47 results on '"Grauso M"'
Search Results
2. Novel putative nicotinic acetylcholine receptor subunit genes, D[alpha]5, D[alpha]6 and D[alpha]7, in Drosophila melanogaster identify a new and highly conserved target of adenosine deaminase acting on RNA-mediated A-to-I pre-mRNA editing
- Author
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Grauso, M., Reenan, R.A., Culetto, E., and Sattelle, D.B.
- Subjects
Nicotinic receptors -- Genetic aspects ,Genomes -- Analysis ,RNA -- Physiological aspects ,Enzymes -- Structure-activity relationship ,Biological sciences - Abstract
Genome analysis of the fruit fly Drosophila melanogaster reveals three new ligand-gated ion channel subunits with the characteristic YXCC motif found only in [alpha]-type nicotinic acetylcholine receptor subunits. The subunits are designated D[alpha]5, D[alpha]6, and D[alpha]7. Cloning of the D[alpha]5 embryonic cDNAs reveals an atypically large N terminus, part of which is without identifiable sequence motifs and is specified by two polymorphic alleles. Embryonic clones from D[alpha]6 contain multiple variant transcripts arising from alternative splicing as well as A-to-I pre-mRNA editing. Alternative splicing in D[alpha]6 involves exons encoding nAChR functional domains. The D[alpha]6 transcript is a target of the Drosophila adenosine de aminase acting on RNA (dADAR). This is the first case for any organism where a nAChR gene is the target of mRNA editing. Seven adenosines could be modified in the extracellular ligand-binding region of D[alpha]6, four of which are also edited in the D[alpha]6 ortholog in the tobacco budworm Heliothis virescens. The conservation of an editing site between the insect orders Diptera and Lepidoptera makes nAChR editing the most evolutionarily conserved invertebrate RNA editing site so far described. These findings add to our understanding of nAChR subunit diversity, which is increased and regulated by mechanisms acting at the genomic and mRNA levels.
- Published
- 2002
3. Molecular Cloning and Characterization of a cDNA Encoding AChE from Optic Lobe of Loligo Opalescences
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Talesa, V., primary, Grauso, M., additional, Arpagaus, M., additional, Giovannini, E., additional, and Rosi, G., additional
- Published
- 1998
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4. Peroxisome proliferator-activated receptor gamma (PPAR gamma) regulates lactase expression and activity in the gut
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Fumery, M. (Mathurin), Speca, S. (Silvia), Langlois, A. (Audrey), Davila, A-M. (Anne-Marie), Dubuquoy, C. (Caroline), Grauso, M. (Marta), Martin Mena, A. (Anthony), Figeac, M. (Martin), Metzger, D. (Daniel), Rousseaux, C. (Christel), Colombel, J-F. (Jean-Frederic), Dubuquoy, L. (Laurent), Desreumaux, P. (Pierre), Bertin, B. (Benjamin), Inserm, Université de Lille, CHU Lille, Lille Inflammation Research International Center - U 995 [LIRIC], Université Paris-Saclay, Physiopathologie des Maladies Inflammatoires de l'Intestin, Université Paris Saclay (COmUE), Plateforme de génomique fonctionnelle et structurelle [Lille], Institut de Génétique et de Biologie Moléculaire et Cellulaire [IGBMC], and Icahn School of Medicine at Mount Sinai [New York] [MSSM]
- Subjects
lactose intolerance ,intestinal epithelial cells ,hypolactasia ,PPARgamma ,lactase - Abstract
ReportPeroxisome proliferator-activated receptor gamma(PPARc) regulates lactase expression and activity inthe gutMathurin Fumery1,2,3,†, Silvia Speca1,2,†, Audrey Langlois1,2, Anne-Marie Davila4, Caroline Dubuquoy5,Marta Grauso4, Anthony Martin Mena1,2, Martin Figeac6, Daniel Metzger7, Christel Rousseaux5,Jean-Frederic Colombel8, Laurent Dubuquoy1,2, Pierre Desreumaux1,2,9& Benjamin Bertin1,2,*Abstract 9
- Published
- 2017
5. Overexpression of the cardiac Na + channel in Scn5a deficient mice using AAVs
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Doisne, N., primary, Grauso, M., additional, Mougenot, N., additional, Clergue, M., additional, Coulombe, A., additional, Charpentier, F., additional, Guicheney, P., additional, and Neyroud, N., additional
- Published
- 2017
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6. Control of Rhythm and Rate278Cx43 hemichannels in ventricular cardiomyocytes can be activated by an elevation of cytoplasmic Ca2+ through a CaM-dependent signaling cascade and are a potent contributor to cardiac arrhythmogenesis279Exploration of the dominant-negative effect of a SCN5A mutation in mice using adeno-associated viruses280Modeling susceptibility to drug-induced long qt syndrome with a panel of subject-specific induced pluripotent stem cells
- Author
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De Smet, M, primary, Doisne, N, primary, Stillitano, F, primary, Lissoni, A, additional, Wang, N, additional, Leybaert, L, additional, Grauso, M, additional, Mougenot, N, additional, Clergue, M, additional, Coulombe, A, additional, Guicheney, P, additional, Neyroud, N, additional, Kong, CW, additional, Hansen, J, additional, Funck-Brentano, C, additional, Jeziorowska, D, additional, Zahr, N, additional, Li, R, additional, Iyengar, R, additional, Hajjar, RJ, additional, and Hulot, JS, additional
- Published
- 2016
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7. 227 - Overexpression of the cardiac Na+ channel in Scn5a deficient mice using AAVs
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Doisne, N., Grauso, M., Mougenot, N., Clergue, M., Coulombe, A., Charpentier, F., Guicheney, P., and Neyroud, N.
