1,933 results on '"Deleage, Gilbert"'
Search Results
2. SuMo: A Tool for Protein Function Inference Based on 3D Structures Comparisons
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Chemelle, Julie-Anne, Bettler, Emmmanuel, Combet, Christophe, Terreux, Raphaël, Geourjon, Christophe, Deléage, Gilbert, and Roterman-Konieczna, Irena, editor
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- 2013
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3. Web Services Interface to Run Protein Sequence Tools on Grid, Testcase of Protein Sequence Alignment
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Blanchet, Christophe, Combet, Christophe, Daric, Vladimir, Deléage, Gilbert, Hutchison, David, editor, Kanade, Takeo, editor, Kittler, Josef, editor, Kleinberg, Jon M., editor, Mattern, Friedemann, editor, Mitchell, John C., editor, Naor, Moni, editor, Nierstrasz, Oscar, editor, Pandu Rangan, C., editor, Steffen, Bernhard, editor, Sudan, Madhu, editor, Terzopoulos, Demetri, editor, Tygar, Dough, editor, Vardi, Moshe Y., editor, Weikum, Gerhard, editor, Istrail, Sorin, editor, Pevzner, Pavel, editor, Waterman, Michael, editor, Maglaveras, Nicos, editor, Chouvarda, Ioanna, editor, Koutkias, Vassilis, editor, and Brause, Rüdiger, editor
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- 2006
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4. NMR structure and molecular dynamics of the in-plane membrane anchor of nonstructural protein 5A from bovine viral diarrhea virus
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Sapay, Nicolas, Montserret, Roland, Chipot, Christophe, Brass, Volker, Moradpour, Darius, Deleage, Gilbert, and Penin, Francois
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Viral diarrhea -- Research ,Hepatitis C virus -- Research ,Nuclear magnetic resonance spectroscopy -- Usage ,Biological sciences ,Chemistry - Abstract
The NMR structure of the membrane anchor 1-28 of nonstructural protein 5A (NS5A) from bovine viral diarrhea virus (BVDV) in the presence of different membrane mimetic media is reported. The structural conservations found in the result point toward conserved roles of the N-terminal in-plane membrane anchors of NS5A in replication complex formation of hepatitis C virus (HCV), BVDV and other related viruses.
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- 2006
5. Genetic capsid modifications allow efficient re-targeting of adeno-associated virus type 2
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Girod, Anne, Ried, Martin, Wobus, Christiane, Lahm, Harald, Leike, Kristin, Kleinschmidt, Jurgen, Deleage, Gilbert, and Hallek, Michael
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Adenoviruses -- Genetic aspects ,Gene therapy -- Research - Abstract
The human parvovirus adeno-associated virus type 2 will accept foreign proteins inserted via genetic engineering. The virus could then be targeted to specific tissue types to deliver therapeutic genes.
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- 1999
6. A comprehensive system for consistent numbering of HCV sequences, proteins and epitopes
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Kuiken, Carla, Combet, Christophe, Bukh, Jens, Shin-I, Tadasu, Deleage, Gilbert, Mizokami, Masashi, Richardson, Russell, Sablon, Erwin, Yusim, Karina, Pawlotsky, Jean-Michel, and Simmonds, Peter
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- 2006
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7. Hepatitis C databases, principles and utility to researchers
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Kuiken, Carla, Mizokami, Masashi, Deleage, Gilbert, Yusim, Karina, Penin, Francois, Shin-I, Tadasu, Charavay, Céline, Tao, Ning, Crisan, Daniel, Grando, Delphine, Dalwani, Anita, Geourjon, Christophe, Agrawal, Ashish, and Combet, Christophe
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- 2006
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8. Low Resolution Structure Determination Shows Procollagen C-Proteinase Enhancer to be an Elongated Multidomain Glycoprotein
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Bernocco, Simonetta, Steiglitz, Barry M., Svergun, Dmitri I., Petoukhov, Maxim V., Ruggiero, Florence, Ricard-Blum, Sylvie, Ebel, Christine, Geourjon, Christophe, Deléage, Gilbert, Font, Bernard, Eichenberger, Denise, Greenspan, Daniel S., and Hulmes, David J.S.
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- 2003
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9. ANALYSIS OF PROSTATE SPECIFIC ANTIGEN AND α1-ANTICHYMOTRYPSIN INTERACTION USING ANTIPEPTIDE MONOCLONAL ANTIBODIES
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MICHEL, SANDRINE, CHARRIER, JEAN-PHILIPPE, DELEAGE, GILBERT, BATTAIL-POIROT, NICOLE, JOLIVET, MICHEL, and JOLIVET-REYNAUD, COLETTE
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- 2001
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10. Use of Class Prediction to Improve Protein Secondary Structure Prediction : Joint Prediction with Methods Based on Sequence Homology
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Deléage, Gilbert, Roux, Bernard, and Fasman, Gerald D., editor
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- 1989
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11. A common mechanism for ATP hydrolysis in ABC transporter and helicase superfamilies
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Geourjon, Christophe, Orelle, Cedric, Steinfels, Emmanuelle, Blanchet, Christophe, Deleage, Gilbert, Di Pietro, Attilio, and Jault, Jean-Michel
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Adenosine triphosphate -- Research ,Protein hydrolysates -- Research ,Biological sciences ,Chemistry - Abstract
ABC (ATP-binding cassette) transporters and helicases are large superfamilies of seemingly unrelated proteins, whose functions depend on the energy provided by ATP hydrolysis. Comparison of the 3D structures of their nucleotide-binding domains reveals that, besides two well-characterized ATP-binding signatures, the folds of their nucleotide-binding sites are similar. Furthermore, there are striking similarities in the positioning of residues thought to be important for ATP binding or hydrolysis. Interestingly, structures have recently been obtained for two ABC proteins that are not involved in transport activities, but that have a function related to DNA modification. These ABC proteins, which contain a nucleotide-binding site akin to those of typical ABC transporters, might constitute the missing link between the two superfamilies.
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- 2001
12. PDB_REDO : automated re-refinement of X-ray structure models in the PDB
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Joosten, Robbie P., Salzemann, Jean, Bloch, Vincent, Stockinger, Heinz, Berglund, Ann-Charlott, Blanchet, Christophe, Bongcam-Rudloff, Erik, Combet, Christophe, Da Costa, L, Deleage, Gilbert, Diarena, Matteo, Fabbretti, Roberto, Fettahi, Geraldine, Flegel, Volker, Gisel, Andreas, Kasam, Vinod, Kervinen, Timo, Korpelainen, Eija, Mattila, Kimmo, Pagni, Marco, Reichstadt, Matthieu, Breton, Vincent, Tickle, J, Vriend, Gert, Joosten, Robbie P., Salzemann, Jean, Bloch, Vincent, Stockinger, Heinz, Berglund, Ann-Charlott, Blanchet, Christophe, Bongcam-Rudloff, Erik, Combet, Christophe, Da Costa, L, Deleage, Gilbert, Diarena, Matteo, Fabbretti, Roberto, Fettahi, Geraldine, Flegel, Volker, Gisel, Andreas, Kasam, Vinod, Kervinen, Timo, Korpelainen, Eija, Mattila, Kimmo, Pagni, Marco, Reichstadt, Matthieu, Breton, Vincent, Tickle, J, and Vriend, Gert
- Abstract
Structural biology, homology modelling and rational drug design require accurate three-dimensional macromolecular coordinates. However, the coordinates in the Protein Data Bank (PDB) have not all been obtained using the latest experimental and computational methods. In this study a method is presented for automated re-refinement of existing structure models in the PDB. A large-scale benchmark with 16 807 PDB entries showed that they can be improved in terms of fit to the deposited experimental X-ray data as well as in terms of geometric quality. The re-refinement protocol uses TLS models to describe concerted atom movement. The resulting structure models are made available through the PDB_REDO databank (http://www.cmbi.ru.nl/pdb_redo/). Grid computing techniques were used to overcome the computational requirements of this endeavour.
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- 2009
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13. PDB_REDO: automated re-refinement of X-ray structure models in the PDB
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Joosten, Robbie P., primary, Salzemann, Jean, additional, Bloch, Vincent, additional, Stockinger, Heinz, additional, Berglund, Ann-Charlott, additional, Blanchet, Christophe, additional, Bongcam-Rudloff, Erik, additional, Combet, Christophe, additional, Da Costa, Ana L., additional, Deleage, Gilbert, additional, Diarena, Matteo, additional, Fabbretti, Roberto, additional, Fettahi, Géraldine, additional, Flegel, Volker, additional, Gisel, Andreas, additional, Kasam, Vinod, additional, Kervinen, Timo, additional, Korpelainen, Eija, additional, Mattila, Kimmo, additional, Pagni, Marco, additional, Reichstadt, Matthieu, additional, Breton, Vincent, additional, Tickle, Ian J., additional, and Vriend, Gert, additional
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- 2009
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14. Grid Deployment of Legacy Bioinformatics Applications with Transparent Data Access
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Blanchet, Christophe, primary, Mollon, Remi, additional, Thain, Douglas, additional, and Deleage, Gilbert, additional
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- 2006
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15. Structural analysis of the heparin-binding site of the NC1 domain of collagen XIV by CD and NMR
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Montserret, Roland, Aubert-Foucher, Elisabeth, McLeish, Michael J., Hill, Joanna M., Ficheux, Damien, Jaquinod, Michel, Rest, Michel van der, Deleage, Gilbert, and Penin, Francois
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Collagen -- Ultrastructure ,Proteins -- Structure ,Heparin -- Research ,Binding sites (Biochemistry) -- Research ,Biological sciences ,Chemistry - Abstract
Amino acids essential for heparin binding were identified by affinity chromatography analysis after proteolytic digestion of the synthetic peptide NC1(84-116) to further characterize interactions in the NC1 domain of chicken collagen XIV. Circular dichroism and NMR were then used to obtain the 3D structure of this peptide. A nativelike conformation was observed in the NC1(84-116) model which presented suitably oriented residues for the interaction with a specific glycosaminoglycan.
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- 1999
16. Characterization of Two Genes Coding for a Similar Four-Cysteine Motif of the Amino-Terminal Propeptide of a Sea Urchin Fibrillar Collagen
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Exposito, Jean-Yves, primary, Boute, Nicolas, additional, Deleage, Gilbert, additional, and Garrone, Robert, additional
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- 1995
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17. Intrinsic tryptophan fluorescence of Schizosaccharomyces pombe mitochondrial F1-ATPase. A powerful probe for phosphate and nucleotide interactions
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Divita, Gilles, primary, Di Pietro, Attilio, additional, Deleage, Gilbert, additional, Roux, Bernard, additional, and Gautheron, Daniele C., additional
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- 1991
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18. Interaction between .delta. and .epsilon. subunits of F1-ATPase from pig heart mitochondria. Circular dichroism and intrinsic fluorescence of purified and reconstituted .delta..epsilon. complex
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Penin, Francois, primary, Deleage, Gilbert, additional, Gagliardi, Dominique, additional, Roux, Bernard, additional, and Gautheron, Daniele C., additional
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- 1990
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19. Structural aspects and orientation mechanism of mitochondrial F1-adenosine triphosphatase. Evidence for a negative electric birefringence due to a permanent moment perpendicular to the long axes of the particle
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Deleage, Gilbert, primary, Roux, Bernard, additional, and Marion, Christian, additional
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- 1986
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20. Epitope of OSCP oligomycin sensitivity conferring protein exposed at the surface of the mitochondrial ATPase-ATPsynthase complex
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Godinot, Catherine, primary, Colorio, Serge, additional, Cretin, François, additional, Inçaurgarat, Brigitte, additional, Deleage, Gilbert, additional, and Roux, Bernard, additional
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- 1989
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21. FVT-1, A Novel Human Transcription Unit Affected by Variant Translocation t(2;18)(p11;q21) of Follicular Lymphoma
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Rimokh, Ruth, Gadoux, Mylène, Berthéas, Marie-Françoise, Berger, Françoise, Garoscio, Monique, Deléage, Gilbert, Germain, Daniel, and Magaud, Jean-Pierre
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- 1993
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22. PHOTOELECTRON-SPECTRA OF BIS-CYCLOPENTADIENYL METAL DIHALIDES
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Ignazio L. Fragalà, John P. Clark, Anthony W. Coleman, Carla Cauletti, Jennifer C. Green, Sally E. Jackson, Enrico Ciliberto, and Deleage, Gilbert
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Radiation ,Chemistry ,Inorganic chemistry ,Condensed Matter Physics ,Atomic and Molecular Physics, and Optics ,Electronic, Optical and Magnetic Materials ,Metal ,Delocalized electron ,Crystallography ,Cyclopentadienyl complex ,Atomic orbital ,Ionization ,visual_art ,Halogen ,[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,visual_art.visual_art_medium ,Molecular orbital ,Physical and Theoretical Chemistry ,Ionization energy ,Spectroscopy - Abstract
He(I) and HE(II) photoelectron spectra are reported for (η-C5H5)2MX2 (M = Ti; X = F, Cl, Br, I: M = Zr, Hf; X = Cl, Br: M = Ta; X = Cl, Br) and (η-MeC5H4)2MX2 (M = Nb, X = Cl: M = Mo; X = Cl, Br, I). A substantial variation is found in the ordering of the halogen and cyclopentadienyl ionizations, the order being dependent on the metal as well as on the halogen. The compounds may be divided into three classes, namely, those in which the electrons in cyclopentadienyl e1 orbitals ionize at a lower energy than those occupying halogen pπ orbitals, those in which halogen pπ electrons have lower ionization energy than cyclopentadienyl e1 electrons and those in which the corresponding electrons arise from extensively delocalized molecular orbitals with significant contributions from both these categories of fragment orbital.
