30 results on '"Nicolas Boute"'
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2. Data from Efficacy of the Antibody–Drug Conjugate W0101 in Preclinical Models of IGF-1 Receptor Overexpressing Solid Tumors
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Nathalie Corvaia, Eric Chetaille, Mariya Pavlyuk, Cyrille Dreyfus, Michel Perez, Alain Beck, Jean-Francois Haeuw, Thierry Champion, Nicolas Boute, Martine Malissard, Charlotte Beau-Larvor, Alain Robert, Noureddine Loukili, Matthieu Broussas, and Barbara Akla more...
- Abstract
The insulin-like growth factor type 1 receptor (IGF-1R) is important in tumorigenesis, and its overexpression occurs in numerous tumor tissues. To date, therapeutic approaches based on mAbs and tyrosine kinase inhibitors targeting IGF-1R have only shown clinical benefit in specific patient populations. We report a unique IGF-1R–targeted antibody–drug conjugate (ADC), W0101, designed to deliver a highly potent cytotoxic auristatin derivative selectively to IGF-1R overexpressing tumor cells. The mAb (hz208F2-4) used to prepare the ADC was selected for its specific binding properties to IGF-1R compared with the insulin receptor, and for its internalization properties. Conjugation of a novel auristatin derivative drug linker to hz208F2-4 did not alter its binding and internalization properties. W0101 induced receptor-dependent cell cytotoxicity in vitro when applied to various cell lines overexpressing IGF-1R, but it did not affect normal cells. Efficacy studies were conducted in several mouse models expressing different levels of IGF-1R to determine the sensitivity of the tumors to W0101. W0101 induced potent tumor regression in certain mouse models. Interestingly, the potency of W0101 correlated with the expression level of IGF-1R evaluated by IHC. In an MCF-7 breast cancer model with high-level IGF-1R expression, a single injection of W0101 3 mg/kg led to strong inhibition of tumor growth. W0101 provides a potential new therapeutic option for patients overexpressing IGF-1R. A first-in-human trial of W0101 is currently ongoing to address clinical safety. more...
- Published
- 2023
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- View/download PDF
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3. Efficacy of the Antibody-Drug Conjugate W0101 in Preclinical Models of IGF-1 Receptor Overexpressing Solid Tumors
- Author
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Nicolas Boute, Mariya Pavlyuk, Matthieu Broussas, Barbara Akla, Cyrille Dreyfus, Michel Perez, Charlotte Beau-Larvor, Thierry Champion, Alain Robert, Eric Chetaille, Alain Beck, Noureddine Loukili, Martine Malissard, Jean-François Haeuw, and Nathalie Corvaia more...
- Subjects
0301 basic medicine ,Cancer Research ,Antibody-drug conjugate ,Immunoconjugates ,media_common.quotation_subject ,medicine.medical_treatment ,Mice, Nude ,medicine.disease_cause ,Receptor, IGF Type 1 ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Cell Line, Tumor ,Neoplasms ,medicine ,Cytotoxic T cell ,Animals ,Humans ,Internalization ,Receptor ,media_common ,biology ,Chemistry ,Growth factor ,Insulin receptor ,030104 developmental biology ,Oncology ,030220 oncology & carcinogenesis ,Cancer research ,biology.protein ,Female ,Carcinogenesis ,Tyrosine kinase - Abstract
The insulin-like growth factor type 1 receptor (IGF-1R) is important in tumorigenesis, and its overexpression occurs in numerous tumor tissues. To date, therapeutic approaches based on mAbs and tyrosine kinase inhibitors targeting IGF-1R have only shown clinical benefit in specific patient populations. We report a unique IGF-1R–targeted antibody–drug conjugate (ADC), W0101, designed to deliver a highly potent cytotoxic auristatin derivative selectively to IGF-1R overexpressing tumor cells. The mAb (hz208F2-4) used to prepare the ADC was selected for its specific binding properties to IGF-1R compared with the insulin receptor, and for its internalization properties. Conjugation of a novel auristatin derivative drug linker to hz208F2-4 did not alter its binding and internalization properties. W0101 induced receptor-dependent cell cytotoxicity in vitro when applied to various cell lines overexpressing IGF-1R, but it did not affect normal cells. Efficacy studies were conducted in several mouse models expressing different levels of IGF-1R to determine the sensitivity of the tumors to W0101. W0101 induced potent tumor regression in certain mouse models. Interestingly, the potency of W0101 correlated with the expression level of IGF-1R evaluated by IHC. In an MCF-7 breast cancer model with high-level IGF-1R expression, a single injection of W0101 3 mg/kg led to strong inhibition of tumor growth. W0101 provides a potential new therapeutic option for patients overexpressing IGF-1R. A first-in-human trial of W0101 is currently ongoing to address clinical safety. more...
- Published
- 2019
4. A novel antagonist anti-cMet antibody with antitumor activities targeting both ligand-dependent and ligand-independent c-Met receptors
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Liliane Goetsch, Alain Beck, Christian Bailly, Matthieu Broussas, Alexandra Gonzalez, Jean-François Haeuw, Alain Robert, Charlotte Beau-Larvor, Nicolas Boute, Nathalie Corvaia, and Thierry Champion
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0301 basic medicine ,A549 cell ,Cancer Research ,C-Met ,biology ,Angiogenesis ,Receptor tyrosine kinase ,In vitro ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,0302 clinical medicine ,Oncology ,chemistry ,In vivo ,030220 oncology & carcinogenesis ,biology.protein ,Cancer research ,Autocrine signalling ,Receptor - Abstract
c-Met is a prototypic member of a sub-family of RTKs. Inappropriate c-Met activation plays a crucial role in tumor formation, proliferation and metastasis. Using a key c-Met dimerization assay, a set of 12 murine whole IgG1 monoclonal antibodies was selected and a lead candidate, m224G11, was humanized by CDR-grafting and engineered to generate a divalent full antagonist humanized IgG1 antibody, hz224G11. Neither m224G11 nor hz224G11 bind to the murine c-Met receptor. Their antitumor activity was investigated in vitro in a set of experiments consistent with the reported pleiotropic effects mediated by c-Met and, in vivo, using several human tumor xenograft models. Both m224G11 and hz224G11 exhibited nanomolar affinities for the receptor and inhibited HGF binding, c-Met phosphorylation, and receptor dimerization in a similar fashion, resulting in a profound inhibition of all c-Met functions in vitro. These effects were presumably responsible for the inhibition of c-Met's major functions including cell proliferation, migration, invasion scattering, morphogenesis and angiogenesis. In addition to these in vitro properties, hz224G11 dramatically inhibits the growth of autocrine, partially autophosphorylated and c-Met amplified cell lines in vivo. Pharmacological studies performed on Hs746T gastric cancer xenografts demonstrate that hz224G11 strongly downregulates c-Met expression and phosphorylation. It also decreases the tumor mitotic index (Ki67) and induces apoptosis. Taken together, the in vitro and in vivo data suggest that hz224G11 is a promising candidate for the treatment of tumors. This antibody, now known as ABT-700 and currently in Phase I clinical trials, may provide a novel therapeutic approach to c-Met-expressing cancers. more...
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- 2016
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5. Abstract 3372: VISTA interaction with PSGL1, a likely VISTA receptor in tumors, is effectively disrupted by K0401-020 anti-VISTA antibody
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Francisco Cruzalegui, Nathalie Corvaia, Nicolas Boute, Noureddine Loukili, Pierre Ferré, Celine Thuilliez, Martine Malissard, Olivier Delfour, Pierre Launay, Eric Chetaille, Thierry Champion, and Alexandre Passioukov more...
