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142 results on '"Canaux calciques , fonctions et pathologies"'

1. FRET-Based Nanobiosensors for Imaging Intracellular Ca2+ and H+ Microdomains

Author

Martin Oheim, Mayeul Collot, Anne Feltz, Jean-Maurice Mallet, Camilla Luccardini, Alsu I. Zamaleeva, Guillaume Despras, Michel De Waard, Institut de biologie de l'ENS Paris (UMR 8197/1024) (IBENS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Département de Biologie - ENS Paris, École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC), Canaux calciques, Fonctions et Pathologies, [GIN] Grenoble Institut des Neurosciences (GIN), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA), Laboratoire de physiologie cérébrale (LPC - UMR 8118), Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie de l'ENS Paris (IBENS), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Département de Biologie - ENS Paris, d'Eggis, Gilles, Département de Biologie - ENS Paris, École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie de l'ENS Paris (UMR 8197/1024) ( IBENS ), École normale supérieure - Paris ( ENS Paris ) -École normale supérieure - Paris ( ENS Paris ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), Université Pierre et Marie Curie - Paris 6 ( UPMC ), Equipe 3 : Canaux calciques, Fonctions et Pathologies, Université Joseph Fourier - Grenoble 1 ( UJF ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -[GIN] Grenoble Institut des Neurosciences, Laboratoire de physiologie cérébrale ( LPC - UMR 8118 ), and Université Paris Diderot - Paris 7 ( UPD7 ) -Université Paris Descartes - Paris 5 ( UPD5 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS )

Subjects
Fluorescence-lifetime imaging microscopy, Intracellular Space, Analytical chemistry, Biosensing Techniques, lcsh:Chemical technology, [ CHIM ] Chemical Sciences, Biochemistry, Analytical Chemistry, chemistry.chemical_compound, FRET-based Ca 2+ and H + probes, [CHIM] Chemical Sciences, Fluorescence Resonance Energy Transfer, lcsh:TP1-1185, Instrumentation, chemistry.chemical_classification, Chemistry, Titrimetry, Endocytosis, Atomic and Molecular Physics, and Optics, Molecular Imaging, nanoparticle surface chemistry, Protons, Fluorophore, intracellular Ca2+ and H+ fluorometry, Endosomes, Article, Biophysical Phenomena, Cell Line, Rhodamine, Nanosensor, Quantum Dots, Animals, [CHIM]Chemical Sciences, Electrical and Electronic Engineering, FRET-based Ca2+ and H+ probes, Fluorescent Dyes, quantum dot nanobiosensors, Ions, Rhodamines, Biomolecule, red-emitting indicator, Acceptor, Förster resonance energy transfer, Quantum dot, Biophysics, Nanoparticles, Calcium, concentration microdomain, intracellular Ca 2+ and H + fluorometry, Lysosomes, cell-penetrating peptide
Abstract

International audience; Semiconductor nanocrystals (NCs) or quantum dots (QDs) are luminous point emitters increasingly being used to tag and track biomolecules in biological/biomedical imaging. However, their intracellular use as highlighters of single-molecule localization and nanobiosensors reporting ion microdomains changes has remained a major challenge. Here, we report the design, generation and validation of FRET-based nanobiosensors for detection of intracellular Ca 2+ and H + transients. Our sensors combine a commercially available CANdot ® 565QD as an energy donor with, as an acceptor, our custom-synthesized red-emitting Ca 2+ or H + probes. These 'Rubies' are based on an extended rhodamine as a OPEN ACCESS Sensors 2015, 15 24663 fluorophore and a phenol or BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid) for H + or Ca 2+ sensing, respectively, and additionally bear a linker arm for conjugation. QDs were stably functionalized using the same SH/maleimide crosslink chemistry for all desired reactants. Mixing ion sensor and cell-penetrating peptides (that facilitate cytoplasmic delivery) at the desired stoichiometric ratio produced controlled multi-conjugated assemblies. Multiple acceptors on the same central donor allow up-concentrating the ion sensor on the QD surface to concentrations higher than those that could be achieved in free solution, increasing FRET efficiency and improving the signal. We validate these nanosensors for the detection of intracellular Ca 2+ and pH transients using live-cell fluorescence imaging.

Published
2015

2. Deletion of the microtubule-associated protein 6 (MAP6) results in skeletal muscle dysfunction

Author

Sylvie Gory-Fauré, Annie Andrieux, Mathilde Chivet, Christophe Bosc, Laura Pietrangelo, Julie Brocard, Muriel Sébastien, Isabelle Marty, Perrine Aubin, Benoît Giannesini, Anne Fourest-Lieuvin, John Rendu, Jacques Brocard, Simona Boncompagni, Julien Fauré, INSERM U836, équipe 4, Muscles et pathologies, Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de résonance magnétique biologique et médicale (CRMBM), Aix Marseille Université (AMU)-Assistance Publique - Hôpitaux de Marseille (APHM)-Centre National de la Recherche Scientifique (CNRS), Université Grenoble Alpes (UGA), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM), University 'G. d'Annunzio' of Chieti-Pescara [Chieti], INSERM U836, équipe 1, Physiopathologie du cytosquelette, Organisation Fonctionnelle du Cytosquelette, Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-IFR27, Université de Montpellier (UM), Canaux calciques , fonctions et pathologies, Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Association Française contre lesMyopathies (AFM-Téléthon) to MS, PA, and IM, and from Italian Ministry ofHealth - GR2011-02352681 to SB, Giannesini, Benoit, Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), [GIN] Grenoble Institut des Neurosciences (GIN), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])

Subjects
Male, 0301 basic medicine, lcsh:Diseases of the musculoskeletal system, [SDV]Life Sciences [q-bio], Muscle Fibers, Skeletal, Triad, Sarcoplasmic reticulum, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Microtubules, Mice, 03 medical and health sciences, Microtubule, medicine, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Animals, Orthopedics and Sports Medicine, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Calcium Signaling, MAP6, Molecular Biology, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, Cells, Cultured, Calcium release, Chemistry, Myogenesis, Microtubule-associated protein, Muscle weakness, Skeletal muscle, Cell Biology, Muscle atrophy, Cell biology, Mice, Inbred C57BL, [SDV] Life Sciences [q-bio], 030104 developmental biology, medicine.anatomical_structure, Muscle contraction, Knockout mouse, Female, medicine.symptom, lcsh:RC925-935, Microtubule-Associated Proteins, Gene Deletion
Abstract

Background The skeletal muscle fiber has a specific and precise intracellular organization which is at the basis of an efficient muscle contraction. Microtubules are long known to play a major role in the function and organization of many cells, but in skeletal muscle, the contribution of the microtubule cytoskeleton to the efficiency of contraction has only recently been studied. The microtubule network is dynamic and is regulated by many microtubule-associated proteins (MAPs). In the present study, the role of the MAP6 protein in skeletal muscle organization and function has been studied using the MAP6 knockout mouse line. Methods The presence of MAP6 transcripts and proteins was shown in mouse muscle homogenates and primary culture using RT-PCR and western blot. The in vivo evaluation of muscle force of MAP6 knockout (KO) mice was performed on anesthetized animals using electrostimulation coupled to mechanical measurement and multimodal magnetic resonance. The impact of MAP6 deletion on microtubule organization and intracellular structures was studied using immunofluorescent labeling and electron microscopy, and on calcium release for muscle contraction using Fluo-4 calcium imaging on cultured myotubes. Statistical analysis was performed using Student’s t test or the Mann-Whitney test. Results We demonstrate the presence of MAP6 transcripts and proteins in skeletal muscle. Deletion of MAP6 results in a large number of muscle modifications: muscle weakness associated with slight muscle atrophy, alterations of microtubule network and sarcoplasmic reticulum organization, and reduction in calcium release. Conclusion Altogether, our results demonstrate that MAP6 is involved in skeletal muscle function. Its deletion results in alterations in skeletal muscle contraction which contribute to the global deleterious phenotype of the MAP6 KO mice. As MAP6 KO mouse line is a model for schizophrenia, our work points to a possible muscle weakness associated to some forms of schizophrenia.

Published
2018

3. Lack of FcRn Impairs Natural Killer Cell Development and Functions in the Tumor Microenvironment

Author

Castaneda, Diana Cadena, Dhommée, Christine, Baranek, Thomas, Dalloneau, Emilie, Lajoie, Laurie, Valayer, Alexandre, Arnoult, Christophe, Demattéi, Marie-Véronique, Fouquenet, Delphine, Parent, Christelle, Heuzé-Vourc'h, Nathalie, Gouilleux-Gruart, Valérie, Université de Tours (UT), Centre d'Immunologie de Marseille - Luminy (CIML), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Immunologie et Embryologie Moléculaires (IEM), Université d'Orléans (UO)-Centre National de la Recherche Scientifique (CNRS), Groupe innovation et ciblage cellulaire (GICC), EA 7501 [2018-...] (GICC EA 7501), Centre d’Etude des Pathologies Respiratoires (CEPR), UMR 1100 (CEPR), Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM), Canaux calciques , fonctions et pathologies, Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire d'Etude des Parasites Génétiques (LEPG), Virus, pseudo-virus: Morphogénèse et Antigénicité, Université de Tours (UT)-EA3856, Pathologies Respiratoires : Protéolyse et Aérosolthérapie, Université de Tours, Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU), Université de Tours-Institut National de la Santé et de la Recherche Médicale (INSERM), Université de Tours-EA3856, Génétique, immunothérapie, chimie et cancer (GICC), UMR 7292 CNRS [2012-2017] (GICC UMR 7292 CNRS), Université de Tours-Centre National de la Recherche Scientifique (CNRS), Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS), LAJOIE, Laurie, Université Francois Rabelais [Tours], and brea, deborah

Subjects
Lung Neoplasms, [SDV.IMM] Life Sciences [q-bio]/Immunology, [SDV]Life Sciences [q-bio], fcgrt knock-out, Immunology, Down-Regulation, [SDV.CAN]Life Sciences [q-bio]/Cancer, Receptors, Fc, NK cells, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, anti-tumor immunity, Cell Degranulation, Statistics, Nonparametric, Interferon-gamma, Mice, CD107a, [SDV.CAN] Life Sciences [q-bio]/Cancer, Cell Line, Tumor, Tumor Microenvironment, Animals, Neoplasm Metastasis, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, IFN-γ, Original Research, Mice, Knockout, Histocompatibility Antigens Class I, Lysosome-Associated Membrane Glycoproteins, Cell Differentiation, [SDV] Life Sciences [q-bio], Killer Cells, Natural, Mice, Inbred C57BL, Disease Models, Animal, FcRn, [SDV.IMM]Life Sciences [q-bio]/Immunology, Transcytosis
Abstract

International audience; The neonatal Fc receptor (FcRn) is responsible for the recycling and transcytosis of IgG and albumin. FcRn level was found altered in cancer tissues and implicated in tumor immunosurveillance and neoplastic cell growth. However, the consequences of FcRn down-regulation in the anti-tumor immune response are not fully elucidated. By using the B16F10 experimental lung metastasis model in an FcRn-deficient microenvironment (FcRn-/- mice), we found lung metastasis associated with an abnormal natural killer (NK) cell phenotype. In FcRn-/- mice, NK cells were immature, as shown by their surface marker profile and their decreased ability to degranulate and synthesize interferon γ after chemical and IL-2 or IL-12, IL-15 and IL-18 activation. These new findings support the critical role of FcRn downregulation in the tumor microenvironment in anti-tumor immunity, via NK cell maturation and activation.

Published
2018

4. From identification to functional characterization of cyriotoxin-1a, an antinociceptive toxin from the spider Cyriopagopus schioedtei

Author

Rémy Béroud, Laurent Bialy, Andrees Bohme, Jean-Marie Chambard, Michel De Waard, Laetitia Lucarain, Lucie Jaquillard, Denis Servent, Stéphane Hourcade, Michel Partiseti, Gerhard Hessler, Stéphanie Combemale, Brigitte Schombert, Michael Kurz, Sophie Fouconnier, Rachid Boukaiba, Tânia C. Gonçalves, Evelyne Benoit, Sanofi R & D, Integrated Drug Discovery, In Vitro Biology & Pharmacology, Service d'Ingénierie Moléculaire pour la Santé (ex SIMOPRO) (SIMoS), Médicaments et Technologies pour la Santé (MTS), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut des Neurosciences Paris-Saclay (NeuroPSI), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Smartox Biotechnology, Université Joseph Fourier - Grenoble 1 (UJF), Sanofi-Aventis Deutschland GmbH, Neuroscience Therapeutic Area, UCB BioPharma, INSERM U836, équipe 3, Canaux calciques, fonctions et pathologies, Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM), unité de recherche de l'institut du thorax UMR1087 UMR6291 (ITX), Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), ANR-11-LABX-0015,ICST,Canaux ioniques d'intérêt thérapeutique(2011), Unité de recherche de l'institut du thorax (ITX-lab), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), BENOIT, Evelyne, and Laboratoires d'excellence - Canaux ioniques d'intérêt thérapeutique - - ICST2011 - ANR-11-LABX-0015 - LABX - VALID

Subjects
0301 basic medicine, [SDV]Life Sciences [q-bio], Narcotic Antagonists, Pain, Spider Venoms, Peptide, Venom, Voltage-Gated Sodium Channels, Pharmacology, Cell Line, 03 medical and health sciences, chemistry.chemical_compound, Mice, 0302 clinical medicine, In vivo, Animals, Humans, [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], chemistry.chemical_classification, Analgesics, Sodium channel, Spiders, [SDV.SP]Life Sciences [q-bio]/Pharmaceutical sciences, Research Papers, In vitro, [SDV] Life Sciences [q-bio], [SDV.SP] Life Sciences [q-bio]/Pharmaceutical sciences, Electrophysiology, Disease Models, Animal, 030104 developmental biology, HEK293 Cells, chemistry, [SDV.SP.PHARMA] Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, Tetrodotoxin, [SDV.SP.PHARMA]Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Female, Inhibitor cystine knot, 030217 neurology & neurosurgery, Sodium Channel Blockers
Abstract

Background and purpose The NaV 1.7 channel is highly expressed in dorsal root ganglia of the sensory nervous system and plays a central role in the pain signalling process. We investigated a library prepared from original venoms of 117 different animals to identify new selective inhibitors of this target. Experimental approach We used high throughput screening of a large venom collection using automated patch-clamp experiments on human voltage-gated sodium channel subtypes and then in vitro and in vivo electrophysiological experiments to characterize the active peptides that have been purified, sequenced, and chemically synthesized. Analgesic effects were evaluated in vivo in mice models. Key results We identified cyriotoxin-1a (CyrTx-1a), a novel peptide isolated from Cyriopagopus schioedtei spider venom, as a candidate for further characterization. This 33 amino acids toxin belongs to the inhibitor cystine knot structural family and inhibits hNaV 1.1-1.3 and 1.6-1.7 channels in the low nanomolar range, compared to the micromolar range for hNaV 1.4-1.5 and 1.8 channels. CyrTx-1a was 920 times more efficient at inhibiting tetrodotoxin (TTX)-sensitive than TTX-resistant sodium currents recorded from adult mouse dorsal root ganglia neurons and in vivo electrophysiological experiments showed that CyrTx-1a was approximately 170 times less efficient than huwentoxin-IV at altering mouse skeletal neuromuscular excitability properties. CyrTx-1a exhibited an analgesic effect in mice by increasing reaction time in the hot-plate assay. Conclusions and implications The pharmacological profile of CyrTx-1a paves the way for further molecular engineering aimed to optimize the potential antinociceptive properties of this peptide.

Published
2018

5. Functional characterization of cell-free expressed K v 1.3 channel using a voltage-sensitive fluorescent dye

Author

Caroline Barette, Sandra Cortès, Rémy Béroud, Michel De Waard, Béatrice Schaack, Synthelis, Groupe Plateforme et Moyens Scientifiques et techniques communs / Centre de Criblage pour Molécules Bio-Actives (GPMS / CMBA), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Smartox Biotechnology, Université Joseph Fourier - Grenoble 1 (UJF), unité de recherche de l'institut du thorax UMR1087 UMR6291 (ITX), Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-CHU Grenoble-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de biologie structurale [1992-2019] (IBS - UMR 5075 [1992-2019]), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Contrôle moléculaire de la réponse immune specifique, Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Genetics and Chemogenomics (GenChem), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), INSERM U836, équipe 3, Canaux calciques, fonctions et pathologies, Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Unité de recherche de l'institut du thorax (ITX-lab)

Subjects
0301 basic medicine, K(V)1.3 channel, [SDV]Life Sciences [q-bio], Cell free, Micelle, Proteoliposome, 03 medical and health sciences, 0302 clinical medicine, Lipid bilayer, ComputingMilieux_MISCELLANEOUS, Cell-free protein synthesis, Liposome, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, Margatoxin, Fluorescence, 3. Good health, Oxonol VI, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], 030104 developmental biology, Membrane protein, Biophysics, 030217 neurology & neurosurgery, Biotechnology
Abstract

International audience; Using a cell-free expression system, we produced the Kv1.3 protein embedded in one step within detergent micelles. The protein was then purified and relipidated into mixed lipid bilayers. These proteoliposomes held an average of 0.8 protein per liposome. We examined channel forming activity using an oxonol VI fluorescent probe and verified its inhibition using margatoxin and ShK toxins. This assay was automatized and optimized so as to get a Z' statistical factor acceptable for venom fraction screening. We obtained a sensible amount of membrane protein using the cell-free assay, that proved to be active when embedded in liposomes. These findings emphasize the quality of the cell-free produced KV1.3 proteoliposomes and the usefulness of a fluorescent probe. This method can benefit the field of channel characterization, as well as provide tools for the development of new inhibitors, so as to reinforce our therapeutic arsenal against autoimmune diseases.