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- 2016
23. Modulation by flavonoids of cell multidrug resistance mediated by P-glycoprotein and related ABC transporters
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A. Di Pietro, Anne-Marie Mariotte, Hélène Baubichon-Cortay, G. Dayan, H de Wet, Francisco Gamarro, André Goffeau, Jean-Michel Jault, Emmanuelle Steinfels, Gilles Comte, David B. McIntosh, Mathias Maitrejean, Denis Barron, Santiago Castanys, Charles Dumontet, Doriane Trompier, G. Conseil, José M. Pérez-Victoria, Ahcène Boumendjel, and Deleage, Gilbert
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ATP-binding cassette transporter ,Models, Biological ,Cellular and Molecular Neuroscience ,Structure-Activity Relationship ,ATP hydrolysis ,Drug Resistance, Multiple, Fungal ,Neoplasms ,[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Animals ,Humans ,ATP Binding Cassette Transporter, Subfamily B, Member 1 ,Molecular Biology ,ATP-binding domain of ABC transporters ,P-glycoprotein ,Pharmacology ,Flavonoids ,biology ,Transporter ,Cell Biology ,Drug Resistance, Multiple ,Multiple drug resistance ,Transmembrane domain ,Biochemistry ,Drug Resistance, Neoplasm ,biology.protein ,Molecular Medicine ,ATP-Binding Cassette Transporters ,Efflux - Abstract
Cancer cell resistance to chemotherapy is often mediated by overexpression of P-glycoprotein, a plasma membrane ABC (ATP-binding cassette) transporter which extrudes cytotoxic drugs at the expense of ATP hydrolysis. P-glycoprotein (ABCB1, according to the human gene nomenclature committee) consists of two homologous halves each containing a transmembrane domain (TMD) involved in drug binding and efflux, and a cytosolic nucleotide-binding domain (NBD) involved in ATP binding and hydrolysis, with an overall (TMD-NBD)2 domain topology. Homologous ABC multidrug transporters, from the same ABCB family, are found in many species such as Plasmodiumfalciparum and Leishmania spp. protozoa, where they induce resistance to antiparasitic drugs. In yeasts, some ABC transporters involved in resistance to fungicides, such as Saccharomyces cerevisiae Pdr5p and Snq2p, display a different (NBD-TMD)2 domain topology and are classified in another family, ABCG. Much effort has been spent to modulate multidrug resistance in the different species by using specific inhibitors, but generally with little success due to additional cellular targets and/or extrusion of the potential inhibitors. This review shows that due to similarities in function and maybe in three-dimensional organization of the different transporters, common potential modulators have been found. An in vitro 'rational screening' was performed among the large flavonoid family using a four-step procedure: (i) direct binding to purified recombinant cytosolic NBD and/or full-length transporter, (ii) inhibition of ATP hydrolysis and energy-dependent drug interaction with transporter-enriched membranes, (iii) inhibition of cell transporter activity monitored by flow cytometry and (iv) chemosensitization of cell growth. The results indicate that prenylated flavonoids bind with high affinity, and strongly inhibit drug interaction and nucleotide hydrolysis. As such, they constitute promising potential modulators of multidrug resistance.Cancer cell resistance to chemotherapy is often mediated by overexpression of P-glycoprotein, a plasma membrane ABC (ATP-binding cassette) transporter which extrudes cytotoxic drugs at the expense of ATP hydrolysis. P-glycoprotein (ABCB1, according to the human gene nomenclature committee) consists of two homologous halves each containing a transmembrane domain (TMD) involved in drug binding and efflux, and a cytosolic nucleotide-binding domain (NBD) involved in ATP binding and hydrolysis, with an overall (TMD-NBD)2 domain topology. Homologous ABC multidrug transporters, from the same ABCB family, are found in many species such as Plasmodiumfalciparum and Leishmania spp. protozoa, where they induce resistance to antiparasitic drugs. In yeasts, some ABC transporters involved in resistance to fungicides, such as Saccharomyces cerevisiae Pdr5p and Snq2p, display a different (NBD-TMD)2 domain topology and are classified in another family, ABCG. Much effort has been spent to modulate multidrug resistance in the different species by using specific inhibitors, but generally with little success due to additional cellular targets and/or extrusion of the potential inhibitors. This review shows that due to similarities in function and maybe in three-dimensional organization of the different transporters, common potential modulators have been found. An in vitro 'rational screening' was performed among the large flavonoid family using a four-step procedure: (i) direct binding to purified recombinant cytosolic NBD and/or full-length transporter, (ii) inhibition of ATP hydrolysis and energy-dependent drug interaction with transporter-enriched membranes, (iii) inhibition of cell transporter activity monitored by flow cytometry and (iv) chemosensitization of cell growth. The results indicate that prenylated flavonoids bind with high affinity, and strongly inhibit drug interaction and nucleotide hydrolysis. As such, they constitute promising potential modulators of multidrug resistance.
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- 2016
24. Sequence requirements of the ATP-binding site within the C-terminal nucleotide-binding domain of mouse P-glycoprotein: structure-activity relationships for flavonoid binding
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D B McIntosh, A. Di Pietro, J M Jault, Denis Barron, Hélène Baubichon-Cortay, Tino Krell, Jean-Baptiste Daskiewicz, H de Wet, G. Conseil, and Deleage, Gilbert
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Salmonella typhimurium ,Molecular Sequence Data ,Photoaffinity Labels ,Biology ,Biochemistry ,Mice ,Structure-Activity Relationship ,chemistry.chemical_compound ,Adenosine Triphosphate ,Chalcone ,Bacterial Proteins ,[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Consensus sequence ,Animals ,Nucleotide ,ATP Binding Cassette Transporter, Subfamily B, Member 1 ,Amino Acid Sequence ,Binding site ,Nuclear Magnetic Resonance, Biomolecular ,Peptide sequence ,Flavonoids ,chemistry.chemical_classification ,Binding Sites ,Sequence Homology, Amino Acid ,Walker motifs ,Membrane Transport Proteins ,Recombinant Proteins ,Kinetics ,Models, Chemical ,chemistry ,Cyclic nucleotide-binding domain ,Amino Acid Transport Systems, Basic ,ATP-Binding Cassette Transporters ,Flavonoid binding ,Sequence Alignment ,Adenosine triphosphate - Abstract
Sequence requirements of the ATP-binding site within the C-terminal nucleotide-binding domain (NBD2) of mouse P-glycoprotein were investigated by using two recombinantly expressed soluble proteins of different lengths and photoactive ATP analogues, 8-azidoadenosine triphosphate (8N(3)-ATP) and 2',3',4'-O-(2,4,6-trinitrophenyl)-8-azidoadenosine triphosphate (TNP-8N(3)-ATP). The two proteins, Thr(1044)-Thr(1224) (NBD2(short)) and Lys(1025)-Ser(1276) (NBD2(long)), both incorporated the four consensus sequences of ABC (ATP-binding cassette) transporters, Walker A and B motifs, the Q-loop, and the ABC signature, while differing in N-terminal and C-terminal extensions. Radioactive photolabeling of both proteins was characterized by hyperbolic dependence on nucleotide concentration and high-affinity binding with K(0.5)(8N(3)-ATP) = 36-37 microM and K(0.5)(TNP-8N(3)-ATP) = 0.8-2.6 microM and was maximal at acidic pH. Photolabeling was strongly inhibited by TNP-ATP (K(D) = 0.1-5 microM) and ATP (K(D) = 0.5-2.7 mM). Since flavonoids display bifunctional interactions at the ATP-binding site and a vicinal steroid-interacting hydrophobic sequence [Conseil, G., Baubichon-Cortay, H., Dayan, G., Jault, J.-M., Barron, D., and Di Pietro, A. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9831-9836], a series of 30 flavonoids from different classes were investigated for structure-activity relationships toward binding to the ATP site, monitored by protection against photolabeling. The 3-OH and aromaticity of conjugated rings A and C appeared important, whereas opening of ring C abolished the binding in all but one case. It can be concluded that the benzopyrone portion of the flavonoids binds at the adenyl site and the phenyl ring B at the ribosyl site. The Walker A and B motifs, intervening sequences, and small segments on both sides are sufficient to constitute the ATP site.Sequence requirements of the ATP-binding site within the C-terminal nucleotide-binding domain (NBD2) of mouse P-glycoprotein were investigated by using two recombinantly expressed soluble proteins of different lengths and photoactive ATP analogues, 8-azidoadenosine triphosphate (8N(3)-ATP) and 2',3',4'-O-(2,4,6-trinitrophenyl)-8-azidoadenosine triphosphate (TNP-8N(3)-ATP). The two proteins, Thr(1044)-Thr(1224) (NBD2(short)) and Lys(1025)-Ser(1276) (NBD2(long)), both incorporated the four consensus sequences of ABC (ATP-binding cassette) transporters, Walker A and B motifs, the Q-loop, and the ABC signature, while differing in N-terminal and C-terminal extensions. Radioactive photolabeling of both proteins was characterized by hyperbolic dependence on nucleotide concentration and high-affinity binding with K(0.5)(8N(3)-ATP) = 36-37 microM and K(0.5)(TNP-8N(3)-ATP) = 0.8-2.6 microM and was maximal at acidic pH. Photolabeling was strongly inhibited by TNP-ATP (K(D) = 0.1-5 microM) and ATP (K(D) = 0.5-2.7 mM). Since flavonoids display bifunctional interactions at the ATP-binding site and a vicinal steroid-interacting hydrophobic sequence [Conseil, G., Baubichon-Cortay, H., Dayan, G., Jault, J.-M., Barron, D., and Di Pietro, A. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9831-9836], a series of 30 flavonoids from different classes were investigated for structure-activity relationships toward binding to the ATP site, monitored by protection against photolabeling. The 3-OH and aromaticity of conjugated rings A and C appeared important, whereas opening of ring C abolished the binding in all but one case. It can be concluded that the benzopyrone portion of the flavonoids binds at the adenyl site and the phenyl ring B at the ribosyl site. The Walker A and B motifs, intervening sequences, and small segments on both sides are sufficient to constitute the ATP site.
- Published
- 2016
25. NATURE OF METAL-METAL INTERACTION IN TETRA-MU-CARBOXYLATOCHROMIUM(II) SYSTEMS
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Garner, Cd, Hillier, Ih, Guest, Mf, Green, Jc, Colemanaw, Xxxx, and Deleage, Gilbert
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[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology - Abstract
xxx
- Published
- 2016
26. Thiomandelic acid, a broad spectrum inhibitor of zinc beta-lactamases: kinetic and spectroscopic studies
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Mollard, C., Moali, C., cyril papamicael, Damblon, C., Vessilier, S., Amicosante, G., Cj Schofield, Galleni, M., Jm Frere, Gc Roberts, Institut de biologie et chimie des protéines [Lyon] (IBCP), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), and Deleage, Gilbert
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Models, Molecular ,Binding Sites ,Magnetic Resonance Spectroscopy ,Arginine ,Kinetics ,Structure-Activity Relationship ,Zinc ,Models, Chemical ,Spectrophotometry ,[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Mandelic Acids ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Sulfhydryl Compounds ,Enzyme Inhibitors ,beta-Lactamase Inhibitors ,Protein Binding - Abstract
International audience; Resistance to beta-lactam antibiotics mediated by metallo-beta-lactamases is an increasingly worrying clinical problem. Candidate inhibitors include mercaptocarboxylic acids, and we report studies of a simple such compound, thiomandelic acid. A series of 35 analogues were synthesized and examined as metallo-beta-lactamase inhibitors. The K(i) values (Bacillus cereus enzyme) are 0.09 microm for R-thiomandelic acid and 1.28 microm for the S-isomer. Structure-activity relationships show that the thiol is essential for activity and the carboxylate increases potency; the affinity is greatest when these groups are close together. Thioesters of thiomandelic acid are substrates for the enzyme, liberating thiomandelic acid, suggesting a starting point for the design of "pro-drugs." Importantly, thiomandelic acid is a broad spectrum inhibitor of metallo-beta-lactamases, with a submicromolar K(i) value for all nine enzymes tested, except the Aeromonas hydrophila enzyme; such a wide spectrum of activity is unprecedented. The binding of thiomandelic acid to the B. cereus enzyme was studied by NMR; the results are consistent with the idea that the inhibitor thiol binds to both zinc ions, while its carboxylate binds to Arg(91). Amide chemical shift perturbations for residues 30-40 (the beta(3)-beta(4) loop) suggest that this small inhibitor induces a movement of this loop of the kind seen for other larger inhibitors.Resistance to beta-lactam antibiotics mediated by metallo-beta-lactamases is an increasingly worrying clinical problem. Candidate inhibitors include mercaptocarboxylic acids, and we report studies of a simple such compound, thiomandelic acid. A series of 35 analogues were synthesized and examined as metallo-beta-lactamase inhibitors. The K(i) values (Bacillus cereus enzyme) are 0.09 microm for R-thiomandelic acid and 1.28 microm for the S-isomer. Structure-activity relationships show that the thiol is essential for activity and the carboxylate increases potency; the affinity is greatest when these groups are close together. Thioesters of thiomandelic acid are substrates for the enzyme, liberating thiomandelic acid, suggesting a starting point for the design of "pro-drugs." Importantly, thiomandelic acid is a broad spectrum inhibitor of metallo-beta-lactamases, with a submicromolar K(i) value for all nine enzymes tested, except the Aeromonas hydrophila enzyme; such a wide spectrum of activity is unprecedented. The binding of thiomandelic acid to the B. cereus enzyme was studied by NMR; the results are consistent with the idea that the inhibitor thiol binds to both zinc ions, while its carboxylate binds to Arg(91). Amide chemical shift perturbations for residues 30-40 (the beta(3)-beta(4) loop) suggest that this small inhibitor induces a movement of this loop of the kind seen for other larger inhibitors.
