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20 results on '"MESH: Rhodamines"'

1. Physiological synaptic activity and recognition memory require astroglial glutamine

Author

Giselle Cheung, Danijela Bataveljic, Josien Visser, Naresh Kumar, Julien Moulard, Glenn Dallérac, Daria Mozheiko, Astrid Rollenhagen, Pascal Ezan, Cédric Mongin, Oana Chever, Alexis-Pierre Bemelmans, Joachim Lübke, Isabelle Leray, Nathalie Rouach, Centre interdisciplinaire de recherche en biologie (CIRB), Labex MemoLife, École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Collège de France (CdF (institution))-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS-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), Ecole doctorale Cerveau Cognition et Comportement [Paris] (ED 158 - 3C), Sorbonne Université (SU), Photophysique et Photochimie Supramoléculaires et Macromoléculaires (PPSM), Institut de Chimie du CNRS (INC)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Ecole Normale Supérieure Paris-Saclay (ENS Paris Saclay), Institut des Neurosciences Paris-Saclay (NeuroPSI), Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Institute of Neuroscience and Medicine [Jülich] (INM-1), Différenciation et communication neuronale et neuroendocrine (DC2N), Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire des Maladies Neurodégénératives - UMR 9199 (LMN), Service MIRCEN (MIRCEN), Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie François JACOB (JACOB), 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 Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie François JACOB (JACOB), 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), Department of Psychiatry, Psychotherapy and Psychosomatics [Aachen], and Rheinisch-Westfälische Technische Hochschule Aachen University (RWTH)

Subjects
Male, MESH: Hippocampus, Intravital Microscopy, MESH: Mice, Transgenic, Glutamine, Science, [SDV.NEU.NB]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, MESH: Neurons, Glutamic Acid, MESH: Stereotaxic Techniques, General Physics and Astronomy, Mice, Transgenic, MESH: Cognition, MESH: Rhodamines, Hippocampus, Synaptic Transmission, General Biochemistry, Genetics and Molecular Biology, Stereotaxic Techniques, Mice, MESH: Intravital Microscopy, Cognition, MESH: Recognition, Psychology, MESH: Synaptic Transmission, Animals, MESH: Animals, MESH: Mice, Fluorescent Dyes, Neurons, MESH: Glutamine, Multidisciplinary, [SDV.NEU.PC]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Psychology and behavior, Rhodamines, MESH: Molecular Probes, [SDV.NEU.SC]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Cognitive Sciences, Recognition, Psychology, MESH: Glutamic Acid, General Chemistry, MESH: Fluorescent Dyes, MESH: Male, MESH: Astrocytes, MESH: Models, Animal, Astrocytes, Molecular Probes, Models, Animal, ddc:500
Abstract

Nature Communications 13, 753 (2022). doi:10.1038/s41467-022-28331-7, Published by Nature Publishing Group UK, [London]

Published
2022
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2. Optimization of Advanced Live-Cell Imaging through Red/Near-Infrared Dye Labeling and Fluorescence Lifetime-Based Strategies

Author

Komuro, Yutaro, Galas, Ludovic, Morozov, Yury, Fahrion, Jennifer, Raoult, Emilie, Lebon, Alexis, Tilot, Amanda, Kikuchi, Shin, Ohno, Nobuhiko, Vaudry, David, Rakic, Pasko, Komuro, Hitoshi, Plate-Forme de Recherche en Imagerie Cellulaire de Haute-Normandie (PRIMACEN), Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Normandie Université (NU)-Institute for Research and Innovation in Biomedicine (IRIB), Normandie Université (NU)-Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-High-tech Research Infrastructures for Life Sciences (HeRacLeS), Normandie Université (NU)-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 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), Département de Pathologie [CHU Caen], Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Normandie Université (NU)-CHU Caen, Normandie Université (NU)-Tumorothèque de Caen Basse-Normandie (TCBN)-Tumorothèque de Caen Basse-Normandie (TCBN), Imagerie et Stratégies Thérapeutiques des pathologies Cérébrales et Tumorales (ISTCT), Normandie Université (NU)-Normandie Université (NU)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Lerner Research Institute [Cleveland, OH, USA], Cleveland Clinic, Lebon, Alexis, Department of Neurosciences [Lerner Research Institute, Cleveland Clinic], Cleveland Clinic-Cleveland Clinic, Department of Neuroscience, Yale University School of Medicine, Yale School of Medicine [New Haven, Connecticut] (YSM), Department of Genomic Medicine [Lerner Research Institute, Cleveland Clinic], Division of Neurobiology and Bioinformatics, National Institute for Physiological Sciences, Aichi 444-8787, Japan, National Institute for Physiological Sciences, School of Medicine [Jichi Medical University, Japan], Jichi Medical University [Tochigi-Ken, Japan], Différenciation et communication neuronale et neuroendocrine (DC2N), Normandie Université (NU)-Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM), Kavli Institute for Neuroscience, Yale School of Medicine, and Yale School of Medicine [New Haven, Connecticut] (YSM)-Yale School of Medicine [New Haven, Connecticut] (YSM)

Subjects
MESH: Photons, [SDV]Life Sciences [q-bio], MESH: Microscopy, Fluorescence, live-cell imaging, law.invention, law, Microscopy, Image Processing, Computer-Assisted, MESH: Microscopy, Confocal, Stimulated emission, Biology (General), MESH: Fluorescein, Spectroscopy, Microscopy, Confocal, STED microscopy, Equipment Design, General Medicine, MESH: Fluorescent Dyes, MESH: Image Processing, Computer-Assisted, Fluorescence, Computer Science Applications, Numerical aperture, STED, [SDV] Life Sciences [q-bio], Chemistry, confocal, Optoelectronics, Fluorescein, MESH: Infrared Rays, FLIM, Photomultiplier, MESH: Cell Line, Tumor, Materials science, QH301-705.5, Infrared Rays, τ separation, MESH: Rhodamines, Article, Catalysis, Inorganic Chemistry, Confocal microscopy, Cell Line, Tumor, fluorescence lifetime, Humans, Physical and Theoretical Chemistry, QD1-999, Molecular Biology, Fluorescent Dyes, Photons, MESH: Humans, Rhodamines, business.industry, MESH: Fluorescence, Organic Chemistry, FLIM-STED, Photon counting, Microscopy, Fluorescence, business, sample preservation, MESH: Equipment Design
Abstract

Fluorescence microscopy is essential for a detailed understanding of cellular processes, however, live-cell preservation during imaging is a matter of debate. In this study, we proposed a guide to optimize advanced light microscopy approaches by reducing light exposure through fluorescence lifetime (τ) exploitation of red/near-infrared dyes. Firstly, we characterized key instrumental elements which revealed that red/near-infrared laser lines with an 86x (Numerical Aperture (NA) = 1.2, water immersion) objective allowed high transmission of fluorescence signals, low irradiance and super-resolution. As a combination of two technologies, i.e., vacuum tubes (e.g., photomultiplier) and semiconductor microelectronics (e.g., avalanche photodiode), type S, X and R of hybrid detectors (HyD-S, HyD-X and HyD-R) were particularly adapted for red/near-infrared photon counting and τ separation. Secondly, we tested and compared lifetime-based imaging including coarse τ separation for confocal microscopy, fitting and phasor plot analysis for fluorescence lifetime microscopy (FLIM), and lifetimes weighting for enhanced stimulated emission depletion (STED) nanoscopy, in light of red/near-infrared multiplexing. Mainly, we showed that the choice of appropriate imaging approach may depend on fluorochrome number, together with their spectral/lifetime characteristics and STED compatibility. Photon-counting mode and sensitivity of HyDs together with phasor plot analysis of fluorescence lifetimes enabled the flexible and fast imaging of multi-labeled living H28 cells. Therefore, a combination of red/near-infrared dyes labeling with lifetime-based strategies offers new perspectives for live-cell imaging by enhancing sample preservation through acquisition time and light exposure reduction.

