28 results on '"Estephan R"'
Search Results
2. Expression and biophysical analysis of a triple-transmembrane domain-containing fragment from a yeast G protein-coupled receptor
- Author
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Caroccia, K E, Estephan, R, Cohen, L S, Arshava, B, Hauser, M, Zerbe, O, Becker, J M, Naider, F, University of Zurich, and Naider, F
- Subjects
10120 Department of Chemistry ,1303 Biochemistry ,Molecular Sequence Data ,Biophysics ,Saccharomyces cerevisiae ,Biochemistry ,Mass Spectrometry ,Protein Structure, Secondary ,Receptors, G-Protein-Coupled ,Biomaterials ,03 medical and health sciences ,GPCR ,540 Chemistry ,Amino Acid Sequence ,Nuclear Magnetic Resonance, Biomolecular ,Chromatography, High Pressure Liquid ,030304 developmental biology ,0303 health sciences ,Circular Dichroism ,2502 Biomaterials ,030302 biochemistry & molecular biology ,Organic Chemistry ,General Medicine ,NMR ,Peptide Fragments ,Electrophoresis, Polyacrylamide Gel ,1304 Biophysics ,1605 Organic Chemistry - Abstract
Structural characterization of G protein-coupled receptors (GPCRs) is hindered by the inherent hydrophobicity, flexibility, and large size of these signaling proteins. Insights into conformational preferences and the three-dimensional (3D) structure of domains of these receptors can be obtained using polypeptide fragments of these proteins. Herein, we report the expression, purification, and biophysical characterization of a three-transmembrane domain-containing 131-residue fragment of the yeast α-factor receptor, Ste2p. Ste2p TM1–TM3 (G31–R161) was expressed as a TrpΔLE fusion protein in Escherichia coli. The expressed protein was subject to CNBr cleavage to remove the fusion tag and TM1–TM3 was purified by reverse-phased HPLC. The cleavage product was isolated in yields of up to 20 mg per liter of culture in both unlabeled and uniformly [15N]-labeled and [15N, 13C, 2H]-labeled forms. The secondary structure of TM1–TM3 was determined to be helical in a number of membrane mimetic environments, including 2,2,2-trifluoroethanol (TFE):water and lysomyristoylphosphatidylglycerol (LMPG) detergent micelles by circular dichroism. Preliminary HSQC analysis in 50% TFE:water and LMPG micelles prepared in sodium phosphate and 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) buffers revealed that this fragment is suitable for structural analysis by nuclear magnetic resonance (NMR). Complete backbone assignments and a detailed localization of the secondary structural elements of TM1–TM3 in 50% TFE:water have been achieved.
- Published
- 2011
- Full Text
- View/download PDF
3. Expression and biophysical analysis of two double transmembrane domain-containing fragments from a yeast G protein-coupled receptor
- Author
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Cohen, L S, Arshava, B, Estephan, R, Englander, J, Kim, H, Hauser, M, Zerbe, O, Ceruso, M, Becker, J M, Naider, F, University of Zurich, and Naider, F
- Subjects
10120 Department of Chemistry ,1303 Biochemistry ,540 Chemistry ,2502 Biomaterials ,1304 Biophysics ,1605 Organic Chemistry ,membrane peptides • double transmembrane domains • GPCRs • expression of peptides • NMR in detergents - Published
- 2008
4. Analyse du comportement d'un réseau élémentaire de micropieux : effet de l'inclinaison
- Author
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Estephan, R., Frank, Roger, Géotechnique (cermes), Laboratoire Navier (navier umr 8205), and École des Ponts ParisTech (ENPC)-Centre National de la Recherche Scientifique (CNRS)-Institut Français des Sciences et Technologies des Transports, de l'Aménagement et des Réseaux (IFSTTAR)-École des Ponts ParisTech (ENPC)-Centre National de la Recherche Scientifique (CNRS)-Institut Français des Sciences et Technologies des Transports, de l'Aménagement et des Réseaux (IFSTTAR)
- Subjects
[SPI]Engineering Sciences [physics] ,ComputingMilieux_MISCELLANEOUS - Abstract
International audience
- Published
- 2004
5. Synthèse des résultats et recommandations du Projet national sur les micropieux FOREVER.Opération du Réseau Génie Civil et Urbain
- Author
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Cyna, H., Schlosser, F., Frank, Roger, Plumelle, C., Estephan, R., Altmayer, F., Goulesco, N., Juran, I., Maurel, C., Shahrour, I., Vezole, P., Géotechnique (cermes), Laboratoire Navier (navier umr 8205), École des Ponts ParisTech (ENPC)-Centre National de la Recherche Scientifique (CNRS)-Institut Français des Sciences et Technologies des Transports, de l'Aménagement et des Réseaux (IFSTTAR)-École des Ponts ParisTech (ENPC)-Centre National de la Recherche Scientifique (CNRS)-Institut Français des Sciences et Technologies des Transports, de l'Aménagement et des Réseaux (IFSTTAR), Presses de l'Ecole nationale des ponts et chaussées, and Institut Français des Sciences et Technologies des Transports, de l'Aménagement et des Réseaux (IFSTTAR)-École des Ponts ParisTech (ENPC)-Centre National de la Recherche Scientifique (CNRS)-Institut Français des Sciences et Technologies des Transports, de l'Aménagement et des Réseaux (IFSTTAR)-École des Ponts ParisTech (ENPC)-Centre National de la Recherche Scientifique (CNRS)
- Published
- 2004
6. Contributions aux méthodes de calcul des groupes et des réseaux de micropieux
- Author
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ESTEPHAN, R and PASTEL, Admin
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chargement vertical ,numerical study ,modèle hybride ,[SPI] Engineering Sciences [physics] ,finite element method ,micropieux ,Load transfer method ,hybrid model ,reticulated ,pieux ,enchevêtrement ,micropile ,group ,groupe ,vertical loading ,FOREVER ,essais expérimentaux ,experimental tests ,réseau ,horizontal loading ,chargement horizontal ,étude numérique ,network ,méthodes des fonctions de transfert de charge ,pile ,méthode des éléments finis - Abstract
The research realized within this thesis concern the study of the behaviour of micropiles groups and networks. The interactions between the micropiles within a group, as well as the effect of micropile inclination within a network, are treated through the three parts of this thesis.The first part concerns the definition, the classification and the application of micropiles. The usual design methods of micropiles (single, groups or networks) are presented herein.The second part presents a synthesis of various experimental tests carried out, within the framework of the French national research project FOREVER, on groups and/or networks of micropiles. These tests are carried out in full-scale or in reduced model (centrifuge, calibration chamber). The third part presents two different numerical approaches for the study of micropiles behavior. The Load transfer method is used through the micropile groups design program (GOUPEG) which is developed to take into consideration the effect of micropiles inclination. The case of an elementary network of 4 micropiles and of an equivalent group is studied. A parametric study on the effect of micropile inclination angle within an elementary network is presented. Finally, the finite element method is used (CESAR-LCPC) for the study of the behaviour of a double A shaped elementary network of 4 micropiles. This last approach provides interesting results on the behaviour of the soil under the battered micropiles., Les travaux de recherche menés dans le cadre de cette thèse concernent l'étude du comportement des groupes et des réseaux de micropieux. L'interaction entre les micropieux d'un groupe, ainsi que l'effet d'inclinaison des micropieux au sein d'un réseau sont traités à travers les trois parties de cette thèse.La première partie est consacrée à la définition, la classification et les domaines d'application des micropieux. Les différentes méthodes usuelles de calcul des micropieux (isolés, en groupes ou en réseaux) sont également présentées.La deuxième partie présente la synthèse de divers essais expérimentaux réalisés, dans le cadre du projet national FOREVER, sur des groupes et/ou des réseaux de micropieux. Ces essais sont réalisés en vraie grandeur ou en modèle réduit (centrifugeuse, chambre d'étalonnage ou cuve expérimentale).La troisième partie traite de deux approches numériques différentes pour l'étude du comportement des micropieux. La méthode des fonctions de transfert de charge est utilisée à travers le programme de calcul des groupes de pieux (GOUPEG) qui est développé pour tenir compte de l'effet de l'inclinaison des micropieux. Le cas d'un réseau élémentaire de 4 micropieux et d'un groupe équivalent est étudié. Une étude paramétrique sur l'effet de l'inclinaison des micropieux au sein d'un réseau élémentaire est présentée. Enfin, la méthode des éléments finis est utilisée (le logiciel CESAR-LCPC) pour l'étude du comportement d'un réseau élémentaire de 4 micropieux (en double chevalet). Cette dernière approche fournit des résultats intéressants sur le comportement du massif du sol sous les micropieux inclinés.