- Subjects
Cancer Research ,Messenger RNA ,biology ,T cell ,Molecular biology ,Transmembrane protein ,Protein–protein interaction ,Transcriptome ,medicine.anatomical_structure ,Oncology ,biology.protein ,medicine ,Antibody ,Receptor ,Gene - Abstract
Background: VISTA (V-domain Ig suppressor of T cell Activation) is a transmembrane B7 family protein that is described as a negative checkpoint of T cell response. K01401-020 is a novel anti-VISTA antibody entering clinical trials (Loukili et al, AACR 2019). Unlike PD1 protein for which PDL1 and PDL2 are clearly demonstrated as its main ligands, several VISTA candidate receptors have been suggested with various degrees of evidence. The prominent candidate receptors published for VISTA are VSIG3/IGSF11 (Wang et al, Immunology, 2018; Mehta et al, Cell Rep, 2019), VSIG8 (WO2016090347), LRIG1 (WO2015187395), and VISTA itself (WO2015179799). PSGL1 recently joined the list based on in vitro biochemical experiments describing VISTA as an acidic pH selective ligand for PSGL1 (Johnston et al, 2019). Methods: Here we report a series of in vitro protein/protein interactions results aimed at testing the VISTA candidate receptors above. mRNA co-expression was analyzed via bioinformatics in a range of human cancer samples. The mRNA and protein of each candidate receptor were also precisely localized in tumor slices using amplified RNA-ISH or protein-IHC staining. Results: We found specific physicochemical conditions, in vitro, for each candidate, to show specific protein/protein interactions with VISTA, mostly occurring at acidic pH and being effectively disrupted by K01401-020 anti-VISTA antibody. In addition, K01401-020 was also confirmed to bind to VISTA whatever the pH conditions tested [6 to 7.4]. Despite the potential for biochemical interaction for all candidates, only VISTA and PSGL1 mRNAs were found strongly correlated across most cancer indications with PSGL1 being one of the best correlated genes to VISTA when compared to the whole transcriptome. In addition, when compared to the other proteins explored (VSIG3, LRIG1 and VSIG8), PSGL1 expression showed much greater correlation of expression with a myeloid-infiltrate identified by a transcriptomic signature. Dual amplified RNA-ISH on human tumors showed co-occurrence for VISTA and PSGL1, and only rare co-occurrence for VISTA and the other receptors. At the protein level, using dual IHC and proximity ligation staining, the proximity of VISTA and PSGL1 was confirmed in tumors, with metrics comparable to PD1 and PDL1 proximity. Potential interactions may still happen between circulating cells outside of tumors. Conclusions: VISTA interactions with other proteins (PSGL1, VISTA, VISG3, VSIG8 and LRIG1) were evidenced in vitro. However, based on RNA correlations and protein spatial distribution in a series of patient tumors, VISTA/PSGL1 may be a biologically relevant interaction in cancer. The likelihood that interactions with VISG3, VSIG8 and LRIG1play a meaningful biological role in tumors is relatively lower. Other potential candidate receptors to VISTA, including VISTA itself, may still need to be revealed. Citation Format: Francisco Cruzalegui, Noureddine Loukili, Olivier Delfour, Nicolas Boute, Céline Thuilliez, Thierry Champion, Martine Malissard, Pierre Launay, Eric Chetaille, Alexandre Passioukov, Nathalie Corvaïa, Pierre Ferré. VISTA interaction with PSGL1, a likely VISTA receptor in tumors, is effectively disrupted by K0401-020 anti-VISTA antibody [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3372. more...
- Published
- 2020
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6. NanoLuc luciferase - A multifunctional tool for high throughput antibody screening
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Nicolas Boute, Alain Robert, Sven Berger, Peter Lowe, Michael Tesar, and Martine Malissard
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0301 basic medicine ,Phage display ,library screening ,NanoLuc® ,03 medical and health sciences ,0302 clinical medicine ,Western blot ,medicine ,Methods ,Bioluminescence ,Pharmacology (medical) ,Luciferase ,expression analysis ,Homogeneous assay ,Bioluminescence resonance energy transfer (BRET) ,Pharmacology ,western blot ,biology ,medicine.diagnostic_test ,lcsh:RM1-950 ,affinity determination ,luciferase ,Molecular biology ,Blot ,030104 developmental biology ,lcsh:Therapeutics. Pharmacology ,Homogeneous ,030220 oncology & carcinogenesis ,biology.protein ,Antibody ,phage display ,Antibody screening - Abstract
Based on the recent development of NanoLuc Luciferase a small (19 kDa), highly stable, ATP independent, bioluminescent protein, an extremely robust and ultra high sensitivity screening system has been developed whereby primary hits of therapeutic antibodies and antibody fragments could be characterized and quantified without purification. This system is very versatile allowing cellular and solid phase ELISA but also homogeneous BRET based screening assays, relative affinity determinations with competition ELISA and direct western blotting. The new NanoLuc Luciferase protein fusion represents a “swiss army knife solution” for today and future high throughput antibody drug screenings. more...
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- 2016
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7. A New Anti-CXCR4 Antibody That Blocks the CXCR4/SDF-1 Axis and Mobilizes Effector Cells
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Alain Beck, Charlotte Beau-Larvor, Christine Klinguer-Hamour, Liliane Goetsch, Sven Berger, Matthieu Broussas, Thomas Matthes, Thierry Champion, Alain Robert, Nathalie Corvaia, Nicolas Boute, Barbara Akla, Christian Bailly, Jean-François Haeuw, Charles Dumontet, 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), Laboratoire de Physique Nucléaire et de Hautes Énergies (LPNHE), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Département de pharmacochimie moléculaire (DPM ), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Centre de Recherche en Cancérologie de Lyon (UNICANCER/CRCL), Centre Léon Bérard [Lyon]-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Université Pierre et Marie Curie - Paris 6 (UPMC) more...
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0301 basic medicine ,Cancer Research ,Mice ,Chemokine receptor ,0302 clinical medicine ,Cell Movement ,Phosphorylation ,Receptor ,ddc:616 ,Leukemia ,Effector ,Antibodies, Monoclonal ,Cell migration ,beta-Arrestin 2 ,Tumor Burden ,3. Good health ,Leukemia, Myeloid, Acute ,Oncology ,030220 oncology & carcinogenesis ,France ,Signal transduction ,Multiple Myeloma ,Switzerland ,Protein Binding ,Signal Transduction ,Receptors, CXCR4 ,Stromal cell ,Patients ,Cell Survival ,Cells ,[SDV.CAN]Life Sciences [q-bio]/Cancer ,Biology ,Binding, Competitive ,Antibodies ,Cell Line ,03 medical and health sciences ,blood ,Cell Line, Tumor ,Animals ,Humans ,Protein kinase B ,Cell Proliferation ,Blood Cells ,Cell growth ,Antibody-Dependent Cell Cytotoxicity ,Complement System Proteins ,Xenograft Model Antitumor Assays ,Chemokine CXCL12 ,Disease Models, Animal ,030104 developmental biology ,Immunology ,Cancer research ,pathology ,Protein Multimerization - Abstract
The type IV C-X-C-motif chemokine receptor (CXCR4) is expressed in a large variety of human cancers, including hematologic malignancies, and this receptor and its ligand, stromal cell–derived factor-1 (SDF-1), play a crucial role in cancer progression. We generated a humanized immunoglobulin G1 mAb, hz515H7, which binds human CXCR4, efficiently competes for SDF-1 binding, and induces a conformational change in CXCR4 homodimers. Furthermore, it inhibits both CXCR4 receptor–mediated G-protein activation and β-arrestin-2 recruitment following CXCR4 activation. The binding of the hz515H7 antibody to CXCR4 inhibits the SDF-1–induced signaling pathway, resulting in reduced phosphorylation of downstream effectors, such as Akt, Erk1/2, p38, and GSK3β. Hz515H7 also strongly inhibits cell migration and proliferation and, while preserving normal blood cells, induces both antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against neoplastic cells. In mouse xenograft models, hz515H7 displays antitumor activities with multiple hematologic tumor cell lines, with its Fc-mediated effector functions proving essential in this context. Furthermore, hz515H7 binds to primary tumor cells from acute myeloid leukemia and multiple myeloma patients. Collectively, our results demonstrate two major mechanisms of action, making hz515H7 unique in this regard. Its potential as a best-in-class molecule is currently under investigation in a phase I clinical trial. Mol Cancer Ther; 15(8); 1890–9. ©2016 AACR. more...