Published
2021
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3. The Triggering Receptor Expressed on Myeloid cells-1: A new player during acute myocardial infarction

Author

Lemarié Jérémie, Derive Marc, Boufenzer Amir, Gibot Sébastien, Service de Réanimation Médicale [CHRU Nancy], Centre Hospitalier Régional Universitaire de Nancy (CHRU Nancy), Défaillance Cardiovasculaire Aiguë et Chronique (DCAC), Centre Hospitalier Régional Universitaire de Nancy (CHRU Nancy)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lorraine (UL), Faculté de Médecine [Nancy], Université de Lorraine (UL), INOTREM SA, and Université de Lorraine (UL)-Université de Lorraine (UL)

Subjects
MESH: Inflammation, Chemokine, TREM-1, Myeloid, MESH: Membrane Glycoproteins, MESH: Lauric Acids, Inflammation, 030204 cardiovascular system & hematology, MESH: Rhodamines, Peripheral blood mononuclear cell, Proinflammatory cytokine, MESH: Leukocytes, 03 medical and health sciences, 0302 clinical medicine, Immune system, [SDV.MHEP.CSC]Life Sciences [q-bio]/Human health and pathology/Cardiology and cardiovascular system, medicine, Leukocytes, Animals, Humans, Myeloid Cells, MESH: Animals, Receptors, Immunologic, Receptor, MESH: Receptors, Immunologic, 030304 developmental biology, Pharmacology, 0303 health sciences, Innate immune system, Membrane Glycoproteins, MESH: Humans, biology, business.industry, Rhodamines, Lauric Acids, MESH: Triggering Receptor Expressed on Myeloid Cells-1, MESH: Myeloid Cells, Triggering Receptor Expressed on Myeloid Cells-1, Myocardial infarction, MESH: Myocardial Infarction, medicine.anatomical_structure, Immunology, Acute Disease, biology.protein, MESH: Acute Disease, medicine.symptom, business, Oligopeptides
Abstract

International audience; Following myocardial ischemia, an intense activation of the immune system occurs that leads to inflammatory cytokines and chemokines production and to the recruitment of neutrophils and mononuclear cells in the infarcted area. Although pro-inflammatory signals initiate the cellular events necessary for scar formation, excessive and prolonged inflammation promotes deleterious cardiac remodeling and dysfunction. The triggering receptor expressed on myeloid cells-1 (TREM-1) is a highly conserved immune-receptor expressed by neutrophils and monocytes that acts as an amplifier of the innate immune response. Blockade of TREM-1 activation protects from hyper-responsiveness and death during severe infections. Here we review the role of TREM-1 in orchestrating the inflammatory response that follows MI. TREM-1 deletion (Trem-1-/-) or modulation by the use of a short inhibitory peptide (LR12) dampens myocardial inflammation, limits leukocyte recruitment, and improves heart function and survival in mice or pigs. Moreover, the soluble form of TREM-1 (sTREM-1) is found in the plasma of patients suffering from an acute MI and its concentration is an independent predictor of death. This suggests that TREM-1 may constitute a new therapeutic target during acute MI.

Published
2015
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4. Biochemical Characterization of a Caspase-3 Far-red Fluorescent Probe for Non-invasive Optical Imaging of Neuronal Apoptosis

Author

Valérie Jolivel, Alain Fournier, Pierre-Yves Renard, Jérôme Leprince, Béatrice Botia, Heidi Ligeret, Omar Touzani, Hubert Vaudry, Bruno Delest, Anthony Romieu, David Vaudry, Marc Massonneau, Sébastien Arthaud, Pauline Noack, Christophe Portal, Guillaume Clavé, Différenciation et communication neuronale et neuroendocrine (DC2N), Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institute for Research and Innovation in Biomedicine (IRIB), Normandie Université (NU)-Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), International Associated laboratory Samuel de Champlain, Institut National de la Recherche Scientifique [Québec] (INRS)-Université de Rouen Normandie (UNIROUEN), QUantitative Imaging in Drug Development (QUIDD), QUantitative Imaging in Drug Development, Plate-Forme de Recherche en Imagerie Cellulaire de Haute-Normandie (PRIMACEN), Normandie Université (NU)-Normandie Université (NU)-Institute for Research and Innovation in Biomedicine (IRIB), Normandie Université (NU)-Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-High-tech Research Infrastructures for Life Sciences (HeRacLeS), Normandie Université (NU)-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 National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Chimie Organique et Bioorganique : Réactivité et Analyse (COBRA), Institut de Chimie Organique Fine (IRCOF), Normandie Université (NU)-Normandie Université (NU)-Institut national des sciences appliquées Rouen Normandie (INSA Rouen Normandie), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Université de Rouen Normandie (UNIROUEN), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Institut Normand de Chimie Moléculaire Médicinale et Macromoléculaire (INC3M), Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Normandie Université (NU)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Université Le Havre Normandie (ULH), Normandie Université (NU)-Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Institut national des sciences appliquées Rouen Normandie (INSA Rouen Normandie), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Caen Normandie (UNICAEN), Normandie Université (NU)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Hypoxie, physiopathologies cérébrovasculaire et tumorale (CERVOxy), Imagerie et Stratégies Thérapeutiques des pathologies Cérébrales et Tumorales (ISTCT), Normandie Université (NU)-Normandie Université (NU)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Normandie Université (NU)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Institut Armand Frappier (INRS-IAF), Institut National de la Recherche Scientifique [Québec] (INRS)-Réseau International des Instituts Pasteur (RIIP), Normandie Université (NU)-Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Normandie Université (NU)-Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Normand de Chimie Moléculaire Médicinale et Macromoléculaire (INC3M), Institut de Chimie du CNRS (INC)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Université Le Havre Normandie (ULH), Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Institut de Chimie du CNRS (INC)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie Organique Fine (IRCOF), and Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)

Subjects
medicine.medical_treatment, MESH: Neurons, Apoptosis, Proteomics, Imaging, Mice, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, MESH: Caspase 3, MESH: Animals, Caspase, Cells, Cultured, Neurons, biology, Caspase 3, Optical Imaging, Brain, General Medicine, Carbocyanines, MESH: Fluorescent Dyes, 3. Good health, Cell biology, Stroke, Caspase-3, [SDV.TOX]Life Sciences [q-bio]/Toxicology, MESH: Oligopeptides, Pituitary Adenylate Cyclase-Activating Polypeptide, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], MESH: Carbocyanines, Oligopeptides, MESH: Cells, Cultured, Programmed cell death, MESH: Rats, MESH: Rhodamines, Cellular and Molecular Neuroscience, MESH: Brain, In vivo, MESH: Mice, Inbred C57BL, medicine, Animals, [CHIM]Chemical Sciences, Rats, Wistar, MESH: Mice, Fluorescent Dyes, MESH: Optical Imaging, Protease, Rhodamines, MESH: Apoptosis, MESH: Pituitary Adenylate Cyclase-Activating Polypeptide, MESH: Rats, Wistar, Molecular biology, In vitro, Rats, Mice, Inbred C57BL, biology.protein, [SDV.SP.PHARMA]Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology
Abstract

International audience; Apoptosis is a regulated process, leading to cell death, which is involved in several pathologies including neurodegenerative diseases and stroke. Caspase-3 is a key enzyme of the apoptotic pathway and is considered as a major target for the treatment of abnormal cell death. Sensitive and non-invasive methods to monitor caspase-3 activity in cells and in the brain of living animals are needed to test the efficiency of novel therapeutic strategies. In the present study, we have biochemically characterized a caspase-3 far-red fluorescent probe, QCASP3.2, that can be used to detect apoptosis in vivo. The specificity of cleavage of QCASP3.2 was demonstrated using recombinant caspases and protease inhibitors. The functionality of the probe was also established in cere-bellar neurons cultured in apoptotic conditions. QCASP3.2 did not exhibit any toxicity and appeared to accurately reflect the induction and inhibition of caspase activity by H 2 O 2 and PACAP, respectively, both in cell lysates and in cultured neurons. Finally, intravenous injection of the probe after cere-bral ischemia revealed activation of caspase-3 in the infarcted hemisphere. Thus, the present study demonstrates that QCASP3.2 is a suitable probe to monitor apoptosis both in vitro and in vivo and illustrates some of the possible applications of this caspase-3 fluorescent probe.