- Published
- 2003
7. Différentes approches du comportement des groupes et des réseaux de micropieux, en modèle réduit, en modèle centrifugé et en vraie grandeur
- Author
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Plumelle, C., Frank, Roger, Canou, Jean, Estephan, R., Gangneux, P., Foray, P., Garnier, J., Centre d'enseignement Cnam Paris (CNAM Paris), Conservatoire National des Arts et Métiers [CNAM] (CNAM), Géotechnique (cermes), Laboratoire Navier (navier umr 8205), Institut Français des Sciences et Technologies des Transports, de l'Aménagement et des Réseaux (IFSTTAR)-École des Ponts ParisTech (ENPC)-Centre National de la Recherche Scientifique (CNRS)-Institut Français des Sciences et Technologies des Transports, de l'Aménagement et des Réseaux (IFSTTAR)-École des Ponts ParisTech (ENPC)-Centre National de la Recherche Scientifique (CNRS), Institut Français des Sciences et Technologies des Transports, de l'Aménagement et des Réseaux (IFSTTAR)-École des Ponts ParisTech (ENPC)-Centre National de la Recherche Scientifique (CNRS), Laboratoire sols, solides, structures - risques [Grenoble] (3SR), Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National Polytechnique de Grenoble (INPG)-Centre National de la Recherche Scientifique (CNRS), Milieux Environnementaux, Transferts et Interactions dans les hydrosystèmes et les Sols (METIS), Université Pierre et Marie Curie - Paris 6 (UPMC)-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), Documentation, Navier, HESAM Université - Communauté d'universités et d'établissements Hautes écoles Sorbonne Arts et métiers université (HESAM)-HESAM Université - Communauté d'universités et d'établissements Hautes écoles Sorbonne Arts et métiers université (HESAM), Université Joseph Fourier - Grenoble 1 (UJF)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Institut National Polytechnique de Grenoble (INPG)-Centre National de la Recherche Scientifique (CNRS), and Université Pierre et Marie Curie - Paris 6 (UPMC)-École Pratique des Hautes Études (EPHE)
- Subjects
[SPI]Engineering Sciences [physics] ,[SPI] Engineering Sciences [physics] ,ComputingMilieux_MISCELLANEOUS - Abstract
International audience
- Published
- 2001
8. Solution-State Structures of Oleate-Liganded LFABP, Protein Only
- Author
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He, Y., primary, Yang, X., additional, Wang, H., additional, Estephan, R., additional, Francis, F., additional, Kodukula, S., additional, Storch, J., additional, and Stark, R.E., additional
- Published
- 2007
- Full Text
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9. Solution-state NMR structures of apo-LFABP (Liver Fatty Acid-Binding Protein)
- Author
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He, Y., primary, Yang, X., additional, Wang, H., additional, Estephan, R., additional, Francis, F., additional, Kodukula, S., additional, Storch, J., additional, and Stark, R.E., additional
- Published
- 2007
- Full Text
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10. Solution-State Structures of Oleate-Liganded LFABP, Major Form of 1:2 Protein-Ligand Complex
- Author
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He, Y., primary, Yang, X., additional, Wang, H., additional, Estephan, R., additional, Francis, F., additional, Kodukula, S., additional, Storch, J., additional, and Stark, R.E., additional
- Published
- 2007
- Full Text
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11. Mast cells are required for the development of renal fibrosis in the rodent unilateral ureteral obstruction model.
- Author
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Veerappan A, Reid AC, O'Connor N, Mora R, Brazin JA, Estephan R, Kameue T, Chen J, Felsen D, Seshan SV, Poppas DP, Maack T, and Silver RB
- Subjects
- Angiotensin II physiology, Animals, Cell Degranulation, Fibrosis, Humans, In Vitro Techniques, Kidney metabolism, Kidney pathology, Kidney Diseases drug therapy, Losartan therapeutic use, Male, Mice, Rats, Renin-Angiotensin System physiology, Kidney Diseases pathology, Mast Cells physiology, Renin physiology, Ureteral Obstruction pathology
- Abstract
Mast cells are associated with inflammation and fibrosis. Whether they protect against or contribute to renal fibrosis is unclear. Based on our previous findings that mast cells can express and secrete active renin, and that angiotensin (ANG II) is profibrotic, we hypothesized that mast cells play a critical role in tubulointerstitial fibrosis. We tested this hypothesis in the 14-day unilateral ureteral obstruction (UUO) model in rats and mast cell-deficient (MCD) mice (WBB6F1-W/Wv) and their congenic controls (CC). In the 14-day UUO rat kidney, mast cell number is increased and they express active renin. Stabilizing mast cells in vivo with administration of cromolyn sodium attenuated the development of tubulointerstitial fibrosis, which was confirmed by measuring newly synthesized pepsin-soluble collagen and blind scoring of fixed trichrome-stained kidney sections accompanied by spectral analysis. Fibrosis was absent in UUO kidneys from MCD mice unlike that observed in the CC mice. Losartan treatment reduced the fibrosis in the CC UUO kidneys. The effects of mast cell degranulation and renin release were tested in the isolated, perfused kidney preparation. Mast cell degranulation led to renin-dependent protracted flow recovery. This demonstrates that mast cell renin is active in situ and the ensuing ANG II can modulate intrarenal vascular resistance in the UUO kidney. Collectively, the data demonstrate that mast cells are critical to the development of renal fibrosis in the 14-day UUO kidney. Since renin is present in human kidney mast cells, our work identifies potential targets in the treatment of renal fibrosis.