Published
2018

6. Srf controls satellite cell fusion through the maintenance of actin architecture

Author

Ulduz Faradova, Laura Collard, Stéphane Vassilopoulos, Emilie Schol, Jean François Decaux, Alessandra Pincini, Aikaterini Papaefthymiou, Athanassia Sotiropoulos, Chiara Noviello, Voahangy Randrianarison-Huetz, Pascal Maire, Gaëlle Herledan, Institut Cochin (IC UM3 (UMR 8104 / U1016)), Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL), Régulation de la survie cellulaire et des allogreffes, Université Paris-Sud - Paris 11 (UP11)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpital Paul Brousse-Institut National de la Santé et de la Recherche Médicale (INSERM), Department of Chemistry, Canaux calciques , fonctions et pathologies, Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), The Francis Crick Institute [London], Adaptation Biologique et Vieillissement = Biological Adaptation and Ageing (B2A), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Paris Seine (IBPS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Centre de recherche en Myologie – U974 SU-INSERM, Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Institut de Myologie, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), and Université Paris-Sud - Paris 11 (UP11)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Hôpital Paul Brousse-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
0301 basic medicine, Serum Response Factor, Satellite Cells, Skeletal Muscle, Mice, Transgenic, macromolecular substances, Biology, Article, Cell Fusion, 03 medical and health sciences, Myoblast fusion, Mice, 0302 clinical medicine, Cell Movement, Serum response factor, Myocyte, Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Cytoskeleton, Actin, Cells, Cultured, Research Articles, ComputingMilieux_MISCELLANEOUS, Cell Proliferation, Cell fusion, Cell growth, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Cell Biology, Actin cytoskeleton, musculoskeletal system, Actins, Cell biology, 030104 developmental biology, tissues, 030217 neurology & neurosurgery
Abstract

This work describes a crucial role for the transcription factor Srf and F-actin scaffold to drive muscle stem cell fusion in vitro and in vivo and provides evidence of how actin cytoskeleton architecture affects myoblast fusion in vertebrates., Satellite cells (SCs) are adult muscle stem cells that are mobilized when muscle homeostasis is perturbed. Here, we show that serum response factor (Srf) is needed for optimal SC-mediated hypertrophic growth. We identified Srf as a master regulator of SC fusion required in both fusion partners, whereas it was dispensable for SC proliferation and differentiation. We show that SC-specific Srf deletion leads to impaired actin cytoskeleton and report the existence of finger-like actin–based protrusions at fusion sites in vertebrates that were notoriously absent in fusion-defective myoblasts lacking Srf. Restoration of a polymerized actin network by overexpression of an α-actin isoform in Srf mutant SCs rescued their fusion with a control cell in vitro and in vivo and reestablished overload-induced muscle growth. These findings demonstrate the importance of Srf in controlling the organization of actin cytoskeleton and actin-based protrusions for myoblast fusion in mammals and its requirement to achieve efficient hypertrophic myofiber growth.

Published
2018

7. Crucial Role for Immune Complexes but Not FcRn in Immunization against Anti–TNF-α Antibodies after a Single Injection in Mice

Author

Arnoult, Christophe, Brachet, Guillaume, Cadena Castaneda, Diana, Azzopardi, Nicolas, Passot, Christophe, Desvignes, Celine, Paintaud, Gilles, Heuzé-Vourc'H, Nathalie, Watier, Hervé, Gouilleux-Gruart, Valérie, Heuzé-Vourc’h, Nathalie, AGeing and IMagery (AGIM), Université Pierre Mendès France - Grenoble 2 (UPMF)-Université Joseph Fourier - Grenoble 1 (UJF)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS), Génétique, immunothérapie, chimie et cancer (GICC), UMR 7292 CNRS [2012-2017] (GICC UMR 7292 CNRS), Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS), Centre d’Etude des Pathologies Respiratoires (CEPR), UMR 1100 (CEPR), Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM), Développement normal et pathologique des lymphocytes et signalisation, Université de Picardie Jules Verne (UPJV)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Canaux calciques , fonctions et pathologies, Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire d’Immunologie, Centre Hospitalier Universitaire de Reims (CHU Reims), Université de Tours-Centre National de la Recherche Scientifique (CNRS), Pathologies Respiratoires : Protéolyse et Aérosolthérapie, Université de Tours-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire d'Immunologie, Centre Hospitalier Régional Universitaire de Tours (CHRU Tours), Université de Picardie Jules Verne (UPJV)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-École pratique des hautes études (EPHE), and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Joseph Fourier - Grenoble 1 (UJF)-Université Pierre Mendès France - Grenoble 2 (UPMF)

Subjects
0301 basic medicine, musculoskeletal diseases, Immunology, Antigen-Antibody Complex, Receptors, Fc, 03 medical and health sciences, Mice, Immune system, Neonatal Fc receptor, [SDV.SP.MED]Life Sciences [q-bio]/Pharmaceutical sciences/Medication, Adalimumab, Immunology and Allergy, Medicine, Animals, Humans, skin and connective tissue diseases, Mice, Knockout, biology, business.industry, Tumor Necrosis Factor-alpha, Immunogenicity, Histocompatibility Antigens Class I, Infliximab, 3. Good health, 030104 developmental biology, Immunization, biology.protein, [SDV.IMM]Life Sciences [q-bio]/Immunology, Tumor necrosis factor alpha, Antibody, business, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology, medicine.drug
Abstract

The immunogenicity of infliximab and adalimumab is a major concern because patients may develop Abs also called antidrug Abs (ADA), directed against these anti–TNF-α Abs after just a few weeks of treatment. These ADAs can lead to a decrease in biologic concentration, which is associated with lower treatment efficacy. Our aim was to study the involvement of immune complexes and neonatal Fc receptor (FcRn) in the emergence of ADAs in the case of anti–TNF-α Abs. Wild type and FcRn knockout mice were injected once with either infliximab or adalimumab, alone or preincubated with TNF-α. Adalimumab cross-reacts with murine TNF-α whereas infliximab is species specific. When injected alone, only adalimumab elicited a humoral response. By preforming immune complexes with TNF-α, an anti-infliximab response was elicited. Surprisingly, both wild type and FcRn knockout mice were able to mount an immune response against anti–TNF-α Abs, suggesting that immune complexes are a major determinant of this immunization.

Published
2017

8. Imaging Fast Calcium Currents beyond the Limitations of Electrode Techniques

Author

Michel De Waard, Nadia Jaafari, Marco Canepari, CIC - Poitiers, Université de Poitiers-Centre hospitalier universitaire de Poitiers (CHU Poitiers)-Direction Générale de l'Organisation des Soins (DGOS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM), INSERM U836, équipe 3, Canaux calciques, fonctions et pathologies, Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Laboratoire Interdisciplinaire de Physique [Saint Martin d’Hères] (LIPhy), Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS), Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), and Canepari, Marco

Subjects
[SDV]Life Sciences [q-bio], Voltage clamp, Calcium imaging, Intracellular Space, Analytical chemistry, Biophysics, Hippocampus, Calcium Channels, T-Type, Mice, voltage-gated calcium channels, Apical dendrite, medicine, Animals, Electrodes, Membrane potential, neuronal dendrites, Voltage-gated ion channel, Voltage-dependent calcium channel, Chemistry, Optical Imaging, Voltage imaging, Electrophysiological Phenomena, [SDV] Life Sciences [q-bio], medicine.anatomical_structure, Cell Biophysics, Electrode, Calcium, Current (fluid)
Abstract

The current understanding of Ca(2+) channel function is derived from the use of the patch-clamp technique. In particular, the measurement of fast cellular Ca(2+) currents is routinely achieved using whole-cell voltage-clamp recordings. However, this experimental approach is not applicable to the study of local native Ca(2+) channels during physiological changes of membrane potential in complex cells, since the voltage-clamp configuration constrains the membrane potential to a given value. Here, we report for the first time to our knowledge that Ca(2+) currents from individual cells can be quantitatively measured beyond the limitations of the voltage-clamp approach using fast Ca(2+) imaging with low-affinity indicators. The optical measurement of the Ca(2+) current was correlated with the membrane potential, simultaneously measured with a voltage-sensitive dye to investigate the activation of Ca(2+) channels along the apical dendrite of the CA1 hippocampal pyramidal neuron during the back-propagation of an action potential. To validate the method, we analyzed the voltage dependence of high- and low-voltage-gated Ca(2+) channels. In particular, we measured the Ca(2+) current component mediated by T-type channels, and we investigated the mechanisms of recovery from inactivation of these channels. This method is expected to become a reference approach to investigate Ca(2+) channels in their native physiological environment.

Published
2014
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9. Influence ofFCGRTgene polymorphisms on pharmacokinetics of therapeutic antibodies

Author

Gilles Paintaud, Hervé Watier, Sylvaine Renault, Erick Gamelin, Marc Ohresser, Michèle Boisdron-Celle, Christophe Passot, Nicolas Azzopardi, Valérie Gouilleux-Gruart, Christophe Arnoult, Nadine Baroukh, Génétique, immunothérapie, chimie et cancer (GICC), UMR 7292 CNRS [2012-2017] (GICC UMR 7292 CNRS), Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS), Canaux calciques , fonctions et pathologies, Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Cytokines : structure, signalisation et prolifération tumorale, Université d'Angers (UA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire d'Immunologie, Centre Hospitalier Régional Universitaire de Tours (CHRU Tours), Développement normal et pathologique des lymphocytes et signalisation, Université de Picardie Jules Verne (UPJV)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université de Tours-Centre National de la Recherche Scientifique (CNRS), and Université de Picardie Jules Verne (UPJV)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
Male, medicine.drug_class, therapeutic drug monitoring, Immunology, Gene Dosage, Antineoplastic Agents, Receptors, Fc, Biology, Antibodies, Monoclonal, Humanized, Monoclonal antibody, Cohort Studies, Jurkat Cells, Neonatal Fc receptor, [SDV.SP.MED]Life Sciences [q-bio]/Pharmaceutical sciences/Medication, Report, cetuximab, Genotype, TaqMan, medicine, Humans, genetic polymorphism, Immunology and Allergy, Copy-number variation, Promoter Regions, Genetic, DNA Copy Number Variation, Polymorphism, Genetic, neonatal Fc receptor, Histocompatibility Antigens Class I, 3. Good health, FcRn, Variable number tandem repeat, Tandem Repeat Sequences, biology.protein, therapeutic monoclonal antibodies, Female, Antibody, pharmacokinetics, Pharmacogenetics
Abstract

International audience; The neonatal Fc receptor (FcRn) encoded by FCGRT is known to be involved in the pharmacokinetics (PK) of therapeutic monoclonal antibodies (mAbs). Variability in the expression of FCGRT gene and consequently in the FcRn protein level could explain differences in PK observed between patients treated with mAbs. We studied whether the previously described variable number tandem repeat (VNTR) or copy number variation (CNV) of FCGRT are associated with individual variations of PK parameters of cetuximab. VNTR and CNV were assessed on genomic DNA of 198 healthy individuals and of 94 patients treated with the therapeutic mAb. VNTR and CNV were analyzed by allele-specific PCR and duplex real-time PCR with Taqman (®) technology, respectively. The relationship between FCGRT polymorphisms (VNTR and CNV) and PK parameters of patients treated with cetuximab was studied. VNTR3 homozygote patients had a lower cetuximab distribution clearance than VNTR2/VNTR3 and VNTR3/VNTR4 patients (p = 0.021). We observed no affects of VNTR genotype on elimination clearance. One healthy person (0.5%) and 1 patient (1.1%) had 3 copies of FCGRT. The PK parameters of this patient did not differ from those of patients with 2 copies. The FCGRT promoter VNTR may influence mAbs' distribution in the body. CNV of FCGRT cannot be used as a relevant pharmacogenetic marker because of its low frequency.

Published
2013

10. Nuclear life of the voltage-gated Cacnb4 subunit and its role in gene transcription regulation

Author

Yasuo Mori, Michel De Waard, Maud Barbado, Michel Ronjat, Shigeki Kiyonaka, INSERM U836, équipe 3, Canaux calciques, fonctions et pathologies, Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratory of Molecular Biology, Kyoto University [Kyoto]-Graduate School of Engineering, LabEX ICST, Kyoto University-Graduate School of Engineering, and Canepari, Marco

Subjects
Voltage-gated calcium channel, HP1γ, Gene isoform, Transcription, Genetic, Tyrosine 3-Monooxygenase, Neurogenesis, Protein subunit, Active Transport, Cell Nucleus, Biophysics, CACNB4, Biochemistry, Mice, tyrosine hydroxylase, medicine, Animals, Humans, phosphatase 2A, Gene Regulatory Networks, [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Protein Phosphatase 2, Cell Nucleus, Neurons, Regulation of gene expression, biology, HEK 293 cells, Protein phosphatase 2, gene transcription regulation, Molecular biology, Article Addendum, Protein Subunits, Cell nucleus, HEK293 Cells, medicine.anatomical_structure, Gene Expression Regulation, β4 subunit, Mutation, biology.protein, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Calcium Channels, Signal transduction, Signal Transduction, thyroid receptor alpha
Abstract

International audience; The pore-forming subunit of voltage-gated calcium channels is associated to auxiliary subunits among which the cytoplasmic β subunit. The different isoforms of this subunit control both the plasma membrane targeting and the biophysical properties of the channel moiety. In a recent study, we demonstrated that the Cacnb4 (β 4) isoform is at the center of a new signaling pathway that connects neuronal excitability and gene transcription. This mechanism relies on nuclear targeting of β 4 triggered by neuronal electrical stimulation. This re-localization of β 4 is promoted by its interaction with Ppp2r5d a regulatory subunit of PP2A in complex with PP2A itself. The formation, as well as the nuclear translocation, of the β 4/ Ppp2r5d/ PP2A complex is totally impaired by the premature R482X stops mutation of β 4 that has been previously associated with juvenile epilepsy. Taking as a case study the tyrosine hydroxylase gene that is strongly upregulated in brain of lethargic mice, deficient for β 4 expression, we deciphered the molecular steps presiding to this signaling pathway. Here we show that expression of wild-type β 4 in HEK293 cells results in the regulation of several genes, while expression of the mutated β 4 (β 1-481) produces a different set of gene regulation. Several genes regulated by β 4 in HEK293 cells were also regulated upon neuronal differentiation of NG108-15 cells that induces nuclear translocation of β 4 suggesting a link between β 4 nuclear targeting and gene regulation.

Published
2013

11. Antipsychotic and sedative effects of the leaf extract of Crassocephalum bauchiense (Hutch.) Milne-Redh (Asteraceae) in rodents

Author

Théophile Dimo, Elisabeth Ngo Bum, Amadou Dawe, G.T. Ngoupaye, Fleur Clarisse Okomolo Moto, Paul Désiré Djomeni Dzeufiet, Michel De Waard, Bernard Dabole, Neteydji Sidiki, Emmanuel Talla, Germain Sotoing Taiwe, INSERM U836, équipe 3, Canaux calciques, fonctions et pathologies, Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Department of Zoology and Animal Physiology, Faculty of Science-University of Buea-Faculty of Science-University of Buea-Department of Animal Biology and Physiology [university of Yaoundé], University of Yaoundé [Cameroun]-University of Yaoundé [Cameroun], Département des Sciences Biologiques [Univ Ngaoundéré] (UN-FS), Faculté de Sciences [Univ Ngaoundéré] (UN-FS), Université de Ngaoundéré/University of Ngaoundéré [Cameroun] (UN)-Université de Ngaoundéré/University of Ngaoundéré [Cameroun] (UN), Département de Chimie [Univ Ngaoundéré] (UN-FS), Department of Chemistry, Higher Teachers' Training College-University of Maroua (UMa), Higher Teachers' Training College, University of Yaoundé [Cameroun], Department of Animal Biology, Faculty of Science-Université de Dschang, Department of Organic Chemistry, Faculty of Science-University of Yaoundé [Cameroun], Department of Animal Biology and Physiology [university of Yaoundé], Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Smartox Biotechnologies, Université Joseph Fourier - Grenoble 1 (UJF)-FLORALIS-FLORALIS, and Canepari, Marco

Subjects
Flumazenil, Male, Receptor complex, Apomorphine, Asteraceae, Pharmacology, Body Temperature, Rats, Sprague-Dawley, Antipsychotic, Mice, chemistry.chemical_compound, 0302 clinical medicine, Drug Discovery, Hypnotics and Sedatives, Crassocephalum bauchiense, Medicine, African Traditional, Pentobarbital, gamma-Aminobutyric Acid, Behavior, Animal, Chemistry, GABAA receptor, Brain, 3. Good health, Competitive antagonist, 030220 oncology & carcinogenesis, Female, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Antipsychotic Agents, medicine.drug, GABA Agents, Mice, Inbred Strains, Motor Activity, FG-7142, 03 medical and health sciences, medicine, Animals, Inverse agonist, [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Catalepsy, Dose-Response Relationship, Drug, Plant Extracts, Valproic Acid, Sedative, Traditional medicine, Bicuculline, Receptors, GABA-A, Plant Leaves, Disease Models, Animal, Psychotic Disorders, Dopamine Antagonists, Sleep, 030217 neurology & neurosurgery, Phytotherapy
Abstract

International audience; ETHNOPHARMACOLOGICAL RELEVANCE: Crassocephalum bauchiense (Hutch.) Milne-Redh (Asteraceae) has been used as a medicine for the treatment of epilepsy, insomnia, dementia and psychotic disorders in Cameroonian traditional medicine. AIM OF THE STUDY: This study was designed to examine whether the aqueous extract and the alkaloid fraction prepared from the leaves of Crassocephalum bauchiense possess antipsychotic and sedative properties in rodents. MATERIALS AND METHODS: The rectal temperature of mice was recorded with a probe thermometer at a constant depth. Novelty-induced rearing behavior is used to evaluate a central excitatory locomotor behavior in mice. The antipsychotic effects of the extracts were assessed using the apomorphine animal model of psychosis. The catalepsy test was tested based on the ability of the leaves extracts of Crassocephalum bauchiense to alter the duration of akinesia by placing the naive mice with both forelegs over a horizontal bar. The extracts of Crassocephalum bauchiense effects were evaluated on sodium pentobarbital-induced sleeping time. In addition, gamma-aminobutyric acid concentrations in the brain treated mice were also estimated. RESULTS: The aqueous extract and the alkaloid fraction from Crassocephalum bauchiense caused dose-dependent inhibition of novelty-induced rearing behavior, decreased the apomorphine-induced stereotypy and fighting, and had significant fall of the body temperature. The aqueous extract prolonged the sodium pentobarbital sleeping time. This prolongation was not reversed by bicuculline, a light-sensitive competitive antagonist of GABA(A) receptors complex. However, the effect of the aqueous extract on sodium pentobarbital-induced sleeping time was blocked by N-methyl-β-carboline-3-carboxamide, a partial inverse agonist of the benzodiazepine site in the GABA(A) receptor complex and flumazenil, a specific antagonist of the benzodiazepine site in the GABAA receptor complex. In biochemical experiments, the concentration of the inhibitory amino acid, gamma-aminobutyric acid, was significantly increased in the brain of animals treated with the aqueous extract of Crassocephalum bauchiense and sodium valproate. CONCLUSIONS: The results show that the antipsychotic and sedative properties of Crassocephalum bauchiense are possibly mediated via the blockade of dopamine D-2 receptors and GABAergic activation, respectively. However, pharmacological and chemical studies are continuing in order to characterize the mechanism(s) responsible for these neuropharmacological actions and also to identify the active substances present in the extracts of Crassocephalum bauchiense.