Published
2014
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5. Protective effect of intravitreal injection of vasoactive intestinal peptide-loaded liposomes on experimental autoimmune uveoretinitis

Author

Duron, Emmanuelle, Vidal, Jean-Sébastien, Funalot, Benoît, Viollet, Cécile, Seux, Marie-Laure, Treluyer, Jean-Marc, Epelbaum, Jacques, Bouc, Yves Le, Hanon, Olivier, Camelo, Serge, Lajavardi, Laure, Bochot, Amélie, Goldenberg, Brigitte, Naud, Marie-Christine, Brunel, Nadège, Lescure, Bernadette, Klein, Christophe, Fattal, Elias, Behar-Cohen, Francine, De Kozak, Yvonne, Centre de Mémoire de Ressources et de Recherche [Paris Broca], AP-HP - Hôpital Broca [Paris], CHU Saint-Antoine [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Service de Génétique Médicale [CHU Necker], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-CHU Necker - Enfants Malades [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Centre de Psychiatrie et Neurosciences (U894), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), CIC-0901, Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpital Cochin [AP-HP], Neurobiologie de la Croissance et de la Senescence, Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Dpt Gériatrie [CHU Broca], AP-HP - Hôpital Cochin Broca Hôtel Dieu [Paris], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Unité de Recherche Clinique, Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpital Lariboisière-Fernand-Widal [APHP], Institut Galien Paris-Sud (IGPS), Université Paris-Sud - Paris 11 (UP11)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Physiopathologie des Maladies Oculaires : Innovations Therapeutiques, Université Pierre et Marie Curie - Paris 6 (UPMC)-IFR58-Institut National de la Santé et de la Recherche Médicale (INSERM), Cancer, Inflammation, Hormones, Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Université Pierre et Marie Curie - Paris 6 (UPMC)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de Recherche des Cordeliers (CRC (UMR_S_1138 / U1138)), École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Service de neurologie [Saint-Antoine], Université Pierre et Marie Curie - Paris 6 (UPMC)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-CHU Saint-Antoine [APHP], CHU Necker - Enfants Malades [AP-HP]-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP), Institut de psychiatrie et neurosciences (U894 / UMS 1266), Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-CHU Cochin [AP-HP]-CHU Necker - Enfants Malades [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Hôpital Lariboisière, Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Université Pierre et Marie Curie - Paris 6 (UPMC)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Physico-chimie, pharmacotechnie, biopharmacie (PCPB), École pratique des hautes études (EPHE)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Université Pierre et Marie Curie - Paris 6 (UPMC)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-CHU Saint-Antoine [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Sorbonne Université (SU), Institut National de la Santé et de la Recherche Médicale (INSERM)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), and École pratique des hautes études (EPHE)

Subjects
MESH: Cytokines, MESH: Rats, Elderly participants, [SDV]Life Sciences [q-bio], MESH: Vasoactive Intestinal Peptide, MESH: Vitreous Body, MESH: Macrophages, MESH: Lymph Nodes, MESH: Rhodamines, Atrial fibrillation, MESH: Male, Insulin-like growth factor binding protein-3, MESH: T-Lymphocytes, MESH: Arrestin, MESH: Autoimmune Diseases, MESH: Cell Proliferation, MESH: Uveitis, MESH: Retinitis, MESH: Injections, MESH: Liposomes, MESH: Animals, MESH: Rats, Inbred Lew, Insulin-like growth factor-I, MESH: Disease Models, Animal
Abstract

International audience; Insulin-like growth factor I (IGF-I) and insulin-like growth factor binding protein 3 (IGFBP-3) are involved in oxidative stress and atherosclerosis; however, the relationship between the IGF-I system and atrial fibrillation (AF) is not known. The objective of this analysis was to assess, the relationship between IGF-I and IGFBP-3 serum levels and AF among elderly participants.

Published
2014
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6. Attenuation of Responses to Endotoxin by the Triggering Receptor Expressed on Myeloid Cells-1 Inhibitor LR12 in Nonhuman Primate

Author

Amir Boufenzer, Sébastien Gibot, Marc Derive, Défaillance Cardiovasculaire Aiguë et Chronique (DCAC), Centre Hospitalier Régional Universitaire de Nancy (CHRU Nancy)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lorraine (UL), Service de Réanimation Médicale [CHRU Nancy], and Centre Hospitalier Régional Universitaire de Nancy (CHRU Nancy)

Subjects
Male, Myeloid, Inflammatory response, MESH: Random Allocation, MESH: Shock, Septic, MESH: Lauric Acids, Blood Pressure, MESH: Rhodamines, MESH: Leukopenia, Inhibitory postsynaptic potential, 03 medical and health sciences, Random Allocation, 0302 clinical medicine, [SDV.MHEP.CSC]Life Sciences [q-bio]/Human health and pathology/Cardiology and cardiovascular system, MESH: Endotoxins, medicine, Animals, Myeloid Cells, MESH: Animals, Receptor, 030304 developmental biology, 0303 health sciences, business.industry, Rhodamines, Lauric Acids, Leukopenia, MESH: Blood Pressure, Shock, Septic, MESH: Injections, Intravenous, MESH: Myeloid Cells, Nonhuman primate, MESH: Male, 3. Good health, Endotoxins, Disease Models, Animal, Macaca fascicularis, Anesthesiology and Pain Medicine, medicine.anatomical_structure, MESH: Macaca fascicularis, 030220 oncology & carcinogenesis, Immunology, Myeloid cells, Injections, Intravenous, MESH: Disease Models, Animal, business, Oligopeptides
Abstract

Background: The triggering receptor expressed on myeloid cells-1 is an immunoreceptor that amplifies the inflammatory response mediated by toll-like receptors engagement. Triggering receptor expressed on myeloid cells-1 inhibitory peptides such LR12 have been shown to prevent hyperresponsiveness and death in several experimental models of septic shock. Methods: Twelve adult male Cynomolgus (Macaca fascicularis) monkeys exposed to an intravenous bolus of endotoxin (10 μg/kg) were randomized to receive LR12 or placebo (n = 6 per group) as an initial intravenous bolus followed by an 8-h continuous intravenous infusion. An additional group of four only received vehicle infusion. Vital signs were monitored for 8 h. Blood was sampled at H0, 1, 2, 4, and 8 for analysis of clinical chemistries, leukocyte count, coagulation parameters, and cytokine plasma concentration. Results: LR12 showed no effect on heart rate and body temperature. By contrast to the placebo group, which experienced a 25 to 40% blood pressure decrease after endotoxin administration, LR12-treated monkeys remained normotensive. Endotoxin induced leukopenia at 2 h (mean leukocyte count, 7.62 g/l vs. 21.1 at H0), which was attenuated by LR12. LR12 also attenuated cytokine production. Conclusions: The triggering receptor expressed on myeloid cells-1 inhibitor LR12 is able to mitigate endotoxin-associated clinical and biological alterations, with no obvious side effects. This study paves the way for future phases Ia and Ib trials in humans.

Published
2014
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7. Specific in vivo staining of astrocytes in the whole brain after intravenous injection of sulforhodamine dyes

Author

Boudewijn van der Sanden, Johannes Römer, Mireille Albrieux, Florence Appaix, Antoine Depaulis, Mathieu Maurin, Isabelle Guillemain, Sylvie Boisseau, Sabine Girod, Jean-Claude Vial, 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 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 10, Dynamique des réseaux neuronaux du mouvement, Laboratoire de Spectrométrie Physique (LSP), Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS), Inserm U836, équipe 7, Nanomédecine et cerveau, FFRE Contrat de projets Etat-région (CPER), and Issartel, Jean-Paul

Subjects
Pathology, Anatomy and Physiology, Mouse, lcsh:Medicine, MESH: Rats, Sprague-Dawley, Biochemistry, Rats, Sprague-Dawley, chemistry.chemical_compound, Mice, 0302 clinical medicine, Molecular Cell Biology, MESH: Animals, Histochemistry, lcsh:Science, Neurons, 0303 health sciences, education.field_of_study, Multidisciplinary, Brain, Electroencephalography, Neurochemistry, Animal Models, MESH: Staining and Labeling, Injections, Intravenous, Cytochemistry, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Cellular Types, Intravital microscopy, Research Article, Genetically modified mouse, medicine.medical_specialty, MESH: Rats, Neural Networks, Transgene, Population, Biology, MESH: Rhodamines, MESH: Calcium Signaling, Stain, Neurological System, 03 medical and health sciences, MESH: Brain, Model Organisms, In vivo, MESH: Mice, Inbred C57BL, MESH: Electroencephalography, medicine, Animals, [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Calcium Signaling, education, MESH: Mice, 030304 developmental biology, Staining and Labeling, Rhodamines, lcsh:R, Sulforhodamine 101, MESH: Injections, Intravenous, Staining, Rats, MESH: Astrocytes, Mice, Inbred C57BL, Neuroanatomy, chemistry, Astrocytes, Cellular Neuroscience, Rat, lcsh:Q, 030217 neurology & neurosurgery, Neuroscience
Abstract

International audience; Fluorescent staining of astrocytes without damaging or interfering with normal brain functions is essential for intravital microscopy studies. Current methods involved either transgenic mice or local intracerebral injection of sulforhodamine 101. Transgenic rat models rarely exist, and in mice, a backcross with GFAP transgenic mice may be difficult. Local injections of fluorescent dyes are invasive. Here, we propose a non-invasive, specific and ubiquitous method to stain astrocytes in vivo. This method is based on iv injection of sulforhodamine dyes and is applicable on rats and mice from postnatal age to adulthood. The astrocytes staining obtained after iv injection was maintained for nearly half a day and showed no adverse reaction on astrocytic calcium signals or electroencephalographic recordings in vivo. The high contrast of the staining facilitates the image processing and allows to quantify 3D morphological parameters of the astrocytes and to characterize their network. Our method may become a reference for in vivo staining of the whole astrocytes population in animal models of neurological disorders.