- Published
- 2012
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12. A nuclear magnetic resonance-based structural rationale for contrasting stoichiometry and ligand binding site(s) in fatty acid-binding proteins.
- Author
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He Y, Estephan R, Yang X, Vela A, Wang H, Bernard C, and Stark RE
- Subjects
- Animals, Binding Sites, Fatty Acid-Binding Proteins genetics, Ligands, Models, Molecular, Mutagenesis, Site-Directed, Mutation, Oleic Acid metabolism, Protein Binding, Protein Conformation, Rats, Static Electricity, Fatty Acid-Binding Proteins chemistry, Fatty Acid-Binding Proteins metabolism, Nuclear Magnetic Resonance, Biomolecular
- Abstract
Liver fatty acid-binding protein (LFABP) is a 14 kDa cytosolic polypeptide, differing from other family members in the number of ligand binding sites, the diversity of bound ligands, and the transfer of fatty acid(s) to membranes primarily via aqueous diffusion rather than direct collisional interactions. Distinct two-dimensional (1)H-(15)N nuclear magnetic resonance (NMR) signals indicative of slowly exchanging LFABP assemblies formed during stepwise ligand titration were exploited, without determining the protein-ligand complex structures, to yield the stoichiometries for the bound ligands, their locations within the protein binding cavity, the sequence of ligand occupation, and the corresponding protein structural accommodations. Chemical shifts were monitored for wild-type LFABP and an R122L/S124A mutant in which electrostatic interactions viewed as being essential to fatty acid binding were removed. For wild-type LFABP, the results compared favorably with the data for previous tertiary structures of oleate-bound wild-type LFABP in crystals and in solution: there are two oleates, one U-shaped ligand that positions the long hydrophobic chain deep within the cavity and another extended structure with the hydrophobic chain facing the cavity and the carboxylate group lying close to the protein surface. The NMR titration validated a prior hypothesis that the first oleate to enter the cavity occupies the internal protein site. In contrast, (1)H and (15)N chemical shift changes supported only one liganded oleate for R122L/S124A LFABP, at an intermediate location within the protein cavity. A rationale based on protein sequence and electrostatics was developed to explain the stoichiometry and binding site trends for LFABPs and to put these findings into context within the larger protein family.
- Published
- 2011
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13. Aldehyde dehydrogenase activation prevents reperfusion arrhythmias by inhibiting local renin release from cardiac mast cells.
- Author
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Koda K, Salazar-Rodriguez M, Corti F, Chan NY, Estephan R, Silver RB, Mochly-Rosen D, and Levi R
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- Animals, Cell Degranulation, Cell Line, Tumor, Enzyme Activation, Guinea Pigs, Humans, Ischemic Preconditioning, Myocardial, Male, Protein Kinase C-epsilon physiology, Receptor, Adenosine A2B physiology, Receptor, Adenosine A3 physiology, Renin metabolism, Renin-Angiotensin System physiology, Aldehyde Dehydrogenase physiology, Arrhythmias, Cardiac prevention & control, Mast Cells physiology, Myocardial Reperfusion Injury prevention & control, Myocytes, Cardiac metabolism, Renin antagonists & inhibitors
- Abstract
Background: Renin released by ischemia/reperfusion from cardiac mast cells activates a local renin-angiotensin system (RAS). This exacerbates norepinephrine release and reperfusion arrhythmias (ventricular tachycardia and fibrillation), making RAS a new therapeutic target in myocardial ischemia., Methods and Results: We investigated whether ischemic preconditioning (IPC) prevents cardiac RAS activation in guinea pig hearts ex vivo. When ischemia/reperfusion (20 minutes of ischemia/30 minutes of reperfusion) was preceded by IPC (two 5-minute ischemia/reperfusion cycles), renin and norepinephrine release and ventricular tachycardia and fibrillation duration were markedly decreased, a cardioprotective anti-RAS effect. Activation and blockade of adenosine A(2b)/A(3) receptors and activation and inhibition of protein kinase Cepsilon (PKCepsilon) mimicked and prevented, respectively, the anti-RAS effects of IPC. Moreover, activation of A(2b)/A(3) receptors or activation of PKCepsilon prevented degranulation and renin release elicited by peroxide in cultured mast cells (HMC-1). Activation and inhibition of mitochondrial aldehyde dehydrogenase type-2 (ALDH2) also mimicked and prevented, respectively, the cardioprotective anti-RAS effects of IPC. Furthermore, ALDH2 activation inhibited degranulation and renin release by reactive aldehydes in HMC-1. Notably, PKCepsilon and ALDH2 were both activated by A(2b)/A(3) receptor stimulation in HMC-1, and PKCepsilon inhibition prevented ALDH2 activation., Conclusions: The results uncover a signaling cascade initiated by A(2b)/A(3) receptors, which triggers PKCepsilon-mediated ALDH2 activation in cardiac mast cells, contributing to IPC-induced cardioprotection by preventing mast cell renin release and the dysfunctional consequences of local RAS activation. Thus, unlike classic IPC in which cardiac myocytes are the main target, cardiac mast cells are the critical site at which the cardioprotective anti-RAS effects of IPC develop.
- Published
- 2010
- Full Text
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14. Expression of double transmembrane domain GPCR fragments for biophysical analysis.
- Author
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Cohen LS, Arshava B, Estephan R, Englander J, Hauser M, Becker JM, and Naider F
- Subjects
- Chromatography, High Pressure Liquid, Crystallography, X-Ray, Electrophoresis, Polyacrylamide Gel, Receptors, G-Protein-Coupled chemistry, Spectrometry, Mass, Electrospray Ionization, Receptors, G-Protein-Coupled metabolism
- Published
- 2009
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15. Cardioprotective effect of histamine H3-receptor activation: pivotal role of G beta gamma-dependent inhibition of voltage-operated Ca2+ channels.