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- 2016
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8. A New Highly Efficient Substrate-trapping Mutant of Protein Tyrosine Phosphatase 1B (PTP1B) Reveals Full Autoactivation of the Insulin Receptor Precursor
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Vladimir Zilberfarb, Samira Boubekeur, P. Pagesy, Névéna Christeff, Nicolas Boute, and Tarik Issad
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Mutant ,Mutation, Missense ,Protein tyrosine phosphatase ,Endoplasmic Reticulum ,Biochemistry ,Receptor tyrosine kinase ,Enzyme activator ,Insulin receptor substrate ,Humans ,Insulin ,Protein Precursors ,Molecular Biology ,Protein Tyrosine Phosphatase, Non-Receptor Type 1 ,biology ,Endoplasmic reticulum ,GRB10 ,Cell Biology ,Receptor, Insulin ,Cell biology ,Enzyme Activation ,Insulin receptor ,HEK293 Cells ,Amino Acid Substitution ,Gene Expression Regulation ,biology.protein ,hormones, hormone substitutes, and hormone antagonists ,Signal Transduction - Abstract
PTP1B is a protein tyrosine-phosphatase located on the cytosolic side of the endoplasmic reticulum that plays an important role in the regulation of the insulin receptor (IR). Replacement of the conserved Asp-181 by alanine is known to convert PTP1B into a substrate-trapping protein that binds to but cannot dephosphorylate its substrates. In this work, we have studied the effect of an additional mutation (Y46F) on the substrate-trapping efficiency of PTP1B-D181A. We observed that this mutation converts PTP1B-D181A into a highly efficient substrate-trapping mutant, resulting in much higher recovery of tyrosine-phosphorylated proteins coimmunoprecipitated with PTP1B. Bioluminescence resonance energy transfer (BRET) experiments were also performed to compare the dynamics of interaction of the IR with these mutants. Basal BRET, which mainly reflects the interaction of PTP1B with the IR precursor during its biosynthesis in the endoplasmic reticulum, was markedly increased with the PTP1B-D181A-Y46F mutant. In contrast, insulin-induced BRET was markedly reduced with PTP1B-D181A-Y46F. I(125) insulin binding experiments indicated that PTP1B-D181-Y46F reduced the expression of IR at the plasma membrane. Reduced expression at the cell surface was associated with higher amounts of the uncleaved IR precursor in the cell. Moreover, we observed that substantial amounts of the uncleaved IR precursor reached the Tris-phosphorylated, fully activated form in an insulin independent fashion. These results support the notion that PTP1B plays a crucial role in the control of the activity of the IR precursor during its biosynthesis. In addition, this new substrate-trapping mutant may be a valuable tool for the identification of new PTP1B substrates. more...
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- 2011
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9. A novel antagonist anti-cMet antibody with antitumor activities targeting both ligand-dependent and ligand-independent c-Met receptors
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Alexandra, Gonzalez, Matthieu, Broussas, Charlotte, Beau-Larvor, Jean-François, Haeuw, Nicolas, Boute, Alain, Robert, Thierry, Champion, Alain, Beck, Christian, Bailly, Nathalie, Corvaïa, and Liliane, Goetsch more...
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Male ,Hepatocyte Growth Factor ,Mice, Nude ,CHO Cells ,Mice, SCID ,Proto-Oncogene Proteins c-met ,Antibodies, Monoclonal, Humanized ,Ligands ,Xenograft Model Antitumor Assays ,Antibodies, Monoclonal, Murine-Derived ,Mice ,Random Allocation ,Cricetulus ,A549 Cells ,Cell Line, Tumor ,Immunoglobulin G ,Neoplasms ,Human Umbilical Vein Endothelial Cells ,MCF-7 Cells ,Animals ,Humans ,Female - Abstract
c-Met is a prototypic member of a sub-family of RTKs. Inappropriate c-Met activation plays a crucial role in tumor formation, proliferation and metastasis. Using a key c-Met dimerization assay, a set of 12 murine whole IgG1 monoclonal antibodies was selected and a lead candidate, m224G11, was humanized by CDR-grafting and engineered to generate a divalent full antagonist humanized IgG1 antibody, hz224G11. Neither m224G11 nor hz224G11 bind to the murine c-Met receptor. Their antitumor activity was investigated in vitro in a set of experiments consistent with the reported pleiotropic effects mediated by c-Met and, in vivo, using several human tumor xenograft models. Both m224G11 and hz224G11 exhibited nanomolar affinities for the receptor and inhibited HGF binding, c-Met phosphorylation, and receptor dimerization in a similar fashion, resulting in a profound inhibition of all c-Met functions in vitro. These effects were presumably responsible for the inhibition of c-Met's major functions including cell proliferation, migration, invasion scattering, morphogenesis and angiogenesis. In addition to these in vitro properties, hz224G11 dramatically inhibits the growth of autocrine, partially autophosphorylated and c-Met amplified cell lines in vivo. Pharmacological studies performed on Hs746T gastric cancer xenografts demonstrate that hz224G11 strongly downregulates c-Met expression and phosphorylation. It also decreases the tumor mitotic index (Ki67) and induces apoptosis. Taken together, the in vitro and in vivo data suggest that hz224G11 is a promising candidate for the treatment of tumors. This antibody, now known as ABT-700 and currently in Phase I clinical trials, may provide a novel therapeutic approach to c-Met-expressing cancers. more...
- Published
- 2015
10. Interaction between the insulin receptor and Grb14: A dynamic study in living cells using BRET
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Anne-Françoise Burnol, Christophe Blanquart, Nicolas Boute, Sébastien Nouaille, Tarik Issad, Vladimir Zilberfarb, and Dominique Perdereau
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medicine.medical_treatment ,Protein tyrosine phosphatase ,Kidney ,Biochemistry ,Cell Line ,Dephosphorylation ,Insulin receptor substrate ,Protein Interaction Mapping ,medicine ,Humans ,Insulin ,Adaptor Proteins, Signal Transducing ,Protein Tyrosine Phosphatase, Non-Receptor Type 1 ,Pharmacology ,Dose-Response Relationship, Drug ,biology ,Receptor, Insulin ,IRS2 ,Cell biology ,Luminescent Proteins ,Insulin receptor ,Luminescent Measurements ,biology.protein ,Phosphorylation ,Mitogen-Activated Protein Kinases ,Protein Tyrosine Phosphatases ,Signal transduction ,Protein Binding ,Signal Transduction - Abstract
Grb14 is a molecular adaptor that binds to the activated insulin receptor (IR) and negatively regulates insulin signaling. We have studied the dynamics of interaction of the IR with Grb14, in real time, in living HEK cells, using bioluminescence resonance energy transfer (BRET). Insulin rapidly and dose-dependently stimulated this interaction. Removing insulin from the incubation medium only resulted in a modest decrease in BRET signal, indicating that the interaction between the IR and Grb14 can remain long after insulin stimulus has disappeared. BRET saturation experiments indicated that insulin markedly increases the affinity between IR and Grb14, resulting in recruitment of the adaptor to the activated IR. In addition, using both BRET and co-immunoprecipitation experiments, we demonstrated that insulin induced the dimerization of Grb14, most likely as a result of simultaneous binding of two Grb14 molecules on the activated IR. We also investigated the relationships between IR, Grb14 and the protein tyrosine phosphatase PTP1B. We observed that insulin-induced BRET between the IR and PTP1B was markedly reduced by Grb14, suggesting that Grb14 regulated this interaction in living cells. Using site-specific antibodies against phosphorylated tyrosines of the insulin receptor, we showed that Grb14 protected the three tyrosines of the kinase loop from dephosphorylation by PTP1B, while favouring dephosphorylation of tyrosine 972. This resulted in decreased IRS-1 binding to the IR and decreased activation of the ERK pathway. Our work suggests that Grb14 may regulate signalling through the insulin receptor by controlling its tyrosine-dephosphorylation in a site-specific manner. more...
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- 2006
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11. Interaction with Grb14 results in site‐specific regulation of tyrosine phosphorylation of the insulin receptor
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Tarik Issad, Johan Roix, Sébastien Nouaille, Dominique Perdereau, Christophe Blanquart, Vladimir Zilberfarb, Anne-Françoise Burnol, and Nicolas Boute
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Luminescence ,Scientific Report ,Protein tyrosine phosphatase ,Biology ,Biochemistry ,Receptor tyrosine kinase ,Cell Line ,Dephosphorylation ,chemistry.chemical_compound ,Bacterial Proteins ,Protein Interaction Mapping ,Genetics ,Animals ,Humans ,Phosphorylation ,Tyrosine ,Molecular Biology ,Adaptor Proteins, Signal Transducing ,Protein Tyrosine Phosphatase, Non-Receptor Type 1 ,Kinase ,Tyrosine phosphorylation ,Receptor, Insulin ,Rats ,Cell biology ,Luminescent Proteins ,Insulin receptor ,chemistry ,biology.protein ,Signal Transduction - Abstract
The dynamics of interaction of the insulin receptor (IR) with Grb14 was monitored, in real time, in living human embryonic kidney cells, using bioluminescence resonance energy transfer (BRET). We observed that insulin rapidly and dose-dependently stimulated this interaction. We also observed that insulin-induced BRET between the IR and protein tyrosine phosphatase 1B (PTP1B) was markedly reduced by Grb14, suggesting that Grb14 regulated this interaction in living cells. Using site-specific antibodies against phosphorylated tyrosines of the IR, we showed that Grb14 protected the three tyrosines of the kinase loop from dephosphorylation by PTP1B, while favouring dephosphorylation of tyrosine 972. This resulted in decreased IRS-1 binding to the IR and decreased activation of the extracellular signal-regulated kinase pathway. Increased Grb14 expression in human liver-derived HuH7 cells also seemed to specifically decrease the phosphorylation of Y972. Our work therefore suggests that Grb14 may regulate signalling through the IR by controlling its tyrosine dephosphorylation in a site-specific manner. more...