Published
2011
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8. Deep in vivo two-photon imaging of blood vessels with a new dye encapsulated in pluronic nanomicelles

Author

Boudewijn van der Sanden, Seth R. Marder, Mathieu Maurin, Jean-Claude Vial, Olivier Stephan, Laboratoire de Spectrométrie Physique (LSP), Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS), School of Chemistry and Biochemistry, and Center for Organic Electronics and Photonics, Georgia Institute of Technology [Atlanta], Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM), This work was supported by a grant from the Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche., and Issartel, Jean-Paul

Subjects
MESH: Photons, Optical Phenomena, 02 engineering and technology, Pluronic nanomicelles, 01 natural sciences, Micelle, chemistry.chemical_compound, Mice, Two-photon excitation microscopy, Blood plasma, MESH: Microscopy, Confocal, MESH: Animals, MESH: Dextrans, Micelles, Microscopy, Confocal, Blood Volume Determination, Chemistry, MESH: Micelles, Brain, Dextrans, 021001 nanoscience & nanotechnology, MESH: Fluorescent Dyes, Atomic and Molecular Physics, and Optics, Extravasation, Electronic, Optical and Magnetic Materials, Dextran, MESH: Neoplasms, Experimental, MESH: Styrenes, Intravital two-photon microscopy, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], 0210 nano-technology, MESH: Optical Phenomena, Preclinical imaging, Diagnostic Imaging, MESH: Cell Line, Tumor, MESH: Blood Volume Determination, Biomedical Engineering, Mice, Nude, Nanotechnology, Poloxamer, MESH: Rhodamines, Article, Styrenes, Biomaterials, MESH: Brain, MESH: Nanostructures, In vivo, Cell Line, Tumor, MESH: Mice, Nude, Animals, Humans, [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], MESH: Mice, Fluorescent Dyes, Photons, MESH: Humans, 010405 organic chemistry, Rhodamines, MESH: Diagnostic Imaging, Neoplasms, Experimental, cerebral and tumor microvasculature, MESH: Blood Vessels, MESH: Poloxamer, 0104 chemical sciences, Nanostructures, Biophysics, Blood Vessels
Abstract

International audience; Our purpose is to test if Pluronic® fluorescent nanomicelles can be used for in vivo two-photon imaging of both the normal and the tumor vasculature. The nanomicelles were obtained after encapsulating a hydrophobic two-photon dye: di-stryl benzene derivative, in Pluronic block copolymers. Their performance with respect to imaging depth, blood plasma staining, and diffusion across the tumor vascular endothelium is compared to a classic blood pool dye Rhodamin B dextran (70 kDa) using two-photon microscopy. Pluronic nanomicelles show, like Rhodamin B dextran, a homogeneous blood plasma staining for at least 1 h after intravenous injection. Their two-photon imaging depth is similar in normal mouse brain, using 10 times less injected mass. In contrast with Rhodamin B dextran, no extravasation is observed in leaky tumor vessels due to their large size: 20-100 nm. In conclusion, Pluronic nanomicelles can be used as a blood pool dye, even in leaky tumor vessels. The use of Pluronic block copolymers is a valuable approach for encapsulating two-photon fluorescent dyes that are hydrophobic and not suitable for intravenous injection.

Published
2011
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9. Multi-confocal fluorescence correlation spectroscopy

Author

Martial Balland, Meike Kloster, Gaetan Herbomel, Antoine Delon, Irène Wang, Catherine Souchier, Jie Gao, Jacques Derouard, Olivier Destaing, Yves Usson, Rémi Galland, MOTIV, 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), Equipe 10, Institut d'oncologie/développement Albert Bonniot de Grenoble (INSERM U823), Université Joseph Fourier - Grenoble 1 (UJF)-CHU Grenoble-EFS-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-CHU Grenoble-EFS-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Joseph Fourier - Grenoble 1 (UJF)-CHU Grenoble-EFS-Institut National de la Santé et de la Recherche Médicale (INSERM), Dynamique des systèmes d'adhérence et différenciation (DySAD), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Dynamique Cellulaire et Tissulaire- Interdisciplinarité, Modèles & Microscopies (TIMC-IMAG-DyCTiM), Techniques de l'Ingénierie Médicale et de la Complexité - Informatique, Mathématiques et Applications, Grenoble - UMR 5525 (TIMC-IMAG), VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF), Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS), Région Rhône-Alpes, Cible 2009, Delon, Antoine, Matériaux, Optique et Techniques Instrumentales pour le Vivant (MOTIV-LIPhy), Dynamiques Cellulaire, Tissulaire & Microscopie fonctionnelle (TIMC-IMAG-DyCTiM), and Université Joseph Fourier - Grenoble 1 (UJF)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Diffraction, 01 natural sciences, law.invention, Mice, correlations, law, MESH: Least-Squares Analysis, Image Processing, Computer-Assisted, MESH: Animals, 0303 health sciences, Spatial light modulator, [PHYS.PHYS.PHYS-BIO-PH] Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], molecular transport, MESH: Image Processing, Computer-Assisted, Fick's laws of diffusion, Fluorescence, 180.1790, 170.6280, [SDV.IB]Life Sciences [q-bio]/Bioengineering, fluorescence, MESH: Spectrometry, Fluorescence, Materials science, [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], Confocal, Green Fluorescent Proteins, Fluorescence correlation spectroscopy, MESH: Rhodamines, MESH: Actins, General Biochemistry, Genetics and Molecular Biology, Cell Line, 010309 optics, 03 medical and health sciences, MESH: Green Fluorescent Proteins, Optics, Confocal microscopy, 0103 physical sciences, Animals, Least-Squares Analysis, MESH: Mice, 030304 developmental biology, [SDV.IB] Life Sciences [q-bio]/Bioengineering, General Immunology and Microbiology, business.industry, Rhodamines, Laser, molecular dynamics, Actins, MESH: Cell Line, Spectrometry, Fluorescence, business
Abstract

International audience; We report a multi-confocal Fluorescence Correlation Spectroscopy (mFCS) technique that combines a Spatial Light Modulator (SLM), with an Electron Multiplying-CCD camera (EM-CCD). The SLM is used to produce a series of laser spots, while the pixels of the EM-CCD play the roles of virtual pinholes. The phase map addressed to the SLM, calculated by using the spherical wave approximation, makes it possible to produce several diffraction limited laser spots. The fastest acquisition mode leads to a time resolution of 100 microseconds. By using solutions of sulforhodamine G we demonstrated that the observation volumes are similar to that of a standard confocal set-up. mFCS experiments have also been conducted on two stable cell lines: mouse embryonic fibroblasts expressing eGFP-actin and H1299 cells expressing the heat shock factor fusion protein HSF1-eGFP. In the first case we could recover the diffusion constant of G-actin within the cytoplasm, although we were also sensitive to interactions with F-actin. Concerning HSF1, we could clearly observe the modifications of the number of molecules and of the HSF1 dynamics during heat shock.

Published
2011
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10. Synthesis of RNA by in vitro transcription

Author

Beckert, Bertrand, Masquida, Benoît, Department of Cellular and Molecular Medicine [Copenhagen], Faculty of Health and Medical Sciences, University of Copenhagen = Københavns Universitet (KU)-University of Copenhagen = Københavns Universitet (KU), Architecture et Réactivité de l'ARN (ARN), Institut de biologie moléculaire et cellulaire (IBMC), and Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)

Subjects
MESH: Microvilli, Base Sequence, Transcription, Genetic, MESH: Antibodies, Molecular Sequence Data, MESH: Astacoidea, MESH: Immunohistochemistry, DNA-Directed RNA Polymerases, MESH: Microscopy, Electron, In Vitro Techniques, MESH: Rhodamines, MESH: Actins, MESH: Drosophila melanogaster, MESH: Photoreceptor Cells, Bacteriophage T7, MESH: Cytoskeleton, RNA, MESH: Myosin Subfragments, MESH: Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, MESH: Microfilaments, Promoter Regions, Genetic, Molecular Biology, MESH: Phalloidine, MESH: Intestines
Abstract

International audience; In vitro transcription is a simple procedure that allows for template-directed synthesis of RNA molecules of any sequence from short oligonucleotides to those of several kilobases in μg to mg quantities. It is based on the engineering of a template that includes a bacteriophage promoter sequence (e.g. from the T7 coliphage) upstream of the sequence of interest followed by transcription using the corresponding RNA polymerase. In vitro transcripts are used in analytical techniques (e.g. hybridization analysis), structural studies (for NMR and X-ray crystallography), in biochemical and genetic studies (e.g. as antisense reagents), and as functional molecules (ribozymes and aptamers).