- Author
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Morrey C, Estephan R, Abbott GW, and Levi R
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- Animals, Calcium antagonists & inhibitors, Calcium metabolism, Calcium Channel Blockers pharmacology, Cardiotonic Agents pharmacology, Cardiovascular Diseases metabolism, Cardiovascular Diseases prevention & control, GTP-Binding Protein beta Subunits agonists, GTP-Binding Protein gamma Subunits agonists, Humans, Nerve Growth Factor pharmacology, PC12 Cells, Rats, Calcium Channels physiology, GTP-Binding Protein beta Subunits physiology, GTP-Binding Protein gamma Subunits physiology, Receptors, Histamine H3 metabolism
- Abstract
We previously showed that activation of G(i/o)-coupled histamine H(3)-receptors (H(3)R) is cardioprotective because it attenuates excessive norepinephrine release from cardiac sympathetic nerves. This action is characterized by a marked decrease in intraneuronal Ca(2+) ([Ca(2+)](i)), as G alpha(i) impairs the adenylyl cyclase-cAMP-protein kinase A (PKA) pathway, and this decreases Ca(2+) influx via voltage-operated Ca(2+) channels (VOCC). Yet, the G(i/o)-derived betagamma dimer could directly inhibit VOCC, and the subsequent reduction in Ca(2+) influx would be responsible for the H(3)R-mediated attenuation of transmitter exocytosis. In this study, we tested this hypothesis in nerve-growth factor-differentiated rat pheochromocytoma cells (PC12) stably transfected with H(3)R (PC12-H(3)) and with the G betagamma scavenger beta-adrenergic receptor kinase 1 (beta-ARK1)-(495-689)-polypeptide (PC12-H(3)/beta-ARK1). Thus, we evaluated the effects of H(3)R activation directly on the following: 1) Ca(2+) current (I(Ca)) using the whole-cell patch-clamp technique; and 2) K(+)-induced exocytosis of endogenous dopamine. H(3)R activation attenuated both peak I(Ca) and dopamine exocytosis in PC12-H(3) but not in PC12-H(3)/beta-ARK1 cells. Moreover, a membrane permeable phosducin-like G betagamma scavenger also prevented the antiexocytotic effect of H(3)R activation. In contrast, the H(3)R-induced attenuation of cAMP accumulation and dopamine exocytosis in response to forskolin were the same in both PC12-H(3) and PC12-H(3)/beta-ARK1 cells. Our findings reveal that although G alpha(i) participates in the H(3)-mediated antiexocytotic effect when the adenylyl cyclase-cAMP-PKA pathway is stimulated, a direct G betagamma-induced inhibition of VOCC, resulting in an attenuation of I(Ca), plays a pivotal role in the H(3)R-mediated decrease in [Ca(2+)](i) and associated cardioprotective antiexocytotic effects. The discovery of this H(3)R-signaling step may offer new therapeutic approaches to cardiovascular diseases characterized by hyperadrenergic activity.
- Published
- 2008
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16. Mast cell renin and a local renin-angiotensin system in the airway: role in bronchoconstriction.
- Author
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Veerappan A, Reid AC, Estephan R, O'Connor N, Thadani-Mulero M, Salazar-Rodriguez M, Levi R, and Silver RB
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- Angiotensin II biosynthesis, Angiotensin II physiology, Animals, Bronchi metabolism, Bronchi physiology, Cell Degranulation physiology, Guinea Pigs, Humans, Lung enzymology, Lung metabolism, Lung physiology, Male, Mast Cells metabolism, Mast Cells physiology, Muscle Contraction physiology, Muscle, Smooth cytology, Muscle, Smooth metabolism, Muscle, Smooth physiology, Rats, Rats, Sprague-Dawley, Receptor, Angiotensin, Type 1 metabolism, Renin chemistry, Renin genetics, Renin physiology, Bronchi enzymology, Bronchoconstriction physiology, Mast Cells enzymology, Renin metabolism, Renin-Angiotensin System physiology
- Abstract
We previously reported that mast cells express renin, the rate-limiting enzyme in the renin-angiotensin cascade. We have now assessed whether mast cell renin release triggers angiotensin formation in the airway. In isolated rat bronchial rings, mast cell degranulation released enzyme with angiotensin I-forming activity blocked by the selective renin inhibitor BILA2157. Local generation of angiotensin (ANG II) from mast cell renin elicited bronchial smooth muscle contraction mediated by ANG II type 1 receptors (AT(1)R). In a guinea pig model of immediate type hypersensitivity, anaphylactic mast cell degranulation in bronchial rings resulted in ANG II-mediated constriction. As in rat bronchial rings, bronchoconstriction (BC) was inhibited by a renin inhibitor, an AT(1)R blocker, and a mast cell stabilizer. Anaphylactic release of renin, histamine, and beta-hexosaminidase from mast cells was confirmed in the effluent from isolated, perfused guinea pig lung. To relate the significance of this finding to humans, mast cells were isolated from macroscopically normal human lung waste tissue specimens. Sequence analysis of human lung mast cell RNA showed 100% homology between human lung mast cell renin and kidney renin between exons 1 and 10. Furthermore, the renin protein expressed in lung mast cells was enzymatically active. Our results demonstrate the existence of an airway renin-angiotensin system triggered by release of mast-cell renin. The data show that locally produced ANG II is a critical factor governing BC, opening the possibility for novel therapeutic targets in the management of airway disease.
- Published
- 2008
- Full Text
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17. Expression and biophysical analysis of two double-transmembrane domain-containing fragments from a yeast G protein-coupled receptor.
- Author
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Cohen LS, Arshava B, Estephan R, Englander J, Kim H, Hauser M, Zerbe O, Ceruso M, Becker JM, and Naider F
- Subjects
- Amino Acid Sequence, Amino Acids chemistry, Amino Acids metabolism, Biophysical Phenomena, Biophysics, Circular Dichroism, Cloning, Molecular, Inclusion Bodies metabolism, Micelles, Molecular Sequence Data, Mutation genetics, Nuclear Magnetic Resonance, Biomolecular, Peptide Fragments chemistry, Peptide Fragments genetics, Peptide Fragments metabolism, Receptors, G-Protein-Coupled chemistry, Receptors, G-Protein-Coupled genetics, Receptors, Mating Factor genetics, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics, Sodium Dodecyl Sulfate, Cell Membrane metabolism, Gene Expression, Receptors, G-Protein-Coupled metabolism, Receptors, Mating Factor chemistry, Receptors, Mating Factor metabolism, Saccharomyces cerevisiae chemistry, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins chemistry, Saccharomyces cerevisiae Proteins metabolism
- Abstract
Fragments of G protein-coupled receptors (GPCRs) are widely used as models to investigate these polytopic integral-membrane, signal-transducing molecules, but have proven difficult to prepare in quantities necessary for NMR analyses. We report on the biosynthesis of two double transmembrane (TM) containing fragments of Ste2p, the alpha-factor GPCR from the yeast Saccharomyces cerevisiae. Ste2p(G31-T110) [TM1-TM2] and Ste2p(R231-S339) [TM6-TM7-CT40] were expressed as TrpDeltaLE fusion proteins in Escherichia coli and released by CNBr cleavage. Expression yields were optimized using different strains and induction parameters, and by performing CNBr cleavage directly on inclusion bodies. Nonlabeled and uniformly labeled [15N]-TM1-TM2 and TM6-TM7-CT40, as well as uniformly labeled [15N,13C]-TM1-TM2 and TM1-TM2 selectively labeled with [15N-Ala], [15N-Phe], [15N-Leu], [15N-Ile], and [15N-Val] were prepared. Yields of target peptides with >95% homogeneity varied from 3 mg/L of fermentation ([15N]-TM6-TM7-CT40) to 20 mg/L (selectively labeled TM1-TM2). The high level biosynthesis and the efficient CNBr processing and purification yields allowed the initiation of a comprehensive biophysical analysis of TM1-TM2 and TM6-TM7-CT40. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis showed that TM1-TM2 was monomeric in this micellar environment, whereas TM6-TM7-CT40 migrated as a dimer. CD analysis indicated that TM1-TM2 was highly helical in SDS and 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-RAC-(1-glycerol)], but had a tendency to aggregate in dodecylphosphocholine micelles. Similar results were found with TM6-TM7-CT40. Conditions for NMR measurements were optimized, and both TM1-TM2 and TM6-TM7-CT40 exhibited more than 90% of the expected crosspeaks in the [15N,1H]-HSQC spectrum. These findings set the stage for the determination of the 3D structure of these large domains of a GPCR in micelles using high-resolution NMR.