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- 2006
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12. Interaction of PTPB with the insulin receptor precursor during its biosynthesis in the endoplasmic reticulum
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Nicolas Boute, Tarik Issad, S Boubekeur, and Danièle Lacasa
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medicine.medical_treatment ,Biology ,Endoplasmic Reticulum ,Biochemistry ,Cell Line ,Green fluorescent protein ,Mice ,chemistry.chemical_compound ,Bacterial Proteins ,Protein biosynthesis ,medicine ,Animals ,Humans ,Insulin ,Luciferase ,Protein Precursors ,Luciferases, Renilla ,Protein Tyrosine Phosphatase, Non-Receptor Type 1 ,Endoplasmic reticulum ,STIM1 ,General Medicine ,Tunicamycin ,Receptor, Insulin ,Cell biology ,Luminescent Proteins ,Insulin receptor ,Energy Transfer ,chemistry ,biology.protein ,Protein Tyrosine Phosphatases ,hormones, hormone substitutes, and hormone antagonists - Abstract
PTP1B is a protein tyrosine-phosphatase predominantly located on the cystosolic surface of the endoplasmic reticulum. This tyrosine-phosphatase plays a major role in the regulation of the activity of the insulin receptor (IR). We have studied the interaction of the IR with PTP1B in living cells using bioluminescence resonance energy transfer (BRET). The IR was fused to Renilla luciferase and a substrate-trapping mutant of PTP1B was fused to the yellow variant of the green fluorescent protein (YFP). When the two partners interacted, an energy transfer occurred between the luciferase and the YFP, and a fluorescent signal, emitted by the YFP, could be detected. The interaction of the IR with PTP1B could be monitored in real time for more than 30 min. Insulin rapidly and dose-dependently stimulated this interaction. The basal (insulin-independent) interaction of IR with PTP1B was much lower with a soluble form than with the endoplasmic reticulum-targeted form of PTP1B, indicating that this basal interaction mainly occurred in the endoplasmic reticulum. In the basal state, PTP1B and the IR indeed co-localized in the endoplasmic reticulum, as demonstrated by confocal microscopy and cell fractionation experiments. Moreover, inhibition of IR processing with tunicamycin indicated that the basal interaction of PTP1B with IR occurred during biosynthesis of the IR precursor in the endoplasmic reticulum. These results strongly suggest that PTP1B not only dephosphorylates the insulin receptor that has been activated by insulin, but also regulates the insulin receptor precursor during its biosynthesis. Localisation of PTP1B to the endoplasmic reticulum may be important to prevent insulin-independent autonomous activity of the immature insulin receptor precursor. more...
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- 2005
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13. Podocin Localizes in the Kidney to the Slit Diaphragm Area
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Séverine Roselli, Olivier Gribouval, Nicolas Boute, Mireille Sich, France Benessy, Tania Attié, Marie-Claire Gubler, and Corinne Antignac
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Pathology ,medicine.medical_specialty ,urogenital system ,Kidney metabolism ,Glomerulonephritis ,Biology ,urologic and male genital diseases ,medicine.disease ,Fusion protein ,Pathology and Forensic Medicine ,Cell biology ,Podocyte ,medicine.anatomical_structure ,Membrane protein ,medicine ,Slit diaphragm ,Podocin ,biology.protein ,Stomatin - Abstract
We recently cloned a novel gene, NPHS2, involved in autosomal recessive steroid-resistant nephrotic syndrome. This gene encodes a novel podocyte protein, podocin. Given its similarity with the stomatin family proteins, podocin is predicted to be an integral membrane protein with a single membrane domain forming a hairpin-like structure placing both N- and C-termini in the cytosol. Here, we show by in situ hybridization, that during development, the NPHS2 transcript is first expressed in mesonephric podocytes from the S-shaped body and, later, in the metanephric kidney, in the future podocytes at the late S-shaped body stage. In the mature kidney, NPHS2 is exclusively expressed in the podocytes of mature glomeruli. We generated rabbit polyclonal antibodies against fusion proteins derived from the N- and the C-terminal regions of podocin which detected a single band of 49-kd in transfected HEK293 cell lysates by immunoprecipitation and Western blotting. By immunohistology, podocin was detected in podocytes from the early capillary loop stage in the developing nephrons, and at the basal pole, along the GBM, in mature glomeruli. By electron microscopy, we demonstrate that podocin is facing the slit diaphragm with its two ends in the cytoplasm of the foot processes, in agreement with its predicted structure. Our results suggest that podocin could serve to anchor directly or indirectly components of the slit diaphragm to the cytoskeleton. more...
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- 2002
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14. How many captains for a ship on electoral drift? Limiting the number of leadership candidates in the Flemish Christian-democratic party (CD&V)
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Jasmien Luypaert, Leen Lingier, Nicolas Bouteca, Audrey Vandeleene, and Bram Wauters
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parties leadership selection ,intra-party democratization ,steering process ,elite interviews ,Belgium ,last person standing ,Political science - Abstract
Many Western parties have opened up the process of leadership selection to party members under the noble premises to democratize the party. Yet, this might just be window-dressing as party leadership selection is often a coronation rather than an open contest. We argue that the preparation phase preceding the actual election phase is crucial in understanding the balance between the impact of party members and the steering of the party elite. This study compares the preparation phase of two leadership contests after losing elections in one party, the Flemish Christian-democratic party in Belgium: one with a single candidate and one with an exceptionally high number of candidates. Our analysis, based on 22 in-depth elite interviews, demonstrates that leadership elections are influenced by a cluster of different influencing actors, but in particular by what we label the “last person standing” whose candidacy is identified as the most effective mechanism to influence the nomination process. Other (slightly less effective) influencing mechanisms include encouragements, discouragements and the diffusion of an ideal profile for the future party leader. more...
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- 2022
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15. Type IV collagen in sponges, the missing link in basement membrane ubiquity*
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Katsutoshi Yoshizato, Koyomi Miyazaki, Jean Vacelet, Jean-Yves Exposito, Robert Garrone, Nicole Boury-Esnault, Nicolas Boute, Nobuhiro Noro, and Deleage, Gilbert
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DNA, Complementary ,Molecular Sequence Data ,Basement Membrane ,law.invention ,Type IV collagen ,Species Specificity ,law ,Complementary DNA ,[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,medicine ,Animals ,Humans ,Amino Acid Sequence ,Cloning, Molecular ,Peptide sequence ,Phylogeny ,Basement membrane ,Genome ,biology ,Cell Biology ,General Medicine ,biology.organism_classification ,Porifera ,Sponge ,Collagen, type I, alpha 1 ,medicine.anatomical_structure ,Biochemistry ,Polyclonal antibodies ,biology.protein ,Recombinant DNA ,Collagen ,Oligonucleotide Probes - Abstract
Basement membrane structures, or their main component, type IV collagen, have been detected in all multicellular animal species, except sponges. We cancel this exception by the demonstration of type IV collagenous sequences in a new marine sponge species by cDNA and genomic DNA studies. One of these sequences is long enough to demonstrate the specific characteristics of type IV collagen chains. The 12 cysteines are at conserved positions in the carboxyl-terminal non-helical NCl domain, as are the interruptions in the carboxyl-terminal end of the triple helical domain. The gene organization of the region coding for the NCl domain is similar to that of the human genes COL4A2, COL4A4 and COL4A6. An additional, shorter sequence suggests the presence of a second chain. The expected tissue localization of this collagen has been confirmed using polyclonal antibodies raised against a sponge recombinant protein. These results demonstrate that type IV collagen is representated in all animal phyla. It is actually the only known ubiquitous collagen and it has at least two different alpha chains in all the species where it has been characterized.Basement membrane structures, or their main component, type IV collagen, have been detected in all multicellular animal species, except sponges. We cancel this exception by the demonstration of type IV collagenous sequences in a new marine sponge species by cDNA and genomic DNA studies. One of these sequences is long enough to demonstrate the specific characteristics of type IV collagen chains. The 12 cysteines are at conserved positions in the carboxyl-terminal non-helical NCl domain, as are the interruptions in the carboxyl-terminal end of the triple helical domain. The gene organization of the region coding for the NCl domain is similar to that of the human genes COL4A2, COL4A4 and COL4A6. An additional, shorter sequence suggests the presence of a second chain. The expected tissue localization of this collagen has been confirmed using polyclonal antibodies raised against a sponge recombinant protein. These results demonstrate that type IV collagen is representated in all animal phyla. It is actually the only known ubiquitous collagen and it has at least two different alpha chains in all the species where it has been characterized. more...