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- 2016
27. Carbohydrate binding sites in a pancreatic α-amylase-substrate complex, derived from X-ray structure analysis at 2.1 Å resolution
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Minxie Qian, R. Haser, Françoise Payan, and Deleage, Gilbert
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Protein Conformation ,Swine ,Stereochemistry ,Oligosaccharides ,Crystal structure ,Crystallography, X-Ray ,Biochemistry ,Protein structure ,Chlorides ,[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Animals ,Molecule ,Binding site ,Pancreas ,Molecular Biology ,chemistry.chemical_classification ,Binding Sites ,biology ,Chemistry ,Substrate (chemistry) ,Active site ,Pyrrolidonecarboxylic Acid ,Crystallography ,Glucose ,Enzyme ,biology.protein ,Calcium ,alpha-Amylases ,Research Article ,Starch binding - Abstract
The X-ray structure analysis of a crystal of pig pancreatic alpha-amylase (PPA, EC 3.2.1.1.) that was soaked with the substrate maltopentaose showed electron density corresponding to two independent carbohydrate recognition sites on the surface of the molecule. Both binding sites are distinct from the active site described in detail in our previous high-resolution study of a complex between PPA and a carbohydrate inhibitor (Qian M, Buisson G, Duee E, Haser H, Payan F, 1994, Biochemistry 33:6284-6294). One of the binding sites previously identified in a 5-A-resolution electron density map, lies at a distance of 20 A from the active site cleft and can accommodate two glucose units. The second affinity site for sugar units is located close to the calcium binding site. The crystal structure of the maltopentaose complex was refined at 2.1 A resolution, to an R-factor of 17.5%, with an RMS deviation in bond distances of 0.007 A. The model includes all 496 residues of the enzyme, 1 calcium ion, 1 chloride ion, 425 water molecules, and 3 bound sugar rings. The binding sites are characterized and described in detail. The present complex structure provides the evidence of an increased stability of the structure upon interaction with the substrate and allows identification of an N-terminal pyrrolidonecarboxylic acid in PPA.The X-ray structure analysis of a crystal of pig pancreatic alpha-amylase (PPA, EC 3.2.1.1.) that was soaked with the substrate maltopentaose showed electron density corresponding to two independent carbohydrate recognition sites on the surface of the molecule. Both binding sites are distinct from the active site described in detail in our previous high-resolution study of a complex between PPA and a carbohydrate inhibitor (Qian M, Buisson G, Duee E, Haser H, Payan F, 1994, Biochemistry 33:6284-6294). One of the binding sites previously identified in a 5-A-resolution electron density map, lies at a distance of 20 A from the active site cleft and can accommodate two glucose units. The second affinity site for sugar units is located close to the calcium binding site. The crystal structure of the maltopentaose complex was refined at 2.1 A resolution, to an R-factor of 17.5%, with an RMS deviation in bond distances of 0.007 A. The model includes all 496 residues of the enzyme, 1 calcium ion, 1 chloride ion, 425 water molecules, and 3 bound sugar rings. The binding sites are characterized and described in detail. The present complex structure provides the evidence of an increased stability of the structure upon interaction with the substrate and allows identification of an N-terminal pyrrolidonecarboxylic acid in PPA.
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- 2008
28. On the Molecular Mechanism of Drug Intercalation into DNA: A Simulation Study of the Intercalation Pathway, Free Energy, and DNA Structural Changes
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Arnab Mukherjee, Richard Lavery, James T. Hynes, Biman Bagchi, and Deleage, Gilbert
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Models, Molecular ,Base pair ,Kinetics ,Intercalation (chemistry) ,Biochemistry ,Catalysis ,symbols.namesake ,chemistry.chemical_compound ,Colloid and Surface Chemistry ,Osmotic Pressure ,[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Computer Simulation ,Hydrogen bond ,Chemistry ,Water ,Energy landscape ,DNA ,General Chemistry ,Intercalating Agents ,Gibbs free energy ,Crystallography ,symbols ,Nucleic Acid Conformation ,Thermodynamics ,Umbrella sampling - Abstract
Intercalation into DNA (insertion between a pair of base pairs) is a critical step in the function of many anticancer drugs. Despite its importance, a detailed mechanistic understanding of this process at the molecular level is lacking. We have constructed, using extensive atomistic computer simulations and umbrella sampling techniques, a free energy landscape for the intercalation of the anticancer drug daunomycin into a twelve base pair B-DNA. A similar free energy landscape has been constructed for a probable intermediate DNA minor groove-bound state. These allow a molecular level understanding of aspects of the thermodynamics, DNA structural changes, and kinetic pathways of the intercalation process. Key DNA structural changes involve opening the future intercalation site base pairs toward the minor groove (positive roll), followed by an increase in the rise, accompanied by hydrogen bonding changes of the minor groove waters. The calculated intercalation free energy change is -12.3 kcal/mol, in reasonable agreement with the experimental estimate -9.4 kcal/mol. The results point to a mechanism in which the drug first binds to the minor groove and then intercalates into the DNA in an activated process, which is found to be in general agreement with experimental kinetic results.Intercalation into DNA (insertion between a pair of base pairs) is a critical step in the function of many anticancer drugs. Despite its importance, a detailed mechanistic understanding of this process at the molecular level is lacking. We have constructed, using extensive atomistic computer simulations and umbrella sampling techniques, a free energy landscape for the intercalation of the anticancer drug daunomycin into a twelve base pair B-DNA. A similar free energy landscape has been constructed for a probable intermediate DNA minor groove-bound state. These allow a molecular level understanding of aspects of the thermodynamics, DNA structural changes, and kinetic pathways of the intercalation process. Key DNA structural changes involve opening the future intercalation site base pairs toward the minor groove (positive roll), followed by an increase in the rise, accompanied by hydrogen bonding changes of the minor groove waters. The calculated intercalation free energy change is -12.3 kcal/mol, in reasonable agreement with the experimental estimate -9.4 kcal/mol. The results point to a mechanism in which the drug first binds to the minor groove and then intercalates into the DNA in an activated process, which is found to be in general agreement with experimental kinetic results.
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- 2008
29. The Crystal Structure of Pectate Lyase PelI from Soft Rot Pathogen Erwinia chrysanthemi in Complex with Its Substrate
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Sandra Castang, Nicole Hugouvieux-Cotte-Pattat, Christophe Creze, Patrice Gouet, Richard Haser, Emmanuel Derivery, Vladimir E. Shevchik, Deleage, Gilbert, Institut de biologie et chimie des protéines [Lyon] (IBCP), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Microbiologie, adaptation et pathogénie (MAP), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Trafic et signalisation membranaires chez les bactéries (MTSB), and Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL)
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Models, Molecular ,Protein Folding ,Stereochemistry ,Molecular Sequence Data ,Mutant ,Arginine ,Crystallography, X-Ray ,Biochemistry ,Catalysis ,03 medical and health sciences ,Protein structure ,Catalytic Domain ,[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Amino Acid Sequence ,Binding site ,Molecular Biology ,Peptide sequence ,Polysaccharide-Lyases ,030304 developmental biology ,0303 health sciences ,Binding Sites ,biology ,030306 microbiology ,Lysine ,Dickeya chrysanthemi ,Active site ,Cell Biology ,Lyase ,Protein Structure, Tertiary ,Kinetics ,Crystallography ,Gene Expression Regulation ,Pectate lyase ,biology.protein ,Protein folding - Abstract
International audience; The crystallographic structure of the family 3 polysaccharide lyase (PL-3) PelI from Erwinia chrysanthemi has been solved to 1.45 A resolution. It consists of an N-terminal domain harboring a fibronectin type III fold linked to a catalytic domain displaying a parallel beta-helix topology. The N-terminal domain is located away from the active site and is not involved in the catalytic process. After secretion in planta, the two domains are separated by E. chrysanthemi proteases. This event turns on the hypersensitive response of the host. The structure of the single catalytic domain determined to 2.1 A resolution shows that the domain separation unveils a "Velcro"-like motif of asparagines, which might be recognized by a plant receptor. The structure of PelI in complex with its substrate, a tetragalacturonate, has been solved to 2.3 A resolution. The sugar binds from subsites -2 to +2 in one monomer of the asymmetric unit, although it lies on subsites -1 to +3 in the other. These two "Michaelis complexes" have never been observed simultaneously before and are consistent with the dual mode of bond cleavage in this substrate. The bound sugar adopts a mixed 2(1) and 3(1) helical conformation similar to that reported in inactive mutants from families PL-1 and PL-10. However, our study suggests that the catalytic base in PelI is not a conventional arginine but a lysine as proposed in family PL-9.The crystallographic structure of the family 3 polysaccharide lyase (PL-3) PelI from Erwinia chrysanthemi has been solved to 1.45 A resolution. It consists of an N-terminal domain harboring a fibronectin type III fold linked to a catalytic domain displaying a parallel beta-helix topology. The N-terminal domain is located away from the active site and is not involved in the catalytic process. After secretion in planta, the two domains are separated by E. chrysanthemi proteases. This event turns on the hypersensitive response of the host. The structure of the single catalytic domain determined to 2.1 A resolution shows that the domain separation unveils a "Velcro"-like motif of asparagines, which might be recognized by a plant receptor. The structure of PelI in complex with its substrate, a tetragalacturonate, has been solved to 2.3 A resolution. The sugar binds from subsites -2 to +2 in one monomer of the asymmetric unit, although it lies on subsites -1 to +3 in the other. These two "Michaelis complexes" have never been observed simultaneously before and are consistent with the dual mode of bond cleavage in this substrate. The bound sugar adopts a mixed 2(1) and 3(1) helical conformation similar to that reported in inactive mutants from families PL-1 and PL-10. However, our study suggests that the catalytic base in PelI is not a conventional arginine but a lysine as proposed in family PL-9.
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- 2008
30. First evidence of the pore-forming properties of a keratin from skin mucus of rainbow trout (Oncorhynchus mykiss, formerly Salmo gairdneri)
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Yannick Bessin, Nathalie Ebran, Virginie Molle, Sylvie Campagna, Nathalie Saint, Gérard Molle, Institut de biologie et chimie des protéines [Lyon] (IBCP), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Université Henri Poincaré - Nancy 1 (UHP), Centre de Biochimie Structurale [Montpellier] (CBS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratory of Solid Tumors Genetics, Nice University Hospital, and Deleage, Gilbert
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DNA, Complementary ,[SDV]Life Sciences [q-bio] ,Biochemistry ,law.invention ,03 medical and health sciences ,law ,Keratin ,[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Animals ,Cloning, Molecular ,Salmo ,Molecular Biology ,Skin ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Base Sequence ,biology ,030302 biochemistry & molecular biology ,Cell Biology ,biology.organism_classification ,[SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology ,Mucus ,Peptide Fragments ,Trout ,[SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology ,chemistry ,Oncorhynchus mykiss ,Recombinant DNA ,Keratins ,Rainbow trout ,Epidermis ,Glycoprotein ,Porosity ,Bacteria - Abstract
The epidermis of fish is covered with a layer of mucus, which contributes to the defence of the species against parasites, bacteria and fungi. We have previously extracted glycoproteins from various mucus samples from fish and have shown that they present pore-forming activities well correlated with strong antibacterial properties [Ebran, Julien, Orange, Saglio, Lemaitre and Molle(2000) Biochim. Biophys. Acta 1467, 271-280]. The present study focuses on the 65 kDa glycoprotein, Tr65, from the rainbow trout (Oncorhynchus mykiss, formerly Salmo gairdneri).Enzymatic digestion of Tr65 yielded a fragment pattern with strong homology with that of trout type II cytokeratin. Sequence analysis of the cDNA clone obtained by PCR confirmed this homology. We thus constructed a plasmid to overproduce the recombinant Tr65. We extracted and purified this recombinant Tr65, using it for multichannel and single-channel experiments in azolectin bilayers. Our results with recombinant Tr65 confirmed the pore-forming properties already shown with native antibacterial Tr65. These findings offer new insights into the function of keratin proteins present in various mucosal surfaces of animals and human beings.The epidermis of fish is covered with a layer of mucus, which contributes to the defence of the species against parasites, bacteria and fungi. We have previously extracted glycoproteins from various mucus samples from fish and have shown that they present pore-forming activities well correlated with strong antibacterial properties [Ebran, Julien, Orange, Saglio, Lemaitre and Molle(2000) Biochim. Biophys. Acta 1467, 271-280]. The present study focuses on the 65 kDa glycoprotein, Tr65, from the rainbow trout (Oncorhynchus mykiss, formerly Salmo gairdneri).Enzymatic digestion of Tr65 yielded a fragment pattern with strong homology with that of trout type II cytokeratin. Sequence analysis of the cDNA clone obtained by PCR confirmed this homology. We thus constructed a plasmid to overproduce the recombinant Tr65. We extracted and purified this recombinant Tr65, using it for multichannel and single-channel experiments in azolectin bilayers. Our results with recombinant Tr65 confirmed the pore-forming properties already shown with native antibacterial Tr65. These findings offer new insights into the function of keratin proteins present in various mucosal surfaces of animals and human beings.