Published
2012

12. Evaluation of antinociceptive effects of Crassocephalum bauchiense Hutch (Asteraceae) leaf extract in rodents

Author

Germain Sotoing Taiwe, Amadou Dawe, Elisabeth Ngo Bum, Neteydji Sidiki, Michel De Waard, Théophile Dimo, Emmanuel Talla, Paul Désiré Djomeni Dzeufiet, Richard Marcel Nguimbou, ANTE-INSERM U836, équipe 3, Canaux calciques, fonctions et pathologies, Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Department of Animal Biology and Physiology, Université de Yaoundé I-Université de Yaoundé I-Department of Zoology and Animal Physiology, University of Buea-University of Buea, Département des Sciences Biologiques [Univ Ngaoundéré] (UN-FS), Faculté de Sciences [Univ Ngaoundéré] (UN-FS), Université de Ngaoundéré/University of Ngaoundéré [Cameroun] (UN)-Université de Ngaoundéré/University of Ngaoundéré [Cameroun] (UN), Département de Chimie [Univ Ngaoundéré] (UN-FS), Department of Animal Biology and Physiology, Université de Yaoundé I, Department of Chemistry, Higher Teachers' Training College-University of Maroua (UMa), Ecole Nationale Supérieure des Sciences Agro-Industrielles [Univ Ngaoundéré] (ENSAI), Université de Ngaoundéré/University of Ngaoundéré [Cameroun] (UN), Smartox Biotechnologies, Université Joseph Fourier - Grenoble 1 (UJF)-FLORALIS-Université Joseph Fourier - Grenoble 1 (UJF)-FLORALIS-Département des Sciences Biologiques [Univ Ngaoundéré] (UN-FS), Université de Ngaoundéré/University of Ngaoundéré [Cameroun] (UN)-Université de Ngaoundéré/University of Ngaoundéré [Cameroun] (UN)-Faculté de Sciences [Univ Ngaoundéré] (UN-FS), Smartox Biotechnologies, Floralis, Biopolis, Université de Ngaoundéré, Cameroun, and Canepari, Marco

Subjects
Male, MESH: Analgesics, MESH: Capsaicin, Hot Temperature, Time Factors, Narcotic Antagonists, Administration, Oral, MESH: Solvents, opioidergic pathway, Asteraceae, Pharmacology, MESH: Formaldehyde, MESH: Naloxone, MESH: Plants, Medicinal, MESH: Analgesics, Opioid, MESH: Dose-Response Relationship, Drug, law.invention, Mice, chemistry.chemical_compound, 0302 clinical medicine, MESH: Asteraceae, Oral administration, law, MESH: Behavior, Animal, Drug Discovery, Medicine, MESH: Animals, Crassocephalum bauchiense, [SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Acetic Acid, Opioidergic, Analgesics, 0303 health sciences, Behavior, Animal, Morphine, Naloxone, Alkaloid, MESH: Pain Threshold, MESH: Glutamic Acid, MESH: Motor Activity, 3. Good health, MESH: Plant Leaves, Analgesics, Opioid, Nociception, MESH: Phytotherapy, MESH: Administration, Oral, MESH: Acetic Acid, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Female, MESH: Narcotic Antagonists, MESH: Pain, Pain Threshold, Analgesic, aqueous extract, MESH: Plant Extracts, Glutamic Acid, Pain, MESH: Hot Temperature, Motor Activity, antinociceptive action, 03 medical and health sciences, Alkaloids, MESH: Alkaloids, Formaldehyde, MESH: Water, Animals, [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], MESH: Mice, 030304 developmental biology, alkaloid fraction, Plants, Medicinal, Dose-Response Relationship, Drug, Plant Extracts, business.industry, MESH: Time Factors, Water, MESH: Male, Acute toxicity, Plant Leaves, Disease Models, Animal, MESH: Morphine, chemistry, Capsaicin, Solvents, MESH: Disease Models, Animal, business, Phytotherapy, MESH: Female, 030217 neurology & neurosurgery
Abstract

International audience; ETHNOPHARMACOLOGICAL RELEVANCE: The leaves of Crassocephalum bauchiense have long been used in traditional Cameroonian medicine for the treatment of epilepsy, pain, inflammatory disorders, arthritis and intestinal pain. AIM OF THE STUDY: In this study, we attempted to identify the possible antinociceptive action of the aqueous extract and the alkaloid fraction prepared from the leaves of Crassocephalum baucheiense. MATERIALS AND METHODS: Using acetic acid induced abdominal constrictions, formalin-, capsaisin- and glutamate-induced nociception, and hot plate assay procedures, the antinociceptive effects of the aqueous extract and the alkaloid fraction was assessed after oral administration in mice. Morphine sulfate was used as reference analgesic agent. Mice were submitted to the rota-rod task and open-field test in order to assess any non-specific muscle-relaxant or sedative effects of the extracts of Crassocephalum bauchiense. Male and female Swiss mice were used to assess acute toxicity of these extracts. RESULTS: The aqueous extract and the alkaloid fraction of Crassocephalum bauchiense produced a significant antinociceptive effects in the acetic acid, formalin, glutamate, capsaicin and hot plate tests. These antinociceptive effects of Crassocephalum bauchiense were significantly attenuated by pretreatment with naloxone. The extracts of Crassocephalum bauchiense did not alter the locomotion of animals in the open-field or rotarod tests, which suggest a lack of a central depressant effect. The animals did not exhibit any acute toxicity to the aqueous extract and the alkaloid fraction, so it was not possible to calculate the LD(50). CONCLUSION: The results confirm the popular use of Crassocephalum bauchiense as an antinociceptive, and contribute to the pharmacological knowledge of this species because it was shown that the aqueous extract and the alkaloid fraction of Crassocephalum bauchiense produced dose related antinociception in models of chemical and thermal nociception through mechanisms that involve an interaction with opioidergic pathway.

Published
2012

13. A Cav3.2/Syntaxin-1A Signaling Complex Controls T-type Channel Activity and Low-threshold Exocytosis

Author

Norbert Weiss, Isabelle Bidaud, Sylvaine Huc-Brandt, José M. Fernández-Fernández, Michel De Waard, Maria Karmazinova, Cathy Poillot, Juliane Proft, Katell Fablet, Philippe Lory, Lina Chen, Lubica Lacinova, Gerald W. Zamponi, Shahid Hameed, Arnaud Monteil, INSERM U836, équipe 3, Canaux calciques, fonctions et pathologies, Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Department of Physiology and Pharmacology, University of Calgary-Hotchkiss Brain Institute-University of Calgary-Hotchkiss Brain Institute, Department of Physiology and Pharmacology, University of Calgary-Hotchkiss Brain Institute, Laboratory of Molecular Physiology and Channelopathies, Universitat Pompeu Fabra [Barcelona] (UPF), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratory of Biophysics, Slovak Academy of Science [Bratislava] (SAS)-Institute of Molecular Physiology and Genetics, Department of Clinical Neurosciences, Institut de Génomique Fonctionnelle (IGF), Université de Montpellier (UM)-Université Montpellier 1 (UM1)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Montpellier 2 - Sciences et Techniques (UM2)-Centre National de la Recherche Scientifique (CNRS), Canepari, Marco, and Université Montpellier 1 (UM1)-Université Montpellier 2 - Sciences et Techniques (UM2)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
MESH: Signal Transduction, T-type, Syntaxin 1, N-type calcium channel, Biology, Biochemistry, Cell Line, SK channel, Calcium Channels, T-Type, 03 medical and health sciences, MESH: Calcium Channels, T-Type, 0302 clinical medicine, Neurobiology, MESH: Syntaxin 1, Humans, [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Cav3.2 channel, Molecular Biology, 030304 developmental biology, Calcium signaling, MESH: Exocytosis, 0303 health sciences, MESH: Humans, Voltage-dependent calcium channel, Voltage-gated ion channel, Calcium channel, T-type calcium channel, Cell Biology, MESH: Multiprotein Complexes, neuron, MPC cell, MESH: Cell Line, Cell biology, R-type calcium channel, SNARE, Multiprotein Complexes, SNAP-25, syntaxin-1A, calcium channel, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], exocytosis, 030217 neurology & neurosurgery, Signal Transduction
Abstract

International audience; T-type calcium channels represent a key pathway for Ca(2+) entry near the resting membrane potential. Increasing evidence supports a unique role of these channels in fast and low-threshold exocytosis in an action potential-independent manner, but the underlying molecular mechanisms have remained unknown. Here, we report the existence of a syntaxin-1A/Ca(v)3.2 T-type calcium channel signaling complex that relies on molecular determinants that are distinct from the synaptic protein interaction site (synprint) found in synaptic high voltage-activated calcium channels. This interaction potently modulated Ca(v)3.2 channel activity, by reducing channel availability. Other members of the T-type calcium channel family were also regulated by syntaxin-1A, but to a smaller extent. Overexpression of Ca(v)3.2 channels in MPC 9/3L-AH chromaffin cells induced low-threshold secretion that could be prevented by uncoupling the channels from syntaxin-1A. Altogether, our findings provide compelling evidence for the existence of a syntaxin-1A/T-type Ca(2+) channel signaling complex and provide new insights into the molecular mechanism by which these channels control low-threshold exocytosis.

Published
2012

14. Utilisation de la maurocalcine dans des applications de délivrance intracellulaire de molécules ou de nanoparticules

Author

Bahembera, Eloi Kamugisha, Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM), Canaux calciques, Fonctions et Pathologies, [GIN] Grenoble Institut des Neurosciences (GIN), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA), Université Grenoble Alpes, Michel De Waard, and STAR, ABES

Subjects
Ca2+ nanobiosensors, Maurocalcine, Quantum dots, Hadrucalcine, Nanobiodétecteurs de Ca2+, Nanoparticules, Nanoparticles, Cell penetration, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, Pénétration cellulaire
Abstract

The Maurocalcine (MCa), an excellent cell penetrating peptide (CPP) from the scorpion venom, presents a real interest for the development of applications for the intracellular delivery of experimental or therapeutic molecules or particles. In order for these applications to be developed, the peptide previously underwent a series of optimization processes, such as the suppression of native biological activity and the removal of intramolecular disulfide bonds. In this research, we carried on the optimization by reducing the size of the MCa and then developed one among other possible applications of this peptide which consists of the vectorization of the intracellular Ca2+ nanobiosensor.The native size of MCa (33 residues) is much longer than the size of popular CPPs such as Tat and penetratin (13 and 16 residues, respectively). To reduce the size of the peptide, we truncated the previously designed linear MCa. Twelve new powerful small CPPs were produced, among which the MCaUF1-9 that was the best in cell penetration at low concentrations. Thus, the MCaUF1-9 appears very interesting for applications that require low concentrations. The MCaUF1-9 was further characterized and happened to be more efficient in cell penetration than the Tat, the penetratin, and other small analogous peptides derived from the peptides belonging to the same family as the MCa's, except from the hadrucalcineUF1-11 (HadUF1-11) and the hadrucalcinUF3-11 which showed best performance.As application, the most efficient analogous in cell penetration, the HadUF1-11, was efficiently utilized as a vector for the intracellular delivery of an intracellular Ca2+ nanobiosensor composed of a quantum dot coated with Ca2+ indicator (CaRuby) molecules.Small MCa analogous (derived from MCa or the other peptides of the calcine family) might be the choice CPPs for diverse intracellular delivery applications. In order to confirm this, their extensive characterization is necessary.Key words: Maurocalcine, hadrucalcine, nanoparticles, quantum dots, cell penetration, Ca2+ nanobiosensor., La Maurocalcine (MCa), excellent peptide de pénétration cellulaire (CPP, Cell Penetrating Peptide) issu du venin de scorpion, présente un grand intérêt pour des applications d'administration intracellulaire de molécules ou de nanoparticules expérimentales ou thérapeutiques. Afin de développer ces applications, le peptide a auparavant subi une série d'optimisations, dont la suppression de l'activité biologique native et l'élimination des ponts disulfures intramoléculaires. Dans le présent travail, nous avons poursuivi l'optimisation du peptide par la réduction de la taille. Ensuite, nous avons développé une des applications possibles du peptide consistant en la vectorisation d'un nanobiodétecteur du Ca2+ intracellulaire.La taille de la MCa native (33 résidus) est plus longue que celle des CPPs populaires, telles que la Tat et la pénétratine (13 et 16 résidus, respectivement). Pour réduire la taille du peptide, nous avons procédé à la troncation de la MCa linéaire antérieurement conçue. Douze nouveaux puissants CPPs de taille réduite ont été produits, dont la MCaUF1-9 qui s'est révélée la plus efficace de tous à de faibles concentrations. Le CPP MCaUF1-9 paraît donc très intéressant pour des applications utilisant de faibles concentrations. La caractérisation approfondie de la MCaUF1-9 a montré sa meilleure efficacité en pénétration cellulaire comparativement à Tat, à pénétratine, et à de petits analogues dérivés des peptides de la même famille de calcine, à l'exception de l'hadrucalcineUF1-11 (HadUF1-11) et de l'hadrucalcineUF3-11 qui se sont révélés plus performants.En guise d'application, l'analogue le plus efficace en pénétration cellulaire, l'HadUF1-11, a été efficacement utilisé comme vecteur pour la délivrance intracellulaire d'un nanobiodétecteur du Ca2+ cytoplasmique formé d'un quantum dot recouvert des molécules indicatrices de Ca2+(CaRuby).Les petits analogues de MCa (dérivés de MCa ou des autres peptides de la famille de calcine) pourraient être des CPPs de choix pour divers types d'applications de délivrance intracellulaire. Pour le confirmer (ou l'infirmer) leur caractérisation extensif est nécessaire.Mots clés: Maurocalcine, hadrucalcine, nanoparticules, quantum dots, pénétration cellulaire, nanobiodétecteur de Ca2+.

Published
2015

15. Heterogeneous distribution of TRPC proteins in the embryonic cortex

Author

Christiane Kunert-Keil, Alexandre Bouron, Silke Lucke, Sylvie Boisseau, Canaux calciques , fonctions et pathologies, Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut fur pathophysiology (EMAU Greifswald), Institut für Pathophysiology, Laboratoire de Chimie et Biologie des Métaux (LCBM - UMR 5249), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), ANR SEST08, Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)

Subjects
Telencephalon, Histology, [SDV.NEU.NB]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, Neurogenesis, Biology, TRPC5, TRPC4, TRPC1, Mice, 03 medical and health sciences, Transient receptor potential channel, 0302 clinical medicine, Cortex (anatomy), medicine, Animals, MESH: Animals, Tissue Distribution, RNA, Messenger, MESH: TRPC Cation Channels, MESH: Tissue Distribution, MESH: Mice, Molecular Biology, TRPC, MESH: RNA, Messenger, TRPC Cation Channels, 030304 developmental biology, Cerebral Cortex, 0303 health sciences, MESH: Embryo, Mammalian, MESH: Immunohistochemistry, Cell Biology, Embryo, Mammalian, Immunohistochemistry, MESH: Cerebral Cortex, MESH: Neurogenesis, Cell biology, Medical Laboratory Technology, Corticogenesis, medicine.anatomical_structure, MESH: Telencephalon, 030217 neurology & neurosurgery
Abstract

International audience; The present study was initiated to gain some information about the tissue distribution of transient receptor potential proteins of C-type (TRPC), a family of voltage-independent cation channels, at the beginning of neurogenesis in the telencephalon of embryonic mice. The mRNAs of all known TRPCs (TRPC1-TRPC7) could be found in the cortex at E13. TRPC1, TRPC3 and TRPC5 were the main isoforms, whereas the mRNAs for TRPC2, TRPC4, TRPC6 and TRPC7 were less abundant. The distribution throughout the cortical wall of TRPC1, TRPC3 and TRPC6 was studied by means of immuno-histochemistry. The data collected pointed to a heterogeneous expression of the channels. Three groups were identified. The first one comprises TRPC1, specifically found in the preplate but only in some post-mitotic neurons. It was mainly observed in a subset of cells distinct from the Cajal-Retzius cells. The second group is composed of TRPC3. It was found in non-neuronal cells and also in dividing (5-bromo-2'-deoxyuridine-positive) cells, indicating that TRPC3 is present in precursor cells. The third group contains TRPC6 detected in neuronal and in dividing non-neuronal cells. Double immunostaining experiments showed that TRPC3-positive cells also express TRPC6. Collectively, this report highlights a specific TRPC expression pattern in the immature cortical wall.