- Published
- 1996
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16. Monitoring the activation state of the insulin-like growth factor-1 receptor and its interaction with protein tyrosine phosphatase 1B using bioluminescence resonance energy transfer
- Author
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Tarik Issad, Nicolas Boute, Danièle Lacasa, and Christophe Blanquart
- Subjects
Yellow fluorescent protein ,medicine.medical_treatment ,Recombinant Fusion Proteins ,Cell Line ,Receptor, IGF Type 1 ,Insulin-like growth factor ,Bacterial Proteins ,Insulin-Like Growth Factor II ,medicine ,Humans ,Insulin ,Luciferase ,Insulin-Like Growth Factor I ,Phosphorylation ,Receptor ,Insulin-like growth factor 1 receptor ,Pharmacology ,Protein Tyrosine Phosphatase, Non-Receptor Type 1 ,biology ,Dose-Response Relationship, Drug ,Autophosphorylation ,HEK 293 cells ,In vitro ,Cell biology ,body regions ,Luminescent Proteins ,Energy Transfer ,Luminescent Measurements ,biology.protein ,Molecular Medicine ,Protein Tyrosine Phosphatases ,Protein Binding - Abstract
We have developed two bioluminescence resonance energy transfer (BRET)-based approaches to monitor 1) ligand-induced conformational changes within partially purified insulin-like growth factor-1 (IGF-1) receptors (IGF1R) and 2) IGF1R interaction with a substrate-trapping mutant of protein tyrosine phosphatase 1B (PTP1B-D181A) in living cells. In the first assay, human IGF1R fused to Renilla reniformis luciferase (Rluc) or yellow fluorescent protein (YFP) were cotransfected in human embryonic kidney (HEK)-293 cells. The chimeric receptors were then partially purified by wheat germ lectin chromatography, and BRET measurements were performed in vitro. In the second assay, BRET measurements were performed on living HEK-293 cells cotransfected with IGF1R-Rluc and YFP-PTP1B-D181A. Ligand-induced conformational changes within the IGF1R and interaction of the IGF1R with PTP1B could be detected as an energy transfer between Rluc and YFP. Dose-response experiments with IGF-1, IGF-2, and insulin demonstrated that the effects of these ligands on BRET correlate well with their known pharmacological properties toward the IGF1R. Inhibition of IGF1R autophosphorylation by the tyrphostin AG1024 (3-bromo-5-t-butyl-4-hydroxy-benzylidenemalonitrile) resulted in the inhibition of IGF1-induced BRET signal between the IGF1R and PTP1B. In addition, an anti-IGF1R antibody known to inhibit the biological effects of IGF-1 inhibited ligand-induced BRET signal within the IGF1R, as well as between IGF1R and PTP1B. This inhibition of BRET signal paralleled the inhibition of the ligand-induced autophosphorylation of the IGF1R by this antibody. In conclusion, these BRET-based assays permit 1) the rapid evaluation of the effects of agonists or inhibitory molecules on IGF1R activation and 2) the analysis of the regulation of IGF1R-PTP1B interaction in living cells. more...
- Published
- 2005
17. Interaction of the insulin receptor with the receptor-like protein tyrosine phosphatases PTPalpha and PTPepsilon in living cells
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Nicolas Boute, Tarik Issad, and Danièle Lacasa
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Pharmacology ,biology ,Protein Conformation ,GRB10 ,Receptor-Like Protein Tyrosine Phosphatases, Class 4 ,Protein tyrosine phosphatase ,Receptor tyrosine kinase ,IRS2 ,Receptor, Insulin ,Insulin receptor ,Biochemistry ,Receptor-Like Protein Tyrosine Phosphatases ,Insulin receptor substrate ,Luminescent Measurements ,biology.protein ,Molecular Medicine ,Humans ,Insulin ,Protein Tyrosine Phosphatases ,Tyrosine kinase ,Cells, Cultured - Abstract
The interactions between the insulin receptor and the two highly homologous receptor-like protein tyrosine phosphatases (PTPase) PTPalpha and PTPepsilon were studied in living cells by using bioluminescence resonance energy transfer. In human embryonic kidney 293 cells expressing the insulin receptor fused to luciferase and substrate-trapping mutants of PTPalpha or PTPepsilon fused to the fluorescent protein Topaz, insulin induces an increase in resonance energy transfer that could be followed in real time in living cells. Insulin effect could be detected at very early time points and was maximal less than 2 min after insulin addition. Bioluminescence resonance energy-transfer saturation experiments indicate that insulin does not stimulate the recruitment of protein tyrosine phosphatase molecules to the insulin receptor but rather induces conformational changes within preassociated insulin receptor/protein tyrosine phosphatase complexes. Physical preassociation of the insulin receptor with these protein tyrosine phosphatases at the plasma membrane, in the absence of insulin, was also demonstrated by chemical cross-linking with a non-cell-permeable agent. These data provide the first evidence that PTPalpha and PTPepsilon associate with the insulin receptor in the basal state and suggest that these protein tyrosine phosphatases may constitute important negative regulators of the insulin receptor tyrosine kinase activity by acting rapidly at the plasma membrane level. more...
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- 2005
18. Dynamics of the interaction between the insulin receptor and protein tyrosine-phosphatase 1B in living cells
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Danièle Lacasa, Samira Boubekeur, Nicolas Boute, and Tarik Issad
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medicine.medical_specialty ,DNA, Complementary ,medicine.medical_treatment ,Recombinant Fusion Proteins ,Scientific Report ,Protein tyrosine phosphatase ,Kidney ,Transfection ,Biochemistry ,src Homology Domains ,chemistry.chemical_compound ,Internal medicine ,Insulin receptor substrate ,Genetics ,medicine ,Humans ,Molecular Biology ,Cells, Cultured ,Protein Tyrosine Phosphatase, Non-Receptor Type 1 ,biology ,GRB10 ,Endoplasmic reticulum ,Insulin ,Tunicamycin ,IRS2 ,Receptor, Insulin ,Insulin receptor ,Kinetics ,Endocrinology ,chemistry ,Luminescent Measurements ,biology.protein ,Protein Tyrosine Phosphatases ,hormones, hormone substitutes, and hormone antagonists - Abstract
The dynamics of the interaction of the insulin receptor with a substrate-trapping mutant of protein-tyrosine phosphatase 1B (PTP1B) were monitored in living human embryonic kidney cells using bioluminescence resonance energy transfer (BRET). Insulin dose-dependently stimulates this interaction, which could be followed in real time for more than 30 minutes. The effect of insulin on the BRET signal could be detected at early time-points (30 seconds), suggesting that in intact cells the tyrosine-kinase activity of the insulin receptor is tightly controlled by PTP1B. Interestingly, the basal (insulin-independent) interaction of the insulin receptor with PTP1B was much weaker with a soluble form of the tyrosine-phosphatase than with the endoplasmic reticulum (ER)-targeted form. Inhibition of insulin-receptor processing using tunicamycin suggests that the basal interaction occurs during insulin-receptor biosynthesis in the ER. Therefore, localization of PTP1B in this compartment might be important for the regulation of insulin receptors during their biosynthesis. more...