Published
2011
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11. In vivo staining of neocortical astrocytes via the cerebral microcirculation using sulforhodamine B

Author

Clément Ricard, Pascale Vérant, Jean-Claude Vial, Raphaël Serduc, Boudewijn van der Sanden, Laboratoire de Spectrométrie Physique (LSP), Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS), Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM), Neuroimagerie Fonctionnelle et Metabolique, and Serduc, Raphael

Subjects
Pathology, Sulforhodamine B, Contrast Media, Neocortex, Mice, chemistry.chemical_compound, MESH: Neocortex, 0302 clinical medicine, Blood plasma, MESH: Microcirculation, MESH: Animals, 0303 health sciences, Chemistry, Fluorescence, Atomic and Molecular Physics, and Optics, Electronic, Optical and Magnetic Materials, Cell biology, MESH: Staining and Labeling, medicine.anatomical_structure, MESH: Microvessels, Injections, Intravenous, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], MESH: Image Enhancement, Preclinical imaging, medicine.medical_specialty, Biomedical Engineering, MESH: Rhodamines, Blood–brain barrier, Stain, Biomaterials, 03 medical and health sciences, In vivo, MESH: Contrast Media, medicine, Animals, [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], MESH: Mice, 030304 developmental biology, Staining and Labeling, Rhodamines, Microcirculation, Image Enhancement, MESH: Injections, Intravenous, Staining, MESH: Astrocytes, Microscopy, Fluorescence, Multiphoton, Astrocytes, Microvessels, MESH: Microscopy, Fluorescence, Multiphoton, 030217 neurology & neurosurgery
Abstract

International audience; Staining and imaging glial cells in vivo while observing the microvasculature could help understand brain physiology, namely neuronal-glial-vascular communication. Two-photon excitation microscopy provides a means to monitor these interactions at the cellular level in living animals, but the cells of interest must be fluorescent. Injecting dyes intravenously is a rapid and quasi noninvasive method to stain cells in the brain. It necessitates that the dye is soluble in the blood plasma and crosses the blood brain barrier (BBB). We demonstrate here, using two-photon imaging, that sulforhodamine B (SRB) crosses the BBB and stains in vivo, specifically mouse astrocytes. This is confirmed by experiments on primary neurons and astrocytes cultures showing the preferential SRB staining of the latter. SRB is rapidly eliminated from the blood, which allows repeated injections in longitudinal studies.

Published
2008
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12. Proteomic analysis of the cellular responses induced in uninfected immune cells by cell-expressed X4 HIV-1 envelope

Author

Martine Biard-Piechaczyk, Claude Granier, Laurence Molina, Christian Devaux, Marina Grimaldi, Véronique Robert-Hebmann, Mihayl Varbanov, Lucile Espert, Modélisation et ingéniérie des systèmes complexes pour le diagnostic (MISCD), BIO-RAD FRANCE HOLDING-Centre National de la Recherche Scientifique (CNRS), Institut de Recherche en Infectiologie de Montpellier (IRIM), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), and Centre d’études d’Agents Pathogènes et Biotechologies pour la Santé (CPBS)

Subjects
CD4-Positive T-Lymphocytes, Proteomics, MESH: Cell Death, Time Factors, Proteome, MESH: HIV Envelope Protein gp41, Cell, MESH: Flow Cytometry, HIV Envelope Protein gp120, Kidney, MESH: Monocytes, Biochemistry, MESH: Peptide Mapping, Mass Spectrometry, Monocytes, MESH: HIV-1, MESH: HIV Envelope Protein gp120, 0302 clinical medicine, Electrophoresis, Gel, Two-Dimensional, MESH: HSP70 Heat-Shock Proteins, 0303 health sciences, Cell Death, MESH: Proteomics, MESH: CD4-Positive T-Lymphocytes, MESH: Reactive Oxygen Species, Fetal Blood, Flow Cytometry, MESH: Fluorescent Dyes, HIV Envelope Protein gp41, 3. Good health, Cell biology, MESH: Proteome, medicine.anatomical_structure, 030220 oncology & carcinogenesis, Receptors, CXCR4, Programmed cell death, Cell type, Biology, MESH: Rhodamines, Gp41, Peptide Mapping, Peripheral blood mononuclear cell, MESH: Receptors, CXCR4, Cell Line, MESH: Coculture Techniques, 03 medical and health sciences, Immune system, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Autophagy, medicine, Humans, MESH: Autophagy, HSP70 Heat-Shock Proteins, MESH: Fetal Blood, Molecular Biology, Fluorescent Dyes, 030304 developmental biology, MESH: Mass Spectrometry, MESH: Humans, Rhodamines, MESH: Time Factors, MESH: Kidney, MESH: Electrophoresis, Gel, Two-Dimensional, Coculture Techniques, MESH: Cell Line, MESH: Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Cell culture, Apoptosis, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, HIV-1, Reactive Oxygen Species
Abstract

HIV-1 envelope gp120 and gp41 glycoproteins (Env), expressed at the cell surface, induce uninfected CD4 T-cell death, but the molecular mechanisms leading to this demise are still largely unknown. To better understand these events, we analyzed by a proteomic approach the differential protein expression profile of two types of uninfected immune cells after their coculture for 1-3 days with cells that express, or not, Env. First, umbilical cord blood mononuclear cells (UCBMCs) were used to approach the in vivo situation, i.e., blood uninfected naive cells that encounter infected cells. Second, we used the A2.01/CD4.403 T-cell line expressing wild type CXCR4 and a truncated form of CD4 that still undergoes Env-mediated apoptosis, independently of CD4 signaling. After coculture with cells expressing Env, 35 and 39 proteins presenting an altered expression in UCBMCs and the A2.01/CD4.403 T-cell line, respectively, were identified by mass-spectrometry. Whatever the cell type analyzed, the majority of these proteins are involved in degradation processes, redox homeostasis, metabolism and cytoskeleton dynamics, and linked to mitochondrial functions. This study provides new insights into the events that sequentially occur in bystander T lymphocytes after contact with HIV-infected cells and leading, finally, to apoptotic cell death.

Published
2007
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13. Consequences of the bleed-through phenomenon in immunofluorescence of proteins forming radiation-induced nuclear foci

Author

Zuzana Bencokova, Catherine Massart, Wendy Rénier, Jérôme Gastaldo, Nicolas Foray, Aurélie Joubert, European Synchrotron Radiation Facility (ESRF), Rayonnement Synchrotron et Recherche Medicale (RSRM), Université Joseph Fourier - Grenoble 1 (UJF)-European Synchrotron Radiation Facility (ESRF)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de Radioprotection et de Sureté Nucléaire, Institut de Radioprotection et de Sûreté Nucléaire (IRSN), Serduc, Raphael, and European Synchrotron Radiation Facility (ESRF)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
DNA Repair, Fluorescent Antibody Technique, Ionizing radiation, Histones, chemistry.chemical_compound, 0302 clinical medicine, Radiation, Ionizing, Phosphorylation, Fluorescein isothiocyanate, MESH: Fluorescent Antibody Technique, MESH: DNA Repair, MESH: Histones, 0303 health sciences, MRE11 Homologue Protein, Radiological and Ultrasound Technology, medicine.diagnostic_test, MESH: Kinetics, MESH: Fluorescent Dyes, Fluorescence, 3. Good health, DNA-Binding Proteins, 030220 oncology & carcinogenesis, Fluorescein-5-isothiocyanate, MESH: Cell Nucleus, DNA damage, DNA repair, Biology, MESH: Radiation, Ionizing, MESH: Rhodamines, Immunofluorescence, 03 medical and health sciences, MESH: Fluorescein-5-isothiocyanate, medicine, Humans, Radiology, Nuclear Medicine and imaging, 030304 developmental biology, Fluorescent Dyes, MESH: DNA Damage, Cell Nucleus, MESH: Humans, MESH: Phosphorylation, Rhodamines, Molecular biology, Hsp70, Kinetics, chemistry, Biophysics, MESH: DNA-Binding Proteins, DNA Damage
Abstract