Published
2008

16. Biomimetic synthesis of Tramadol

Author

Marcos Marçal Ferreira Queiroz, Germain Sotoing Taiwe, Romain Haudecoeur, Thierry Lomberget, Jean-Luc Wolfender, Marc Le Borgne, Soura Challal, Laurence Marcourt, Ahcène Boumendjel, Florine Lecerf-Schmidt, Basile Pérès, Richard J. Robins, Emerson Ferreira Queiroz, Michel De Waard, Département de Chimie Moléculaire - Chimie Inorganique Redox Biomimétique (DCM - CIRE), Département de Chimie Moléculaire (DCM), Université Joseph Fourier - Grenoble 1 (UJF)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), School of Pharmaceutical Sciences, Université de Lausanne (UNIL)-University of Geneva [Switzerland], INSERM U836, équipe 3, Canaux calciques, fonctions et pathologies, Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Department of Zoology and Animal Physiology, Faculty of Science-University of Buea-Faculty of Science-University of Buea-Department of Animal Biology and Physiology [university of Yaoundé], University of Yaoundé [Cameroun]-University of Yaoundé [Cameroun], Méthodologie de synthèse et molécules bioactives (MSMB), Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon, Biomolécules Cancer et Chimiorésistances (B2C), Université Claude Bernard Lyon 1 (UCBL), School Pharmaceutical Sciences, Phytochemistry and Bioactive Natural Products, Université de Lausanne, Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM), Chimie Et Interdisciplinarité : Synthèse, Analyse, Modélisation (CEISAM), Université de Nantes - UFR des Sciences et des Techniques (UN UFR ST), Université de Nantes (UN)-Université de Nantes (UN)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Département de pharmacochimie moléculaire (DPM), and Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF)

Subjects
Nauclea, Magnetic Resonance Spectroscopy, Stereochemistry, [CHIM.THER]Chemical Sciences/Medicinal Chemistry, Catalysis, Mass Spectrometry, [SDV.SP.MED]Life Sciences [q-bio]/Pharmaceutical sciences/Medication, In vivo, Biomimetics, Biomimetic synthesis, Tramadol/chemical synthesis, Materials Chemistry, medicine, ComputingMilieux_MISCELLANEOUS, Tramadol, Chromatography, High Pressure Liquid, ddc:615, biology, Chemistry, Metals and Alloys, General Chemistry, [SDV.BV.BOT]Life Sciences [q-bio]/Vegetal Biology/Botanics, [SDV.SP]Life Sciences [q-bio]/Pharmaceutical sciences, biology.organism_classification, Surfaces, Coatings and Films, Electronic, Optical and Magnetic Materials, Analgesics, Opioid, Ceramics and Composites, Opioid analgesics, Analgesics, Opioid/chemical synthesis, medicine.drug
Abstract

Tramadol has recently been isolated from the roots and bark of Nauclea latifolia. A plausible biosynthetic pathway has been proposed and the product-precursor relationship has been probed by (13)C position-specific isotope analysis. By further exploring this pathway, we demonstrate that a key step of the proposed pathway can be achieved using mild conditions that mimic in vivo catalysis.

Published
2015

17. Muscle imaging in dominant core myopathies linked or unlinked to the ryanodine receptor 1 gene

Author

Michel Fardeau, Héctor Manuel Barragán-Campos, Nicole Monnier, Norma B. Romero, Svetlana Maugenre, Joël Lunardi, Jean-Paul Leroy, Dirk Fischer, Ana Ferreiro, Jacques Chiras, Muriel Herasse, Pascale Guicheney, Louis Viollet, Physiopathologie et thérapie du muscle strié, Université Pierre et Marie Curie - Paris 6 (UPMC)-IFR14-Institut National de la Santé et de la Recherche Médicale (INSERM), Muskellabor, Rheinische Friedrich-Wilhelms-Universität Bonn, Muskelzentrum/ALS Clinic, Kantonsspital St. Gallen, Institut de Myologie, Université Pierre et Marie Curie - Paris 6 (UPMC)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Service de Neuroradiologie [CHU Pitié-Salpêtrière], CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Service de pédiatrie, Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpital Raymond Poincaré [AP-HP], Service d'anatomie pathologique, Centre Hospitalier Régional Universitaire de Brest (CHRU Brest)-Hôpital Morvan [Brest], Laboratoire de biochimie et génétique moléculaire, CHU Grenoble, Canaux calciques , fonctions et pathologies, Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), DFG, BONFOR, Association Française contre les Myopathies, PHRC., Collaboration, Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Hôpital Morvan [Brest]-Centre Hospitalier Régional Universitaire de Brest (CHRU Brest), and Roux-Buisson, Nathalie

Subjects
medicine.medical_specialty, Statistics as Topic, MESH: Myopathy, Central Core, Chromosome Disorders, [SDV.GEN] Life Sciences [q-bio]/Genetics, [SDV.GEN.GH] Life Sciences [q-bio]/Genetics/Human genetics, Gene mutation, MESH: Ryanodine Receptor Calcium Release Channel, 03 medical and health sciences, 0302 clinical medicine, Internal medicine, medicine, Humans, Genetic Predisposition to Disease, Myopathy, Central Core, Muscle, Skeletal, Myopathy, MESH: Statistics as Topic, RYR1, MESH: Chromosome Disorders, MESH: Muscle, Skeletal, [SDV.GEN]Life Sciences [q-bio]/Genetics, 0303 health sciences, MESH: Humans, biology, Genetic heterogeneity, Ryanodine receptor, Gluteus minimus, 030305 genetics & heredity, MESH: Genetic Predisposition to Disease, Ryanodine Receptor Calcium Release Channel, musculoskeletal system, medicine.disease, biology.organism_classification, Endocrinology, [SDV.GEN.GH]Life Sciences [q-bio]/Genetics/Human genetics, MYH7, Neurology (clinical), medicine.symptom, tissues, 030217 neurology & neurosurgery, Central core disease
Abstract

Objective: To characterize the muscle involvement of patients with central core disease (CCD) caused by mutations in the ryanodine receptor 1 gene ( RYR1 ) and to compare these findings with those from patients with core myopathies unlinked to the RYR1 gene. Methods: We performed a systematic muscular imaging assessment in 11 patients with an RYR1 gene mutation and compared these findings with those of 5 patients from two unrelated families with autosomal dominant core myopathies not linked to RYR1 , ACTA1 , or MYH7 gene loci. Results: All patients with RYR1 CCD had a characteristic pattern with predominant involvement of the gluteus maximus, adductor magnus, sartorius, vastus intermediolateralis, soleus, and lateral gastrocnemius muscles. In contrast, muscle CT in the first family not linked to RYR1 showed predominant affection of the gluteus minimus and hamstring muscles, whereas the second family presented with predominant involvement of the gluteus minimus, vastus intermediolateralis, tibialis anterior, and medial gastrocnemius muscles. In addition to muscle imaging data, we present detailed information on the clinical and pathologic findings of these novel phenotypes of core myopathies not linked to RYR1 . Conclusions: Our data suggest genetic heterogeneity in autosomal dominant core myopathies and the existence of additional unidentified genes.

Published
2006

18. Importance of voltage-dependent inactivation in N-type calcium channel regulation by G-proteins

Author

Norbert Weiss, Michel Ronjat, Michel De Waard, Mohamad A. Mikati, Abir Tadmouri, Canaux calciques , fonctions et pathologies, Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Department of Pediatrics, American University of Beirut Medical Center, Inserm, and Collaboration

Subjects
Time Factors, Physiology, Clinical Biochemistry, Receptors, Opioid, mu, MESH: Rabbits, MESH: Protein Isoforms, N-type calcium channel, µ-opioid receptor, beta subunit, TRPC1, Calcium Channels, N-Type, 0302 clinical medicine, Protein Isoforms, MESH: Animals, Cav2.2 subunit, 0303 health sciences, MESH: Electrophysiology, MESH: Kinetics, Voltage-dependent calcium channel, Chemistry, Electrophysiology, R-type calcium channel, Biochemistry, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Rabbits, MESH: GTP-Binding Proteins, G-protein, MESH: Rats, G protein, MESH: Electric Conductivity, MESH: Receptors, Opioid, mu, Article, 03 medical and health sciences, GTP-binding protein regulators, GTP-Binding Proteins, MESH: Calcium Channels, N-Type, Physiology (medical), Animals, inactivation, 030304 developmental biology, G protein-coupled receptor, MESH: Time Factors, Electric Conductivity, Rats, Kinetics, G-protein coupled receptor, Biophysics, 030217 neurology & neurosurgery
Abstract

International audience; Direct regulation of N-type calcium channels by G-proteins is essential to control neuronal excitability and neurotransmitter release. Binding of the G(betagamma) dimer directly onto the channel is characterized by a marked current inhibition ("ON" effect), whereas the pore opening- and time-dependent dissociation of this complex from the channel produce a characteristic set of biophysical modifications ("OFF" effects). Although G-protein dissociation is linked to channel opening, the contribution of channel inactivation to G-protein regulation has been poorly studied. Here, the role of channel inactivation was assessed by examining time-dependent G-protein de-inhibition of Ca(v)2.2 channels in the presence of various inactivation-altering beta subunit constructs. G-protein activation was produced via mu-opioid receptor activation using the DAMGO agonist. Whereas the "ON" effect of G-protein regulation is independent of the type of beta subunit, the "OFF" effects were critically affected by channel inactivation. Channel inactivation acts as a synergistic factor to channel activation for the speed of G-protein dissociation. However, fast inactivating channels also reduce the temporal window of opportunity for G-protein dissociation, resulting in a reduced extent of current recovery, whereas slow inactivating channels undergo a far more complete recovery from inhibition. Taken together, these results provide novel insights on the role of channel inactivation in N-type channel regulation by G-proteins and contribute to the understanding of the physiological consequence of channel inactivation in the modulation of synaptic activity by G-protein coupled receptors.

Published
2006

19. Transient Loss of Voltage Control of Ca2+ Release in the Presence of Maurocalcine in Skeletal Muscle

Author

László Csernoch, Michel De Waard, Michel Ronjat, Vincent Jacquemond, Sandrine Pouvreau, Bruno Allard, Jean-Marc Sabatier, Physiologie intégrative, cellulaire et moléculaire (PICM), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Department of Physiology, Medical and Health Science Center, University of Debrecen Egyetem [Debrecen], ERT 62, Université de la Méditerranée - Aix-Marseille 2-Ambrilia Biopharma S.A., Canaux calciques , fonctions et pathologies, Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), CNRS, University Claude Bernard Lyon 1, Association Française contre les Myopathies., Collaboration, Canepari, Marco, Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon, and University of Debrecen

Subjects
Patch-Clamp Techniques, [SDV.MHEP.PHY] Life Sciences [q-bio]/Human health and pathology/Tissues and Organs [q-bio.TO], MESH: Muscle Contraction, Muscle Fibers, Skeletal, Calcium in biology, MESH: Ryanodine Receptor Calcium Release Channel, Membrane Potentials, Mice, MESH: Scorpion Venoms, 0302 clinical medicine, MESH: Animals, Elméleti orvostudományok, Membrane potential, MESH: Muscle, Skeletal, 0303 health sciences, MESH: Muscle Fibers, Skeletal, Ryanodine receptor, Orvostudományok, musculoskeletal system, Sarcoplasmic Reticulum, medicine.anatomical_structure, MESH: Calcium, MESH: Sarcoplasmic Reticulum, cardiovascular system, Maurocalcine, medicine.symptom, Ion Channel Gating, Muscle Contraction, Muscle contraction, medicine.medical_specialty, Biophysics, Scorpion Venoms, chemistry.chemical_element, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, In Vitro Techniques, Calcium, 03 medical and health sciences, Internal medicine, MESH: Patch-Clamp Techniques, [SDV.MHEP.PHY]Life Sciences [q-bio]/Human health and pathology/Tissues and Organs [q-bio.TO], medicine, MESH: Membrane Potentials, Animals, Muscle, Skeletal, MESH: Mice, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, 030304 developmental biology, Membranes, Cell Membrane, Skeletal muscle, Ryanodine Receptor Calcium Release Channel, MESH: Ion Channel Gating, Membrane repolarization, Endocrinology, chemistry, 030217 neurology & neurosurgery, MESH: Cell Membrane
Abstract

International audience; In skeletal muscle, sarcoplasmic reticulum (SR) calcium release is controlled by the plasma membrane voltage through interactions between the voltage-sensing dihydropyridine receptor (DHPr) and the ryanodine receptor (RYr) calcium release channel. Maurocalcine (MCa), a scorpion toxin peptide presenting some homology with a segment of a cytoplasmic loop of the DHPr, has been previously shown to strongly affect the activity of the isolated RYr. We injected MCa into mouse skeletal muscle fibers and measured intracellular calcium under voltage-clamp conditions. Voltage-activated calcium transients exhibited similar properties in control and in MCa-injected fibers during the depolarizing pulses, and the voltage dependence of calcium release was similar under the two conditions. However, MCa was responsible for a pronounced sustained phase of Ca(2+) elevation that proceeded for seconds following membrane repolarization, with no concurrent alteration of the membrane current. The magnitude of the underlying uncontrolled extra phase of Ca(2+) release correlated well with the peak calcium release during the pulse. Results suggest that MCa binds to RYr that open on membrane depolarization and that this interaction specifically alters the process of repolarization-induced closure of the channels.

Published
2006

20. Hourglass SiO2 coating increases the performance of planar patch-clamp

Author

Nathalie Picollet-D'hahan, Christophe Arnoult, Thomas Sordel, Frédérique Marcel, Fabien Sauter, Stéphanie Garnier-Raveaud, Francois Chatelain, Michel Vivaudou, Michel De Waard, Catherine Pudda, Laboratoire Biopuces (BIOPUCES), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire Composants intégrés pour le vivant (LCIV), Commissariat à l'énergie atomique et aux énergies alternatives - Laboratoire d'Electronique et de Technologie de l'Information (CEA-LETI), Direction de Recherche Technologique (CEA) (DRT (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Technologique (CEA) (DRT (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Canaux calciques , fonctions et pathologies, Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire de biophysique moléculaire et cellulaire (LBMC), Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'Energie Atomique, Collaboration, Canepari, Marco, and Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)

Subjects
Patch-Clamp Techniques, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, MESH: Cricetinae, 02 engineering and technology, Chemical vapor deposition, Microscopy, Atomic Force, Applied Microbiology and Biotechnology, Ion Channels, Membrane Potentials, Planar, MESH: Cricetulus, Coating, Plasma-enhanced chemical vapor deposition, Cricetinae, Surface roughness, MESH: Animals, PECVD coating, [INFO.INFO-BT]Computer Science [cs]/Biotechnology, MESH: Microscopy, Atomic Force, 0303 health sciences, SEM, Pipette, General Medicine, Silicon Dioxide, 021001 nanoscience & nanotechnology, MESH: Reproducibility of Results, Optoelectronics, AFM, 0210 nano-technology, Plasmids, Biotechnology, Materials science, MESH: Microscopy, Electron, Scanning, Bioengineering, Nanotechnology, CHO Cells, engineering.material, Transfection, Capacitance, MESH: Silicon Dioxide, Cell Line, 03 medical and health sciences, Cricetulus, MESH: CHO Cells, MESH: Plasmids, MESH: Patch-Clamp Techniques, Animals, Humans, MESH: Membrane Potentials, [SDV.IB.BIO]Life Sciences [q-bio]/Bioengineering/Biomaterials, 030304 developmental biology, MESH: Humans, business.industry, MESH: Transfection, Reproducibility of Results, MESH: Cell Line, [SDV.BIO] Life Sciences [q-bio]/Biotechnology, [SDV.IB.BIO] Life Sciences [q-bio]/Bioengineering/Biomaterials, Surface coating, planar patch-clamp, [INFO.INFO-BT] Computer Science [cs]/Biotechnology, BK(Ca) channels, MESH: Ion Channels, Microscopy, Electron, Scanning, engineering, IRK1 channels, business
Abstract

International audience; Obtaining high-throughput electrophysiological recordings is an ongoing challenge in ion channel biophysics and drug discovery. One particular area of development is the replacement of glass pipettes with planar devices in order to increase throughput. However, successful patch-clamp recordings depend on a surface coating which ideally should promote and stabilize giga-seal formation. Here, we present data supporting the use of a structured SiO(2) coating to improve the ability of cells to form a "seal" with a planar patch-clamp substrate. The method is based on a correlation study taking into account structure and size of the pores, surface roughness and chip capacitance. The influence of these parameters on the quality of the seal was assessed. Plasma-enhanced chemical vapour deposition (PECVD) of SiO(2) led to an hourglass structure of the pore and a tighter seal than that offered by a flat, thermal SiO(2) surface. The performance of PECVD chips was validated by recording recombinant potassium channels, BK(Ca), expressed in stable HEK-293 cell lines and in inducible CHO cell lines and low conductance IRK1, and endogenous cationic currents from CHO cells. This multiparametric investigation led to the production of improved chips for planar patch-clamp applications which allow electrophysiological recordings from a wide range of cell lines.

Published
2006

21. Functional properties of ryanodine receptors carrying three amino acid substitutions identified in patients affected by multi-minicore disease and central core disease, expressed in immortalized lymphocytes

Author

Clemens R. Müller, Susan Treves, Francesco Muntoni, Ana Ferreiro, Francesco Zorzato, Nicole Monnier, Heinz Jungbluth, Sylvie Ducreux, Department of Anaesthesia and Research, University Hospital Basel [Basel], Dipartimento di Medicina Sperimentale e Diagnostica, Università degli Studi di Ferrara (UniFE), Physiopathologie et thérapie du muscle strié, Université Pierre et Marie Curie - Paris 6 (UPMC)-IFR14-Institut National de la Santé et de la Recherche Médicale (INSERM), The Dubowitz Neuromuscular Centre, Imperial College London, Canaux calciques , fonctions et pathologies, Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut für Humangenetik, Julius-Maximilians-Universität Würzburg [Wurtzbourg, Allemagne] (JMU), Swiss National Science Foundation, Department of Anaesthesia, Basel University Hospital, Association Française contre les Myopathies, Muscular Dystrophy Campaign, German MD-Net Bundesministerium für Bildung und Forschung., Università degli Studi di Ferrara = University of Ferrara (UniFE), Julius-Maximilians-Universität Würzburg (JMU), and Roux-Buisson, Nathalie

Subjects
Herpesvirus 4, Human, DNA Mutational Analysis, [SDV.GEN] Life Sciences [q-bio]/Genetics, MESH: Thapsigargin, Biochemistry, MESH: Caffeine, MESH: Ryanodine Receptor Calcium Release Channel, MESH: Dose-Response Relationship, Drug, Calcium channel, Central core disease, Lymphoblastoid cell line, Malignant hyperthermia, Multi-minicore disease, Ryanodine receptor, Cresols, 0302 clinical medicine, Lymphocytes, Myopathy, Central Core, MESH: DNA Mutational Analysis, Receptor, Cells, Cultured, chemistry.chemical_classification, 0303 health sciences, MESH: Drug Screening Assays, Antitumor, MESH: Amino Acid Substitution, MESH: Case-Control Studies, 3. Good health, Amino acid, MESH: Cresols, medicine.anatomical_structure, Thapsigargin, Intracellular, MESH: Cells, Cultured, Research Article, medicine.medical_specialty, MESH: Mutation, MESH: Myopathy, Central Core, [SDV.GEN.GH] Life Sciences [q-bio]/Genetics/Human genetics, Biology, MESH: Calcium Signaling, 03 medical and health sciences, Caffeine, Internal medicine, medicine, Humans, Calcium Signaling, Molecular Biology, 030304 developmental biology, [SDV.GEN]Life Sciences [q-bio]/Genetics, MESH: Humans, Dose-Response Relationship, Drug, Skeletal muscle, MESH: Herpesvirus 4, Human, Ryanodine Receptor Calcium Release Channel, Cell Biology, medicine.disease, Molecular biology, Endocrinology, [SDV.GEN.GH]Life Sciences [q-bio]/Genetics/Human genetics, Amino Acid Substitution, chemistry, Case-Control Studies, Mutation, MESH: Lymphocytes, Drug Screening Assays, Antitumor, 030217 neurology & neurosurgery, Homeostasis
Abstract

International audience; More than 80 mutations in the skeletal muscle ryanodine receptor gene have been found to be associated with autosomal dominant forms of malignant hyperthermia and central core disease, and with recessive forms of multi-minicore disease. Studies on the functional effects of pathogenic dominant mutations have shown that they mostly affect intracellular Ca2+ homoeostasis, either by rendering the channel hypersensitive to activation (malignant hyperthermia) or by altering the amount of Ca2+ released subsequent to physiological or pharmacological activation (central core disease). In the present paper, we show, for the first time, data on the functional effect of two recently identified recessive ryanodine receptor 1 amino acid substitutions, P3527S and V4849I, as well as that of R999H, another substitution that was identified in two siblings that were affected by multi-minicore disease. We studied the intracellular Ca2+ homoeostasis of EBV (Epstein-Barr virus)-transformed lymphoblastoid cells from the affected patients, their healthy relatives and control individuals. Our results show that the P3527S substitution in the homozygous state affected the amount of Ca2+ released after pharmacological activation with 4-chloro-m-cresol and caffeine, but did not affect the size of the thapsigargin-sensitive Ca2+ stores. The other substitutions had no effect on either the size of the intracellular Ca2+ stores, or on the amount of Ca2+ released after ryanodine receptor activation; however, both the P3527S and V4849I substitutions had a small but significant effect on the resting Ca2+ concentration.