- Published
- 2003
19. The activity of the insulin receptor assessed by bioluminescence resonance energy transfer
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Tarik Issad, Karine Pernet, and Nicolas Boute
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biology ,Chemistry ,General Neuroscience ,Insulin ,medicine.medical_treatment ,Energy transfer ,Resonance ,Transfection ,General Biochemistry, Genetics and Molecular Biology ,Receptor, Insulin ,Insulin receptor ,Cnidaria ,History and Philosophy of Science ,Energy Transfer ,Genes, Reporter ,Luminescent Measurements ,medicine ,biology.protein ,Biophysics ,Bioluminescence ,Animals ,Humans ,Receptor ,Luciferases - Abstract
This work describes a new assay, based on the bioluminescence resonance energy transfer (BRET) methodology, to monitor the activation state of the insulin receptor.
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- 2002
20. The use of resonance energy transfer in high-throughput screening: BRET versus FRET
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Ralf Jockers, Tarik Issad, and Nicolas Boute
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Pharmacology ,Drug discovery ,High-throughput screening ,Green Fluorescent Proteins ,Biology ,Toxicology ,Fluorescence ,Resonance (particle physics) ,Green fluorescent protein ,Luminescent Proteins ,Förster resonance energy transfer ,Biochemistry ,Luminescent Measurements ,Biophysics ,Bioluminescence ,Animals ,Humans ,Luciferase ,Luciferases ,Molecular Biology ,Nuclear Magnetic Resonance, Biomolecular - Abstract
Bioluminescence resonance energy transfer has developed in recent years as a new technique to study protein-protein interactions. Protein partners of interest are tagged with either luciferase or green fluorescent protein (GFP). Non-radiative energy transfer between the excited luciferase and the GFP permits the study of spatial relationships between the two partners. This technique constitutes an important tool for the study of the functional activity of different types of receptors, and can be used in sensitive, homogenous high-throughput screening assays. more...
- Published
- 2002
21. A homogenous assay to monitor the activity of the insulin receptor using Bioluminescence Resonance Energy Transfer
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Nicolas Boute, Karine Pernet, and Tarik Issad
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Pharmacology ,biology ,GRB10 ,Autophosphorylation ,Biochemistry ,Receptor tyrosine kinase ,IRS2 ,Receptor, Insulin ,Insulin receptor ,Energy Transfer ,Insulin receptor substrate ,Luminescent Measurements ,biology.protein ,Diabetes Mellitus ,Methods ,Humans ,Obesity ,Tyrosine kinase ,Insulin-like growth factor 1 receptor - Abstract
Insulin exerts its biological effects through a plasma membrane receptor that possesses a tyrosine kinase activity. Binding of insulin to its receptor induces a conformational change that stimulates the autophosphorylation of the receptor on tyrosine residues. This autophosphorylation stimulates the tyrosine kinase activity of the receptor toward intracellular substrates involved in the transmission of the signal. The discovery of pharmacological agents that specifically activate the tyrosine kinase activity of the insulin receptor will be of great importance for the treatment of insulin-resistant or insulin-deficient patients. We have developed a procedure based on Bioluminescence Resonance Energy Transfer (BRET) to monitor the activation state of the insulin receptor. Human insulin receptor cDNA, was fused to either Renilla luciferase or yellow fluorescent protein coding sequences. Fusion insulin receptors were partially purified by wheat-germ lectin chromatography from HEK-293 cells co-transfected with these constructs. The conformational change induced by insulin on its receptor could be detected as an energy transfer (BRET signal) between Renilla luciferase and yellow fluorescent protein. BRET signal paralleled insulin-induced autophosphorylation of the fusion receptor. Dose-dependent effects of insulin, insulin-like growth factor 1 and epidermal growth factor on BRET signal were in agreement with known pharmacological properties of these ligands. Moreover, an antibody, which activated the autophosphorylation of the receptor, had similar effects on BRET signal. This methodology allows for rapid analysis of the effects of agonists on insulin receptor activity and could, therefore, be used in high-throughput screening for the discovery of molecules with insulin-like properties. more...
- Published
- 2002
22. With respect for the core business: the impact of party ideology on the odds of government participation among regionalist parties
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Lorenzo Terrière and Nicolas Bouteca
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regionalist parties ,government participation ,party ideology ,quantitative manifesto research ,western multi-level democracies ,comparative research ,Political science - Abstract
An increasing number of regionalist parties have participated in regional or national executive office. This article examines the specific conditions under which this party type increases its odds of successful cabinet entry – with a focus on ideological party change. Their programmatic profile is mapped before and after government entry by applying quantitative content analysis on coded electoral manifestos. The binary logistic regression analyses provide empirical evidence that regionalist parties that compromise on their territorial core business are more likely to enter (regional) government. Regionalist parties are also more likely to cross the threshold of (regional) governance when they operate in more decentralized countries and when they are a larger electoral factor in the regional political arena. Other relevant control variables, such as economic growth, national electoral score and party age, do not generate a significant effect on the odds of government participation. more...
- Published
- 2020
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23. Expression of PKD1 and PKD2 transcripts and proteins in human embryo and during normal kidney development
- Author
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Olivier Devuyst, Nicolas Boute, Yiqiang Cai, Marie Claire Gubler, Luis F. Onuchic, Tania Attié-Bitach, Véronique Chauvet, Bunyong Phakdeekitacharoen, Gregory G. Germino, Feng Qian, Liliane Guicharnaud, and UCL - MD/MINT - Département de médecine interne more...
- Subjects
Adult ,medicine.medical_specialty ,TRPP Cation Channels ,Transcription, Genetic ,Kidney development ,Biology ,urologic and male genital diseases ,Kidney ,Pathology and Forensic Medicine ,Pregnancy ,Internal medicine ,Gene expression ,Polycystic kidney disease ,medicine ,Human embryogenesis ,Humans ,Northern blot ,PKD1 ,urogenital system ,Gene Expression Regulation, Developmental ,Membrane Proteins ,Proteins ,medicine.disease ,Embryo, Mammalian ,female genital diseases and pregnancy complications ,Cell biology ,Endocrinology ,Membrane protein ,Ureteric bud ,Protein Biosynthesis ,embryonic structures ,Female ,Regular Articles - Abstract
Autosomal-dominant polycystic kidney disease, one of the most frequent human genetic disorders, is genetically heterogeneous. Most cases result from mutations of PKD1 or PKD2 encoding polycystin-1 or polycystin-2, respectively. Polycystin-1 is a large transmembrane protein containing several domains involved in cell-cell and/or cell-matrix interactions. Polycystin-2 is transmembrane glycoprotein sharing homology with some families of cation channels. Despite a large number of reports, the tissue distribution of these two proteins, especially of polycystin-1, is still debated. We investigated the expression pattern of PKD1 and PKD2 transcripts and proteins during human embryogenesis and kidney development, using Northern blot analysis, in situ hybridization, and immunohistochemical methods. For each gene, the expression pattern of transcripts and protein was concordant. In human 5- to 6-week-old embryos, both genes are widely expressed, mainly in neural tissue, cardiomyocytes, endodermal derivatives, and mesonephros. At this age, PKD2 but not PKD1 expression is observed in the ureteric bud and the uninduced metanephros. Thereafter, PKD2 is diffusely expressed at all stages of nephron development, whereas high PKD1 expression first appears in differentiated proximal tubules. Proximal tubule expression of both genes decreases from weeks 20 to 24 onwards. PKD1 transcripts, later restricted to distal tubules in fetal nephrogenesis, are no longer detected in adult kidneys, which nevertheless maintain a faint expression of polycystin-1, whereas persistent expression of PKD2 transcripts and protein is observed throughout nephrogenesis. Overall, contrary to previous observations, we found profound differences in the spatiotemporal expression of PKD1 and PKD2 during nephrogenesis, PKD2 being expressed earlier and more diffusely than PKD1. These data suggest that polycystins could interact with different partners, at least during kidney development. more...