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- 2008
31. Conformational Change Induced by ATP Binding in the Multidrug ATP-Binding Cassette Transporter BmrA
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Sergio Marco, Jean-Michel Jault, Philippe Gros, Anne Durand, and Attilio Di Pietro, Daniel Lévy, Francesca Gubellini, Cédric Orelle, and Deleage, Gilbert
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chemistry.chemical_classification ,Conformational change ,Protein Conformation ,Mutant ,Walker motifs ,Lysine ,Membrane Transport Proteins ,Water ,ATP-binding cassette transporter ,Transporter ,Models, Biological ,Biochemistry ,Protein Structure, Tertiary ,Structure-Activity Relationship ,Adenosine Triphosphate ,Bacterial Proteins ,chemistry ,Catalytic cycle ,Mutagenesis, Site-Directed ,[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,ATP-Binding Cassette Transporters ,Mutant Proteins ,Nucleotide ,Dimerization - Abstract
ATP-binding cassette (ABC) transporters are involved in the transport of a wide variety of substrates, and ATP-driven dimerization of their nucleotide binding domains (NBDs) has been suggested to be one of the most energetic steps of their catalytic cycle. Taking advantage of the propensity of BmrA, a bacterial multidrug resistance ABC transporter, to form stable, highly ordered ring-shaped structures [Chami et al. (2002) J. Mol. Biol. 315, 1075-1085], we show here that addition of ATP in the presence of Mg2+ prevented ring formation or destroyed the previously formed rings. To pinpoint the catalytic step responsible for such an effect, two classes of hydrolysis-deficient mutants were further studied. In contrast to hydrolytically inactive glutamate mutants that behaved essentially as the wild-type, lysine Walker A mutants formed ring-shaped structures even in the presence of ATP-Mg. Although the latter mutants still bound ATP-Mg, and even slowly hydrolyzed it for the K380R mutant, they were most likely unable to undergo a proper NBD dimerization upon ATP-Mg addition. The ATP-driven dimerization step, which was still permitted in glutamate mutants and led to a stable conformation suitable to monitor the growth of 2D crystals, appeared therefore responsible for destabilization of the BmrA ring structures. Our results provide direct visual evidence that the ATP-induced NBD dimerization triggers a conformational change large enough in BmrA to destabilize the rings, which is consistent with the assumption that this step might constitute the "power stroke" for ABC transporters.ATP-binding cassette (ABC) transporters are involved in the transport of a wide variety of substrates, and ATP-driven dimerization of their nucleotide binding domains (NBDs) has been suggested to be one of the most energetic steps of their catalytic cycle. Taking advantage of the propensity of BmrA, a bacterial multidrug resistance ABC transporter, to form stable, highly ordered ring-shaped structures [Chami et al. (2002) J. Mol. Biol. 315, 1075-1085], we show here that addition of ATP in the presence of Mg2+ prevented ring formation or destroyed the previously formed rings. To pinpoint the catalytic step responsible for such an effect, two classes of hydrolysis-deficient mutants were further studied. In contrast to hydrolytically inactive glutamate mutants that behaved essentially as the wild-type, lysine Walker A mutants formed ring-shaped structures even in the presence of ATP-Mg. Although the latter mutants still bound ATP-Mg, and even slowly hydrolyzed it for the K380R mutant, they were most likely unable to undergo a proper NBD dimerization upon ATP-Mg addition. The ATP-driven dimerization step, which was still permitted in glutamate mutants and led to a stable conformation suitable to monitor the growth of 2D crystals, appeared therefore responsible for destabilization of the BmrA ring structures. Our results provide direct visual evidence that the ATP-induced NBD dimerization triggers a conformational change large enough in BmrA to destabilize the rings, which is consistent with the assumption that this step might constitute the "power stroke" for ABC transporters.
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- 2008
32. Designer amphiphiles based on para-acyl-calix[8]arenes
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Anthony W. Coleman, Floriane Devoge, Said Jebors, Sebastien Cecillon, Fabienne Fache, Sylvain Balme, Melany Monachino, and Deleage, Gilbert
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Erythrocytes ,Time Factors ,Stereochemistry ,Specific adsorption ,Hemolysis ,Biochemistry ,Medicinal chemistry ,Structure-Activity Relationship ,Surface-Active Agents ,Dynamic light scattering ,Monolayer ,Amphiphile ,[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Animals ,Humans ,Particle Size ,Physical and Theoretical Chemistry ,Blood Coagulation ,Molecular Structure ,Chemistry ,Organic Chemistry ,Anticoagulants ,Serum Albumin, Bovine ,Stereoisomerism ,Solubility ,Cattle ,Calixarenes - Abstract
The synthesis of a series of fully O-derivatised para-acyl-calix[8]arenes is described, where the acyl function is either octanoyl or hexadecanoyl. The groups attached at the phenolic face are, carboxymethoxy (anionic), carboxypropoxy (anionic), 4-sulfonatobutoxy (anionic), ethoxycarboxymethoxy (neutral), ethoxycarboxypropoxy (neutral), 2-methoxyethoxy (neutral) and 2-(2-methoxy)diethoxy (neutral). The use of specific synthetic routes has allowed complete substitution in high yields for all the compounds obtained. The interfacial properties of the compounds have been studied and stable monolayers have been obtained for certain compounds in the series having para-octanoyl substituents; all compounds studied in the series having para-hexadecanoyl substituents formed stable monolayers at the air-water interface. The interactions between O-4-sulfonatobutoxy-para-ocatanoylcalix[8]arene and a series of serum albumins have been studied by dynamic light scattering and specific adsorption of the calix-[8]-arene derivative onto the proteins observed. The anionic derivatives O-4-sulfonatobutoxy-para-ocatanoylcalix[8]arene and O-carboxymethoxy-para-ocatanoylcalix[8]arene have been shown to possess anticoagulant properties but to have no haemolytic toxicity.The synthesis of a series of fully O-derivatised para-acyl-calix[8]arenes is described, where the acyl function is either octanoyl or hexadecanoyl. The groups attached at the phenolic face are, carboxymethoxy (anionic), carboxypropoxy (anionic), 4-sulfonatobutoxy (anionic), ethoxycarboxymethoxy (neutral), ethoxycarboxypropoxy (neutral), 2-methoxyethoxy (neutral) and 2-(2-methoxy)diethoxy (neutral). The use of specific synthetic routes has allowed complete substitution in high yields for all the compounds obtained. The interfacial properties of the compounds have been studied and stable monolayers have been obtained for certain compounds in the series having para-octanoyl substituents; all compounds studied in the series having para-hexadecanoyl substituents formed stable monolayers at the air-water interface. The interactions between O-4-sulfonatobutoxy-para-ocatanoylcalix[8]arene and a series of serum albumins have been studied by dynamic light scattering and specific adsorption of the calix-[8]-arene derivative onto the proteins observed. The anionic derivatives O-4-sulfonatobutoxy-para-ocatanoylcalix[8]arene and O-carboxymethoxy-para-ocatanoylcalix[8]arene have been shown to possess anticoagulant properties but to have no haemolytic toxicity.
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- 2008
33. In vitro comparison of human fibroblasts from intact and ruptured ACL for use in tissue engineering
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T Brune, Thomas W. Gilbert, P Sommer, J P Franceschi, A Borel, Stephen F. Badylak, and Deleage, Gilbert
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Male ,Integrins ,Pathology ,lcsh:Diseases of the musculoskeletal system ,Sus scrofa ,Cell ,Biocompatible Materials ,Cell morphology ,extracted fibroblasts ,Extracellular matrix ,Tissue engineering ,Absorbable Implants ,Anterior Cruciate Ligament ,Cells, Cultured ,in vitro models ,Aged, 80 and over ,Tissue Scaffolds ,Graft Survival ,Anatomy ,Middle Aged ,musculoskeletal system ,SMA ,Extracellular Matrix ,surgical procedures, operative ,medicine.anatomical_structure ,Connective Tissue ,Female ,Collagen ,small intestinal submucosa scaffold ,Adult ,medicine.medical_specialty ,Integrin ,lcsh:Surgery ,Connective tissue ,Knee Injuries ,Biology ,Transplantation, Autologous ,[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Cell Adhesion ,medicine ,Animals ,Humans ,Regeneration ,Cell Shape ,Actin ,Aged ,Rupture ,Bioartificial Organs ,Tissue Engineering ,Guided Tissue Regeneration ,Anterior Cruciate Ligament Injuries ,lcsh:RD1-811 ,Fibroblasts ,Actins ,biology.protein ,elastic network ,lcsh:RC925-935 ,human activities - Abstract
The present study compares fibroblasts extracted from intact and ruptured human anterior cruciate ligaments (ACL) for creation of a tissue engineered ACL-construct, made of porcine small intestinal submucosal extracellular matrix (SIS-ECM) seeded with these ACL cells. The comparison is based on histological, immunohistochemical and RT-PCR analyses. Differences were observed between cells in a ruptured ACL (rACL) and cells in an intact ACL (iACL), particularly with regard to the expression of integrin subunits and smooth muscle actin (SMA). Despite these differences in the cell source, both cell populations behaved similarly when seeded on an SIS-ECM scaffold, with similar cell morphology, connective tissue organization and composition, SMA and integrin expression. This study shows the usefulness of naturally occurring scaffolds such as SIS-ECM for the study of cell behaviour in vitro, and illustrates the possibility to use autologous cells extracted from ruptured ACL biopsies as a source for tissue engineered ACL constructs.The present study compares fibroblasts extracted from intact and ruptured human anterior cruciate ligaments (ACL) for creation of a tissue engineered ACL-construct, made of porcine small intestinal submucosal extracellular matrix (SIS-ECM) seeded with these ACL cells. The comparison is based on histological, immunohistochemical and RT-PCR analyses. Differences were observed between cells in a ruptured ACL (rACL) and cells in an intact ACL (iACL), particularly with regard to the expression of integrin subunits and smooth muscle actin (SMA). Despite these differences in the cell source, both cell populations behaved similarly when seeded on an SIS-ECM scaffold, with similar cell morphology, connective tissue organization and composition, SMA and integrin expression. This study shows the usefulness of naturally occurring scaffolds such as SIS-ECM for the study of cell behaviour in vitro, and illustrates the possibility to use autologous cells extracted from ruptured ACL biopsies as a source for tissue engineered ACL constructs.
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- 2007
34. Investigating the Mechanism of the Nucleocapsid Protein Chaperoning of the Second Strand Transfer during HIV-1 DNA Synthesis
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Yves Mély, Hugues de Rocquigny, Jean-Luc Darlix, Damien Ficheux, Nick Ramalanjaona, Aurélie Millet, Institut Gilbert-Laustriat : Biomolécules, Biotechnologie, Innovation Thérapeutique, Université Louis Pasteur - Strasbourg I-Centre National de la Recherche Scientifique (CNRS), Barthel, Ingrid, Deleage, Gilbert, Institut de biologie et chimie des protéines [Lyon] (IBCP), Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)
- Subjects
Kinetics ,[SDV.BBM.BP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics ,03 medical and health sciences ,Nucleic acid thermodynamics ,chemistry.chemical_compound ,Structural Biology ,[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Humans ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Molecular Biology ,030304 developmental biology ,Zinc finger ,0303 health sciences ,biology ,030302 biochemistry & molecular biology ,Reverse Transcription ,Nucleocapsid Proteins ,Fluorescence ,Molecular biology ,Reverse transcriptase ,[SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics ,chemistry ,Chaperone (protein) ,HIV-1 ,Biophysics ,biology.protein ,Nucleic Acid Conformation ,Primer binding site ,DNA ,Molecular Chaperones ,Virus Physiological Phenomena - Abstract
International audience; Conversion of the human immunodeficiency virus type 1 (HIV-1) genomic RNA into the proviral DNA by reverse transcriptase involves two obligatory strand transfers that are chaperoned by the nucleocapsid protein (NC). The second strand transfer relies on the annealing of the (-) and (+) copies of the primer binding site, (-)PBS and (+) PBS, which fold into complementary stem-loops (SLs) with terminal single-stranded overhangs. To understand how NC chaperones their hybridization, we investigated the annealing kinetics of fluorescently labelled (+)PBS with various (-)PBS derivatives. In the absence of NC, the (+)/(-)PBS annealing was governed by a second-order pathway nucleated mainly by the single-stranded overhangs of the two PBS SLs. The annealing reaction appeared to be rate-limited by the melting of the stable G.C-rich stem subsequent to the formation of the partially annealed intermediate. A second pathway nucleated through the loops could be detected, but was very minor. NC(11-55), which consists primarily of the zinc finger domain, increased the (-)/(+) PBS annealing kinetics by about sixfold, by strongly activating the interaction between the PBS loops. NC(11-55) also activated (-)/(+) PBS annealing through the single-strand overhangs, but by a factor of only 2. Full-length NC(1-55) further increased the (-)/(+)PBS annealing kinetics by tenfold. The NC-promoted (-)/(+)PBS mechanism proved to be similar with extended (-)DNA molecules, suggesting that it is relevant in the context of proviral DNA synthesis. These findings favour the notion that the ubiquitous role of NC in the viral life-cycle probably relies on the ability of NC to chaperone nucleic acid hybridization via different mechanisms.Conversion of the human immunodeficiency virus type 1 (HIV-1) genomic RNA into the proviral DNA by reverse transcriptase involves two obligatory strand transfers that are chaperoned by the nucleocapsid protein (NC). The second strand transfer relies on the annealing of the (-) and (+) copies of the primer binding site, (-)PBS and (+) PBS, which fold into complementary stem-loops (SLs) with terminal single-stranded overhangs. To understand how NC chaperones their hybridization, we investigated the annealing kinetics of fluorescently labelled (+)PBS with various (-)PBS derivatives. In the absence of NC, the (+)/(-)PBS annealing was governed by a second-order pathway nucleated mainly by the single-stranded overhangs of the two PBS SLs. The annealing reaction appeared to be rate-limited by the melting of the stable G.C-rich stem subsequent to the formation of the partially annealed intermediate. A second pathway nucleated through the loops could be detected, but was very minor. NC(11-55), which consists primarily of the zinc finger domain, increased the (-)/(+) PBS annealing kinetics by about sixfold, by strongly activating the interaction between the PBS loops. NC(11-55) also activated (-)/(+) PBS annealing through the single-strand overhangs, but by a factor of only 2. Full-length NC(1-55) further increased the (-)/(+)PBS annealing kinetics by tenfold. The NC-promoted (-)/(+)PBS mechanism proved to be similar with extended (-)DNA molecules, suggesting that it is relevant in the context of proviral DNA synthesis. These findings favour the notion that the ubiquitous role of NC in the viral life-cycle probably relies on the ability of NC to chaperone nucleic acid hybridization via different mechanisms.