- Published
- 2008
- Full Text
- View/download PDF
18. Immediate hypersensitivity elicits renin release from cardiac mast cells.
- Author
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Kano S, Tyler E, Salazar-Rodriguez M, Estephan R, Mackins CJ, Veerappan A, Reid AC, Silver RB, and Levi R
- Subjects
- Animals, Anti-Allergic Agents pharmacology, Anti-Asthmatic Agents pharmacology, Cell Degranulation drug effects, Cromolyn Sodium pharmacology, Guinea Pigs, Hypersensitivity, Immediate pathology, In Vitro Techniques, Male, Mast Cells drug effects, Mast Cells enzymology, Mast Cells physiology, Myocardium pathology, Norepinephrine immunology, Ovalbumin immunology, Ovalbumin pharmacology, Oxamic Acid analogs & derivatives, Oxamic Acid pharmacology, Pyridines pharmacology, Renin antagonists & inhibitors, Thiazoles pharmacology, beta-N-Acetylhexosaminidases metabolism, Cell Degranulation immunology, Hypersensitivity, Immediate immunology, Mast Cells immunology, Myocardium immunology, Renin immunology
- Abstract
Background: We recently reported that murine and cavian heart mast cells are a unique extrarenal source of renin. Ischemia/reperfusion releases this renin leading to local angiotensin formation and norepinephrine release. As mast cells are a primary target of hypersensitivity, we assessed whether anaphylactic mast cell degranulation also results in renin and norepinephrine release., Methods: Hearts isolated from presensitized guinea pigs were challenged with antigen., Results: Cardiac anaphylaxis was characterized by mast cell degranulation, evidenced by beta-hexosaminidase release and associated with renin and norepinephrine release. Mast cell stabilization with cromolyn or lodoxamide markedly attenuated the release of beta-hexosaminidase, renin and norepinephrine. Renin inhibition with BILA2157 did not affect mast cell degranulation, but attenuated norepinephrine release., Conclusions: Our findings disclose that immediate-type hypersensitivity elicits renin release from mast cells, activating a local renin-angiotensin system, thereby promoting norepinephrine release. As renin is stored in human heart mast cells, allergic reactions could initiate renin release, leading to local angiotensin formation and hyperadrenergic dysfunction., (Copyright 2007 S. Karger AG, Basel.)
- Published
- 2008
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19. Solution-state molecular structure of apo and oleate-liganded liver fatty acid-binding protein.
- Author
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He Y, Yang X, Wang H, Estephan R, Francis F, Kodukula S, Storch J, and Stark RE
- Subjects
- Animals, Ligands, Models, Molecular, Molecular Sequence Data, Nuclear Magnetic Resonance, Biomolecular, Oleic Acid chemistry, Protein Binding, Protein Structure, Secondary, Protein Structure, Tertiary, Rats, Sequence Homology, Amino Acid, Solutions pharmacology, Fatty Acid-Binding Proteins chemistry, Fatty Acid-Binding Proteins metabolism, Molecular Structure, Oleic Acid metabolism
- Abstract
Rat liver fatty acid-binding protein (LFABP) is distinctive among intracellular lipid-binding proteins (iLBPs): more than one molecule of long-chain fatty acid and a variety of diverse ligands can be bound within its large cavity, and in vitro lipid transfer to model membranes follows a mechanism that is diffusion-controlled rather than mediated by protein-membrane collisions. Because the apoprotein has proven resistant to crystallization, nuclear magnetic resonance spectroscopy offers a unique route to functionally informative comparisons of molecular structure and dynamics for LFABP in free (apo) and liganded (holo) forms. We report herein the solution-state structures determined for apo-LFABP at pH 6.0 and for holoprotein liganded to two oleates at pH 7.0, as well as the structure of the complex including locations of the ligands. 1H, 13C, and 15N resonance assignments revealed very similar types and locations of secondary structural elements for apo- and holo-LFABP as judged from chemical shift indices. The solution-state tertiary structures of the proteins were derived with the CNS/ARIA computational protocol, using distance and angular restraints based on 1H-1H nuclear Overhauser effects (NOEs), hydrogen-bonding networks, 3J(HNHA) coupling constants, intermolecular NOEs, and residual dipolar (NH) couplings. The holo-LFABP solution-state conformation is in substantial agreement with a previously reported X-ray structure [Thompson, J., Winter, N., Terwey, D., Bratt, J., and Banaszak, L. (1997) The crystal structure of the liver fatty acid-binding protein. A complex with two bound oleates, J. Biol. Chem. 272, 7140-7150], including the typical beta-barrel capped by a helix-turn-helix portal. In the solution state, the internally bound oleate has the expected U-shaped conformation and is tethered electrostatically, but the extended portal ligand can adopt a range of conformations based on the computationally refined structures, in contrast to the single conformation observed in the crystal structure. The apo-LFABP also has a well-defined beta-barrel structural motif typical of other members of the iLBP protein family, but the portal region that is thought to facilitate ligand entry and exit exhibits conformational variability and an unusual "open cap" orientation with respect to the barrel. These structural results allow us to propose a model in which ligand binding to LFABP occurs through conformational fluctuations that adjust the helix-turn-helix motif to open or close the top of the beta-barrel, and solvent accessibility to the protein cavity favors diffusion-controlled ligand transport.
- Published
- 2007
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20. Histamine H3-receptor signaling in cardiac sympathetic nerves: Identification of a novel MAPK-PLA2-COX-PGE2-EP3R pathway.