Published
2006

22. Pharmacological Profiling of Orthochirus scrobiculosus Toxin 1 Analogs with a Trimmed N-Terminal Domain

Author

Heike Wulff, Yingliang Wu, Violeta Visan, Stéphanie Mouhat, Michel De Waard, Stephan Grissmer, Daniel Homerick, Hervé Darbon, Jean-Marc Sabatier, Georgeta Teodorescu, Canepari, Marco, Laboratoire Cellpep S.A., Ingénierie des protéines (IP), Université de la Méditerranée - Aix-Marseille 2-Centre National de la Recherche Scientifique (CNRS), Universität Ulm - Ulm University [Ulm, Allemagne], Department of Medical Pharmacology and Toxicology, University of California [Davis] (UC Davis), University of California (UC)-University of California (UC), College of Life Sciences, Wuhan University [China], Architecture et fonction des macromolécules biologiques (AFMB), Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Canaux calciques , fonctions et pathologies, Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Collaboration, University of California-University of California, and Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA)

Subjects
MESH: Sequence Homology, Amino Acid, Stereochemistry, Molecular Sequence Data, Scorpion Venoms, Peptide, Venom, MESH: Amino Acid Sequence, medicine.disease_cause, Cell Line, Mice, 03 medical and health sciences, MESH: Scorpion Venoms, 0302 clinical medicine, MESH: Nuclear Magnetic Resonance, Biomolecular, medicine, Animals, Humans, Potency, MESH: Animals, Amino Acid Sequence, Nuclear Magnetic Resonance, Biomolecular, MESH: Mice, Structural analog, Toxins, Biological, 030304 developmental biology, Pharmacology, chemistry.chemical_classification, 0303 health sciences, MESH: Humans, MESH: Molecular Sequence Data, Sequence Homology, Amino Acid, MESH: Peptides, Chemistry, Toxin, MESH: Toxins, Biological, Disulfide bond, [SDV.SP]Life Sciences [q-bio]/Pharmaceutical sciences, Orthochirus scrobiculosus, MESH: Cell Line, 3. Good health, [SDV.SP] Life Sciences [q-bio]/Pharmaceutical sciences, Molecular Medicine, Peptides, Selectivity, 030217 neurology & neurosurgery
Abstract

International audience; OSK1, a toxin from the venom of the Asian scorpion Orthochirus scrobiculosus, is a 38-residue peptide cross-linked by three disulfide bridges. A structural analog of OSK1, [Lys(16),Asp(20)]-OSK1, was found previously to be one of the most potent blockers of the voltage-gated K(+) channel Kv1.3 hitherto characterized. Here, we demonstrate that progressive trimming of the N-terminal domain of [Lys(16),Asp(20)]-OSK1 results in marked changes in its pharmacological profile, in terms of both K(+) channel affinity and selectivity. Whereas the affinity to Kv1.1 and Kv1.3 did not change significantly, the affinity to Kv1.2 and K(Ca)3.1 was drastically reduced with the truncations. It is surprising that a striking gain in potency was observed for Kv3.2. In contrast, a truncation of the C-terminal domain, expected to partially disrupt the toxin beta-sheet structure, resulted in a significant decrease or a complete loss of activity on all channel types tested. These data highlight the value of structure-function studies on the extended N-terminal domain of [Lys(16),Asp(20)]-OSK1 to identify new analogs with unique pharmacological properties.

Published
2005

23. How do G proteins directly control neuronal Ca2+ channel function?

Author

Julien Hering, Norbert Weiss, Michel De Waard, Anne Feltz, Canaux calciques , fonctions et pathologies, Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire de Neurobiologie (UMR 8544) (NEURO), École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and École normale supérieure - Paris (ENS-PSL)

Subjects
Models, Molecular, MESH: GTP-Binding Proteins, MESH: Sequence Homology, Amino Acid, G protein, Molecular Sequence Data, MESH: Neurons, MESH: Amino Acid Sequence, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Plasma protein binding, Toxicology, Bioinformatics, 03 medical and health sciences, 0302 clinical medicine, GTP-Binding Proteins, KCNJ5, MESH: Protein Binding, Animals, Humans, MESH: Animals, Amino Acid Sequence, Binding site, Receptor, 030304 developmental biology, G protein-coupled receptor, Neurons, Pharmacology, 0303 health sciences, MESH: Humans, MESH: Molecular Sequence Data, Binding Sites, Sequence Homology, Amino Acid, biology, fungi, MESH: Binding Sites, MESH: Calcium Channels, biology.protein, Biophysics, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Calcium Channels, MESH: Models, Molecular, 030217 neurology & neurosurgery, Function (biology), Protein Binding, Communication channel
Abstract

Ca2+ entry into neuronal cells is modulated by the activation of numerous G-protein-coupled receptors (GPCRs). Much effort has been invested in studying direct G-protein-mediated inhibition of voltage-dependent CaV2 Ca2+ channels. This inhibition occurs through a series of convergent modifications in the biophysical properties of the channels. An integrated view of the structural organization of the Gbetagamma-dimer binding-site pocket within the channel is emerging. In this review, we discuss how variable geometry of the Gbetagamma binding pocket can yield distinct sets of channel inhibition. In addition, we propose specific mechanisms for the regulation of the channel by G proteins that take into account the regulatory input of each Gbetagamma binding element.

Published
2005

24. Firing of hippocampal neurogliaform cells induces suppression of synaptic inhibition

Author

Marco Capogna, Robert Stewart, Marco Canepari, Gengyu Li, Canepari, Marco, Anatomical Neuropharmacology Unit (MRC), University of Oxford, INSERM U836, équipe 3, Canaux calciques, fonctions et pathologies, Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Laboratoire Interdisciplinaire de Physique [Saint Martin d’Hères] (LIPhy), Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Medical Research Council, UK (MRC award U138197106), University of Oxford [Oxford], and Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Male, Patch-Clamp Techniques, Interneuron, Mice, Transgenic, Neurotransmission, Hippocampal formation, Biology, Nitric Oxide, Hippocampus, Synaptic Transmission, Interneurons, Postsynaptic potential, Journal Article, medicine, Animals, Patch clamp, [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Neurotransmitter Agents, Neuronal Plasticity, Research Support, Non-U.S. Gov't, General Neuroscience, Excitatory Postsynaptic Potentials, Neural Inhibition, Articles, Rats, medicine.anatomical_structure, nervous system, Synapses, Excitatory postsynaptic potential, Retrograde signaling, Biophysics, Female, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Neuron, Neuroscience
Abstract

Little is known about how neuron firing recordedin vivoretrogradely influences synaptic strength. We injected the firing of a rat hippocampal neurogliaform cell (NGFC), a widely expressed GABAergic neuron type, detectedin vivoduring theta rhythm, into NGFCs of rat or neuronal nitric oxide synthase (nNOS)-Cre-tdTomato mouse recordedin vitro. We found that the “in vivofiring pattern” produced a transient firing-induced suppression of synaptic inhibition (FSI) evoked by a presynaptic NGFC. Imaging experiments demonstrate that FSI was associated with action potential backpropagation (bAP) and a supralinear increase in dendritic Ca2+. The application of the L-type Ca2+channel antagonist nimodipine blocked FSI. Further pharmacological experiments, such as the application of a nitric oxide-sensitive guanylyl cyclase (NO-sGC) receptor antagonist, a NOS inhibitor, and NO donors, suggested that NO released from postsynaptic cells mediated FSI and likely activated presynaptic receptors to inhibit GABA release. Thein vivofiring pattern modulated the size of unitary EPSPs impinging on NGFCs through FSI and not via a direct effect on excitatory synaptic transmission. Our data demonstrate: (1) retrograde signaling initiated byin vivofiring pattern, (2) interneuron bAPs detected with fast temporal resolution, and (3) a novel role for NO expressed by specific interneuron types.

Published
2014

25. Nauclea latifolia Smith (Rubiaceae) exerts antinociceptive effects in neuropathic pain induced by chronic constriction injury of the sciatic nerve

Author

Germain Sotoing Taiwe, Elisabeth Ngo Bum, Amadou Dawe, Bruno Bonaz, Théophile Dimo, Michel De Waard, Valérie Sinniger, Ahcène Boumendjel, Emmanuel Talla, Canepari, Marco, INSERM U836, équipe 3, Canaux calciques, fonctions et pathologies, Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Department of Zoology and Animal Physiology, University of Buea-University of Buea, Département des Sciences Biologiques [Univ Ngaoundéré] (UN-FS), Faculté de Sciences [Univ Ngaoundéré] (UN-FS), Université de Ngaoundéré/University of Ngaoundéré [Cameroun] (UN)-Université de Ngaoundéré/University of Ngaoundéré [Cameroun] (UN), Département de Chimie [Univ Ngaoundéré] (UN-FS), Department of Animal Biology and Physiology [university of Yaoundé], University of Yaoundé [Cameroun], Department of Chemistry, Higher Teachers' Training College-University of Maroua (UMa), INSERM U836, équipe 8, Stress et interactions neurodigestives, Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM), Département de pharmacochimie moléculaire (DPM), Université Joseph Fourier - Grenoble 1 (UJF)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Smartox Biotechnologies, Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Smartox Biotechnologies, Smartox Biotechnologies-Smartox Biotechnologies, Agence Universitaire de la Francophonie (Bourse Internationale de Formation à la Recherche Doctorale, AUF 2009/2010, Ref.: 1021FR/277/BAC 2009/JGZ/AS), and Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF)

Subjects
Male, Nauclea, MESH: Analgesics, MESH: Neuralgia, MESH: Plant Roots, Rubiaceae, Pharmacology, anti-allodynic, Plant Roots, MESH: Dose-Response Relationship, Drug, Mice, Drug Discovery, Convulsion, Nauclea latifolia, MESH: Animals, Analgesics, Morphine, biology, Alkaloid, anti-hyperalgesic, Sciatic Nerve, 3. Good health, MESH: Sciatic Nerve, Nociception, MESH: Cell Survival, Anesthesia, Hyperalgesia, Neuropathic pain, Female, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Sciatic nerve, medicine.symptom, MESH: Rats, Cell Survival, Catalepsy, traditional medicine, Alkaloids, MESH: Alkaloids, medicine, Animals, [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Rats, Wistar, MESH: Mice, Dose-Response Relationship, Drug, business.industry, MESH: Chronic Disease, MESH: Rats, Wistar, biology.organism_classification, medicine.disease, MESH: Male, Rats, MESH: Rubiaceae, MESH: Morphine, Chronic Disease, Neuralgia, neuropthic pain, business, MESH: Female
Abstract

International audience; ETHNOPHARMACOLOGICAL RELEVANCE: The roots of Nauclea latifolia Smith (Rubiaceae) popularly known as "koumkouma" is used in traditional Cameroonian medicine as neuropathic pain remedy and for the treatment of headache, inflammatory pain and convulsion. This study was conducted to evaluate the antinociceptive effects of the alkaloid fraction isolated from Nauclea latifolia in neuropathic pain induced by chronic constriction injury (CCI) of the sciatic nerve in rat. MATERIALS AND METHODS: Bioactive-guided fractionation of the root extracts of Nauclea latifolia using the Von Frey in a rat model of neuropathic pain (Benett model), afforded a potent anti-hyperalgesic fraction IV. Further fractionation of this fraction was performed by high-performance liquid chromatography (HPLC), yielded eight sub-fractions (F1-F8) which were tested for antinociceptive effects. The alkaloid fraction (F3) collected by HPLC, exhibited potent antinociceptive effects, and the anti-allodynic and anti-hyperalgesic effects of this fraction (8, 16, 40 and 80 mg/kg) were determined using the von Frey and acetone tests respectively in a rat model of neuropathic pain. Rota-rod performance and catalepsy tests were used for the assessment of motor coordination. RESULTS: The alkaloid fraction (80 mg/kg) administered intraperitoneally induced a completely decreased hyperalgesia 90 min post-dosing. In the acetone test, the Nauclea latifolia fraction at 80mg/kg showed its maximal anti-allodynic effects 120 min post-injection. The areas under the curve (AUC) of the anti-allodynic or anti-hyperalgesic effects produced by the alkaloid fraction at 80 mg/kg were significantly (p

Published
2014

26. Combining Ca2+ imaging with -glutamate photorelease. : Ca2+ Imaging and Glutamate Photorelease

Author

David Ogden, Marco Canepari, Michel De Waard, INSERM U836, équipe 3, Canaux calciques, fonctions et pathologies, Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire de physiologie cérébrale (LPC - UMR 8118), Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Joseph Fourier - Grenoble 1 (UJF) - CHU Grenoble - Institut National de la Santé et de la Recherche Médicale (INSERM) - Université Joseph Fourier - Grenoble 1 (UJF) - CHU Grenoble - Institut National de la Santé et de la Recherche Médicale (INSERM), and Université Paris Diderot - Paris 7 (UPD7) - Université Paris Descartes - Paris 5 (UPD5) - Institut National de la Santé et de la Recherche Médicale (INSERM) - Centre National de la Recherche Scientifique (CNRS)

Subjects
Cations, Divalent, Glutamic Acid, General Biochemistry, Genetics and Molecular Biology, Article, Photostimulation, 03 medical and health sciences, 0302 clinical medicine, Postsynaptic potential, Microscopy, [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Physiological Phenomena, 030304 developmental biology, Neurons, 0303 health sciences, Chemistry, Optical Imaging, Glutamate receptor, Brain, 3. Good health, Linear optics, Microscopy, Fluorescence, Synapses, Biophysics, Calcium, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], 030217 neurology & neurosurgery, Ca2 imaging
Abstract

This article describes simple configurations and methods for measuring optical Ca2+ signals in response to photorelease of ʟ-glutamate. This photostimulation allows activation of postsynaptic glutamate receptors without activation of voltage-gated Ca2+ channels, thereby permitting the separation and analysis of different Ca2+ components. We give details of basic microscopy configurations and recommend tools for efficiently illuminating the preparation while preserving the healthy condition of the tissue. We also suggest methodological procedures and discuss linear optics for achieving simultaneous imaging and uncaging using two-photon illumination.

Published
2013

27. Combining Ca2+ and membrane potential imaging in single neurons

Author

Canepari, Marco, Vogt, Kaspar, De Waard, Michel, Zecevic, Dejan, INSERM U836, équipe 3, Canaux calciques, fonctions et pathologies, Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM), Department of Pharmacology and Neurobiology, University of Basel (Unibas), Department of Cellular and Molecular Physiology, and Yale University School of Medicine

Subjects
[SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]
Abstract

International audience; The ability to monitor Ca(2+) signals and membrane potential simultaneously at multiple locations on the same neuron facilitates further progress in our understanding of neuronal function. In particular, this method allows correlation of electrical and chemical signals from multiple sites, including those inaccessible to microelectrodes. This protocol describes a procedure for loading cells with two indicators, a Ca(2+)-sensitive Fura dye and voltage-sensitive JPW1114, together with the equipment required for detecting and imaging the two signals. Potential problems are discussed as well as the capabilities and limitations of the technique.

Published
2013

28. Combining calcium imaging with other optical techniques.: Ca 2+ Imaging and Other Optical Techniques

Author

David Ogden, Marco Canepari, Dejan Zecevic, Michel De Waard, Kaspar E. Vogt, INSERM U836, équipe 3, Canaux calciques, fonctions et pathologies, Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM), Department of Cellular and Molecular Physiology, Yale University School of Medicine, Division of Pharmacology and Neurobiology, University of Basel (Unibas), Laboratoire de physiologie cérébrale (LPC - UMR 8118), Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Joseph Fourier - Grenoble 1 (UJF) - CHU Grenoble - Institut National de la Santé et de la Recherche Médicale (INSERM) - Université Joseph Fourier - Grenoble 1 (UJF) - CHU Grenoble - Institut National de la Santé et de la Recherche Médicale (INSERM), Yale School of Medicine, and Université Paris Diderot - Paris 7 (UPD7) - Université Paris Descartes - Paris 5 (UPD5) - Institut National de la Santé et de la Recherche Médicale (INSERM) - Centre National de la Recherche Scientifique (CNRS)

Subjects
0303 health sciences, Materials science, Staining and Labeling, Optical Imaging, General Biochemistry, Genetics and Molecular Biology, Article, Photostimulation, Electrophysiological Phenomena, Membrane Potentials, 03 medical and health sciences, 0302 clinical medicine, Calcium imaging, Electrode, High spatial resolution, Calcium, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Calcium Signaling, Image resolution, 030217 neurology & neurosurgery, 030304 developmental biology, Biomedical engineering
Abstract

Ca2+ imaging is a commonly used approach for measuring Ca2+ signals at high spatial resolution. The method is often combined with electrode recordings to correlate electrical and chemical signals or to investigate Ca2+ signals following an electrical stimulation. To obtain information on electrical activity at the same spatial resolution, Ca2+ imaging must be combined with membrane potential imaging. Similarly, stimulation of subcellular compartments requires photostimulation. Thus, combining Ca2+ imaging with an additional optical technique facilitates the study of a number of physiological questions. The aim of this article is to introduce some basic principles regarding the combination of Ca2+ imaging with other optical techniques. We discuss the design of the optics, the design of experimental protocols, the optical characteristics of Ca2+ indicators used in combination with an optical probe, and the affinity of the Ca2+ indicator in relation to the type of measurement. This information will enable the reader to devise an optimal strategy for combined optical experiments.