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- 2007
35. Calix[n]arenes as components for the construction of micellar systems: synthesis and self-assembly properties of 5,11,17-Tris[(dimethylamino)methyl]-25-monoalkoxy-26,27,28-trihydroxycalix[4]arene derivatives
- Author
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Oleksandr Shkurenko, Cyrille Mbemba, Kinga Suwinska, Katia Sigaud, Anthony W. Coleman, Florent Perret, and Deleage, Gilbert
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chemistry.chemical_classification ,Inorganic chemistry ,Iodide ,General Chemistry ,Crystal structure ,Condensed Matter Physics ,Caesium fluoride ,Potassium carbonate ,chemistry.chemical_compound ,chemistry ,Dynamic light scattering ,Critical micelle concentration ,Polymer chemistry ,[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Weak base ,Alkyl ,Food Science - Abstract
A series of mono-O-alkylated calix [4] arenes derivatives, with alkyl chain lengths of between 1 and 12 carbon atoms are reported. Monoalkylation is best achieved using potassium carbonate as the weak base and the respective alkyl iodide for chain lengths of one to three carbon atoms and using caesium fluoride as the base and the respective alkyl iodide for longer chain lengths. The mono-alkylated derivatives were converted into the tri-para-dimethylaminomethylene derivatives by the para-quinonemethide reaction in good yields. Surface tension measurements showed that at pH 2, 4, 6 and 8 all the tri-dimethylaminomethylene derivatives showed surfactant behaviour, and at pH 2 all show a Critical Micellar Concentration values. No correlation between Critical Micellar Concentration values and chain length is observed. Dynamic Light Scattering measurements showed that the CMC behaviour may be correlated with the observed aggregate sizes. The solid state structure of mono-O-ethoxy-calix[4]arene is described, in this structure a 1-D inclusion polymer is observed.A series of mono-O-alkylated calix [4] arenes derivatives, with alkyl chain lengths of between 1 and 12 carbon atoms are reported. Monoalkylation is best achieved using potassium carbonate as the weak base and the respective alkyl iodide for chain lengths of one to three carbon atoms and using caesium fluoride as the base and the respective alkyl iodide for longer chain lengths. The mono-alkylated derivatives were converted into the tri-para-dimethylaminomethylene derivatives by the para-quinonemethide reaction in good yields. Surface tension measurements showed that at pH 2, 4, 6 and 8 all the tri-dimethylaminomethylene derivatives showed surfactant behaviour, and at pH 2 all show a Critical Micellar Concentration values. No correlation between Critical Micellar Concentration values and chain length is observed. Dynamic Light Scattering measurements showed that the CMC behaviour may be correlated with the observed aggregate sizes. The solid state structure of mono-O-ethoxy-calix[4]arene is described, in this structure a 1-D inclusion polymer is observed.
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- 2007
36. Generation of specific Th1 and CD8+ T-cell responses by immunization with mouse CD8+ dendritic cells loaded with HIV-1 viral lysate or envelope glycoproteins
- Author
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Bernard Verrier, Delphine Fouquenet, Daniel Bout, Denys Brand, Isabelle Dimier-Poisson, Josette Pierre, Fleur Aline, Institut de biologie et chimie des protéines [Lyon] (IBCP), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Inconnu, Immunologie Parasitaire et Vaccinologie, Institut National de la Recherche Agronomique (INRA)-Université de Tours (UT)-UMR 483, Institut National de la Recherche Agronomique (INRA)-Université de Tours-UMR 483, and Deleage, Gilbert
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[SDV]Life Sciences [q-bio] ,medicine.medical_treatment ,Immunology ,Population ,HIV Core Protein p24 ,CD8-Positive T-Lymphocytes ,Biology ,Immunotherapy, Adoptive ,Microbiology ,Interferon-gamma ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Immune system ,Viral Envelope Proteins ,Antigen ,[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,medicine ,Animals ,Cytotoxic T cell ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,education ,ComputingMilieux_MISCELLANEOUS ,030304 developmental biology ,0303 health sciences ,education.field_of_study ,env Gene Products, Human Immunodeficiency Virus ,Gene Products, env ,Dendritic Cells ,Dendritic cell ,Immunotherapy ,Th1 Cells ,Interleukin-12 ,Virology ,3. Good health ,Infectious Diseases ,Cell culture ,Antibody Formation ,HIV-1 ,Mice, Inbred CBA ,Female ,Interleukin-4 ,Spleen ,CD8 ,T-Lymphocytes, Cytotoxic ,030215 immunology - Abstract
International audience; Immunization with antigen-pulsed dendritic cells (DCs) can be used to elicit optimal immune responses. We developed the SRDC cell line, with a morphology, phenotype and activity similar to mouse splenic CD4(-)CD8alpha(+)CD205(+)CD11b(-) dendritic cells, which induce a polarized Th1 immune response. We evaluated the ability of SRDCs pulsed with HIV-1 viral lysate, oligomeric soluble gp140 or capsid p24 to induce specific antibody and T-cell responses in CBA/J mice. Immunization with all loaded SRDCs elicited antibody responses against the antigens tested. However, only HIV-1 viral lysate and gp140-pulsed SRDCs elicited specific CD4(+) and CD8(+) T-cell responses. These findings demonstrate the value of well characterized DC lines for optimizing the antigen-loading mixture, according to the DC population targeted. Our data suggest that splenic DCs pulsed with complex antigens, such as HIV-1 viral lysate or oligomeric soluble gp140, could be used as vaccines, eliciting strong primary Th1-polarized and humoral immune responses against HIV proteins in vivo.Immunization with antigen-pulsed dendritic cells (DCs) can be used to elicit optimal immune responses. We developed the SRDC cell line, with a morphology, phenotype and activity similar to mouse splenic CD4(-)CD8alpha(+)CD205(+)CD11b(-) dendritic cells, which induce a polarized Th1 immune response. We evaluated the ability of SRDCs pulsed with HIV-1 viral lysate, oligomeric soluble gp140 or capsid p24 to induce specific antibody and T-cell responses in CBA/J mice. Immunization with all loaded SRDCs elicited antibody responses against the antigens tested. However, only HIV-1 viral lysate and gp140-pulsed SRDCs elicited specific CD4(+) and CD8(+) T-cell responses. These findings demonstrate the value of well characterized DC lines for optimizing the antigen-loading mixture, according to the DC population targeted. Our data suggest that splenic DCs pulsed with complex antigens, such as HIV-1 viral lysate or oligomeric soluble gp140, could be used as vaccines, eliciting strong primary Th1-polarized and humoral immune responses against HIV proteins in vivo.
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- 2007
37. Expression of the alternative reading frame protein of Hepatitis C virus induces cytokines involved in hepatic injuries
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Geneviève Inchauspé, Gláucia Paranhos-Baccalà, Anne Fournillier, Marc Fiorucci, Jean Pierre Lavergne, Christine Bain, Steeve Boulant, Jean Daniel Abraham, and Deleage, Gilbert
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Chemokine ,Hepacivirus ,medicine.medical_treatment ,p38 mitogen-activated protein kinases ,Hepatitis C virus ,Genetic Vectors ,Molecular Sequence Data ,medicine.disease_cause ,Monocytes ,Virus ,Adenoviridae ,Cell Line ,Pathogenesis ,Transduction, Genetic ,Virology ,[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,medicine ,Humans ,Amino Acid Sequence ,Cells, Cultured ,biology ,Macrophages ,Viral Core Proteins ,Wild type ,Dendritic Cells ,Flow Cytometry ,biology.organism_classification ,Cytokine ,Microscopy, Fluorescence ,Hepatocytes ,biology.protein ,Cytokines - Abstract
Hepatitis C virus (HCV) Core has been implicated in immune-mediated mechanisms associated with the development of chronic hepatic diseases. Discovery of different alternative reading frame proteins (ARFPs) expressed from the HCV Core coding sequence challenges properties assigned to Core. This study was designed to evaluate the immunomodulatory functions of Core and ARFPs in monocytes, dendritic cells (DCs), macrophages (Mphi) and hepatocytes, cells that are all capable of supporting HCV replication. THP-1 cells, monocyte-derived Mphi and DCs, and Huh7 cells were infected by using adenoviruses (Ad) encoding Core, CE1E2 and a Core sequence modified so that the Core protein is wild type, but no ARFPs are expressed (CDeltaARFP). THP-1 cells and DCs infected with Ad encoding Core or CE1E2 produced significant levels of interleukin-6 (IL-6), IL-8, MCP-1 and MIP-1beta, whereas production of these chemokines with AdCDeltaARFP was reduced or abolished. Similar effects on IL-8 production were observed in Huh7 cells and on IL-6 and MIP-1beta in Mphi. Wild-type Core sequence, but not CDeltaARFP, could trans-activate the IL-8 promoter and this activation was not associated with activation of p38/p42-44MAPK. This study illustrates, for the first time, the critical importance of ARFP expression in immunomodulatory functions attributed to Core expression and suggests a potential involvement of ARFP in mechanisms associated with HCV pathogenesis.Hepatitis C virus (HCV) Core has been implicated in immune-mediated mechanisms associated with the development of chronic hepatic diseases. Discovery of different alternative reading frame proteins (ARFPs) expressed from the HCV Core coding sequence challenges properties assigned to Core. This study was designed to evaluate the immunomodulatory functions of Core and ARFPs in monocytes, dendritic cells (DCs), macrophages (Mphi) and hepatocytes, cells that are all capable of supporting HCV replication. THP-1 cells, monocyte-derived Mphi and DCs, and Huh7 cells were infected by using adenoviruses (Ad) encoding Core, CE1E2 and a Core sequence modified so that the Core protein is wild type, but no ARFPs are expressed (CDeltaARFP). THP-1 cells and DCs infected with Ad encoding Core or CE1E2 produced significant levels of interleukin-6 (IL-6), IL-8, MCP-1 and MIP-1beta, whereas production of these chemokines with AdCDeltaARFP was reduced or abolished. Similar effects on IL-8 production were observed in Huh7 cells and on IL-6 and MIP-1beta in Mphi. Wild-type Core sequence, but not CDeltaARFP, could trans-activate the IL-8 promoter and this activation was not associated with activation of p38/p42-44MAPK. This study illustrates, for the first time, the critical importance of ARFP expression in immunomodulatory functions attributed to Core expression and suggests a potential involvement of ARFP in mechanisms associated with HCV pathogenesis.
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- 2007
38. The upregulation of lysyl oxidase in oral submucous fibrosis and squamous cell carcinoma
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C. Trivedy, Newell W. Johnson, Vinay Hazarey, P. Sommer, Mahvash Tavassoli, K. A. A. S. Warnakulasuriya, and Deleage, Gilbert
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Cancer Research ,Pathology ,medicine.medical_specialty ,Stromal cell ,Oral Submucous Fibrosis ,Lysyl oxidase ,Pathology and Forensic Medicine ,Protein-Lysine 6-Oxidase ,Extracellular matrix ,Polyps ,Hepatolenticular Degeneration ,Fibrosis ,[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,medicine ,Humans ,Lamina propria ,biology ,business.industry ,medicine.disease ,Immunohistochemistry ,Up-Regulation ,stomatognathic diseases ,medicine.anatomical_structure ,Liver ,Otorhinolaryngology ,Epidermoid carcinoma ,Oral submucous fibrosis ,Carcinoma, Squamous Cell ,biology.protein ,Periodontics ,Mouth Neoplasms ,Oral Surgery ,business ,Elastin - Abstract
Lysyl oxidase (LO) takes part in the initial steps of converting soluble monomers of collagen and elastin into insoluble fibres in the extracellular matrix. We have studied the immunolocalization of LO as a marker of fibrogenesis in oral submucous fibrosis (OSF). Oral biopsies from 13 subjects with OSF, 6 with histologically confirmed squamous cell carcinoma (SCC) arising in OSF and 10 SCC nonrelated to OSF, were examined. Strong positive staining was observed in 7/13 OSF samples in the cytoplasmic processes of fibroblasts and extracellularly in the upper third of the lamina propria. Furthermore, LO was found to co-localize in the areas stained strongly for collagen and elastin by histochemical stains. Examination of SCC tissues showed localization of LO adjacent to invading epithelial islands as evidence of a stromal reaction both in carcinomas arising from OSF and in SCC from non-OSF cases. These findings suggest that upregulation of LO may be an important factor in the pathogenesis of OSF and in the early stromal reaction of oral cancer.Lysyl oxidase (LO) takes part in the initial steps of converting soluble monomers of collagen and elastin into insoluble fibres in the extracellular matrix. We have studied the immunolocalization of LO as a marker of fibrogenesis in oral submucous fibrosis (OSF). Oral biopsies from 13 subjects with OSF, 6 with histologically confirmed squamous cell carcinoma (SCC) arising in OSF and 10 SCC nonrelated to OSF, were examined. Strong positive staining was observed in 7/13 OSF samples in the cytoplasmic processes of fibroblasts and extracellularly in the upper third of the lamina propria. Furthermore, LO was found to co-localize in the areas stained strongly for collagen and elastin by histochemical stains. Examination of SCC tissues showed localization of LO adjacent to invading epithelial islands as evidence of a stromal reaction both in carcinomas arising from OSF and in SCC from non-OSF cases. These findings suggest that upregulation of LO may be an important factor in the pathogenesis of OSF and in the early stromal reaction of oral cancer.