International audience; PURPOSE: By allowing the visualization of the proteins inside cells, the immunofluorescence technique has revolutionized our view of events that follow radiation response. Particularly, the formation of nuclear foci, their kinetic of appearance and disappearance, and the association-dissociation of protein partners are useful endpoints to better understand the effects of ionizing radiation. Recently, the technique based on the phosphorylation of the histone 2A family, member X (H2AX) has generated a plethora of reports concerning the interaction between the major proteins involved in DNA repair and stress signaling pathways. However, some unavoidable overlaps of excitation and emission wavelength spectra (the so-called bleed-through phenomenon) of the available fluorescent markers are still generating discrepancies and misinterpretations in the choreography of DNA damage response. Biases are particularly strong with the fluorescein isothiocyanate (FITC)-rhodamine couple, tetramethyl rhodamine iso-thiocyanate (TRITC), the most extensively used markers. METHOD AND RESULTS: Here, two representative examples of biased co-immunofluorescence with pH2AX proteins that form radiation-induced nuclear foci or not are presented. A brief review of literature points out differences in kinetic of appearance and association-dissociation of radiation-induced pH2AX and MRE11 foci. CONCLUSION: Through this report, we would like authors to consider more carefully protein co-localizations by performing systematically, before any co-immunofluorescence, immunofluorescence of each protein separately to avoid bleed-through artifacts.

Published
2007
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14. Uptake of host cell transforming growth factor-ß by Trypanosoma cruzi amastigotes in cardiomyocytes: potential role in parasite cycle completion

Author

Waghabi, Mariana, Keramidas, Michelle, Bailly, Sabine, Degrave, Wim, Mendonça-Lima, Leila, Soeiro, Maria De Nazaré, Meirelles, Maria De Nazareth, Paciornik, Sidnei, Araújo-Jorge, Tania, Feige, Jean-Jacques, Fundação Oswaldo Cruz (FIOCRUZ), Réseau International des Instituts Pasteur (RIIP), Département réponse et dynamique cellulaire (DRDC), Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Laboratory of Digital Image Processing (Pontifica Universidade Catolica), PUC, Angiogenèse hormono-regulée et angiogenèse tumorale (LAPV), 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), INSERM, CEA, FIOCRUZ, INSERM-FIOCRUZ joint program, PAPES/FIOCRUZ, FAPERJSupported by an Institut National de la Santé et de la Recherche Médicale-Fundação Instituto Oswaldo Cruz (FIOCRUZ) collaborative research program, by grants from Programa de Apoio à Pesquisa Estratégica em Saúde/FIOCRUZ, Instituto Oswaldo Cruz, Conselho Nacional de Desenvolvimento Científico e Techológico, Fundação de Amparo a Perquisa do Estado do Rio de Janeiro to the Brazilian laboratories, and by recurrent funding from Commissariat à l'Energie Atomique and INSERM to the French laboratory., We thank Marcos Meuser for his technical help in parasite preparations, Dr. Didier Grunwald for his advice in confocal microscopy analysis, and Dr. Solange L. Castro and Thais Souto-Padrón for critical reading of the manuscript., and Fundação Oswaldo Cruz / Oswaldo Cruz Foundation (FIOCRUZ)

Subjects
Chagas disease, MESH: Recombinant Proteins/metabolism, Trypanosoma cruzi, MESH: Trypanosoma cruzi/ultrastructure, MESH: Trypanosoma cruzi/growth & development, cardiomyocyte, MESH: Transforming Growth Factor beta/metabolism, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, MESH: Rhodamines, MESH: Fluorescein-5-isothiocyanate, MESH: Transforming Growth Factor beta/immunology, parasitic diseases, MESH: Recombinant Proteins/immunology, MESH: Microscopy, Confocal, MESH: Fluorescent Antibody Technique, Indirect, MESH: Animals, MESH: Mice, MESH: Chagas Disease/metabolism, MESH: Indoles, MESH: Myocytes, Cardiac/metabolism, MESH: Actins/metabolism, MESH: Time Factors, MESH: Embryo, Mammalian, MESH: Embryo, Nonmammalian, MESH: Immunohistochemistry, MESH: Fluorescent Dyes, MESH: Myocytes, Cardiac/parasitology, MESH: Trypanosoma cruzi/parasitology, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, TGF-ß, MESH: Transforming Growth Factor beta/genetics, MESH: Life Cycle Stages, MESH: Phalloidine/metabolism, MESH: Cells, Cultured
Abstract

11 pages; International audience; Transforming growth factor-β (TGF-β) is a cytokine that plays various functions in the control of Trypanosoma cruzi infectivity and in the progression of Chagas disease. When we immunostained Trypanosoma cruzi-infected cardiomyocytes (following either in vivo or in vitro infections) for TGF-β, we observed stronger immunoreactivity in parasites than in host cells. TGF-β immunoreactivity evolved during parasite cycle progression: intense staining in amastigotes versus very faint staining in trypomastigotes. TGF-β was present on the surface of amastigotes, in the flagellar pocket and in intraparasitic vesicles as revealed by electron microscopy. However, no ortholog TGF-β gene could be identified in the genome of Trypanosoma cruzi by in silico analysis or by extensive PCR and RT-PCR studies. Immunoreactive TGF-β was most probably taken up by the parasite from the host cell cytoplasm since such an internalization process of biotinylated TGF-β could be observed in axenic amastigotes in vitro. These observations represent the first example of a novel mechanism by which a primitive unicellular protozoan can use host cell TGF-β to control its own intracellular cycle.

Published
2005
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15. Distribution of ganglion cells in the pigeon retina labeled via retrograde transneuronal transport of the fluorescent dye rhodamine beta-isothiocyanate from the telencephalic visual Wulst

Author

Jean-Paul Rio, Dom Miceli, Monique Médina, Jacques Repérant, Michel Volle, Evolution des régulations endocriniennes (ERE), and Muséum national d'Histoire naturelle (MNHN)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Retinal Ganglion Cells, Telencephalon, genetic structures, MESH: Neurons, Biological Transport, Active, Biology, MESH: Rhodamines, Retinal ganglion, Functional Laterality, Rhodamine, chemistry.chemical_compound, Thalamus, medicine, Animals, MESH: Animals, MESH: Functional Laterality, Columbidae, Molecular Biology, Fluorescent Dyes, MESH: Thalamus, Neurons, Retina, MESH: Columbidae, Rhodamines, General Neuroscience, [SDV.BA]Life Sciences [q-bio]/Animal biology, Retinal, MESH: Retinal Ganglion Cells, Anatomy, MESH: Fluorescent Dyes, eye diseases, Visual field, Ganglion, medicine.anatomical_structure, Retinal ganglion cell, chemistry, MESH: Telencephalon, Biophysics, MESH: Biological Transport, Active, Neurology (clinical), Brainstem, sense organs, Developmental Biology
Abstract

The distribution of retinal ganglion cells (RGCs) providing input to the thalamofugal visual system in the pigeon was studied with an anatomical transneuronal transport technique using the fluorescent dye rhodamine beta-isothiocyanate (RITC). Unilateral injections of RITC made into the telencephalic visual Wulst resulted in the retrograde (1) first-order labeling (FOL) of dorsal thalamic (n. dorsolateralis anterior and n. superficialis parvocellularis: SPC) and brainstem somata as well as (2) second-order labeling of other cell populations within the brain and of retinal ganglion cells in both eyes obtained after transneuronal transfer of the tracer from neurons labeled directly via FOL. The mapping and counting of labeled RGCs in retinal flat-mounts showed that they were mainly distributed within the nasal portion of the retinal yellow field (YF) and that their total numbers were consistently higher (averaging 57%) in the eye contralateral to the tracer injection. Labeled RGCs in the retinal red field (RF) represented 13.4% and 12.0% of total labeled cells in the ipsilateral and contralateral eye, respectively. Moreover, the average densities of labeled cells/mm(2) in the RF and YF were respectively 8.4 and 42.8 (ipsilateral) and 17.9 and 54.0 (contralateral). The preferential distribution of labeled RGCs within the nasal YF supports the notion that the thalamofugal visual system in the lateral-eyed pigeon is mainly concerned with viewing in the lateral visual field. Conversely, the relatively low numbers of labeled RGCs observed within the specialized RF indicate that, unlike the case in frontal-eyed bird species and mammals, this system does not appear to be involved in binocular visual processing.