- Published
- 2002
24. NPHS2, encoding the glomerular protein podocin, is mutated in autosomal recessive steroid-resistant nephrotic syndrome
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Nicolas Boute, Olivier Gribouval, Séverine Roselli, France Benessy, Hyunjoo Lee, Arno Fuchshuber, Karin Dahan, Marie-Claire Gubler, Patrick Niaudet, and Corinne Antignac
- Subjects
medicine.medical_specialty ,Nephrotic Syndrome ,Positional cloning ,Genetic Linkage ,DNA Mutational Analysis ,Kidney Glomerulus ,Molecular Sequence Data ,Genes, Recessive ,urologic and male genital diseases ,Focal segmental glomerulosclerosis ,Fetus ,Internal medicine ,Genetics ,medicine ,Animals ,Humans ,Cloning, Molecular ,Caenorhabditis elegans ,Caenorhabditis elegans Proteins ,Congenital nephrotic syndrome ,In Situ Hybridization ,Expressed Sequence Tags ,biology ,Sequence Homology, Amino Acid ,urogenital system ,Reverse Transcriptase Polymerase Chain Reaction ,Intracellular Signaling Peptides and Proteins ,Membrane Proteins ,Glomerulonephritis ,Blood Proteins ,Helminth Proteins ,medicine.disease ,Physical Chromosome Mapping ,Steroid-resistant nephrotic syndrome ,Pedigree ,Endocrinology ,Organ Specificity ,Multigene Family ,Mutation ,Podocin ,biology.protein ,Kidney disorder ,Nephrotic syndrome - Abstract
Familial idiopathic nephrotic syndromes represent a heterogeneous group of kidney disorders, and include autosomal recessive steroid-resistant nephrotic syndrome, which is characterized by early childhood onset of proteinuria, rapid progression to end-stage renal disease and focal segmental glomerulosclerosis. A causative gene for this disease, NPHS2, was mapped to 1q25-31 and we report here its identification by positional cloning. NPHS2 is almost exclusively expressed in the podocytes of fetal and mature kidney glomeruli, and encodes a new integral membrane protein, podocin, belonging to the stomatin protein family. We found ten different NPHS2 mutations, comprising nonsense, frameshift and missense mutations, to segregate with the disease, demonstrating a crucial role for podocin in the function of the glomerular filtration barrier. more...
- Published
- 2000
25. Characterization of two genes coding for a similar four-cysteine motif of the amino-terminal propeptide of a sea urchin fibrillar collagen
- Author
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Robert Garrone, Jean-Yves Exposito, Nicolas Boute, Gilbert Deléage, and Deleage, Gilbert
- Subjects
Genetics ,biology ,Base Sequence ,Sequence Homology, Amino Acid ,Sequence analysis ,Alternative splicing ,Molecular Sequence Data ,Intron ,biology.organism_classification ,Biochemistry ,Biological Evolution ,Paracentrotus lividus ,Exon ,biology.animal ,Sea Urchins ,Sequence Homology, Nucleic Acid ,[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Animals ,Amino Acid Sequence ,Collagen ,Cysteine ,Cloning, Molecular ,Sea urchin ,Peptide sequence ,Gene - Abstract
We report the characterization of the 5' region of the gene coding for the 2 alpha fibrillar collagen chain of the sea urchin Paracentrotus lividus. This sequence analysis identified the intron/exon organization of the region of the gene coding for the signal peptide, the cysteine-rich domain and the 12 repeats of the four-cysteine module of the unusually long amino-propeptide. This still unknown four-cysteine motif is generally encoded by one exon, which confirms that the distinct amino-propeptide structures of the fibrillar collagens arise from the shuffling of several exon-encoding modules. Moreover, Southern-blot analysis of the sea urchin genome and sequencing of selected genomic clones allowed us to demonstrate that several sea urchin genes could potentially code for the four-cysteine module. Curiously, one of these genes lacks the exons coding for four repeats of this motif while, in another gene, the same exons are submitted to an alternative splicing event. more...
- Published
- 1995
26. Abstract C56: Characterization of hz515H7, a novel humanized anti-CXCR4 antibody: In vitro efficacy on CXCR4-associated signaling pathways and in vivo antitumor activity
- Author
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Christine Klinguer-Hamour, Barbara Akla, Jean-François Haeuw, Alain Robert, Liliane Goetsch, Nathalie Corvaia, Charlotte Beau-Larvor, Matthieu Broussas, Christian Bailly, Nicolas Boute, and Sven Berger more...
- Subjects
A549 cell ,Cancer Research ,medicine.medical_specialty ,Stromal cell ,Cell growth ,Angiogenesis ,Growth factor ,medicine.medical_treatment ,Cancer ,Biology ,medicine.disease ,Endocrinology ,Oncology ,Growth factor receptor ,Internal medicine ,Cancer cell ,Cancer research ,medicine - Abstract
Chemokines are small, secreted peptides that control the migration of leukocytes along a chemical gradient of ligand, especially during immune and inflammatory reactions. They are divided into two major subfamilies, CC and CXC, based on the position of their NH2-terminal cysteine residues, and bind to G protein coupled receptors, whose two major sub families are designated CCR and CXCR. More than 50 human chemokines and 18 chemokine receptors have been discovered so far. The chemokine receptor CXCR4 and its ligand Stromal cell-derived factor-1 (SDF-1) play a central role in various physiological and pathological processes, including cancer. CXCR4 is over-expressed in a large number of tumors: colon, breast, prostate, lung, ovary, pancreas. CXCR4/SDF-1 axis is directly implicated in migration, invasion leading to metastases, cell proliferation and angiogenesis. Moreover, CXCR4 overexpression correlated with poor prognosis in many types of cancer. A novel humanized monoclonal antibody hz515H7 was raised against the human CXCR4. It displayed efficacious antagonist properties for all major pathways associated with SDF-1-induced CXCR4 signaling in vitro. Its antitumor activity was investigated in vivo using several human tumor models. Materials and Methods: CHOK1 and NIH3T3 cells were stably transfected with human CXCR4. [125I]SDF1 and [35S]GTP S binding assays were performed on cell membranes containing CXCR4, using SPA-WGA beads. Calcium mobilization was monitored using Fluo-4NW dye. BRET assays were developed upon genetic fusion of CXCR4 to Rluc and β-arrestin-2 to YFP and transient co-expression in HEK293 cells. Xenograft model: 10.106 Ramos cells (B-cell lymphoma) were implanted s.c. into the right flank region of each SCID mouse and allowed to grow to the designated size before administration of antibodies. The mice were followed twice a week for the observation of xenograft growth. Tumor volume was calculated using the formula: π/6 × length × width × height. Results: Hz515H7 Mab was found to strongly inhibit SDF-1 binding, Gα protein activation and -arrestin-2 recruitment. It also inhibited calcium release, and constrained by itself CXCR4 homodimers conformation. Hz515H7 reduced SDF-1-induced cell migration in vitro. Moreover we also demonstrated that hz515H7 Mab was able to significantly inhibit growth of xenograft tumors in mice. Altogether, the data demonstrate that Mab hz515H7 behaves as a potent and efficacious antagonist of all major CXCR4-controlled signaling pathways and suggest that targeting CXCR4 is a promising way for the treatment of tumors. 285-286 C57 Targeting stromal platelet-derived growth factor receptorα (PDGFRα) inhibits lung cancer growth independent of tumor cell PDGFRα expression. Colleen Burns1, David Gerber2, Puja Gupta2, Michael T. Dellinger2, Jason E. Toombs2, Michael Peyton2, Inga Dunignan1, Jennifer Malaby1, Timothy Bailey1, Rolf A. Brekken2, Nick Loizos1. 1ImClone Systems, New York, NY; 2University of Texas Southwestern Medical Center, Dallas, TX. Platelet-derived growth factor receptor alpha (PDGFRα) is a type III receptor tyrosine kinase that is normally expressed on cells of mesenchymal origin (e.g. fibroblasts and smooth muscle cells) as well as on a variety of tumor types. In lung cancer, PDGFRα is expressed frequently by tumor associated stromal cells and, in a subset of tumors, by cancer cells themselves. The anti-human PDGFRα mAb, IMC-3G3, blocks the binding of PDGF-AA, -BB & - CC to PDGFRα, and was previously shown to inhibit tumor growth of glioblastoma, prostate, and sarcoma xenografts in mice. In our analysis, IMC-3G3 administered to mice 2× per week at 40 mg/kg significantly inhibited the growth of human H1703 non-small cell lung cancer (NSCLC) tumors with a T/C value of 35%. The PDGFRα-positive H1703 lung cancer line contains both PDGFRα and PDGF-CC gene amplifications and shows a co-dependency on this axis for proliferation. Co-dependence of this kind is a rare event in human lung cancer cell lines, suggesting that only a limited number of lung tumor patients might benefit from PDGFRα inhibition. However, this experimental system does not account for the potential therapeutic effects of stromal PDGFRα inhibition. In tumor stroma, the PDGF-PDGFRα axis functions in fibroblast activation, modulation of tumor interstitial pressure, and production and secretion of vascular endothelial growth factor (VEGF). To target stromal PDGFRα, an anti-mouse PDGFRα mAb (1E10) was generated and shown to inhibit PDGF-AA from binding to murine PDGFRα with an IC50 of 8.51 × 10−9M. MAb 1E10 inhibited PDGF-AA-induced phosphorylation of mouse PDGFRα but not human PDGFRα in cell-based assays; thus demonstrating the species specificity of 1E10. Tumor xenograft studies were performed using the PDGFRα-negative NSCLC cell lines Calu-6, H1993, and A549. MAb 1E10 treatment attenuated the tumor growth of A549 and Calu-6 xenografts in nude mice with T/C values of 67% and 51%, respectfully. In Calu-6 xenografts, the combination of 1E10 plus an anti-VEGF antibody (S12) significantly inhibited tumor growth compared to control. Microvessel density was significantly decreased by both 1E10 and S12 given alone or in combination. MAb 1E10 enhanced the anti-tumor activity of cisplatin-gemcitabine chemotherapy in A549 xenografts. However, MAb 1E10 monotherapy or when combined with cisplatin-gemcitabline chemotherapy showed no anti-tumor effect on H1993 xenograft growth. PDGF-AA and VEGF-A levels were determined in cell lines resistant and sensitive to 1E10 in vivo treatment. Elevated cancer cell expression of PDGF-AA and low expression of VEGF were associated with response to stromal PDGFRα targeting. Specifically, sensitive cell lines had relatively low VEGF-A expression (VEGF-A/PDGF-AA ratio 1.96 in Calu-6 cells and 10.87 in A549 cells), while the resistant cell line H1993 had elevated VEGF-A (VEGF-A/PDGF-AA ratio 65.86) when grown in cell culture. This difference may suggest that the 1E10-sensitive tumors have a relatively greater dependence on PDGF-AA-induced production of stromal VEGF. Therefore, inhibition of stromal PDGFRα represents a means for enhancing control of lung cancer growth in some cases, independent of tumor cell PDGFRα expression. 286 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C56. more...