Published
2013

29. Strategies toward structural and functional ‘optimization’ of animal peptide toxins

Author

Andreotti N, Mouhat S, Sabatier J-M, Michael Joanna Publications, Ziadi H, Canepari, Marco, Gènes HLA-DR, Autoanticorps et Microchimérisme dans la Polyarthrite Rhumatoïde et la Sclérodermie (HLA-DR), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), INSERM U836, équipe 3, Canaux calciques, fonctions et pathologies, Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU)

Subjects
chemistry.chemical_classification, 0303 health sciences, Venom, toxins, peptide, activity, drugs, business.industry, activity, 030302 biochemistry & molecular biology, toxins, Peptide, Venom, Computational biology, Biology, Functional optimization, peptide, drugs, Biotechnology, 03 medical and health sciences, Functional diversity, chemistry, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Chemotherapeutic drugs, [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Animal species, business, 030304 developmental biology
Abstract

Background: venoms of a variety of animal species (i.e. scorpions, snakes, spiders, sea anemones, marine cone snails, worms, and insects) are rich sources of bioactive compounds that possess obvious pharmacological, therapeutic and/or biotechnological values. A majority of these compounds are peptides that mainly target enzymes, membrane receptors or ion channels. Aim: In recent years, much efforts of researchers have been focused on characterizing and ‘improving’ the pharmacological profile of some venom toxins, mainly because of their structural/functional diversity and potential value as chemotherapeutic drugs in the treatment of specific human pathologies. Materials and methods: This study reviews the strategies towards optimization of animal peptide toxins. Results: These peptides are most often in a size range that allows their production in vitro by chemical synthesis or genetic engineering. Unfortunately, they rarely display the required characteristics in terms of selectivity, affinity, stability and targeting with regard to the desired application. During the last decade, a number of successful structural approaches or strategies have been developed to improve the intrinsic potential of venom peptides. Using different strategies (reviewed herein), a number of highly potent and selective toxinderived candidate drugs were designed and produced by chemical synthesis. Conclusion: Such strategies proved to be effective as several toxin-based drugs are already marketed while an increasing number of candidate chemotherapeutics are currently being developed in clinical phase. Keywords: Venom, toxins, peptide, activity, drugs

Published
2013

30. Light sources and cameras for standard in vitro membrane potential and high-speed ion imaging

Author

Davies, R., Graham, J., Canepari, M., Fluorescence Imaging, Electrophysiology and Microscopy, Cairn Research Limited, INSERM U836, équipe 3, Canaux calciques, fonctions et pathologies, Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Laboratoire Interdisciplinaire de Physique [Saint Martin d’Hères] (LIPhy), Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS), Labex ICST, Canepari, Marco, and Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Ions, Microscopy, Light, [SDV]Life Sciences [q-bio], Optical Imaging, LED, [INFO.INFO-IM] Computer Science [cs]/Medical Imaging, Calcium imaging, Optical Devices, Voltage imaging, Membrane Potentials, laser, [SDV] Life Sciences [q-bio], CCD cameras, [INFO.INFO-IM]Computer Science [cs]/Medical Imaging, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], CMOS cameras
Abstract

International audience; Membrane potential and fast ion imaging are now standard optical techniques routinely used to record dynamic physiological signals in several preparations in vitro. Although detailed resolution of optical signals can be improved by confocal or two-photon microscopy, high spatial and temporal resolution can be obtained using conventional microscopy and affordable light sources and cameras. Thus, standard wide-field imaging methods are still the most common in research laboratories and can often produce measurements with a signal-to-noise ratio that is superior to other optical approaches. This paper seeks to review the most important instrumentation used in these experiments, with particular reference to recent technological advances. We analyse in detail the optical constraints dictating the type of signals that are obtained with voltage and ion imaging and we discuss how to use this information to choose the optimal apparatus. Then, we discuss the available light sources with specific attention to light emitting diodes and solid state lasers. We then address the current state-of-the-art of available charge coupled device, electron multiplying charge coupled device and complementary metal oxide semiconductor cameras and we analyse the characteristics that need to be taken into account for the choice of optimal detector. Finally, we conclude by discussing prospective future developments that are likely to further improve the quality of the signals expanding the capability of the techniques and opening the gate to novel applications.

Published
2013

31. Economic and simple system to combine single-spot photolysis and whole-field fluorescence imaging

Author

Mark Henson, Jeremy Graham, Marco Canepari, Nadia Jaafari, CIC - Poitiers, Université de Poitiers-Centre hospitalier universitaire de Poitiers (CHU Poitiers)-Direction Générale de l'Organisation des Soins (DGOS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Fluorescence Imaging, Electrophysiology and Microscopy, Cairn Research Limited, INSERM U836, équipe 3, Canaux calciques, fonctions et pathologies, Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Laboratoire Interdisciplinaire de Physique [Saint Martin d’Hères] (LIPhy), Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS), Canepari, Marco, Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire Interdisciplinaire de Physique [Saint Martin d’Hères] (LIPhy), Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS), Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Labex Ion Channels Science and Therapeutics, and Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF)

Subjects
Fluorescence-lifetime imaging microscopy, Optics and Photonics, Rhodopsin, Materials science, Microscope, N-Methylaspartate, Light, [SDV]Life Sciences [q-bio], Biomedical Engineering, [INFO.INFO-IM] Computer Science [cs]/Medical Imaging, Hippocampus, Receptors, N-Methyl-D-Aspartate, Fluorescence, Article, law.invention, Biomaterials, 03 medical and health sciences, Mice, 0302 clinical medicine, Optics, law, Fluorescence microscope, [INFO.INFO-IM]Computer Science [cs]/Medical Imaging, Animals, [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], 030304 developmental biology, Neurons, 0303 health sciences, Photolysis, business.industry, Brain, Laser, Atomic and Molecular Physics, and Optics, Electronic, Optical and Magnetic Materials, Rats, [SDV] Life Sciences [q-bio], Mice, Inbred C57BL, Microscopy, Fluorescence, Optoelectronics, Pinhole (optics), [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Calcium, Luminescence, business, 030217 neurology & neurosurgery, Light-emitting diode
Abstract

International audience; ABSTRACT. In recent years, the use of light emitting diodes (LEDs) has become commonplace in fluorescence microscopy. LEDs are economical and easy to couple to commercial microscopes, and they provide powerful and stable light that can be triggered by transistor-transistor logic pulses in the range of tens of microseconds or shorter. LEDs are usually installed on the epifluorescence port of the microscope to obtain whole-field illumination, which is ideal for fluorescence imaging. In contrast, photolysis or channelrhodopsin stimulation often requires localized illumination, typically achieved using lasers. Here we show that insertion of a long-pass (>411 nm) filter with an appropriately sized pinhole in the epifluorescence pathway, combined with dual UV/visible illumination, can produce efficient whole-field visible illumination and spot UV illumination of 15 to 20 μm. We tested our system by performing calcium imaging experiments combined with L-glutamate or N-methyl-D-aspartic acid (NMDA) photorelease in hippocampal neurons from brain slices or dissociated cultures, demonstrating the ability to obtain local activation of NMDA receptors exclusively in the illuminated spot. The very inexpensive and simple system that we report here will allow many laboratories with limited budgets to run similar experiments in a variety of physiological applications.

Published
2013

32. Two conserved arginine residues from the SK3 potassium channel outer vestibule control selectivity of recognition by scorpion toxins.: Key Residues of SK3 Channel for Selective Toxin Recognition

Author

Feng, Jing, Hu, Youtian, Yi, Hong, Yin, Shijin, Han, Song, Hu, Jun, Chen, Zongyun, Yang, Weishan, Cao, Zhijian, De Waard, Michel, Sabatier, Jean-Marc, Li, Wenxin, Wu, Yingliang, State Key Laboratory of Virology, Wuhan University [China], INSERM U836, équipe 3, Canaux calciques, fonctions et pathologies, Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Smartox Biotechnologies, Université Joseph Fourier - Grenoble 1 (UJF)-FLORALIS-FLORALIS, Gènes HLA-DR, Autoanticorps et Microchimérisme dans la Polyarthrite Rhumatoïde et la Sclérodermie (HLA-DR), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU), Labex ICST, National Basic Research Program of China Grant N°. 2010CB529800, National High Technology Research and Development Program of China : Grant N°. 2012AA020304, National Natural Sciences Foundation of China : Grants Nos. 30530140, 30973636, 31170789, New Century Excellent Talents in Wuhan University by Ministry of Education of China : Grant N° NCET-10-0651, and Hubei Province Natural Sciences Foundation of China : Grant No. 2009CDA076

Subjects
[SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology
Abstract

International audience; Potassium channel functions are often deciphered by using selective and potent scorpion toxins. Among these toxins, only a limited subset is capable of selectively blocking small conductance Ca(2+)-activated K(+) (SK) channels. The structural bases of this selective SK channel recognition remain unclear. In this work, we demonstrate the key role of the electric charges of two conserved arginine residues (Arg-485 and Arg-489) from the SK3 channel outer vestibule in the selective recognition by the SK3-blocking BmP05 toxin. Indeed, individually substituting these residues with histidyl or lysyl (maintaining the positive electric charge partially or fully), although decreasing BmP05 affinity, still preserved the toxin sensitivity profile of the SK3 channel (as evidenced by the lack of recognition by many other types of potassium channel-sensitive charybdotoxin). In contrast, when Arg-485 or Arg-489 of the SK3 channel was mutated to an acidic (Glu) or alcoholic (Ser) amino acid residue, the channel lost its sensitivity to BmP05 and became susceptible to the "new" blocking activity by charybdotoxin. In addition to these SK3 channel basic residues important for sensitivity, two acidic residues, Asp-492 and Asp-518, also located in the SK3 channel outer vestibule, were identified as being critical for toxin affinity. Furthermore, molecular modeling data indicate the existence of a compact SK3 channel turret conformation (like a peptide screener), where the basic rings of Arg-485 and Arg-489 are stabilized by strong ionic interactions with Asp-492 and Asp-518. In conclusion, the unique properties of Arg-485 and Arg-489 (spatial orientations and molecular interactions) in the SK3 channel account for its toxin sensitivity profile.

Published
2013

33. Two conserved arginine residues from the SK3 potassium channel outer vestibule control selectivity of recognition by scorpion toxins.: Key Residues of SK3 Channel for Selective Toxin Recognition

Author

Wenxin Li, Michel De Waard, Shijin Yin, Song Han, Hong Yi, Jean-Marc Sabatier, Weishan Yang, Jing Feng, Yingliang Wu, Youtian Hu, Zongyun Chen, Zhijian Cao, Jun Hu, State Key Laboratory of Virology, Wuhan University [China], INSERM U836, équipe 3, Canaux calciques, fonctions et pathologies, Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Smartox Biotechnologies, Université Joseph Fourier - Grenoble 1 (UJF)-FLORALIS-FLORALIS, Gènes HLA-DR, Autoanticorps et Microchimérisme dans la Polyarthrite Rhumatoïde et la Sclérodermie (HLA-DR), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Labex ICST, National Basic Research Program of China Grant N°. 2010CB529800, National High Technology Research and Development Program of China : Grant N°. 2012AA020304, National Natural Sciences Foundation of China : Grants Nos. 30530140, 30973636, 31170789, New Century Excellent Talents in Wuhan University by Ministry of Education of China : Grant N° NCET-10-0651, Hubei Province Natural Sciences Foundation of China : Grant No. 2009CDA076, and Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU)

Subjects
Models, Molecular, animal structures, Arginine, Charybdotoxin, Small-Conductance Calcium-Activated Potassium Channels, Scorpion Venoms, Biochemistry, complex mixtures, SK channel, 03 medical and health sciences, KCNN4, chemistry.chemical_compound, 0302 clinical medicine, Protein structure, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, Ion channel, 030304 developmental biology, KCNN2, 0303 health sciences, Cell Biology, Potassium channel, HEK293 Cells, chemistry, Protein Structure and Folding, Biophysics, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], 030217 neurology & neurosurgery
Abstract

International audience; Potassium channel functions are often deciphered by using selective and potent scorpion toxins. Among these toxins, only a limited subset is capable of selectively blocking small conductance Ca(2+)-activated K(+) (SK) channels. The structural bases of this selective SK channel recognition remain unclear. In this work, we demonstrate the key role of the electric charges of two conserved arginine residues (Arg-485 and Arg-489) from the SK3 channel outer vestibule in the selective recognition by the SK3-blocking BmP05 toxin. Indeed, individually substituting these residues with histidyl or lysyl (maintaining the positive electric charge partially or fully), although decreasing BmP05 affinity, still preserved the toxin sensitivity profile of the SK3 channel (as evidenced by the lack of recognition by many other types of potassium channel-sensitive charybdotoxin). In contrast, when Arg-485 or Arg-489 of the SK3 channel was mutated to an acidic (Glu) or alcoholic (Ser) amino acid residue, the channel lost its sensitivity to BmP05 and became susceptible to the "new" blocking activity by charybdotoxin. In addition to these SK3 channel basic residues important for sensitivity, two acidic residues, Asp-492 and Asp-518, also located in the SK3 channel outer vestibule, were identified as being critical for toxin affinity. Furthermore, molecular modeling data indicate the existence of a compact SK3 channel turret conformation (like a peptide screener), where the basic rings of Arg-485 and Arg-489 are stabilized by strong ionic interactions with Asp-492 and Asp-518. In conclusion, the unique properties of Arg-485 and Arg-489 (spatial orientations and molecular interactions) in the SK3 channel account for its toxin sensitivity profile.

Published
2013

34. Redox-sensitive stimulation of type-1 ryanodine receptors by the scorpion toxin maurocalcine

Author

Ronjat, Michel, Finkelstein, José Pablo, Llanos, Paola, Montecinos, Luis, Bichraoui, Hicham, De Waard, Michel, Hidalgo, Cecilia, Bull, Ricardo, INSERM U836, équipe 3, Canaux calciques, fonctions et pathologies, Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM), Programa de Fisiología y Biofísica, Instituto de Ciencias Biomedicas-Centro FONDAP de Estudios Moleculares de la Célula - CEMC, and LabEX ICST

Subjects
channel sub-conductance, Ca2+ release, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], ryanodine binding, planar lipid bilayers, sulfhydryl oxidation, Mg2+ inhibition
Abstract

International audience; The scorpion toxin maurocalcine acts as a high affinity agonist of the type-1 ryanodine receptor expressed in skeletal muscle. Here, we investigated the effects of the reducing agent dithiothreitol or the oxidizing reagent thimerosal on type-1 ryanodine receptor stimulation by maurocalcine. Maurocalcine addition to sarcoplasmic reticulum vesicles actively loaded with calcium elicited Ca(2+) release from native vesicles and from vesicles pre-incubated with dithiothreitol; thimerosal addition to native vesicles after Ca(2+) uptake completion prevented this response. Maurocalcine enhanced equilibrium [(3)H]-ryanodine binding to native and to dithiothreitol-treated reticulum vesicles, and increased 5-fold the apparent Ki for Mg(2+) inhibition of [(3)H]-ryanodine binding to native vesicles. Single calcium release channels incorporated in planar lipid bilayers displayed a long-lived open sub-conductance state after maurocalcine addition. The fractional time spent in this sub-conductance state decreased when lowering cytoplasmic [Ca(2+)] from 10μM to 0.1μM or at cytoplasmic [Mg(2+)]≥30μM. At 0.1μM [Ca(2+)], only channels that displayed poor activation by Ca(2+) were readily activated by 5nM maurocalcine; subsequent incubation with thimerosal abolished the sub-conductance state induced by maurocalcine. We interpret these results as an indication that maurocalcine acts as a more effective type-1 ryanodine receptor channel agonist under reducing conditions.

Published
2013

35. Cell penetration properties of a highly efficient mini maurocalcine peptide

Author

Lucie Dardevet, Eloi Bahembera, Céline Tisseyre, Michel De Waard, Jean-Marc Sabatier, Michel Ronjat, INSERM U836, équipe 3, Canaux calciques, fonctions et pathologies, Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM), Gènes HLA-DR, Autoanticorps et Microchimérisme dans la Polyarthrite Rhumatoïde et la Sclérodermie (HLA-DR), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Smartox Biotechnology, Université Joseph Fourier - Grenoble 1 (UJF), Inserm, Grenoble Hospital, Région Rhône-Alpes, Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and Canepari, Marco

Subjects
lcsh:Medicine, lcsh:RS1-441, Pharmaceutical Science, Peptide, F98 cells, Biology, Article, lcsh:Pharmacy and materia medica, 03 medical and health sciences, maurocalcine, hadrucalcin, toxin, cell penetrating peptide, glioma, analogs, In vivo, Drug Discovery, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Site-directed mutagenesis, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, lcsh:R, 030302 biochemistry & molecular biology, Penetration (firestop), chemistry, Biochemistry, Cancer cell, Cell-penetrating peptide, Molecular Medicine, Maurocalcine, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Cysteine
Abstract

International audience; Maurocalcine is a highly potent cell-penetrating peptide isolated from the Tunisian scorpion Maurus palmatus. Many cell-penetrating peptide analogues have been derived from the full-length maurocalcine by internal cysteine substitutions and sequence truncation. Herein we have further characterized the cell-penetrating properties of one such peptide, MCaUF1-9, whose sequence matches that of the hydrophobic face of maurocalcine. This peptide shows very favorable cell-penetration efficacy compared to Tat, penetratin or polyarginine. The peptide appears so specialized in cell penetration that it seems hard to improve bydirected mutagenesis. A comparative analysis of the efficacies of similar peptides isolated from other toxin members of the same family leads to the identification of hadrucalcin's hydrophobic face as an even better CPP. Protonation of the histidine residue at position 6 renders the cell penetration of MCaUF1-9 pH-sensitive. Greater cell penetration at acidic pH suggests that MCaUF1-9 can be used to specifically target cancer cells in vivo where tumor masses grow in more acidic environments.