Published
2005
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16. Uptake of host cell transforming growth factor-ß by Trypanosoma cruzi amastigotes in cardiomyocytes: potential role in parasite cycle completion

Author

Waghabi, Mariana, Keramidas, Michelle, Bailly, Sabine, Degrave, Wim, Mendonça-Lima, Leila, Soeiro, Maria de Nazaré, Meirelles, Maria de Nazareth, Paciornik, Sidnei, Araújo-Jorge, Tania, Feige, Jean-Jacques, Feige, Jean-Jacques, Fundação Oswaldo Cruz (FIOCRUZ), Réseau International des Instituts Pasteur (RIIP), Département réponse et dynamique cellulaire (DRDC), Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Laboratory of Digital Image Processing (Pontifica Universidade Catolica), PUC, Angiogenèse hormono-regulée et angiogenèse tumorale (LAPV), 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), INSERM, CEA, FIOCRUZ, INSERM-FIOCRUZ joint program, PAPES/FIOCRUZ, FAPERJSupported by an Institut National de la Santé et de la Recherche Médicale-Fundação Instituto Oswaldo Cruz (FIOCRUZ) collaborative research program, by grants from Programa de Apoio à Pesquisa Estratégica em Saúde/FIOCRUZ, Instituto Oswaldo Cruz, Conselho Nacional de Desenvolvimento Científico e Techológico, Fundação de Amparo a Perquisa do Estado do Rio de Janeiro to the Brazilian laboratories, and by recurrent funding from Commissariat à l'Energie Atomique and INSERM to the French laboratory., and We thank Marcos Meuser for his technical help in parasite preparations, Dr. Didier Grunwald for his advice in confocal microscopy analysis, and Dr. Solange L. Castro and Thais Souto-Padrón for critical reading of the manuscript.

Subjects
Chagas disease, MESH: Recombinant Proteins/metabolism, Trypanosoma cruzi, MESH: Trypanosoma cruzi/ultrastructure, MESH: Trypanosoma cruzi/growth & development, cardiomyocyte, MESH: Transforming Growth Factor beta/metabolism, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, MESH: Rhodamines, MESH: Fluorescein-5-isothiocyanate, MESH: Transforming Growth Factor beta/immunology, parasitic diseases, MESH: Recombinant Proteins/immunology, MESH: Microscopy, Confocal, MESH: Fluorescent Antibody Technique, Indirect, MESH: Animals, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, [SDV.MP] Life Sciences [q-bio]/Microbiology and Parasitology, MESH: Mice, MESH: Chagas Disease/metabolism, MESH: Indoles, MESH: Myocytes, Cardiac/metabolism, MESH: Actins/metabolism, MESH: Time Factors, MESH: Embryo, Mammalian, MESH: Embryo, Nonmammalian, MESH: Immunohistochemistry, MESH: Fluorescent Dyes, MESH: Myocytes, Cardiac/parasitology, MESH: Trypanosoma cruzi/parasitology, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, TGF-ß, MESH: Transforming Growth Factor beta/genetics, MESH: Life Cycle Stages, MESH: Phalloidine/metabolism, MESH: Cells, Cultured
Abstract

11 pages; International audience; Transforming growth factor-β (TGF-β) is a cytokine that plays various functions in the control of Trypanosoma cruzi infectivity and in the progression of Chagas disease. When we immunostained Trypanosoma cruzi-infected cardiomyocytes (following either in vivo or in vitro infections) for TGF-β, we observed stronger immunoreactivity in parasites than in host cells. TGF-β immunoreactivity evolved during parasite cycle progression: intense staining in amastigotes versus very faint staining in trypomastigotes. TGF-β was present on the surface of amastigotes, in the flagellar pocket and in intraparasitic vesicles as revealed by electron microscopy. However, no ortholog TGF-β gene could be identified in the genome of Trypanosoma cruzi by in silico analysis or by extensive PCR and RT-PCR studies. Immunoreactive TGF-β was most probably taken up by the parasite from the host cell cytoplasm since such an internalization process of biotinylated TGF-β could be observed in axenic amastigotes in vitro. These observations represent the first example of a novel mechanism by which a primitive unicellular protozoan can use host cell TGF-β to control its own intracellular cycle.

Published
2005
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17. Spontaneous Membrane Transfer Through Homotypic Synapses Between Lymphoma Cells

Author

Mary Poupot, Jean-Jacques Fournié, Centre de Physiopathologie de Toulouse-Purpan (INSERM U563 - CNRS UMR1037), Centre National de la Recherche Scientifique (CNRS)-Centre de lutte contre le cancer (CLCC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Hôpital Purpan [Toulouse], CHU Toulouse [Toulouse]-CHU Toulouse [Toulouse]-Institut Claudius Regaud, Poupot, Mary, and Centre Hospitalier Universitaire de Toulouse (CHU Toulouse)-Institut Claudius Regaud-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre de lutte contre le cancer (CLCC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
[SDV.MHEP.HEM] Life Sciences [q-bio]/Human health and pathology/Hematology, Lymphoma, B-Cell, MESH: Cell Line, Tumor, Immunology, B-cell receptor, Biological Transport, Active, Receptors, Antigen, B-Cell, Cell Communication, Biology, MESH: Rhodamines, Immunological synapse, MESH: Receptors, Antigen, B-Cell, 03 medical and health sciences, Cytosol, 0302 clinical medicine, MESH: Cytosol, Cell Line, Tumor, MESH: Cell Communication, Humans, Immunology and Allergy, Organic Chemicals, Kinase activity, Protein kinase C, Fluorescent Dyes, 030304 developmental biology, MESH: Lymphoma, B-Cell, 0303 health sciences, MESH: Humans, Rhodamines, Cell Membrane, MESH: Organic Chemicals, [SDV.MHEP.HEM]Life Sciences [q-bio]/Human health and pathology/Hematology, MESH: Fluorescent Dyes, Burkitt Lymphoma, Immunological Synapses, Microvesicles, Cell biology, MESH: Burkitt Lymphoma, Intercellular Junctions, Cell culture, MESH: Biological Transport, Active, Intracellular, 030215 immunology, MESH: Intercellular Junctions, MESH: Cell Membrane
Abstract

Formation of an immunological synapse by T, B, or NK cells is associated with an intercellular transfer of some membrane fragments from their respective target cells. This capture is thought to require effector cell activation by surface recognition of stimulatory ligand(s). However, spontaneous synaptic transfers between homotypic lymphoid cells has never been described. In this study, we show that without adding Ag, resting healthy lymphoid cells and several tumor cell lines are inactive. Conversely, however, some leukemia cell lines including the Burkitt’s lymphoma Daudi continuously uptake patches of autologous cell membranes. This intercellular transfer does not involve cytosol molecules or exosomes, but requires cell contact. In homotypic Daudi cell conjugates, this occurs through immunological synapses, involves constitutive protein kinase C and mitogen-activated protein/extracellular signal-regulated kinase kinase activity and strongly increases upon B cell receptor activation. Thus, spontaneous homosynaptic transfer may reflect the hitherto unsuspected autoreactivity of some leukemia cell lines.

Published
2003
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18. Sphingoid base signaling via Pkh kinases is required for endocytosis in yeast

Author

Sylvie Friant, Michael N. Hall, Ruben Lombardi, Howard Riezman, Tobias Schmelzle, Génétique moléculaire, génomique, microbiologie (GMGM), and Université Louis Pasteur - Strasbourg I-Centre National de la Recherche Scientifique (CNRS)

Subjects
MESH: Signal Transduction, Time Factors, Phalloidine, MESH: Microscopy, Fluorescence, MESH: Dose-Response Relationship, Drug, MESH: Genotype, MESH: Saccharomyces cerevisiae Proteins, Sphingosine, MESH: Precipitin Tests, Phosphorylation, Internalization, Cytoskeleton, media_common, Kinase, General Neuroscience, MESH: Saccharomyces cerevisiae, Endocytosis, Cell biology, Biochemistry, MESH: Endocytosis, MESH: Sphingosine, Signal transduction, MESH: Phalloidine, Plasmids, Signal Transduction, MESH: Mutation, Saccharomyces cerevisiae Proteins, Genotype, media_common.quotation_subject, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Saccharomyces cerevisiae, MESH: Rhodamines, Biology, Protein Serine-Threonine Kinases, MESH: Actins, MESH: Protein-Serine-Threonine Kinases, General Biochemistry, Genetics and Molecular Biology, Article, 3-Phosphoinositide-Dependent Protein Kinases, MESH: Plasmids, MESH: Cytoskeleton, MESH: Protein Kinases, Molecular Biology, Eisosome, Protein-Serine-Threonine Kinases, MESH: Phosphorylation, General Immunology and Microbiology, Dose-Response Relationship, Drug, Rhodamines, MESH: Time Factors, Actin cytoskeleton, Precipitin Tests, Actins, Microscopy, Fluorescence, Mutation, Protein Kinases
Abstract