- Author
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Levi R, Seyedi N, Schaefer U, Estephan R, Mackins CJ, Tyler E, and Silver RB
- Subjects
- Animals, Dinoprostone physiology, Enzyme Activation, Exocytosis, Guinea Pigs, Male, Mitogen-Activated Protein Kinases metabolism, Norepinephrine metabolism, Prostaglandin-Endoperoxide Synthases metabolism, Subcellular Fractions, Heart innervation, Receptors, Histamine H3 physiology, Signal Transduction physiology, Sympathetic Nervous System physiology
- Abstract
We hypothesized that the histamine H(3)-receptor (H(3)R)-mediated attenuation of norepinephrine (NE) exocytosis from cardiac sympathetic nerves results not only from a Galpha(i)-mediated inhibition of the adenylyl cyclase-cAMP-PKA pathway, but also from a Gbetagamma(i)-mediated activation of the MAPK-PLA(2) cascade, culminating in the formation of an arachidonate metabolite with anti-exocytotic characteristics (e.g., PGE(2)). We report that in Langendorff-perfused guinea-pig hearts and isolated sympathetic nerve endings (cardiac synaptosomes), H(3)R-mediated attenuation of K(+)-induced NE exocytosis was prevented by MAPK and PLA(2) inhibitors, and by cyclooxygenase and EP(3)-receptor (EP(3)R) antagonists. Moreover, H(3)R activation resulted in MAPK phosphorylation in H(3)R-transfected SH-SY5Y neuroblastoma cells, and in PLA(2) activation and PGE(2) production in cardiac synaptosomes; H(3)R-induced MAPK phosphorylation was prevented by an anti-betagamma peptide. Synergism between H(3)R and EP(3)R agonists (i.e., imetit and sulprostone, respectively) suggested that PGE(2) may be a downstream effector of the anti-exocytotic effect of H(3)R activation. Furthermore, the anti-exocytotic effect of imetit and sulprostone was potentiated by the N-type Ca(2+)-channel antagonist omega-conotoxin GVIA, and prevented by an anti-Gbetagamma peptide. Our findings imply that an EP(3)R Gbetagamma(i)-induced decrease in Ca(2+) influx through N-type Ca(2+)-channels is involved in the PGE(2)/EP(3)R-mediated attenuation of NE exocytosis elicited by H(3)R activation. Conceivably, activation of the Gbetagamma(i) subunit of H(3)R and EP(3)R may also inhibit Ca(2+) entry directly, independent of MAPK intervention. As heart failure, myocardial ischemia and arrhythmic dysfunction are associated with excessive local NE release, attenuation of NE release by H(3)R activation is cardioprotective. Accordingly, this novel H(3)R signaling pathway may ultimately bear therapeutic significance in hyper-adrenergic states.
- Published
- 2007
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- View/download PDF
21. Selective labeling of a membrane peptide with 15N-amino acids using cells grown in rich medium.
- Author
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Englander J, Cohen L, Arshava B, Estephan R, Becker JM, and Naider F
- Subjects
- Amino Acids metabolism, Culture Media, Escherichia coli growth & development, Escherichia coli metabolism, Membrane Proteins biosynthesis, Nitrogen Isotopes, Nuclear Magnetic Resonance, Biomolecular, Peptides metabolism, Recombinant Fusion Proteins biosynthesis, Recombinant Fusion Proteins chemistry, Amino Acids analysis, Isotope Labeling methods, Membrane Proteins chemistry, Peptides chemistry
- Abstract
Nuclear magnetic resonance spectra of membrane proteins containing multiple transmembrane helices have proven difficult to resolve due to the redundancy of aliphatic and Ser/Thr residues in transmembrane domains and the low chemical shift dispersity exhibited by residues in alpha-helical structures. Although (13)C- and (15)N-labeling are useful tools in the biophysical analysis of proteins, selective labeling of individual amino acids has been used to help elucidate more complete structures and to probe ligand-protein interactions. In general, selective labeling has been performed in Escherichia coli expression systems using minimal media supplemented with a single labeled amino acid and nineteen other unlabeled amino acids and/or by using auxotrophs for specific amino acids. Growth in minimal media often results in low yields of cells or expression products. We demonstrate a method in which one labeled amino acid is added to a rich medium. These conditions resulted in high expression (> or =100 mg/L) of a test fusion protein and milligram quantities of the selectively labeled membrane peptide after cyanogen bromide cleavage to release the peptide from the fusion protein. High levels of (15)N incorporation and acceptable levels of cross-labeling into other amino acid residues of the peptide were achieved. Growth in rich media is a simple and convenient alternative to growth in supplemented minimal media and is readily applicable to the expression of proteins selectively labeled with specific amino acids., ((c) 2006 Wiley Periodicals, Inc.)
- Published
- 2006
- Full Text
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22. Biosynthesis and NMR analysis of a 73-residue domain of a Saccharomyces cerevisiae G protein-coupled receptor.
- Author
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Estephan R, Englander J, Arshava B, Samples KL, Becker JM, and Naider F
- Subjects
- Amino Acid Sequence, Chromatography, High Pressure Liquid, Cyanogen Bromide, Mass Spectrometry, Molecular Sequence Data, Nitrogen Isotopes, Nuclear Magnetic Resonance, Biomolecular, Protein Conformation, Protein Structure, Secondary, Protein Structure, Tertiary, Recombinant Fusion Proteins isolation & purification, Saccharomyces cerevisiae metabolism, Receptors, G-Protein-Coupled biosynthesis, Receptors, G-Protein-Coupled chemistry, Receptors, Mating Factor biosynthesis, Receptors, Mating Factor chemistry, Recombinant Fusion Proteins biosynthesis, Recombinant Fusion Proteins chemistry, Saccharomyces cerevisiae Proteins biosynthesis, Saccharomyces cerevisiae Proteins chemistry
- Abstract
The yeast Saccharomyces cerevisiae alpha-factor pheromone receptor (Ste2p) was used as a model G protein-coupled receptor (GPCR). A 73-mer multidomain fragment of Ste2p (residues 267-339) containing the third extracellular loop, the seventh transmembrane domain, and 40 residues of the cytosolic tail (E3-M7-24-T40) was biosynthesized fused to a carrier protein. The multidomain fusion protein (designated M7FP) was purified to near homogeneity as judged by HPLC and characterized by mass spectrometry. In minimal medium, 30-40 mg of M7FP were obtained per liter of culture. The 73-residue peptide was released from its carrier by CNBr and obtained in wild-type, (15)N, and (13)C/(15)N forms. The E3-M7-24-T40 peptide integrated into 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] and dodecylphosphocholine micelles at concentrations (200-500 microM) suitable for NMR investigations. HSQC experiments performed in organic solvents and detergent micelles on (15)N-labeled E3-M7-24-T40 showed a clear dispersion of the nitrogen-amide proton correlation cross-peaks indicative of a pure, uniformly labeled molecule that assumed a partially ordered structure. NOE connectivities, chemical shift indices, J-coupling analysis, and structural modeling suggested that in trifluoroethanol/water (1:1) helical subdomains existed in both the transmembrane and cytoslic tail of the multidomain peptide. Similar conclusions were reached in chloroform/methanol/water (4:4:1). As the cytosolic tail participates in down-regulation of Ste2p, the helical regions in the Ste2p tail may play a role in protein-protein interactions involved in endocytosis.
- Published
- 2005
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23. Sexual conjugation in yeast: A paradigm to study G-protein-coupled receptor domain structure.