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- 2007
39. The influence of nitrogen-15 proton-driven spin diffusion on the measurement of nitrogen-15 longitudinal relaxation times
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Anja Böckmann, Lyndon Emsley, Nicolas Giraud, Martin Blackledge, Institut de biologie et chimie des protéines [Lyon] (IBCP), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), and Deleage, Gilbert
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Models, Molecular ,Nuclear and High Energy Physics ,Magnetic Resonance Spectroscopy ,Proton ,Biophysics ,Analytical chemistry ,chemistry.chemical_element ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Molecular physics ,Diffusion ,Magnetization ,Bacterial Proteins ,relaxation ,[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Computer Simulation ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Spin-½ ,SIMPLE (dark matter experiment) ,Nitrogen Isotopes ,010405 organic chemistry ,Chemistry ,Relaxation (NMR) ,Phosphoproteins ,Condensed Matter Physics ,Nitrogen ,0104 chemical sciences ,Kinetics ,MAS ,Models, Chemical ,Deuterium ,Spin diffusion ,Protons ,Artifacts - Abstract
International audience; The effect of nitrogen-15 proton-driven spin diffusion on quantitative (15)N T(1) measurements in solid proteins is investigated, and the impact on the measurement of dynamic parameters is assessed. A simple model of exchange between neighboring nitrogens is used to reproduce the evolution of (15)N spin systems whose longitudinal relaxation rates and exchange rates are compatible with experimental measurements. We show that the induced error in the measured T(1) and its effect on the determination of dynamics parameters is likely to be less than the current experimental error. The use of deuterated protein samples is shown to have a small but sometimes visible effect, and may also considerably slow down or even suppress the exchange of magnetization due to spin diffusion.The effect of nitrogen-15 proton-driven spin diffusion on quantitative (15)N T(1) measurements in solid proteins is investigated, and the impact on the measurement of dynamic parameters is assessed. A simple model of exchange between neighboring nitrogens is used to reproduce the evolution of (15)N spin systems whose longitudinal relaxation rates and exchange rates are compatible with experimental measurements. We show that the induced error in the measured T(1) and its effect on the determination of dynamics parameters is likely to be less than the current experimental error. The use of deuterated protein samples is shown to have a small but sometimes visible effect, and may also considerably slow down or even suppress the exchange of magnetization due to spin diffusion.
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- 2007
40. Modulation of P-glycoprotein activity by acridones and coumarins fromCitrus sinensis
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Christine Bayet, Imad Raad, O. Berger, David Guilet, C. Fazio, Annie Chaboud, Charles Dumontet, Nicole Darbour, Deleage, Gilbert, Institut de biologie et chimie des protéines [Lyon] (IBCP), Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)
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Limonin ,Pharmacognosy ,Plant Roots ,chemistry.chemical_compound ,Coumarins ,Cell Line, Tumor ,[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Humans ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,ATP Binding Cassette Transporter, Subfamily B, Member 1 ,P-glycoprotein ,Pharmacology ,chemistry.chemical_classification ,Molecular Structure ,biology ,biology.organism_classification ,Terpenoid ,Rutaceae ,chemistry ,Biochemistry ,biology.protein ,Acridines ,Efflux ,Lactone ,Citrus × sinensis ,Acridones ,Citrus sinensis - Abstract
International audience; Bioguided fractionation of the roots of Citrus sinensis (Rutaceae) led to the isolation and identification of five coumarins, namely, clausarin, suberosin, poncitrin, xanthyletin and thamnosmonin, seven acridones, namely, acrimarine B, 2-methoxycitpressine I, citpressine I, buntanine, acrimarine E, honyumine and acrimarine C, and one terpenoid, namely, limonin. Among these compounds, clausarin, 2-methoxycitpressine I and acrimarine E inhibited P-glycoprotein-mediated drug efflux in K562/R7 human leukemic cells over-expressing P-glycoprotein.Bioguided fractionation of the roots of Citrus sinensis (Rutaceae) led to the isolation and identification of five coumarins, namely, clausarin, suberosin, poncitrin, xanthyletin and thamnosmonin, seven acridones, namely, acrimarine B, 2-methoxycitpressine I, citpressine I, buntanine, acrimarine E, honyumine and acrimarine C, and one terpenoid, namely, limonin. Among these compounds, clausarin, 2-methoxycitpressine I and acrimarine E inhibited P-glycoprotein-mediated drug efflux in K562/R7 human leukemic cells over-expressing P-glycoprotein.
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- 2007
41. Survival after primary enucleation for choroidal melanoma: changes induced by the introduction of conservative therapies
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Michel Rivoire, J. Gambrelle, Loris G. Baggetto, M. Devouassoux Shisheboran, J. Fleury, B. Jean-Louis, L. Kodjikian, Jean-Daniel Grange, Deleage, Gilbert, Institut de biologie et chimie des protéines [Lyon] (IBCP), Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)
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Adult ,Male ,Choroidal melanoma ,medicine.medical_specialty ,Multivariate analysis ,Enucleation ,Eye Enucleation ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,0302 clinical medicine ,[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,medicine ,Humans ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Melanoma ,Survival rate ,Aged ,Retrospective Studies ,Aged, 80 and over ,business.industry ,Proportional hazards model ,Choroid Neoplasms ,Retrospective cohort study ,Middle Aged ,Prognosis ,medicine.disease ,Sensory Systems ,3. Good health ,Surgery ,Survival Rate ,Ophthalmology ,030220 oncology & carcinogenesis ,030221 ophthalmology & optometry ,Female ,business ,Epithelioid cell - Abstract
International audience; BACKGROUND: Most uveal melanomas are currently treated by eye-preserving radiotherapies. However, for melanomas of the largest size or with initial complications, enucleation remains the reference treatment. Enucleation is called primary when it is proposed as the only local treatment option for a melanoma. There is very little literature on the use of primary enucleation after the introduction of conservative treatments. Our main goal was to evaluate the survival of melanoma patients treated by primary enucleation since the introduction of proton-beam therapy in France in 1991. METHODS: All melanoma patients undergoing primary enucleation in our department between 1991 and 2002 were included in this retrospective study. The 5-year melanoma-specific survival rate was calculated using the Kaplan-Meier method. The multivariate prognostic analysis was performed using the Cox proportional hazards model. RESULTS: Forty patients, representing 8% of all patients with choroidal uveal melanoma diagnosed and followed up in our department during an 11-year period, were included in the study. No patient was lost to follow-up. The 5-year melanoma-specific survival rate was 31.45% (SE: 7.8) after primary enucleation. Significant prognosis factors in the multivariate analysis were: tumor thickness > 12 mm (p = 0.03), anterior margin of the tumor involving the iris (p = 0.018), and presence of epithelioid cells (p = 0.02). CONCLUSIONS: The very low survival rate reported reflects the evolution of primary enucleation, which is currently indicated only for melanomas with the worst prognosis. The knowledge of current post-enucleation survival rates represents an essential achievement for both correct assessment of conservative therapies and patient counseling.BACKGROUND: Most uveal melanomas are currently treated by eye-preserving radiotherapies. However, for melanomas of the largest size or with initial complications, enucleation remains the reference treatment. Enucleation is called primary when it is proposed as the only local treatment option for a melanoma. There is very little literature on the use of primary enucleation after the introduction of conservative treatments. Our main goal was to evaluate the survival of melanoma patients treated by primary enucleation since the introduction of proton-beam therapy in France in 1991. METHODS: All melanoma patients undergoing primary enucleation in our department between 1991 and 2002 were included in this retrospective study. The 5-year melanoma-specific survival rate was calculated using the Kaplan-Meier method. The multivariate prognostic analysis was performed using the Cox proportional hazards model. RESULTS: Forty patients, representing 8% of all patients with choroidal uveal melanoma diagnosed and followed up in our department during an 11-year period, were included in the study. No patient was lost to follow-up. The 5-year melanoma-specific survival rate was 31.45% (SE: 7.8) after primary enucleation. Significant prognosis factors in the multivariate analysis were: tumor thickness > 12 mm (p = 0.03), anterior margin of the tumor involving the iris (p = 0.018), and presence of epithelioid cells (p = 0.02). CONCLUSIONS: The very low survival rate reported reflects the evolution of primary enucleation, which is currently indicated only for melanomas with the worst prognosis. The knowledge of current post-enucleation survival rates represents an essential achievement for both correct assessment of conservative therapies and patient counseling.
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- 2006
42. A comprehensive study of the spatial and temporal expression of the col5a1 gene in mouse embryos: a clue for understanding collagen V function in developing connective tissues
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Muriel Roulet, Dominique LeGuellec, Florence Ruggiero, Gérard Karsenty, Institut de biologie et chimie des protéines [Lyon] (IBCP), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), and Deleage, Gilbert
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Pathology ,medicine.medical_specialty ,Time Factors ,Histology ,Stromal cell ,Mesenchyme ,Mammalian embryology ,Mice, Inbred Strains ,Biology ,Bone and Bones ,Umbilical Cord ,Pathology and Forensic Medicine ,Cornea ,Mesoderm ,Extracellular matrix ,Mice ,03 medical and health sciences ,Dorsal aorta ,0302 clinical medicine ,[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,medicine ,Animals ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Musculoskeletal System ,In Situ Hybridization ,Skin ,030304 developmental biology ,0303 health sciences ,Gene Expression Regulation, Developmental ,Cell Biology ,Embryo, Mammalian ,Surface ectoderm ,Cell biology ,Collagen, type I, alpha 1 ,medicine.anatomical_structure ,Animals, Newborn ,Connective Tissue ,Connective tissue metabolism ,030220 oncology & carcinogenesis ,embryonic structures ,Collagen Type V - Abstract
International audience; Collagen V is a quantitatively minor component of collagen I fibrils and the defective product of classic Ehlers-Danlos syndrome (EDS). To provide new insights into its embryonic function, a continuous evaluation of the expression pattern of proalpha1(V), a chain common to all collagen V molecular forms, was performed by in situ hybridization of developing mouse from 7.5 days after conception (dpc) to birth. Proalpha1(V) transcripts were first detected at 8.5 dpc, signals being considerably augmented at 16.5 dpc and declining at birth. Hybridization signals were, at first, exclusively detected in the dorsal aorta wall, heart, and adnexa. At 10.5 dpc, col5a1 expression was found in the heart, dorsal aorta wall, branchial arches, mesonephrotic tubules, and intestinal mesenchyme and coincided with proalpha1(I) developmental expression. Later stages exhibited an intense signal in more restricted regions, notably the skin, the bones and vertebral column, the cornea, the tendons and ligaments, the peritoneal membranes, the umbilical cord, and the salivary gland. The data revealed the important contribution of collagen V to the development of functional connective tissues. Proalpha1(V) signals were exclusively detected in the flattened cells of the surface ectoderm at 10.5 dpc. By 12.5 dpc, when cells had become cuboidal, the signal switched to the dermal fibroblasts. Thus, type V collagen appears to contribute to epidermis differentiation. Our data also suggest that collagen V participates in bone formation and/or mineralization and in the renewal of stromal cells in the cornea. The results underscore the role of collagen V in developing embryos and provide important clues for analyzing the phenotype of mouse models for EDS.Collagen V is a quantitatively minor component of collagen I fibrils and the defective product of classic Ehlers-Danlos syndrome (EDS). To provide new insights into its embryonic function, a continuous evaluation of the expression pattern of proalpha1(V), a chain common to all collagen V molecular forms, was performed by in situ hybridization of developing mouse from 7.5 days after conception (dpc) to birth. Proalpha1(V) transcripts were first detected at 8.5 dpc, signals being considerably augmented at 16.5 dpc and declining at birth. Hybridization signals were, at first, exclusively detected in the dorsal aorta wall, heart, and adnexa. At 10.5 dpc, col5a1 expression was found in the heart, dorsal aorta wall, branchial arches, mesonephrotic tubules, and intestinal mesenchyme and coincided with proalpha1(I) developmental expression. Later stages exhibited an intense signal in more restricted regions, notably the skin, the bones and vertebral column, the cornea, the tendons and ligaments, the peritoneal membranes, the umbilical cord, and the salivary gland. The data revealed the important contribution of collagen V to the development of functional connective tissues. Proalpha1(V) signals were exclusively detected in the flattened cells of the surface ectoderm at 10.5 dpc. By 12.5 dpc, when cells had become cuboidal, the signal switched to the dermal fibroblasts. Thus, type V collagen appears to contribute to epidermis differentiation. Our data also suggest that collagen V participates in bone formation and/or mineralization and in the renewal of stromal cells in the cornea. The results underscore the role of collagen V in developing embryos and provide important clues for analyzing the phenotype of mouse models for EDS.
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- 2006
43. A novel role for protein-tyrosine kinase Etk from Escherichia coli K-12 related to polymyxin resistance
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Alain J. Cozzone, Christophe Grangeasse, Patricia Doublet, Brice Obadia, Soline Lacour, Deleage, Gilbert, Institut de biologie et chimie des protéines [Lyon] (IBCP), Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)
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medicine.drug_class ,Polymyxin ,Microbial Sensitivity Tests ,Biology ,medicine.disease_cause ,Polymerase Chain Reaction ,Microbiology ,03 medical and health sciences ,Plasmid ,Drug Resistance, Bacterial ,[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,medicine ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Protein phosphorylation ,Polymyxins ,Molecular Biology ,Escherichia coli ,030304 developmental biology ,0303 health sciences ,Escherichia coli K12 ,030306 microbiology ,Kinase ,Escherichia coli Proteins ,Membrane Proteins ,General Medicine ,Protein-Tyrosine Kinases ,Molecular biology ,Anti-Bacterial Agents ,Complementation ,Tyrosine kinase ,Gene Deletion ,Polymyxin B ,medicine.drug - Abstract
International audience; The role of protein-tyrosine kinases in bacterial polymyxin resistance was assessed by both genetic and biochemical approaches. Each of the two genes, wzc and etk, encoding protein-tyrosine kinases in Escherichia coli, was knocked out by using the PCR-based method of one-step inactivation of chromosomal genes, and the corresponding mutant strain was assayed in each case for resistance to different concentrations of polymyxin B by measuring the percentage of surviving cells. The resistance of a double knock-out wzc-etk-mutant was also analyzed and complementation experiments were performed by checking the effect of plasmid vectors expressing either Wzc or Etk. Our results concurred in showing that protein-kinase Wzc is not essential for polymyxin resistance, whereas protein-kinase Etk appears to play a key role in such antibiotic resistance. This newly found specific function of Etk reinforces the concept that protein-tyrosine kinases are involved in distinct facets of bacterial physiology.The role of protein-tyrosine kinases in bacterial polymyxin resistance was assessed by both genetic and biochemical approaches. Each of the two genes, wzc and etk, encoding protein-tyrosine kinases in Escherichia coli, was knocked out by using the PCR-based method of one-step inactivation of chromosomal genes, and the corresponding mutant strain was assayed in each case for resistance to different concentrations of polymyxin B by measuring the percentage of surviving cells. The resistance of a double knock-out wzc-etk-mutant was also analyzed and complementation experiments were performed by checking the effect of plasmid vectors expressing either Wzc or Etk. Our results concurred in showing that protein-kinase Wzc is not essential for polymyxin resistance, whereas protein-kinase Etk appears to play a key role in such antibiotic resistance. This newly found specific function of Etk reinforces the concept that protein-tyrosine kinases are involved in distinct facets of bacterial physiology.