Published
2013

36. Peptide binding to ochratoxin A mycotoxin: a new approach in conception of biosensors

Author

M. De Waard, Jean-Marc Sabatier, A. Ibn Hadj Hassine, Nicolas Andreotti, Catherine Gonzalez, Ingrid Bazin, IMT - MINES ALES (IMT - MINES ALES), Institut Mines-Télécom [Paris] (IMT), Gènes HLA-DR, Autoanticorps et Microchimérisme dans la Polyarthrite Rhumatoïde et la Sclérodermie (HLA-DR), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), INSERM U836, équipe 3, Canaux calciques, fonctions et pathologies, Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM), Labex ICST, Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU), and Canepari, Marco

Subjects
Ochratoxin A, MESH: Equipment Failure Analysis, Peptide, Peptide binding, Biosensing Techniques, 02 engineering and technology, 01 natural sciences, Chemical synthesis, mycotoxin, chemistry.chemical_compound, Protein Interaction Mapping, Electrochemistry, chemistry.chemical_classification, Mimotope, MESH: Peptides, MESH: Enzyme-Linked Immunosorbent Assay, MESH: Ochratoxins, Equipment Design, General Medicine, 021001 nanoscience & nanotechnology, Ochratoxins, MESH: Reproducibility of Results, Biochemistry, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], 0210 nano-technology, ochratoxin A, MESH: Biosensing Techniques, Biotechnology, Biomedical Engineering, Biophysics, Enzyme-Linked Immunosorbent Assay, MESH: Mycotoxins, Sensitivity and Specificity, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Mycotoxin, peptide-based assay, Ochratoxin, Chromatography, 010401 analytical chemistry, MESH: Protein Interaction Mapping, Reproducibility of Results, Mycotoxins, MESH: Sensitivity and Specificity, 0104 chemical sciences, Equipment Failure Analysis, Enzyme, chemistry, Peptides, peptide binding, MESH: Equipment Design
Abstract

International audience; Ochratoxin A (OTA) is a widespread and abundant natural carcinogenic mycotoxin produced by several species of Aspergillus and Penicillium fungi. Due to the ubiquitous presence of these fungi in food and potential risk for human health, a rapid and sensitive in vitro detection assay is required. Analytical methods for OTA detection/identification are generally based on liquid-liquid extraction, clean-up using an immunoaffinity column (IAC), and identification by reversed-phase high pressure liquid chromatography with fluorescence detection (HPLC-FLD). However, IACs are costly and have a short lifespan. Therefore, an interesting approach would appear to be the design and chemical synthesis of a mimotope peptide simulating mycotoxin-specific antibodies. We have developed a promising alternative method that is based on the use of peptides which are able to bind to specific chemical functions and/or molecular structures. Accordingly, a number of peptides (derived from the structures of major redox proteins) were selected and produced by chemical solid phase syntheses. The ability of such peptides to bind to ochratoxin A was evaluated by HPLC. The peptide NF04 (structurally derived from an oxidoreductase enzyme), which was found to be the sole potently reactive compound among tested molecules, was further evaluated in a peptide-based enzyme-linked immunosorbent assay (peptide-based ELISA), thus confirming its specific interaction with ochratoxin A.

Published
2013

37. Combining Ca2+ imaging with -glutamate photorelease

Author

Canepari, Marco, de Waard, Michel, Ogden, David, Canepari, Marco, INSERM U836, équipe 3, Canaux calciques, fonctions et pathologies, Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire de physiologie cérébrale (LPC - UMR 8118), and Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
[SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]
Abstract

International audience; This article describes simple configurations and methods for measuring optical Ca(2+) signals in response to photorelease of -glutamate. This photostimulation allows activation of postsynaptic glutamate receptors without activation of voltage-gated Ca(2+) channels, thereby permitting the separation and analysis of different Ca(2+) components. We give details of basic microscopy configurations and recommend tools for efficiently illuminating the preparation while preserving the healthy condition of the tissue. We also suggest methodological procedures and discuss linear optics for achieving simultaneous imaging and uncaging using two-photon illumination.

Published
2013

38. Animal Toxins

Author

Sabatier, Jean-Marc, De Waard, Michel, Canepari, Marco, Abba J. Kastin, Ingéniérie des peptides à visée thérapeutique (ERT 62), Université de la Méditerranée - Aix-Marseille 2-Faculté de Médecine Nord, INSERM U836, équipe 3, Canaux calciques, fonctions et pathologies, Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Smartbox Biotechnologies, and Bâtiment Biopolis-Bâtiment Biopolis

Subjects
cell penetrating peptide, drug delivery, ryanodine receptor, animal toxin, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], in vivo imaging, biotechnology
Abstract

Animal toxins are renowned for their ability to regulate ion channel activity and hence cell function. Therefore, they have been investigated as lead compounds for the development of analogues with toxic activity or conversely with therapeutic potential. Besides the pharmacological usefulness of animal toxins, there is a second emerging field in which toxins may soon reveal all their potential. The advent of modern biology witnesses the development of novel biotechnological applications of interest for which toxins present tremendous advantages. In this review, we will illustrate how toxins can be derived to carry on novel functions that make them become tools of choice for diagnostic, imaging and therapeutic applications. Among the examples developed, the case of maurocalcine will be detailed. This toxin, discovered for its activity on the ryanodine receptor, a calcium channel, is now entering a new phase of development for in vivo drug or imaging agent cell delivery.

Published
2013

39. Occurrence of the Synthetic Analgesic Tramadol in an African Medicinal Plant

Author

Bruno Bonaz, Chantal Beney, Ahcène Boumendjel, Romain Haudecoeur, Antoine Depaulis, Florence Souard, Marc Le Borgne, Tanguy Chabrol, Michel De Waard, Richard J. Robins, Elisabeth Ngo Bum, Soura Challal, Thierry Lomberget, Catherine Lavaud, Laurence Marcourt, Germain Sotoing Taiwe, Jean-Luc Wolfender, Emerson Ferreira Queiroz, Valérie Sinniger, Canepari, Marco, Département de pharmacochimie moléculaire (DPM), Université Joseph Fourier - Grenoble 1 (UJF)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), INSERM U836, équipe 3, Canaux calciques, fonctions et pathologies, Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Department of Zoology and Animal Physiology, University of Buea-University of Buea-Department of Animal Biology and Physiology, University of Yaoundé [Cameroun]-Faculty of Science-University of Yaoundé [Cameroun]-Faculty of Science, Département des Sciences Biologiques [Univ Ngaoundéré] (UN-FS), Faculté de Sciences [Univ Ngaoundéré] (UN-FS), Université de Ngaoundéré/University of Ngaoundéré [Cameroun] (UN)-Université de Ngaoundéré/University of Ngaoundéré [Cameroun] (UN), INSERM U836, équipe 9, Dynamique des réseaux synchrones épileptiques, Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM), INSERM U836, équipe 8, Stress et interactions neurodigestives, School of Pharmaceutical Sciences, Université de Lausanne = University of Lausanne (UNIL)-Université de Genève = University of Geneva (UNIGE), Biomolécules Cancer et Chimiorésistances (B2C), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon, Fonctions Normales et Pathologiques de la Barrière Cutanée [Hôpital Edouard Herriot - HCL], Université de Lyon-Université de Lyon-Hôpital Edouard Herriot [CHU - HCL], Hospices Civils de Lyon (HCL)-Hospices Civils de Lyon (HCL), Laboratoire de Pharmacognosie, Université de Reims Champagne-Ardenne (URCA), Chimie Et Interdisciplinarité : Synthèse, Analyse, Modélisation (CEISAM), Université de Nantes - UFR des Sciences et des Techniques (UN UFR ST), Université de Nantes (UN)-Université de Nantes (UN)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Inserm, LabEx Ion Channels, ISPB of Lyon, Agence Française pour la Francophonie, Labex ARCANE, Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF), Université de Lausanne (UNIL)-University of Geneva [Switzerland], and Université de Nantes (UN)-Université de Nantes (UN)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)

Subjects
Moderate to severe, Analgesic effect, Phytochemicals, Analgesic, Pain relief, Pain, Pharmacology, 010402 general chemistry, 01 natural sciences, Catalysis, medicine, [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Medicinal plants, Tramadol, Plants, Medicinal, Plant Extracts, business.industry, 010405 organic chemistry, General Chemistry, General Medicine, Opioid chemistry, 3. Good health, 0104 chemical sciences, Analgesics, Opioid, Morphine, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], business, medicine.drug
Abstract

moderate to severe pain without any known side effects. [8, 9] It was designed by a simplification of the structure of morphine that kept the pharmacophoric elements responsible for the analgesic effect. Herein, we describe the isolation of tramadol from the root bark of N. latifolia, and the different methods used to prove the authenticity of its natural origin. The

Published
2013

40. Combining calcium imaging with other optical techniques

Author

Canepari, Marco, Zecevic, Dejan, Vogt, Kaspar, Ogden, David, de Waard, Michel, Canepari, Marco, INSERM U836, équipe 3, Canaux calciques, fonctions et pathologies, Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM), Department of Cellular and Molecular Physiology, Yale School of Medicine [New Haven, Connecticut] (YSM), Division of Pharmacology and Neurobiology, University of Basel (Unibas), Laboratoire de physiologie cérébrale (LPC - UMR 8118), and Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
[SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]
Abstract

International audience; Ca(2+) imaging is a commonly used approach for measuring Ca(2+) signals at high spatial resolution. The method is often combined with electrode recordings to correlate electrical and chemical signals or to investigate Ca(2+) signals following an electrical stimulation. To obtain information on electrical activity at the same spatial resolution, Ca(2+) imaging must be combined with membrane potential imaging. Similarly, stimulation of subcellular compartments requires photostimulation. Thus, combining Ca(2+) imaging with an additional optical technique facilitates the study of a number of physiological questions. The aim of this article is to introduce some basic principles regarding the combination of Ca(2+) imaging with other optical techniques. We discuss the design of the optics, the design of experimental protocols, the optical characteristics of Ca(2+) indicators used in combination with an optical probe, and the affinity of the Ca(2+) indicator in relation to the type of measurement. This information will enable the reader to devise an optimal strategy for combined optical experiments.

Published
2013

41. Animal Toxins: Toxins in Biotechnology

Author

Michel De Waard, Jean-Marc Sabatier, Ingéniérie des peptides à visée thérapeutique (ERT 62), Université de la Méditerranée - Aix-Marseille 2-Faculté de Médecine Nord, INSERM U836, équipe 3, Canaux calciques, fonctions et pathologies, Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Smartbox Biotechnologies, Bâtiment Biopolis-Bâtiment Biopolis, and Abba J. Kastin

Subjects
0303 health sciences, 03 medical and health sciences, cell penetrating peptide, Chemistry, drug delivery, 030302 biochemistry & molecular biology, ryanodine receptor, animal toxin, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], in vivo imaging, biotechnology, 030304 developmental biology, 3. Good health
Abstract

Animal toxins are renowned for their ability to regulate ion channel activity and hence cell function. Therefore, they have been investigated as lead compounds for the development of analogues with toxic activity or conversely with therapeutic potential. Besides the pharmacological usefulness of animal toxins, there is a second emerging field in which toxins may soon reveal all their potential. The advent of modern biology witnesses the development of novel biotechnological applications of interest for which toxins present tremendous advantages. In this review, we will illustrate how toxins can be derived to carry on novel functions that make them become tools of choice for diagnostic, imaging and therapeutic applications. Among the examples developed, the case of maurocalcine will be detailed. This toxin, discovered for its activity on the ryanodine receptor, a calcium channel, is now entering a new phase of development for in vivo drug or imaging agent cell delivery.

Published
2013

42. Redox-sensitive stimulation of type-1 ryanodine receptors by the scorpion toxin maurocalcine

Author

Michel Ronjat, Cecilia Hidalgo, Luis Montecinos, Hicham Bichraoui, Ricardo Bull, Michel De Waard, Paola Llanos, José Pablo Finkelstein, INSERM U836, équipe 3, Canaux calciques, fonctions et pathologies, Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM), Programa de Fisiología y Biofísica, Instituto de Ciencias Biomedicas-Centro FONDAP de Estudios Moleculares de la Célula - CEMC, and LabEX ICST

Subjects
channel sub-conductance, Physiology, Lipid Bilayers, chemistry.chemical_element, Scorpion Venoms, ryanodine binding, Calcium, Dithiothreitol, Ca2+ release, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Animals, Muscle, Skeletal, Molecular Biology, 030304 developmental biology, RYR1, 0303 health sciences, Scorpion toxin, Ryanodine receptor, Ryanodine, Endoplasmic reticulum, Vesicle, Thimerosal, Cytoplasmic Vesicles, Ryanodine Receptor Calcium Release Channel, Cell Biology, Sarcoplasmic Reticulum, Biochemistry, chemistry, Biophysics, Maurocalcine, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Rabbits, planar lipid bilayers, sulfhydryl oxidation, Mg2+ inhibition, Ion Channel Gating, Oxidation-Reduction, 030217 neurology & neurosurgery, Protein Binding
Abstract

International audience; The scorpion toxin maurocalcine acts as a high affinity agonist of the type-1 ryanodine receptor expressed in skeletal muscle. Here, we investigated the effects of the reducing agent dithiothreitol or the oxidizing reagent thimerosal on type-1 ryanodine receptor stimulation by maurocalcine. Maurocalcine addition to sarcoplasmic reticulum vesicles actively loaded with calcium elicited Ca(2+) release from native vesicles and from vesicles pre-incubated with dithiothreitol; thimerosal addition to native vesicles after Ca(2+) uptake completion prevented this response. Maurocalcine enhanced equilibrium [(3)H]-ryanodine binding to native and to dithiothreitol-treated reticulum vesicles, and increased 5-fold the apparent Ki for Mg(2+) inhibition of [(3)H]-ryanodine binding to native vesicles. Single calcium release channels incorporated in planar lipid bilayers displayed a long-lived open sub-conductance state after maurocalcine addition. The fractional time spent in this sub-conductance state decreased when lowering cytoplasmic [Ca(2+)] from 10μM to 0.1μM or at cytoplasmic [Mg(2+)]≥30μM. At 0.1μM [Ca(2+)], only channels that displayed poor activation by Ca(2+) were readily activated by 5nM maurocalcine; subsequent incubation with thimerosal abolished the sub-conductance state induced by maurocalcine. We interpret these results as an indication that maurocalcine acts as a more effective type-1 ryanodine receptor channel agonist under reducing conditions.

Published
2012

43. How do T-type calcium channels control low-threshold exocytosis?

Author

Michel De Waard, Gerald W. Zamponi, Norbert Weiss, Department of Physiology and Pharmacology, University of Calgary-Hotchkiss Brain Institute, INSERM U836, équipe 3, Canaux calciques, fonctions et pathologies, Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM), N.W is supported by a postdoctoral fellowship from Alberta Innovates Health Solutions (AIHS) and Hotchkiss Brain Institute. G.W.Z is funded by the Canadian Institutes of Health Research, is a Canada Research Chair and AIHS Scientist., and Canepari, Marco

Subjects
T-type, N-type calcium channel, Ribbon synapse, Biology, Bioinformatics, Exocytosis, 03 medical and health sciences, 0302 clinical medicine, chromaffin cell, [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Cav3.2 channel, 030304 developmental biology, Membrane potential, 0303 health sciences, Voltage-dependent calcium channel, STX1A, Calcium channel, T-type calcium channel, neuron, Cell biology, Article Addendum, MPC cell, SNARE, SNAP-25, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], calcium channel, syntaxin-1A, General Agricultural and Biological Sciences, exocytosis, 030217 neurology & neurosurgery
Abstract

International audience; Low-voltage-activated T-type calcium channels act as a major pathway for calcium entry near the resting membrane potential in a wide range of neuronal cell types. Several reports have uncovered an unrecognized feature of T-type channels in the control of vesicular neurotransmitter and hormone release, a process so far thought to be mediated exclusively by high-voltage-activated calcium channels. However, the underlying molecular mechanisms linking T-type calcium channels to vesicular exocytosis have remained enigmatic. In a recent study, we have reported that Ca(v)3.2 T-type channel forms a signaling complex with the neuronal Q-SNARE syntaxin-1A and SNAP-25. This interaction that relies on specific Ca(v)3.2 molecular determinants, not only modulates T-type channel activity, but was also found essential to support low-threshold exocytosis upon Ca(v)3.2 channel expression in MPC 9/3L-AH chromaffin cells. Overall, we have indentified an unrecognized regulation pathway of T-type calcium channels by SNARE proteins, and proposed the first molecular mechanism by which T-type channels could mediate low-threshold exocytosis.

Published
2012

44. Small Cell Penetrating Maurocalcine Peptides

Author

Cathy Poillot, Hicham Bichraoui, Céline Tisseyre, Eloi Bahemberae, Nicolas Andreotti, Jean-Marc Sabatier, Michel Ronjat, Michel De Waard, INSERM U836, équipe 3, Canaux calciques, fonctions et pathologies, Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM), Gènes HLA-DR, Autoanticorps et Microchimérisme dans la Polyarthrite Rhumatoïde et la Sclérodermie (HLA-DR), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Smartox Biotechnologies, Université Joseph Fourier - Grenoble 1 (UJF)-FLORALIS-FLORALIS, This work was supported by grants from Technology pour la Santé (Program TIMOMA2 of the Commissariat à l'Energie Atomique) and from Agence Nationale pour la Recherche PNANO (Programs SYNERGIE and NanoFret)., and Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU)

Subjects
Cell, Scorpion Venoms, MESH: Cricetinae, cell penetrating peptides, CHO Cells, Cell-Penetrating Peptides, Biology, complex mixtures, Biochemistry, 03 medical and health sciences, Cricetulus, MESH: Scorpion Venoms, cell delivery, MESH: Cricetulus, MESH: CHO Cells, Cricetinae, medicine, Animals, MESH: Animals, Molecular Biology, 030304 developmental biology, MESH: Cell-Penetrating Peptides, 0303 health sciences, Scorpion toxin, Peptide chemical synthesis, Chinese hamster ovary cell, 030302 biochemistry & molecular biology, Cell Biology, Molecular biology, humanities, maurocalcine, medicine.anatomical_structure, Drug delivery, Maurocalcine, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], fluorescence, Intracellular
Abstract

International audience; Maurocalcine is the first demonstrated example of an animal toxin peptide with efficient cell penetration properties. Although it is a highly competitive cell-penetrating peptide (CPP), its relatively large size of 33 amino acids and the presence of three internal disulfide bridges may hamper its development for in vitro and in vivo applications. Here, we demonstrate that several efficient CPPs can be derived from maurocalcine by replacing Cys residues by isosteric 2-aminobutyric acid residues and sequence truncation down to peptides of up to 9 residues in length. A surprising finding is that all of the truncated maurocalcine analogues possessed cell penetration properties, indicating that the maurocalcine is a highly specialized CPP. Careful examination of the cell penetration properties of the truncated analogues indicates that several maurocalcine-derived peptides should be of great interest for cell delivery applications where peptide size matters.