- Author
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Naider F, Estephan R, Englander J, Suresh Babu VV, Arevalo E, Samples K, and Becker JM
- Subjects
- Amino Acid Sequence, Circular Dichroism, Conjugation, Genetic, Escherichia coli metabolism, Models, Molecular, Molecular Sequence Data, Protein Structure, Tertiary, Receptors, G-Protein-Coupled biosynthesis, Receptors, G-Protein-Coupled genetics, Recombinant Fusion Proteins biosynthesis, Recombinant Fusion Proteins chemistry, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics, Receptors, G-Protein-Coupled chemistry, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism
- Abstract
The yeast Saccharomyces cerevisiae undergoes cell fusion during sexual conjugation to form diploid cells. The haploids participating in this process signal each other through secreted peptide-mating factors (alpha-factor and a-factor) that are recognized by G-protein-coupled receptors. The receptor (Ste2p) recognizing the tridecapeptide alpha-factor is used as a model system in our laboratory to understand various aspects of peptide-receptor interactions and receptor structure. Using chemical procedures we have synthesized peptides corresponding to the seven transmembrane domains of Ste2p and studied their structures in membrane mimetic environments. Extension of these studies requires preparation of longer fragments of Ste2p. This article discusses strategies used in our laboratory to prepare peptides containing multiple domains of Ste2p. Data are presented on the use of chemical synthesis, biosynthesis, and native chemical ligation. Using biosynthetic approaches fusion proteins have been expressed that contain single receptor domains, two transmembrane domains connected by the contiguous loop, and the tail connected to the seventh transmembrane domain. Tens of milligrams of fusion protein were obtained per liter, and multimilligram quantities of the isotopically labeled target peptides were isolated using such biosynthetic approaches. Initial circular dichroism results on a chemically synthesized 64-residue peptide containing a portion of the cytosolic tail and the complete seventh transmembrane domain showed that the tail portion and the hydrophobic core of this peptide maintained individual conformational preferences. Moreover, this peptide could be studied at near millimolar concentrations in the presence of micelles and did not aggregate under these conditions. Thus, these constructs can be investigated using high-resolution nuclear magnetic resonance techniques, and the cytosolic tail of Ste2p can be used as a hydrophilic template to improve solubility of transmembrane peptides for structural analysis., (Copyright 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2004)
- Published
- 2004
- Full Text
- View/download PDF
24. Appearance of voltage-gated calcium channels following overexpression of ATPase II cDNA in neuronal HN2 cells.
- Author
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Chin G, El-Sherif Y, Jayman F, Estephan R, Wieraszko A, and Banerjee P
- Subjects
- Adenosine Triphosphatases genetics, Blotting, Western, Cadmium Chloride pharmacology, Calcium Channel Blockers pharmacology, Calcium Channels, N-Type genetics, Carrier Proteins metabolism, Cell Line, Hippocampus cytology, Hippocampus metabolism, Membrane Potentials drug effects, Membrane Proteins metabolism, Mitogen-Activated Protein Kinase 1 metabolism, Mitogen-Activated Protein Kinase 3, Mitogen-Activated Protein Kinases metabolism, Neuroblastoma, Neurons drug effects, Patch-Clamp Techniques, Protein Isoforms metabolism, Transfection, omega-Conotoxins pharmacology, Adenosine Triphosphatases metabolism, Calcium Channels, N-Type metabolism, DNA, Complementary metabolism, Neurons metabolism, Phospholipid Transfer Proteins
- Abstract
ATPase II (a Mg2+-ATPase) is also believed to harbor aminophospholipid translocase (APTL) activity, which is responsible for the translocation of phosphatidylserine (PS) from the outer leaflet of the plasma membrane to the inner. To test this hypothesis we overexpressed the mouse ATPase II cDNA in the neuronal HN2 cells. In addition to a dramatic increase in APTL activity, we also made the unexpected observation that expression of the mouse ATPase II cDNA from the vector pCMV6 resulted in the appearance of calcium current. Although the hybrid cell line HN2 or a line (HN2V32) obtained by expressing a heterologous gene from the same expression vector showed no calcium current, both ATPase II-overexpressing clones (HN2A12 and HN2A22) showed significant barium conductance. This current was due to calcium channels because it was blocked almost completely by 100 microM CdCl2 and it had a significant N-type component since it was blocked by 38.5% in the presence of 5 microM omega-conotoxin (omega-CTX). Western blot analysis using an antibody against the N-type calcium-channel alpha1B subunit revealed a dramatic increase in expression of this protein in the HN2A12 and HN2A22 cell lines. Our results suggest that ATPase II also harbors APTL activity. In view of the prior knowledge that APTL activity is inhibited by an increase in calcium, our results also suggest that APTL expression exerts a negative feedback regulation on itself by inducing expression of channels that cause an influx of calcium ions. The mechanism of this regulation could reveal important information on a possible cross-regulation between these two families of proteins in neuronal cells.
- Published
- 2003
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25. Apoptosis is associated with an inhibition of aminophospholipid translocase (APTL) in CNS-derived HN2-5 and HOG cells and phosphatidylserine is a recognition molecule in microglial uptake of the apoptotic HN2-5 cells.
- Author
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Das P, Estephan R, and Banerjee P
- Subjects
- Animals, Animals, Newborn, Apoptosis drug effects, Caspase 3, Caspases analysis, Cell Hypoxia physiology, DNA Fragmentation, Dose-Response Relationship, Drug, Humans, Immunohistochemistry, Mice, Mice, Inbred Strains, Microglia cytology, Microglia drug effects, Neurons cytology, Neurons drug effects, Phagocytosis drug effects, Phosphatidylserines pharmacology, Tumor Cells, Cultured, Apoptosis physiology, Carrier Proteins antagonists & inhibitors, Membrane Proteins antagonists & inhibitors, Microglia enzymology, Neurons enzymology, Phagocytosis physiology, Phosphatidylserines metabolism, Phospholipid Transfer Proteins, Phospholipids antagonists & inhibitors
- Abstract
A balance of the activities of multiple enzymes maintains the typical asymmetry of plasma membrane lipids in healthy cells. Such enzyme activities are (a) the aminophopholipid translocase (APTL) (a lipid-selective P-type ATPase that catalyzes inward movement of aminophospholipids), (b) the scramblase (a calcium-dependent and ATP-independent enzyme that catalyzes both inward and outward movement of lipids), (c) the floppase (an ATP-dependent enzyme that catalyzes only outward movement of lipids). Activation or inhibition of any one of these enzymes would lead to a loss in this asymmetry. Apoptosis-associated externalization of phophatidylserine has been reported for many different cell-types, but the exact mechanism involved in this loss of membrane asymmetry has not been identified yet. In this report we demonstrate concurrence of APTL inhibition, caspase-3 activation and apoptosis in CNS-derived HN2-5 and HOG cells. Additionally, we provide data to demonstrate that the phagocytosis of apoptotic, CNS-derived HN2-5 cells by the microglial cells requires recognition through phosphatidylserine (PS). Thus the enzyme aminopholipid translocase is inhibited during apoptosis of CNS-derived cells and this alone could account for the loss of plasma membrane lipid-asymmetry observed in these cells.