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- 2006
44. Major Endothelial Loss From Corneas in Organ Culture
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Nicolas Builles, Laurent Kodjikian, Odile Damour, Carole Burillon, and Deleage, Gilbert
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Adult ,Graft Rejection ,medicine.medical_specialty ,Adolescent ,Endothelium ,Cell Count ,Organ culture ,Polymerase Chain Reaction ,Corneal Transplantation ,Organ Culture Techniques ,Risk Factors ,Cornea ,[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,medicine ,Humans ,Simplexvirus ,Child ,Aged ,Retrospective Studies ,Aged, 80 and over ,Tissue Survival ,business.industry ,Incidence (epidemiology) ,Endothelium, Corneal ,Middle Aged ,Prognosis ,eye diseases ,Surgery ,Ophthalmology ,medicine.anatomical_structure ,DNA, Viral ,Keratitis, Herpetic ,sense organs ,business ,Follow-Up Studies - Abstract
PURPOSE: The aim of this study was to show that major losses can still occur on corneas judged suitable for grafting at the first count. In addition, we studied the frequency of these losses on 1992 corneas over a period of 4 years to evaluate the risk incurred. METHODS: We evaluated the incidence of these major losses and the associated risk factors. An Ishigawa diagram was created with the Cornea Bank team and the ophthalmologists involved in organ retrieval. Endothelial losses caused by bacterial or fungicidal contamination were excluded from the study. For the 29 corneas that suffered major losses, we analyzed the donor files for donor age, clinical file, geographical origins of the corneas, the person who did the retrieval, the length of time the cornea was stored, the data resulting from examining the endothelium at the bank by optical microscope, and the method used for sterilizing the material used. Specific analyses in cases of major loss of endothelial content: anatomopathologic examination of the corneas and search for the herpes simplex virus (HSV; type 1 or 2) by polymerase chain reaction (PCR). We carried out a statistical analysis using a chi(2) test on the 1992 corneas studied to see if the presence of diabetes (type 1 or 2) in the donor led to reduction levels different from those of corneas originating from nondiabetic donors. RESULTS: The incidence was evaluated at between 0.4% and 3% of corneas sampled, and the associated risk factor was between 0.8% and 6% of grafted corneas. The occurrence of major losses was independent of donor age and was independent of the person who did the retrieval. The occurrence of major losses was independent of geographical origin. We tested our media for endotoxin before use and found levels from 0.22 to 3.9 UI/mL. We verified the absence of a chronological relationship between the batches of media used in the bank and the number of major losses observed, showing that the pyrogenicity limit was independent of cytotoxicity limits. Data analysis showed no difference in reduction levels between diabetic and nondiabetic donors (P < 0.05). Results on the detection of HSV-1 by PCR on the storage media were all negative, and these results agree with the anatomopathologic examinations that showed no signs of viral infection. CONCLUSION: Total endothelial losses amounted to 1.4%/yr. Without the double endothelial counts, we would have had 29 primary graft rejections over that period. During storage, this loss has not been linked to a specific cause, but risk factors such as traumatic death, herpes infections, and badly controlled endotoxin levels should be considered when taking preventative actions. For the moment, a second endothelial count before grafting should be carried out, because all these problem grafts conformed to grafting criteria after the first count. The possibility of carrying out this second count is one of the recognized advantages of storage in organ culture.PURPOSE: The aim of this study was to show that major losses can still occur on corneas judged suitable for grafting at the first count. In addition, we studied the frequency of these losses on 1992 corneas over a period of 4 years to evaluate the risk incurred. METHODS: We evaluated the incidence of these major losses and the associated risk factors. An Ishigawa diagram was created with the Cornea Bank team and the ophthalmologists involved in organ retrieval. Endothelial losses caused by bacterial or fungicidal contamination were excluded from the study. For the 29 corneas that suffered major losses, we analyzed the donor files for donor age, clinical file, geographical origins of the corneas, the person who did the retrieval, the length of time the cornea was stored, the data resulting from examining the endothelium at the bank by optical microscope, and the method used for sterilizing the material used. Specific analyses in cases of major loss of endothelial content: anatomopathologic examination of the corneas and search for the herpes simplex virus (HSV; type 1 or 2) by polymerase chain reaction (PCR). We carried out a statistical analysis using a chi(2) test on the 1992 corneas studied to see if the presence of diabetes (type 1 or 2) in the donor led to reduction levels different from those of corneas originating from nondiabetic donors. RESULTS: The incidence was evaluated at between 0.4% and 3% of corneas sampled, and the associated risk factor was between 0.8% and 6% of grafted corneas. The occurrence of major losses was independent of donor age and was independent of the person who did the retrieval. The occurrence of major losses was independent of geographical origin. We tested our media for endotoxin before use and found levels from 0.22 to 3.9 UI/mL. We verified the absence of a chronological relationship between the batches of media used in the bank and the number of major losses observed, showing that the pyrogenicity limit was independent of cytotoxicity limits. Data analysis showed no difference in reduction levels between diabetic and nondiabetic donors (P < 0.05). Results on the detection of HSV-1 by PCR on the storage media were all negative, and these results agree with the anatomopathologic examinations that showed no signs of viral infection. CONCLUSION: Total endothelial losses amounted to 1.4%/yr. Without the double endothelial counts, we would have had 29 primary graft rejections over that period. During storage, this loss has not been linked to a specific cause, but risk factors such as traumatic death, herpes infections, and badly controlled endotoxin levels should be considered when taking preventative actions. For the moment, a second endothelial count before grafting should be carried out, because all these problem grafts conformed to grafting criteria after the first count. The possibility of carrying out this second count is one of the recognized advantages of storage in organ culture.
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- 2006
45. Activation by IL-1 of bovine articular chondrocytes in culture within a 3D collagen-based scaffold. An in vitro model to address the effect of compounds with therapeutic potential in osteoarthritis
- Author
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M.-C. Ronzière, Philippe Msika, Frédéric Mallein-Gerin, C.F. Rousseau, Jérôme Gouttenoire, Anne-Marie Freyria, Nathalie Piccardi, D. Herbage, D. Cortial, and Deleage, Gilbert
- Subjects
Cartilage, Articular ,Integrins ,Integrin ,Biomedical Engineering ,Matrix metalloproteinase ,Chondrocyte ,Glycosaminoglycan ,Extracellular matrix ,Chondrocytes ,Rheumatology ,Osteoarthritis ,[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Cytokine (IL-1) ,medicine ,Animals ,Orthopedics and Sports Medicine ,Zymography ,Aggrecans ,Aggrecan ,Tissue Engineering ,biology ,Chemistry ,Cartilage construct ,Cartilage ,Bovine chondrocyte ,Tissue Inhibitor of Metalloproteinases ,Molecular biology ,Matrix Metalloproteinases ,ADAM Proteins ,medicine.anatomical_structure ,Immunology ,biology.protein ,Cattle ,Collagen ,Interleukin-1 - Abstract
OBJECTIVE: To determine the best protocol for the preparation of a tissue-engineered cartilage to investigate the potential anti-arthritic and/or anti-osteoarthritic effects of drugs. METHODS: Calf articular chondrocytes, seeded in collagen sponges were grown in culture for up to 1 month. At day 14 cultures received interleukin (IL)-1beta (ranging from 0.1 to 20 ng/ml) for 1 to 3 days. Analyses of gene expression for extracellular matrix proteins, collagen-binding integrins, matrix metalloproteinases (MMPs), aggrecanases, TIMPs, IL-1Ra and Ikappa-Balpha were carried out using real-time polymerase chain reaction (PCR). Metalloproteinase activities were analysed in the culture medium using both zymography and fluorogenic peptide substrates. RESULTS: We selected a culture for 15 or 17 days with collagen sponges seeded with 10(7) chondrocytes showing a minimal cell proliferation, a maximal sulphated glycosaminoglycan (sGAG) deposition and a high expression of COL2A1, aggrecan and the alpha10 integrin sub-unit and low expression of COL1A2 and the alpha11 integrin sub-unit. In the presence of 1 ng/ml IL-1beta, we observed at day 15 up-regulations of 450-fold for MMP-1, 60-fold for MMP-13, 54-fold for ADAMTS-4 and MMP-3 and 10-fold for ADAMTS-5 and IL-1Ra. Down-regulations of 2.5-fold for COL2A1 and aggrecan were observed only at day 17. At the protein level a dose-dependent increase of total MMP-1 and MMP-13 was noted with less than 15% in the active form. CONCLUSIONS: This in vitro model of chondrocyte culture in three dimensional (3D) seems well adapted to investigate the responses of these cells to inflammatory cytokines and to evaluate the potential anti-inflammatory effects of drugs.OBJECTIVE: To determine the best protocol for the preparation of a tissue-engineered cartilage to investigate the potential anti-arthritic and/or anti-osteoarthritic effects of drugs. METHODS: Calf articular chondrocytes, seeded in collagen sponges were grown in culture for up to 1 month. At day 14 cultures received interleukin (IL)-1beta (ranging from 0.1 to 20 ng/ml) for 1 to 3 days. Analyses of gene expression for extracellular matrix proteins, collagen-binding integrins, matrix metalloproteinases (MMPs), aggrecanases, TIMPs, IL-1Ra and Ikappa-Balpha were carried out using real-time polymerase chain reaction (PCR). Metalloproteinase activities were analysed in the culture medium using both zymography and fluorogenic peptide substrates. RESULTS: We selected a culture for 15 or 17 days with collagen sponges seeded with 10(7) chondrocytes showing a minimal cell proliferation, a maximal sulphated glycosaminoglycan (sGAG) deposition and a high expression of COL2A1, aggrecan and the alpha10 integrin sub-unit and low expression of COL1A2 and the alpha11 integrin sub-unit. In the presence of 1 ng/ml IL-1beta, we observed at day 15 up-regulations of 450-fold for MMP-1, 60-fold for MMP-13, 54-fold for ADAMTS-4 and MMP-3 and 10-fold for ADAMTS-5 and IL-1Ra. Down-regulations of 2.5-fold for COL2A1 and aggrecan were observed only at day 17. At the protein level a dose-dependent increase of total MMP-1 and MMP-13 was noted with less than 15% in the active form. CONCLUSIONS: This in vitro model of chondrocyte culture in three dimensional (3D) seems well adapted to investigate the responses of these cells to inflammatory cytokines and to evaluate the potential anti-inflammatory effects of drugs.
