mutagenesis, Site-Directed, ras Proteins, ATP-Binding Cassette Transporters, ral GTP-Binding Proteins, Guanine nucleotide exchange factor, Carrier Proteins, Sequence Alignment, Protein Binding
Abstract

International audience; The Ral effector protein RLIP76 (also called RIP/RalBP1) binds to Ral.GTP via a region that shares no sequence homology with the Ras-binding domains of the Ser/Thr kinase c-Raf-1 and the Ral-specific guanine nucleotide exchange factors. Whereas the Ras-binding domains have a similar ubiquitin-like structure, the Ral-binding domain of RLIP was predicted to comprise a coiled-coil region. In order to obtain more information about the specificity and the structural mode of the interaction between Ral and RLIP, we have performed a sequence space and a mutational analysis. The sequence space analysis of a comprehensive nonredundant assembly of Ras-like proteins strongly indicated that positions 36 and 37 in the core of the effector region are tree-determinant positions for all subfamilies of Ras-like proteins and dictate the specificity of the interaction of these GTPases with their effector proteins. Indeed, we could convert the specific interaction with Ras effectors and RLIP by mutating these residues in Ras and Ral. We therefore conclude that positions 36 and 37 are critical for the discrimination between Ras and Ral effectors and that, despite the absence of sequence homology between the Ral-binding and the Ras-binding domains, their mode of interaction is most probably similar.

Published
1999

32. Characterization of pig liver glutathione S-transferases using HPLC-electrospray-ionization mass spectrometry

Author

Patrick Rouimi, Patricia Anglade, Jacques Tulliez, Laurent Debrauwer, Xénobiotiques, Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, Laboratoire de biochimie et technologie laitières, Institut National de la Recherche Agronomique (INRA), ProdInra, Migration, Génotoxicité & Signalisation (ToxAlim-GS), ToxAlim (ToxAlim), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Bactéries Lactiques et Pathogènes Opportunistes (UBLO), Ecole Nationale Vétérinaire de Toulouse (ENVT), and Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA)

Subjects
Male, Carbonyl Reductase, Swine, [SDV]Life Sciences [q-bio], Electrospray ionization, Blotting, Western, Molecular Sequence Data, Mass spectrometry, Peptide Mapping, Biochemistry, Mass Spectrometry, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Animals, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Amino Acid Sequence, Molecular Biology, Peptide sequence, Chromatography, High Pressure Liquid, Binding selectivity, ComputingMilieux_MISCELLANEOUS, Glutathione Transferase, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, Chromatography, Sequence Homology, Amino Acid, Molecular mass, Cell Biology, Glutathione, Isoenzymes, Molecular Weight, Enzyme, Liver, chemistry, 030217 neurology & neurosurgery, Research Article
Abstract

International audience; We have characterized 11 porcine liver cytosolic glutathione S-transferase (GST) subunits from their precise molecular mass, immunoreactivity and partial amino acid sequence. Four Alpha-, six Mu- and one unexpected Pi-class GST subunits were found with average molecular masses of 24.984–25.228 kDa, 25.039–25.657 kDa and 23.510 kDa respectively. Molecular masses were established using electrospray-ionization mass spectrometry, with a precision of ±3–4 mass units. Glutathione (GSH) and S-hexylglutathione (ShGSH) were tested as affinity ligands in the purification procedure. The binding selectivity of GSH was better than that of ShGSH, although non-GST proteins were retained on both matrices. As already described in other studies, a number of non-GST proteins bound to the affinity resins. Two of them were tentatively identified as mevalonate kinase and carbonyl reductase. The characterization of pig liver cytosolic GST subunits pattern achieved in this work should constitute a useful tool for rapid evaluation of these enzymes' expression in modulation studies.

Published
1996

33. Ionizing-radiation induced DNA double-strand breaks: A direct and indirect lighting up

Author

Bernard Salles, Julien Vignard, Gladys Mirey, Génotoxicité & Signalisation (ToxAlim-GS), ToxAlim (ToxAlim), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA), and ANR program [ANR-10-CESA-011]

Subjects
DNA damage, DNA repair, [SDV]Life Sciences [q-bio], Radiosensitivity, 03 medical and health sciences, chemistry.chemical_compound, Radiation dosage, 0302 clinical medicine, Radiation, Ionizing, Humans, DNA Breaks, Double-Stranded, Radiology, Nuclear Medicine and imaging, DNA-PKcs, DNA Single Strand Break, 030304 developmental biology, 0303 health sciences, Radiation, Chemistry, Gamma rays, Hematology, Biomarker, Chromatin, Cell biology, Non-homologous end joining, Oncology, Radiology Nuclear Medicine and imaging, Double-strand break, 030220 oncology & carcinogenesis, Homologous recombination, DNA
Abstract

International audience; The occurrence of DNA double-strand breaks (DSBs) induced by ionizing radiation has been extensively studied by biochemical or cell imaging techniques. Cell imaging development relies on technical advances as well as our knowledge of the cell DNA damage response (DDR) process. The DDR involves a complex network of proteins that initiate and coordinate DNA damage signaling and repair activities. As some DDR proteins assemble at DSBs in an established spatio-temporal pattern, visible nuclear foci are produced. In addition, post-translational modifications are important for the signaling and the recruitment of specific partners at damaged chromatin foci. We briefly review here the most widely used methods to study DSBs. We also discuss the development of indirect methods, using reporter expression or intra-nuclear antibodies, to follow the production of DSBs in real time and in living cells.

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34. Toutes les procréations artificielles sont dans la nature

Author

Jacques Testart, Gametogenèse et génotoxicité (UMR_S_566 ), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris Diderot - Paris 7 (UPD7)-IFR13-Institut National de la Santé et de la Recherche Médicale (INSERM), Maylin, Françoise, and IFR13-Université Paris Diderot - Paris 7 (UPD7)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
0303 health sciences, 03 medical and health sciences, [SDV.MHEP.GEO] Life Sciences [q-bio]/Human health and pathology/Gynecology and obstetrics, MESH: Reproductive Techniques, Assisted, MESH: Humans, 050902 family studies, 05 social sciences, General Medicine, 0509 other social sciences, Biology, [SDV.MHEP.GEO]Life Sciences [q-bio]/Human health and pathology/Gynecology and obstetrics, General Biochemistry, Genetics and Molecular Biology, 030304 developmental biology

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