- Published
- 2011
- Full Text
- View/download PDF
27. Abstract 4571: 515H7, a novel anti-CXCR4 antibody: In vitro efficacy on CXCR4-associated signaling pathways and in vivo anti-tumor activity
- Author
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Charlotte Beau-Larvor, Jean-François Haeuw, Matthieu Broussas, Liliane Goetsch, Christine Klinguer-Hamour, Thierry Wurch, Alain Robert, Barbara Akla, Nathalie Corvaia, Sven Berger, Christian Bailly, and Nicolas Boute more...
- Subjects
Cancer Research ,Chemokine ,Stromal cell ,biology ,Cell growth ,Angiogenesis ,Cell migration ,Chemokine receptor ,Oncology ,Immunology ,Cancer research ,biology.protein ,Signal transduction ,G protein-coupled receptor - Abstract
Chemokines are small, secreted peptides that control the migration of leukocytes along a chemical gradient of ligand, especially during immune and inflammatory reactions. They are divided into two major subfamilies, CC and CXC, based on the position of their NH2-terminal cysteine residues, and bind to G protein coupled receptors, whose two major sub families are designated CCR and CXCR. More than 50 human chemokines and 18 chemokine receptors have been discovered so far. Chemokine receptor CXCR4/[Stromal cell-derived factor (SDF)-1] axis plays a central role in various physiological and pathological processes, including cancer. CXCR4 is over-expressed in a large number of tumors: colon, breast, prostate, lung, ovary, pancreas. CXCR4/SDF-1 axis is directly implicated in migration, invasion leading to metastases, cell proliferation and angiogenesis. Moreover, CXCR4 overexpression correlated with poor prognosis in many types of cancer. A novel monoclonal antibody (Mab 515H7) was raised against the human CXCR4. It displayed efficacious antagonist properties for all major pathways associated with SDF-1-induced CXCR4 signaling in vitro. It was able to efficiently bind CXCR4 on both transfected cells and human tumor cell lines. It was found to strongly inhibit SDF-1 binding, Gα protein activation and beta-arrestin2 recruitment. It also blocked second messenger release (calcium, cAMP) and constrained by itself the formation of CXCR4 homodimers. It also inhibits SDF-1-induced cell migration in vitro. 515H7 Mab antitumor activities were also investigated in vivo using several human tumor models. We demonstrated that 515H7 Mab was able to significantly inhibit growth of xenograft tumors in mice. In addition to its effect on tumor growth, 515H7 Mab was also able to significantly improve mice survival in the lethal U937 model. The herewith data demonstrate that Mab 515H7 behaves as a potent and efficacious antagonist of all major CXCR4-controlled signaling pathways and suggest that targeting CXCR4 is a promising way for the treatment of tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4571. doi:10.1158/1538-7445.AM2011-4571 more...
- Published
- 2011
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28. Origin of basement membrane collagen genes: Characterization of the most primitive gene in a marine sponge
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Robert Garrone, Jean-Yves Exposito, Nicolas Boute, and Christophe Geourjon
- Subjects
Basement membrane ,Sponge ,medicine.anatomical_structure ,biology ,medicine ,Cell Biology ,General Medicine ,Anatomy ,biology.organism_classification ,Gene ,Cell biology - Published
- 1995
- Full Text
- View/download PDF
29. Looking for an insulin pill? Use the BRET methodology!
- Author
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K Pernet, S Boubekeur, Danièle Lacasa, Tarik Issad, and Nicolas Boute
- Subjects
Yellow fluorescent protein ,Recombinant Fusion Proteins ,Endocrinology, Diabetes and Metabolism ,medicine.medical_treatment ,Administration, Oral ,Dephosphorylation ,Endocrinology ,Cell surface receptor ,Internal Medicine ,medicine ,Humans ,Insulin ,Luciferase ,Receptor ,biology ,Research ,Autophosphorylation ,General Medicine ,Receptor, Insulin ,Cell biology ,Luminescent Proteins ,Insulin receptor ,Diabetes Mellitus, Type 1 ,Biochemistry ,biology.protein - Abstract
Summary Insulin exerts its biological effects through a plasma membrane receptor that possesses a tyrosine-kinase activity. This tyrosine-kinase activity depends on the autophosphorylation of the receptor on tyrosine residues and on its dephosphorylation by protein tyrosine-phosphatases. The discovery of pharmacological agents that specifically stimulate the autophosphorylation of the insulin receptor or inhibit its dephosphorylation will be of great importance for the treatment of insulin resistant or insulin deficient patients. Bioluminescence Resonance Energy Transfer (BRET) has developed in recent years as a new technique to study protein-protein interactions. In the BRET technique, one partner is fused to Renilla luciferase, whereas the other partner is fused to a fluorescent protein (e.g. YFP, Yellow Fluorescent Protein). The luciferase is excited by addition of its substrate, cœlenterazine. If the two partners interact, resonance energy transfer occurs between the luciferase and the YFP, and a fluorescent signal, emitted by the YFP, can be detected. Our work indicates that this methodology could be an important tool for the search of molecules that activate insulin receptor autophosphorylation or that inhibit its dephosphorylation. Indeed, we first showed that the activation of the insulin receptor by different ligands can be monitored using a chimeric receptor with one β-subunit fused to Renilla luciferase and the other β-subunit fused to YFP. The conformational changes induced by different ligands could be detected as an energy transfer (BRET signal) between the luciferase and the YFP, that reflects the activation state of the receptor. This methodology allows for rapid analysis of the effects of agonists on insulin receptor activity and may therefore be used in high-throughput screening for the discovery of molecules with insulin-like properties. More recently, we demonstrated that the BRET methodology could also be used to monitor the interaction of the insulin receptor with protein tyrosine-phosphatase 1B, one of the main tyrosine-phosphatase that controls its activity. HEK cells were co-transfected with the insulin receptor fused to Renilla luciferase and a substrate-trapping mutant of PTP1B (PTP1B-D181A) fused to YFP. Insulin-induced BRET signal could be followed in real time for more than 30 min. Therefore, this methodology can also be used in high-throughput screening for the search of molecules that will specifically disrupt the interaction between the insulin receptor and PTP1B. more...
30. Brussel-Halle-Vilvoorde: Voeren van de 21ste eeuw
- Author
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Carl Devos and Nicolas Boutera
- Subjects
Political institutions and public administration (General) ,JF20-2112 ,Political science (General) ,JA1-92 - Published
- 2008
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