Published
2012

45. Small efficient cell-penetrating peptides derived from scorpion toxin maurocalcine

Author

Poillot, Cathy, Bichraoui, Hicham, Tisseyre, Céline, Bahemberae, Eloi, Andreotti, Nicolas, Sabatier, Jean-Marc, Ronjat, Michel, De Waard, Michel, INSERM U836, équipe 3, Canaux calciques, fonctions et pathologies, Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM), Gènes HLA-DR, Autoanticorps et Microchimérisme dans la Polyarthrite Rhumatoïde et la Sclérodermie (HLA-DR), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Smartox Biotechnologies, Université Joseph Fourier - Grenoble 1 (UJF)-FLORALIS-FLORALIS, and This work was supported by grants from Technology pour la Santé (Program TIMOMA2 of the Commissariat à l'Energie Atomique) and from Agence Nationale pour la Recherche PNANO (Programs SYNERGIE and NanoFret).

Subjects
MESH: Cell-Penetrating Peptides, MESH: Scorpion Venoms, cell delivery, MESH: Cricetulus, MESH: CHO Cells, MESH: Cricetinae, cell penetrating peptides, MESH: Animals, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], fluorescence, maurocalcine
Abstract

International audience; Maurocalcine is the first demonstrated example of an animal toxin peptide with efficient cell penetration properties. Although it is a highly competitive cell-penetrating peptide (CPP), its relatively large size of 33 amino acids and the presence of three internal disulfide bridges may hamper its development for in vitro and in vivo applications. Here, we demonstrate that several efficient CPPs can be derived from maurocalcine by replacing Cys residues by isosteric 2-aminobutyric acid residues and sequence truncation down to peptides of up to 9 residues in length. A surprising finding is that all of the truncated maurocalcine analogues possessed cell penetration properties, indicating that the maurocalcine is a highly specialized CPP. Careful examination of the cell penetration properties of the truncated analogues indicates that several maurocalcine-derived peptides should be of great interest for cell delivery applications where peptide size matters.

Published
2012

46. Estrogenic and anti-estrogenic activity of 23 commercial textile dyes

Author

Wissem Mnif, Yosra Haj Hamouda, Ahgleb Bartegi, Aziza Ibn Hadj Hassine, Ingrid Bazin, Miguel López-Ferber, Michel De Waard, Catherine Gonzalez, Canepari, Marco, Laboratoire de Génie de l'Environnement Industriel (LGEI), IMT - MINES ALES (IMT - MINES ALES), Institut Mines-Télécom [Paris] (IMT)-Institut Mines-Télécom [Paris] (IMT), Laboratory of Biochemistry, Institute of Biotechnology, Department of Biology, King Faisal University (KFU), INSERM U836, équipe 3, Canaux calciques, fonctions et pathologies, Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM), and LabEX ICST

Subjects
Textile, Anti estrogenic, Health, Toxicology and Mutagenesis, 02 engineering and technology, 010501 environmental sciences, Endocrine Disruptors, Wastewater, MESH: Estrogens, 01 natural sciences, Genes, Reporter, Yeasts, Bioassay, industrial textile effluent, Coloring Agents, [SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Chemistry, MESH: Yeasts, Textiles, Estrogen Antagonists, General Medicine, 021001 nanoscience & nanotechnology, Pollution, 6. Clean water, MESH: Endocrine Disruptors, MESH: Water Pollutants, Chemical, textile dyes, Environmental chemistry, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Biological Assay, 0210 nano-technology, MESH: Coloring Agents, MESH: Estrogen Antagonists, MESH: Biological Assay, MESH: Waste Water, Competitive binding, [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], estrogenic activity, Effluent, 0105 earth and related environmental sciences, business.industry, MESH: Genes, Reporter, Public Health, Environmental and Occupational Health, Estrogens, Yeast, Tamoxifen, anti-estrogenic activity, 13. Climate action, Benzopurpurine 4B, MESH: Tamoxifen, MESH: Textiles, business, Water Pollutants, Chemical
Abstract

International audience; The presence of dyes in wastewater effluent of textile industry is well documented. In contrast, the endocrine disrupting effects of these dyes and wastewater effluent have been poorly investigated. Herein, we studied twenty-three commercial dyes, usually used in the textile industry, and extracts of blue jean textile wastewater samples were evaluated for their agonistic and antagonistic estrogen activity. Total estrogenic and anti-estrogenic activities were measured using the Yeast Estrogen Screen bioassay (YES) that evaluates estrogen receptor binding-dependent transcriptional and translational activities. The estrogenic potencies of the dyes and wastewater samples were evaluated by dose-response curves and compared to the dose-response curve of 17β-estradiol (E2), the reference compound. The dose-dependent anti-estrogenic activities of the dyes and wastewater samples were normalized to the known antagonistic effect of 4-hydroxytamoxifen (4-OHT) on the induction of the lac Z reporter gene by E2. About half azo textile dyes have anti-estrogenic activity with the most active being Blue HFRL. Most azo dyes however have no or weak estrogenic activity. E2/dye or E2/waste water ER competitive binding assays show activity of Blue HFRL, benzopurpurine 4B, Everzol Navy Blue FBN, direct red 89 BNL 200% and waste water samples indicating a mechanism of action common to E2. Our results indicate that several textile dyes are potential endocrine disrupting agents. The presence of some of these dyes in textile industry wastewater may thus impact the aquatic ecosystem.

Published
2012

47. What does maurocalcine tell us about the process of excitation-contraction coupling?

Author

Michel Ronjat, Michel De Waard, INSERM U836, équipe 3, Canaux calciques, fonctions et pathologies, Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Canepari, Marco

Subjects
0303 health sciences, Voltage-dependent calcium channel, Chemistry, Skeletal muscle, Depolarization, Coupling (electronics), 03 medical and health sciences, Cytosol, 0302 clinical medicine, medicine.anatomical_structure, medicine, Biophysics, Maurocalcine, Myocyte, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], medicine.symptom, 030217 neurology & neurosurgery, 030304 developmental biology, Muscle contraction
Abstract

Cardiac and skeletal muscle contraction is triggered by the arrival of an action potential that locally and transiently depolarizes the plasma membrane. The entire chain of molecular events that link the arrival of the action potential and muscle contraction is called excitation-contraction coupling. This chapter will focus on the way membrane depolarization is sensed and how it triggers a massive increase in the cytosolic Ca2+ concentration. Plasma membrane depolarization is detected by L-type voltage-gated calcium channels whose activation allows Ca2+ entry into muscle cells. Cardiac and skeletal muscle voltage-gated calcium channels differ by their Ca2+ permeability. While cardiac channels lead to an important and rapid Ca2+ influx, skeletal muscle channel activation allows a moderate and rather slow Ca2+ entry (Bean, 1989). In fact, the molecular identity of the pore-forming channel subunit differs in both tissues, cardiac fibers expressing the Cav1.2 isoform while skeletal muscles express the Cav1.1 isoform. Nevertheless, the overall subunit composition of both channels, illustrated in Figure 1, bears interesting similarities.

Published
2012

48. Familial hemiplegic migraine type 1 mutations W1684R and V1696I alter G protein-mediated regulation of Ca(V)2.1 voltage-gated calcium channels

Author

Michel De Waard, Alejandro Sandoval, Maria A. Gandini, Ricardo González-Ramírez, Arn M. J. M. van den Maagdenberg, Edgar Garza-López, Ricardo Felix, Canepari, Marco, Department of Cell Biology, Center for Research and Advanced Studies of the National Polytechnic Institute (CINVESTAV-IPN), School of Medicine FES Istacala, Universidad Nacional Autónoma de México = National Autonomous University of Mexico (UNAM), Department of Molecular Biology and Histocompatibility, Dr. Manuel Gea González General Hospital, Departments of Human Genetics & Neurology, Leiden University Medical Center (LUMC), INSERM U836, équipe 3, Canaux calciques, fonctions et pathologies, Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM), Conacyt (128707), PAPIIT (IN221011), and National Autonomous University of Mexico

Subjects
medicine.medical_specialty, Cerebellar Ataxia, Genotype, G protein, Protein subunit, Migraine Disorders, Receptors, Opioid, mu, Transfection, Cell Line, 03 medical and health sciences, Mice, 0302 clinical medicine, Calcium Channels, N-Type, GTP-Binding Proteins, Internal medicine, medicine, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Animals, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Receptor, Molecular Biology, Familial hemiplegic migraine, Migraine, 030304 developmental biology, 0303 health sciences, Cerebellar ataxia, Voltage-dependent calcium channel, Chemistry, Calcium channel, G-protein regulation, medicine.disease, FHM-1, Migraine with aura, Rats, Endocrinology, HEK293 Cells, Mutation, Molecular Medicine, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], medicine.symptom, Ion Channel Gating, 030217 neurology & neurosurgery, Genome-Wide Association Study
Abstract

International audience; Familial hemiplegic migraine type 1 (FHM-1) is a monogenic form of migraine with aura that is characterized by recurrent attacks of a typical migraine headache with transient hemiparesis during the aura phase. In a subset of patients, additional symptoms such as epilepsy and cerebellar ataxia are part of the clinical phenotype. FHM-1 is caused by missense mutations in the CACNA1A gene that encodes the pore-forming subunit of Ca(V)2.1 voltage-gated Ca(2+) channels. Although the functional effects of an increasing number of FHM-1 mutations have been characterized, knowledge on the influence of most of these mutations on G protein regulation of channel function is lacking. Here, we explored the effects of G protein-dependent modulation on mutations W1684R and V1696I which cause FHM-1 with and without cerebellar ataxia, respectively. Both mutations were introduced into the human Ca(V)2.1α(1) subunit and their functional consequences investigated after heterologous expression in human embryonic kidney 293 (HEK-293) cells using patch-clamp recordings. When co-expressed along with the human μ-opioid receptor, application of the agonist [d-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) inhibited currents through both wild-type (WT) and mutant Ca(V)2.1 channels, which is consistent with the known modulation of these channels by G protein-coupled receptors. Prepulse facilitation, which is a way to characterize the relief of direct voltage-dependent G protein regulation, was reduced by both FHM-1 mutations. Moreover, the kinetic analysis of the onset and decay of facilitation showed that the W1684R and V1696I mutations affect the apparent dissociation and reassociation rates of the Gβγ dimer from the channel complex, suggesting that the G protein-Ca(2+) channel affinity may be altered by the mutations. These biophysical studies may shed new light on the pathophysiology underlying FHM-1.

Published
2012

49. Cacnb4 directly couples electrical activity to gene expression, a process defective in juvenile epilepsy

Author

Tadmouri, Abir, Kiyonaka, Shigeki, Barbado, Maud, Rousset, Matthieu, Fablet, Katell, Sawamura, Seishiro, Bahembera, Eloi, Pernet-Gallay, Karin, Arnoult, Christophe, Miki, Takafumi, Sadoul, Karin, Gory-Faure, Sylvie, Lambrecht, Caroline, Lesage, Florian, Akiyama, Satoshi, Khochbin, Saadi, Baulande, Sylvain, Janssens, Veerle, Andrieux, Annie, Dolmetsch, Ricardo, Ronjat, Michel, Mori, Yasuo, De Waard, Michel, INSERM U836, équipe 3, Canaux calciques, fonctions et pathologies, Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratory of Molecular Biology, Kyoto University [Kyoto]-Graduate School of Engineering, Department of Neurobiology [Stanford], Stanford Medicine, Stanford University-Stanford University, Centre de recherche en Biologie Cellulaire (CRBM), Université Montpellier 1 (UM1)-Université Montpellier 2 - Sciences et Techniques (UM2)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut d'oncologie/développement Albert Bonniot de Grenoble (INSERM U823), Institut National de la Santé et de la Recherche Médicale (INSERM)-EFS-CHU Grenoble-Université Joseph Fourier - Grenoble 1 (UJF), INSERM U836, équipe 1, Physiopathologie du cytosquelette, Protein Phosphorylation and Proteomics Group, Catholic University of Leuven, Institut de pharmacologie moléculaire et cellulaire (IPMC), Centre National de la Recherche Scientifique (CNRS)-Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Côte d'Azur (UCA), PartnerChip, Génopole, Canepari, Marco, Kyoto University-Graduate School of Engineering, Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Université Joseph Fourier - Grenoble 1 (UJF)-CHU Grenoble-EFS-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Nice Sophia Antipolis (1965 - 2019) (UNS), and COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA)

Subjects
HP1γ, Transcription, Genetic, Green Fluorescent Proteins, Active Transport, Cell Nucleus, Biophysics, Epilepsies, Myoclonic, Article, Gene regulation, Electrophysiology, Histones, Mice, HEK293 Cells, Gene Expression Regulation, β4 subunit, Mutation, phosphatase 2A, Animals, Humans, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Calcium Channels, Protein Phosphatase 2, thyroid receptor alpha, Signal Transduction, Thyroid Hormone Receptors alpha
Abstract

International audience; Calcium current through voltage-gated calcium channels (VGCC) controls gene expression. Here, we describe a novel signalling pathway in which the VGCC Cacnb4 subunit directly couples neuronal excitability to transcription. Electrical activity induces Cacnb4 association to Ppp2r5d, a regulatory subunit of PP2A phosphatase, followed by (i) nuclear translocation of Cacnb4/Ppp2r5d/PP2A, (ii) association with the tyrosine hydroxylase (TH) gene promoter through the nuclear transcription factor thyroid hormone receptor alpha (TRα), and (iii) histone binding through association of Cacnb4 with HP1γ concomitantly with Ser(10) histone H3 dephosphorylation by PP2A. This signalling cascade leads to TH gene repression by Cacnb4 and is controlled by the state of interaction between the SH3 and guanylate kinase (GK) modules of Cacnb4. The human R482X CACNB4 mutation, responsible for a form of juvenile myoclonic epilepsy, prevents association with Ppp2r5 and nuclear targeting of the complex by altering Cacnb4 conformation. These findings demonstrate that an intact VGCC subunit acts as a repressor recruiting platform to control neuronal gene expression.

Published
2011

50. Cell-permeable Ln(III) chelate-functionalized InP quantum dots as multimodal imaging agents

Author

Céline Tisseyre, Pascal H. Fries, Graeme J. Stasiuk, Michel De Waard, Emmanuel L. Barbier, Peter Reiss, Marco Giardiello, Sudarsan Tamang, Cathy Poillot, Marinella Mazzanti, Daniel Imbert, Reconnaissance Ionique et Chimie de Coordination (RICC), SYstèmes Moléculaires et nanoMatériaux pour l’Energie et la Santé (SYMMES), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Structures et propriétés d'architectures moléculaire (SPRAM - UMR 5819), Institut Nanosciences et Cryogénie (INAC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), INSERM U836, équipe 3, Canaux calciques, fonctions et pathologies, Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM), Neuro-imagerie fonctionnelle et métabolique (ANTE-INSERM U836, équipe 5), French Research Agency (PNANO-07- 581 NANO-044) CEA Technologies pour la Santé 'TIMO- 582 MA2', Canepari, Marco, AII - Amsterdam institute for Infection and Immunity, APH - Amsterdam Public Health, Global Health, Institut de Chimie du CNRS (INC)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Institut Nanosciences et Cryogénie (INAC), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])

Subjects
Lanthanide, Optical Phenomena, Gadolinium, General Physics and Astronomy, 02 engineering and technology, Photochemistry, 01 natural sciences, Indium, Lanthanoid Series Elements, Cricetinae, magnetic resonance imaging, General Materials Science, Cellular localization, Chelating Agents, General Engineering, 021001 nanoscience & nanotechnology, Fluorescence, 3. Good health, Molecular Imaging, imaging agents, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], fluorescence, 0210 nano-technology, lanthanide complexes, Materials science, Phosphines, Surface Properties, MRI contrast agent, chemistry.chemical_element, Nanotechnology, quantum dots, CHO Cells, Sulfides, 010402 general chemistry, Permeability, Cricetulus, Organometallic Compounds, Animals, Chelation, [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], technology, industry, and agriculture, Biological Transport, equipment and supplies, 0104 chemical sciences, Rats, Spectrometry, Fluorescence, chemistry, Quantum dot, Zinc Compounds, gadolinium, Luminescence
Abstract

Quantum dots (QDs) are ideal scaffolds for the development of multimodal imaging agents, but their application in clinical diagnostics is limited by the toxicity of classical CdSe QDs. A new bimodal MRI/optical nanosized contrast agent with high gadolinium payload has been prepared through direct covalent attachment of up to 80 Gd(III) chelates on fluorescent nontoxic InP/ZnS QDs. It shows a high relaxivity of 900 mM-1 s-1 (13 mM -1s-1 per Gd ion) at 35 MHz (0.81 T) and 298 K, while the bright luminescence of the QDs is preserved. Eu(III) and Tb(III) chelates were also successfully grafted to the InP/ZnS QDs. The absence of energy transfer between the QD and lanthanide emitting centers results in a multicolor system. Using this convenient direct grafting strategy additional targeting ligands can be included on the QD. Here a cell-penetrating peptide has been co-grafted in a one-pot reaction to afford a cell-permeable multimodal multimeric MRI contrast agent that reports cellular localization by fluorescence and provides high relaxivity and increased tissue retention with respect to commercial contrast agents. © 2011 American Chemical Society.

Published
2011

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