- Published
- 2003
- Full Text
- View/download PDF
26. Biosynthesis and biophysical analysis of domains of a yeast G protein-coupled receptor.
- Author
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Arevalo E, Estephan R, Madeo J, Arshava B, Dumont M, Becker JM, and Naider F
- Subjects
- Amino Acid Sequence, Biophysical Phenomena, Biophysics, Cell Membrane chemistry, Cell Membrane metabolism, Chromatography, High Pressure Liquid, Circular Dichroism, Cytoplasm metabolism, Electrophoresis, Polyacrylamide Gel, Escherichia coli metabolism, Fungal Proteins, Genetic Vectors, Magnetic Resonance Spectroscopy, Mass Spectrometry, Molecular Sequence Data, Nitrogen chemistry, Peptides chemistry, Plasmids metabolism, Protein Sorting Signals, Protein Structure, Tertiary, Receptors, Mating Factor, Receptors, Peptide chemistry, GTP-Binding Proteins chemistry, Receptors, Cell Surface biosynthesis, Receptors, Cell Surface chemistry, Transcription Factors
- Abstract
The alpha-factor receptor(Ste2p) is required for the sexual conjugation of the yeast Saccharomyces cerevisiae. Ste2p belongs to the G protein-coupled receptor (GPCR) family sharing a common heptahelical transmembrane structure. Biological synthesis of regions of Ste2p fused to a leader protein in Escherichia coli yielded milligram quantities of polypeptides that corresponded to one or two transmembrane domains. Fusion proteins were characterized by polyacrylamide gel electrophoresis, high performance liquid chromatography, and mass spectrometry. CD studies on the fusion proteins in trifluoroethanol:water mixtures indicated the existence of alpha-helical structures in the single- and the double-transmembrane domains. NMR experiments were performed in CDCl(3):CD(3)OH:H(2)O (4:4:1) on the (15)N-labeled single-transmembrane peptide showing a clear dispersion of the nitrogen-amide proton correlation cross peaks indicative of a high-purity, uniformly labeled molecule. The results indicate that single- and double-transmembrane domains of a GPCR can be produced by biosynthetic methods in quantities and purity sufficient for biophysical studies., (Copyright 2003 Wiley Periodicals, Inc.)
- Published
- 2003
- Full Text
- View/download PDF
27. The influence of water on the nanomechanical behavior of the plant biopolyester cutin as studied by AFM and solid-state NMR.
- Author
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Round AN, Yan B, Dang S, Estephan R, Stark RE, and Batteas JD
- Subjects
- Biomechanical Phenomena, Biophysical Phenomena, Biophysics, Biopolymers chemistry, Elasticity, Solanum lycopersicum chemistry, Magnetic Resonance Spectroscopy, Microscopy, Atomic Force, Rheology, Thermodynamics, Water chemistry, Membrane Lipids chemistry
- Abstract
Atomic force microscopy and solid-state nuclear magnetic resonance have been used to investigate the effect of water absorption on the nanoscale elastic properties of the biopolyester, cutin, isolated from tomato fruit cuticle. Changes in the humidity and temperature at which fruits are grown or stored can affect the plant surface (cuticle) and modify its susceptibility to pathogenic attack by altering the cuticle's rheological properties. In this work, atomic force microscopy measurements of the surface mechanical properties of isolated plant cutin have been made as a first step to probing the impact of water uptake from the environment on surface flexibility. A dramatic decrease in surface elastic modulus (from approximately 32 to approximately 6 MPa) accompanies increases in water content as small as 2 wt %. Complementary solid-state nuclear magnetic resonance measurements reveal enhanced local mobility of the acyl chain segments with increasing water content, even at molecular sites remote from the covalent cross-links that are likely to play a crucial role in cutin's elastic properties.
- Published
- 2000
- Full Text
- View/download PDF
28. Externalization of phosphatidylserine may not be an early signal of apoptosis in neuronal cells, but only the phosphatidylserine-displaying apoptotic cells are phagocytosed by microglia.
- Author
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Adayev T, Estephan R, Meserole S, Mazza B, Yurkow EJ, and Banerjee P
- Subjects
- Amoeba cytology, Animals, Annexin A5, Caspase 3, Caspases metabolism, Cells, Cultured, Flow Cytometry, Fluorescein-5-isothiocyanate, Fluorescent Dyes, Mice, Microglia cytology, Neurons metabolism, Oxygen metabolism, Staining and Labeling, Apoptosis physiology, Microglia physiology, Neurons physiology, Phagocytosis physiology, Phosphatidylserines metabolism
- Abstract
Earlier reports on nonneural cells have shown that the normally inner plasma membrane lipid, phosphatidylserine (PS), flip-flops out during the early stages of apoptosis, whereas DNA laddering and plasma membrane permeabilization occur during the late stages. In this study, the applicability of these parameters to CNS-derived neuronal cells was tested using hippocampal HN2-5, cells that undergo apoptosis under anoxia. Because such insults on unsynchronized cells, e.g., undifferentiated HN2-5 cells, result in both early and late apoptotic cells, we mechanically separated these cells into three fractions containing (a) cells that had completely detached during anoxia, (b) cells that remained weakly attached to the tissue culture dish and, once detached by trituration in serum-containing medium, did not reattach, and (c) cells that reattached in 2-3 h. Fractions a and b contained cells that showed pronounced DNA laddering, whereas cells in fraction c did not show any DNA laddering. Double staining with fluorescein isothiocyanate-annexin V (which binds to PS) and propidium iodide (which stains the DNA in cells with a permeable cell membrane) revealed that all cells in fraction a had a permeable cell membrane (propidium iodide-positive) and PS molecules in the outer leaflet of the plasma membrane (fluorescein isothiocyanate-annexin V-positive). By contrast, fractions b and c contained cells with no externalized PS molecules. Cells in fractions a-c also showed, respectively, 50-, 21-, and 5.5-fold higher caspase-3 (CPP32) activity than that in healthy control cells. All these results show that fraction a contained late apoptotic cells, which also had the highest CPP32 activity; cells in fraction b were at an intermediate stage, when DNA laddering had already occurred; and fraction c contained very early apoptotic cells, in which no DNA laddering had yet occurred. Therefore, in the neuronal HN2-5 cells, externalization of PS occurs only during the final stages of apoptosis when the cells have completely lost their adhesion properties. Further experiments showed that ameboid microglial cells isolated from neonatal mouse brain phagocytosed only the cells in fraction a. These results show that in CNS-derived HN2-5 cells, (a) PS externalization is a late apoptotic event and is concomitant with a complete loss of surface adhesion of the apoptotic cells and (b) PS externalization is crucial for microglial recognition and phagocytosis of the apoptotic HN2-5 cells. Thus, PS externalization could be causally linked to the final detachment of apoptotic neuronal cells, which in turn prepares them for rapid phagocytosis by microglia.
- Published
- 1998
- Full Text
- View/download PDF
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