In yeast, sphingoid base synthesis is required for the internalization step of endocytosis and organization of the actin cytoskeleton. We show that overexpression of either one of the two kinases Pkh1p or Pkh2p, that are homologous to mammalian 3-phosphoinositide-dependent kinase-1 (PDK1), can specifically suppress the sphingoid base synthesis requirement for endocytosis. Pkh1p and Pkh2p have an overlapping function because only a mutant with impaired function of both kinases is defective for endocytosis. Pkh1/2p kinases are activated in vitro by nanomolar concentrations of sphingoid base. These results suggest that Pkh1/2p kinases are part of a sphingoid base-mediated signaling pathway that is required for the internalization step of endocytosis. The Pkc1p kinase that is phosphorylated by Pkh1/2p kinases and plays a role in endocytosis was identified as one of the downstream effectors of this signaling cascade.

Published
2001
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19. Catalytically active bovine serum amine oxidase bound to fluorescent and magnetically drivable nanoparticles

Author

Radek Zboril, Eris Bidollari, Sara Cardillo, Fabio Vianello, Giovanni Miotto, Enzo Agostinelli, Giulietta Sinigaglia, Massimiliano Magro, Department of Biological Chemistry, Universita degli Studi di Padova, Department of Biochemical Sciences 'Rossi Fanelli', Institut Pasteur, Fondation Cenci Bolognetti - Istituto Pasteur Italia, Fondazione Cenci Bolognetti, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Università degli Studi di Roma 'La Sapienza' = Sapienza University [Rome], Institute of Molecular Biology and Pathology (IBPM), Consiglio Nazionale delle Ricerche (CNR), Regional Centre of Advanced Technologies and Materials [Olomouc] (RCPTM), and This work was funded in part by Progetti di Ateneo (A.0ED00.0487PRAT08), by the University of Padua, by the Italian MIUR (Ministero dell'Istruzione, dell'Università e della Ricerca), by Istituto Superiore di Sanità 'Project Italy-USA', by Istituto Pasteur -Fondazione Cenci Bolognetti, and by funds MIUR-PRIN (Cofin). The work was supported by the Operational Program Research and Development for Innovations-European Social Fund (CZ.1.05/2.1.00/03.0058).

Subjects
Medicine (General), rhodamine isothiocyanate, Pharmaceutical Science, Nanoparticle, MESH: Amino Acid Sequence, 02 engineering and technology, fluorescent nanocatalyst, Coated Materials, Biocompatible, MESH: Coated Materials, Biocompatible, International Journal of Nanomedicine, Drug Discovery, MESH: Animals, Bovine serum albumin, Magnetite Nanoparticles, Original Research, 0303 health sciences, biology, Amine oxidase (copper-containing), General Medicine, MESH: Fluorescent Dyes, 021001 nanoscience & nanotechnology, Fluorescence, MESH: Cattle, Nanomedicine, MESH: Colloids, Amine Oxidase (Copper-Containing), 0210 nano-technology, Superparamagnetism, Amine oxidase, amine oxidase, Materials science, fluorescent nanoparticles, hydrogen peroxide production, superparamagnetic nanoparticles, Molecular Sequence Data, Inorganic chemistry, Biophysics, MESH: Magnetite Nanoparticles, Bioengineering, MESH: Rhodamines, Adduct, Biomaterials, 03 medical and health sciences, R5-920, Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Amino Acid Sequence, Colloids, Fluorescent Dyes, 030304 developmental biology, MESH: Molecular Sequence Data, Rhodamines, Organic Chemistry, MESH: Nanomedicine, biology.protein, Magnetic nanoparticles, Cattle, MESH: Amine Oxidase (Copper-Containing), Nuclear chemistry
Abstract

Giulietta Sinigaglia1, Massimiliano Magro1, Giovanni Miotto1, Sara Cardillo1, Enzo Agostinelli2,3, Radek Zboril4, Eris Bidollari2,3, Fabio Vianello11Department of Biological Chemistry, University of Padua, Padua, Italy; 2Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Biochemical Sciences "A. Rossi Fanelli", SAPIENZA University of Rome, Rome, Italy; 3CNR, Institute Biology and Molecular Pathology, Rome, Italy; 4Regional Centre of Advanced Technologies and Materials, Department of Physical Chemistry, Palacky University, Olomouc, Czech RepublicAbstract: Novel superparamagnetic surface-active maghemite nanoparticles (SAMNs) characterized by a diameter of 10 ± 2 nm were modified with bovine serum amine oxidase, which used rhodamine B isothiocyanate (RITC) adduct as a fluorescent spacer-arm. A fluorescent and magnetically drivable adduct comprised of bovine serum copper-containing amine oxidase (SAMN–RITC–BSAO) that immobilized on the surface of specifically functionalized magnetic nanoparticles was developed. The multifunctional nanomaterial was characterized using transmission electron microscopy, infrared spectroscopy, mass spectrometry, and activity measurements. The results of this study demonstrated that bare magnetic nanoparticles form stable colloidal suspensions in aqueous solutions. The maximum binding capacity of bovine serum amine oxidase was approximately 6.4 mg g-1 nanoparticles. The immobilization procedure reduced the catalytic activity of the native enzyme to 30% ± 10% and the Michaelis constant was increased by a factor of 2. We suggest that the SAMN–RITC–BSAO complex, characterized by a specific activity of 0.81 IU g-1, could be used in the presence of polyamines to create a fluorescent magnetically drivable H2O2 and aldehydes-producing system. Selective tumor cell destruction is suggested as a potential future application of this system.Keywords: amine oxidase, hydrogen peroxide production, superparamagnetic nanoparticles, rhodamine isothiocyanate, fluorescent nanoparticles, fluorescent nanocatalyst

Published
2012
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20. In vivo imaging of elastic fibers using sulforhodamine B

Author

Clément Ricard, Boudewijn van der Sanden, Jean-Claude Vial, Julien Douady, Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire de Spectrométrie Physique (LSP), Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS), Collaboration, and Serduc, Raphael

Subjects
Sulforhodamine B, MESH: Elastic Tissue, Mice, chemistry.chemical_compound, 0302 clinical medicine, MESH: Collagen, Microscopy, MESH: Animals, MESH: Muscle, Skeletal, 0303 health sciences, biology, Anatomy, MESH: Fluorescent Dyes, Fluorescence, Atomic and Molecular Physics, and Optics, 3. Good health, Electronic, Optical and Magnetic Materials, medicine.anatomical_structure, Female, Collagen, Preclinical imaging, Materials science, MESH: Rats, MESH: Elastin, Biomedical Engineering, Mice, Nude, MESH: Rhodamines, Biomaterials, 03 medical and health sciences, In vivo, MESH: Mice, Nude, medicine, Animals, Rats, Wistar, Muscle, Skeletal, MESH: Mice, Fluorescent Dyes, 030304 developmental biology, Rhodamines, Epimysium, MESH: Blood Vessels, MESH: Rats, Wistar, Elastic Tissue, Elastin, Rats, Staining, Microscopy, Fluorescence, Multiphoton, chemistry, MESH: Microscopy, Fluorescence, Multiphoton, biology.protein, Blood Vessels, MESH: Female, 030217 neurology & neurosurgery, Biomedical engineering
Abstract

International audience; Until now, the imaging of elastic fibers was restricted to tissue sections using the endofluorescence properties of elastin or histological dyes. Methods to study their morphology in vivo and in situ have been lacking. We present and characterize a new application of a fluorescent dye for two-photon microscopy: sulforhodamine B (SRB), which is shown to specifically stain elastic fibers in vivo. SRB staining of elastic fibers is demonstrated to be better than using elastin endofluorescence for two-photon microscopy. Our imaging method of elastic fibers is shown to be suitable for simultaneous imaging with both other fluorescent intravital dyes and second-harmonic generation (SHG). We illustrate these findings with intravital imaging of elastic and collagen fibers in muscle epimysium and endomysium and in blood vessel walls. We expect SRB staining to become a key method to study elastic fibers in vivo.

Published
2007
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