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- 2006
46. Production of a recombinantly expressed laminin fragment by HEK293-EBNA cells cultured in suspension in a dialysis-based bioreactor
- Author
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Patricia Rousselle, Virginie Belin, and Deleage, Gilbert
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DNA, Complementary ,Time Factors ,Recombinant Fusion Proteins ,Genetic Vectors ,Culture Media, Serum-Free ,Cell Line ,law.invention ,Extracellular matrix ,Bioreactors ,law ,Laminin ,[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Protein biosynthesis ,medicine ,Bioreactor ,Humans ,Cells, Cultured ,biology ,HEK 293 cells ,Transfection ,Molecular biology ,Epithelium ,Kinetics ,medicine.anatomical_structure ,Epstein-Barr Virus Nuclear Antigens ,Recombinant DNA ,biology.protein ,Electrophoresis, Polyacrylamide Gel ,Dialysis ,Biotechnology - Abstract
Laminin 5 is a multifunctional extracellular matrix protein, which supports epithelial cell adhesion through multiple cell binding sites. For elaborate studies, a 35 kDa fragment localized at the C-terminal extremity of the molecule, the LG4/5 fragment was recombinantly expressed in mammalian HEK293-EBNA cells. As the production of the LG4/5 fragment by adherent cell monolayers was very low (
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- 2006
47. Structure of the Epstein-Barr Virus Oncogene BARF1
- Author
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Mireille de Turenne-Tessier, Nicolas Tarbouriech, Wim P. Burmeister, Tadamasa Ooka, Florence Ruggiero, Deleage, Gilbert, Institut de biologie et chimie des protéines [Lyon] (IBCP), Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)
- Subjects
Models, Molecular ,Glycosylation ,Protein Conformation ,Viral protein ,Molecular Sequence Data ,Population ,Immunoglobulins ,Plasma protein binding ,Immunoglobulin domain ,Biology ,Crystallography, X-Ray ,medicine.disease_cause ,Cell Line ,Viral Proteins ,Protein structure ,Microscopy, Electron, Transmission ,Structural Biology ,[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,medicine ,Humans ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Amino Acid Sequence ,education ,Molecular Biology ,chemistry.chemical_classification ,education.field_of_study ,medicine.disease ,Epstein–Barr virus ,Molecular biology ,Protein Structure, Tertiary ,chemistry ,Nasopharyngeal carcinoma ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,Glycoprotein ,Dimerization ,Protein Binding - Abstract
International audience; The Epstein-Barr virus is a human gamma-herpesvirus that persistently infects more than 90% of the human population. It is associated with numerous epithelial cancers, principally undifferentiated nasopharyngeal carcinoma and gastric carcinoma. The BARF1 gene is expressed in a high proportion of these cancers. An oncogenic, mitogenic and immortalizing activity of the BARF1 protein has been shown. We solved the structure of the secreted BARF1 glycoprotein expressed in a human cell line by X-ray crystallography at a resolution of 2.3A. The BARF1 protein consists of two immunoglobulin (Ig)-like domains. The N-terminal domain belongs to the subfamily of variable domains whereas the C-terminal one is related to a constant Ig-domain. BARF1 shows an unusual hexamerisation involving two principal contacts, one between the C-terminal domains and one between the N-terminal domains. The C-terminal contact with an uncommonly large contact surface extends the beta-sandwich of the Ig-domain through the second molecule. The N-terminal contact involves Ig-domains with an unusual relative orientation but with a more classical contact surface with a size in the range of dimer interactions of Ig-domains. The structure of BARF1 is most closely related to CD80 or B7-1, a co-stimulatory molecule present on antigen presenting cells, from which BARF1 must have been derived during evolution. Still, domain orientation and oligomerization differ between BARF1 and CD80. It had been shown that BARF1 binds to hCSF-1, the human colony-stimulating factor 1, but this interaction has to be principally different from the one between CSF-1 and CSF-1 receptor.The Epstein-Barr virus is a human gamma-herpesvirus that persistently infects more than 90% of the human population. It is associated with numerous epithelial cancers, principally undifferentiated nasopharyngeal carcinoma and gastric carcinoma. The BARF1 gene is expressed in a high proportion of these cancers. An oncogenic, mitogenic and immortalizing activity of the BARF1 protein has been shown. We solved the structure of the secreted BARF1 glycoprotein expressed in a human cell line by X-ray crystallography at a resolution of 2.3A. The BARF1 protein consists of two immunoglobulin (Ig)-like domains. The N-terminal domain belongs to the subfamily of variable domains whereas the C-terminal one is related to a constant Ig-domain. BARF1 shows an unusual hexamerisation involving two principal contacts, one between the C-terminal domains and one between the N-terminal domains. The C-terminal contact with an uncommonly large contact surface extends the beta-sandwich of the Ig-domain through the second molecule. The N-terminal contact involves Ig-domains with an unusual relative orientation but with a more classical contact surface with a size in the range of dimer interactions of Ig-domains. The structure of BARF1 is most closely related to CD80 or B7-1, a co-stimulatory molecule present on antigen presenting cells, from which BARF1 must have been derived during evolution. Still, domain orientation and oligomerization differ between BARF1 and CD80. It had been shown that BARF1 binds to hCSF-1, the human colony-stimulating factor 1, but this interaction has to be principally different from the one between CSF-1 and CSF-1 receptor.
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- 2006
48. An adhesion molecule in free‐living Dictyostelium amoebae with integrin β features
- Author
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Prashant Nair, Leigh Gebbie, Mohammed Benghezal, Franz Bruckert, Steve J. Charette, Sophie Cornillon, Sébastien Keller, Pierre Cosson, Bernhard Wehrle-Haller, François Letourneur, and Deleage, Gilbert
- Subjects
Talin ,Cytosol/metabolism ,Integrins ,L1 ,Genes, Protozoan ,Amino Acid Motifs ,Integrins/chemistry/genetics/metabolism ,Biochemistry ,Cytosol ,Cell Movement ,Dictyostelium ,Dictyostelium/genetics ,Conserved Sequence ,Glutathione Transferase ,Recombination, Genetic ,biology ,Cell adhesion molecule ,Adhesion ,Cell biology ,Cell Adhesion Molecules/chemistry/genetics/metabolism ,Protozoan ,Glutathione Transferase/metabolism ,Integrin, beta 6 ,Protein Structure ,Recombinant Fusion Proteins ,Scientific Report ,Molecular Sequence Data ,Integrin ,macromolecular substances ,Protein Sorting Signals ,Genetic ,[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Cell Adhesion ,Genetics ,Animals ,Amino Acid Sequence ,ddc:612 ,Cell adhesion ,Molecular Biology ,biology.organism_classification ,Actin cytoskeleton ,Recombination ,Protein Structure, Tertiary ,Genes ,Mutation ,Talin/metabolism ,biology.protein ,Cell Adhesion Molecules ,Recombinant Fusion Proteins/chemistry/metabolism ,Tertiary - Abstract
The study of free-living amoebae has proven valuable to explain the molecular mechanisms controlling phagocytosis, cell adhesion and motility. In this study, we identified a new adhesion molecule in Dictyostelium amoebae. The SibA (Similar to Integrin Beta) protein is a type I transmembrane protein, and its cytosolic, transmembrane and extracellular domains contain features also found in integrin beta chains. In addition, the conserved cytosolic domain of SibA interacts with talin, a well-characterized partner of mammalian integrins. Finally, genetic inactivation of SIBA affects adhesion to phagocytic particles, as well as cell adhesion and spreading on its substrate. It does not visibly alter the organization of the actin cytoskeleton, cellular migration or multicellular development. Our results indicate that the SibA protein is a Dictyostelium cell adhesion molecule presenting structural and functional similarities to metazoan integrin beta chains. This study sheds light on the molecular mechanisms controlling cell adhesion and their establishment during evolution.The study of free-living amoebae has proven valuable to explain the molecular mechanisms controlling phagocytosis, cell adhesion and motility. In this study, we identified a new adhesion molecule in Dictyostelium amoebae. The SibA (Similar to Integrin Beta) protein is a type I transmembrane protein, and its cytosolic, transmembrane and extracellular domains contain features also found in integrin beta chains. In addition, the conserved cytosolic domain of SibA interacts with talin, a well-characterized partner of mammalian integrins. Finally, genetic inactivation of SIBA affects adhesion to phagocytic particles, as well as cell adhesion and spreading on its substrate. It does not visibly alter the organization of the actin cytoskeleton, cellular migration or multicellular development. Our results indicate that the SibA protein is a Dictyostelium cell adhesion molecule presenting structural and functional similarities to metazoan integrin beta chains. This study sheds light on the molecular mechanisms controlling cell adhesion and their establishment during evolution.
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- 2006
49. Analysis of hepatitis C virus RNA dimerization and core–RNA interactions
- Author
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Igor Kanevsky, Philippe Fossé, Jean-Pierre Lavergne, Jean-Luc Darlix, François Penin, Caroline Gabus, Roland Ivanyi-Nagy, Damien Ficheux, Deleage, Gilbert, Virologie humaine, École normale supérieure - Lyon (ENS Lyon)-IFR128-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire de Biologie et de Pharmacologie Appliquée (LBPA), École normale supérieure - Cachan (ENS Cachan)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie et chimie des protéines [Lyon] (IBCP), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), and École normale supérieure de Lyon (ENS de Lyon)-IFR128-Institut National de la Santé et de la Recherche Médicale (INSERM)
- Subjects
MESH: Viral Core Proteins ,Untranslated region ,MESH: Mutation ,Hepatitis C virus ,Molecular Sequence Data ,RNA-dependent RNA polymerase ,Hepacivirus ,MESH: Base Sequence ,Biology ,medicine.disease_cause ,Article ,MESH: Protein Structure, Tertiary ,03 medical and health sciences ,Protein structure ,Genetics ,medicine ,[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,MESH: Hepacivirus ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,3' Untranslated Regions ,030304 developmental biology ,0303 health sciences ,MESH: Molecular Sequence Data ,Base Sequence ,Point mutation ,Viral Core Proteins ,030302 biochemistry & molecular biology ,RNA ,MESH: 3' Untranslated Regions ,Molecular biology ,3. Good health ,Protein Structure, Tertiary ,MESH: Nucleic Acid Conformation ,MESH: Dimerization ,MESH: RNA, Viral ,Chaperone (protein) ,Mutation ,Nucleic acid ,biology.protein ,Nucleic Acid Conformation ,RNA, Viral ,MESH: Molecular Chaperones ,Dimerization ,Molecular Chaperones - Abstract
The core protein of hepatitis C virus (HCV) has been shown previously to act as a potent nucleic acid chaperone in vitro, promoting the dimerization of the 3'-untranslated region (3'-UTR) of the HCV genomic RNA, a process probably mediated by a small, highly conserved palindromic RNA motif, named DLS (dimer linkage sequence) [G. Cristofari, R. Ivanyi-Nagy, C. Gabus, S. Boulant, J. P. Lavergne, F. Penin and J. L. Darlix (2004) Nucleic Acids Res., 32, 2623-2631]. To investigate in depth HCV RNA dimerization, we generated a series of point mutations in the DLS region. We find that both the plus-strand 3'-UTR and the complementary minus-strand RNA can dimerize in the presence of core protein, while mutations in the DLS (among them a single point mutation that abolished RNA replication in a HCV subgenomic replicon system) completely abrogate dimerization. Structural probing of plus- and minus-strand RNAs, in their monomeric and dimeric forms, indicate that the DLS is the major if not the sole determinant of UTR RNA dimerization. Furthermore, the N-terminal basic amino acid clusters of core protein were found to be sufficient to induce dimerization, suggesting that they retain full RNA chaperone activity. These findings may have important consequences for understanding the HCV replicative cycle and the genetic variability of the virus.The core protein of hepatitis C virus (HCV) has been shown previously to act as a potent nucleic acid chaperone in vitro, promoting the dimerization of the 3'-untranslated region (3'-UTR) of the HCV genomic RNA, a process probably mediated by a small, highly conserved palindromic RNA motif, named DLS (dimer linkage sequence) [G. Cristofari, R. Ivanyi-Nagy, C. Gabus, S. Boulant, J. P. Lavergne, F. Penin and J. L. Darlix (2004) Nucleic Acids Res., 32, 2623-2631]. To investigate in depth HCV RNA dimerization, we generated a series of point mutations in the DLS region. We find that both the plus-strand 3'-UTR and the complementary minus-strand RNA can dimerize in the presence of core protein, while mutations in the DLS (among them a single point mutation that abolished RNA replication in a HCV subgenomic replicon system) completely abrogate dimerization. Structural probing of plus- and minus-strand RNAs, in their monomeric and dimeric forms, indicate that the DLS is the major if not the sole determinant of UTR RNA dimerization. Furthermore, the N-terminal basic amino acid clusters of core protein were found to be sufficient to induce dimerization, suggesting that they retain full RNA chaperone activity. These findings may have important consequences for understanding the HCV replicative cycle and the genetic variability of the virus.
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- 2006
50. Control of carbon flux through enzymes of central and intermediary metabolism during growth of Escherichia coli on acetate
- Author
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Mansi El-Mansi, Alain J. Cozzone, Joseph Shiloach, Bernhard J. Eikmanns, and Deleage, Gilbert
- Subjects
Microbiology (medical) ,Isocitrates ,IDH1 ,Dehydrogenase ,Acetates ,Biology ,medicine.disease_cause ,Microbiology ,Phosphate Acetyltransferase ,chemistry.chemical_compound ,Biosynthesis ,Acetyl Coenzyme A ,[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Escherichia coli ,medicine ,Ketoglutarate Dehydrogenase Complex ,Enzyme Inhibitors ,Phosphorylation ,Escherichia coli Proteins ,Glyoxylates ,Isocitrate lyase ,Metabolism ,Isocitrate Lyase ,Carbon ,Isocitrate Dehydrogenase ,Culture Media ,Infectious Diseases ,Isocitrate dehydrogenase ,chemistry ,Biochemistry ,Flux (metabolism) - Abstract
During aerobic growth of Escherichia coli on acetate, the component parts of the 'acetate switch' are turned-on as a consequence of direct competition, on the one hand, between phosphotransacetylase (PTA) and alpha-ketoglutarate dehydrogenase (alpha-KGDH) for their common co-factor free-CoA (HS-CoA) and, on the other hand, between isocitrate lyase (ICL) and isocitrate dehydrogenase (ICDH) for their common substrate isocitrate. Flux analysis revealed that competitions at both junctions in central metabolism are resolved in a precise way, so that the fraction of HS-CoA flux processed through PTA for biosynthesis relative to that processed through alpha-KGDH for energy generation, matches that observed for isocitrate flux through ICL relative to ICDH at the junction of isocitrate. Whereas the mechanism involved in the partition of carbon flux at the level of HS-CoA in central metabolism remains to be unravelled, the competition at the junction of isocitrate is resolved by the reversible phosphorylation/inactivation of ICDH and the operation of the glyoxylate bypass, the expression of which is subject to regulation at the transcriptional and translational levels as well as being dependent on growth rate.During aerobic growth of Escherichia coli on acetate, the component parts of the 'acetate switch' are turned-on as a consequence of direct competition, on the one hand, between phosphotransacetylase (PTA) and alpha-ketoglutarate dehydrogenase (alpha-KGDH) for their common co-factor free-CoA (HS-CoA) and, on the other hand, between isocitrate lyase (ICL) and isocitrate dehydrogenase (ICDH) for their common substrate isocitrate. Flux analysis revealed that competitions at both junctions in central metabolism are resolved in a precise way, so that the fraction of HS-CoA flux processed through PTA for biosynthesis relative to that processed through alpha-KGDH for energy generation, matches that observed for isocitrate flux through ICL relative to ICDH at the junction of isocitrate. Whereas the mechanism involved in the partition of carbon flux at the level of HS-CoA in central metabolism remains to be unravelled, the competition at the junction of isocitrate is resolved by the reversible phosphorylation/inactivation of ICDH and the operation of the glyoxylate bypass, the expression of which is subject to regulation at the transcriptional and translational levels as well as being dependent on growth rate.
- Published
- 2006
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