- Published
- 2017
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- View/download PDF
8. Novel putative nicotinic acetylcholine receptor subunit genes, Dalpha5, Dalpha6 and Dalpha7, in Drosophila melanogaster identify a new and highly conserved target of adenosine deaminase acting on RNA-mediated A-to-I pre-mRNA editing
- Author
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Grauso, M, Reenan, R A, Culetto, E, and Sattelle, D B
- Subjects
Polymorphism, Genetic ,Adenosine Deaminase ,Molecular Sequence Data ,Gene Expression ,Exons ,Sequence Analysis, DNA ,Receptors, Nicotinic ,Evolution, Molecular ,Drosophila melanogaster ,Animals ,RNA ,Amino Acid Sequence ,RNA Editing ,Cloning, Molecular ,Sequence Alignment ,Phylogeny ,Research Article - Abstract
Genome analysis of the fruit fly Drosophila melanogaster reveals three new ligand-gated ion channel subunits with the characteristic YXCC motif found only in alpha-type nicotinic acetylcholine receptor subunits. The subunits are designated Dalpha5, Dalpha6, and Dalpha7. Cloning of the Dalpha5 embryonic cDNAs reveals an atypically large N terminus, part of which is without identifiable sequence motifs and is specified by two polymorphic alleles. Embryonic clones from Dalpha6 contain multiple variant transcripts arising from alternative splicing as well as A-to-I pre-mRNA editing. Alternative splicing in Dalpha6 involves exons encoding nAChR functional domains. The Dalpha6 transcript is a target of the Drosophila adenosine deaminase acting on RNA (dADAR). This is the first case for any organism where a nAChR gene is the target of mRNA editing. Seven adenosines could be modified in the extracellular ligand-binding region of Dalpha6, four of which are also edited in the Dalpha6 ortholog in the tobacco budworm Heliothis virescens. The conservation of an editing site between the insect orders Diptera and Lepidoptera makes nAChR editing the most evolutionarily conserved invertebrate RNA editing site so far described. These findings add to our understanding of nAChR subunit diversity, which is increased and regulated by mechanisms acting at the genomic and mRNA levels.
- Published
- 2002
9. Acetilcolinesterasi resistenti agli organofosfati:identificazione dei residui aminoacisici implicati nei meccanismi di resistenza. IV
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Talesa, Vincenzo Nicola, Grauso, M., Giovannini, Elvio, Romani, Rita, and Rosi, Gabriella
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acetylcholinesterase organophosphate oprganophosphate resistance - Published
- 1999
10. Molecular cloning and characterization of a cDNA encoding AChE from optic lobe of Loligo opalescences: a new member of the cholinesterase family resistant to diisopropyl fluorophosphate
- Author
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Talesa, Vincenzo Nicola, Giovannini, Elvio, Romani, Rita, Rosi, Gabriella, and Grauso, M.
- Published
- 1999
11. Cerebral venous thrombosis: Clinical, genetic and neuroradiological study
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Sparaco, M., primary, Addonizio, M.C., additional, Apice, G., additional, Ciannella, L., additional, D'Alessio, A., additional, D'Argenio, M.R., additional, Di Muccio, L., additional, Fucci, S., additional, Grauso, M., additional, Ricci, M., additional, and Feleppa, M., additional
- Published
- 2013
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12. Expression of a single dimeric membrane-bound acetylcholinesterase in Parascaris equorum
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Talesa, Vincenzo Nicola, Romani, Rita, Grauso, M., Rosi, Gabriella, and Giovannini, Elvio
- Published
- 1997
13. Acetylcholinesterase in tentacles of Octopus vulgaris. (Cephalopoda). Histochemical localization and characterization of a specific high-salt-soluble and heparin-soluble fraction of globular forms
- Author
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Talesa, Vincenzo Nicola, Grauso, M., Giovannini, E., Rosi, Gabriella, and Toutant, J. P.
- Published
- 1995
14. Human Genetic Polymorphisms in T1R1 and T1R3 Taste Receptor Subunits Affect Their Function
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Raliou, M., primary, Grauso, M., additional, Hoffmann, B., additional, Schlegel-Le-Poupon, C., additional, Nespoulous, C., additional, Debat, H., additional, Belloir, C., additional, Wiencis, A., additional, Sigoillot, M., additional, Preet Bano, S., additional, Trotier, D., additional, Pernollet, J.-C., additional, Montmayeur, J.-P., additional, Faurion, A., additional, and Briand, L., additional
- Published
- 2011
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15. Expression of a single dimeric membrane-bound acetylcholinesterase in Parascaris equorum
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TALESA, V., primary, ROMANI, R., additional, GRAUSO, M., additional, ROSI, G., additional, and GIOVANNINI, E., additional
- Published
- 1997
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16. Solubilization, molecular forms, purification and substrate specificity of two acetylcholinesterases in the medicinal leech (Hirudo medicinalis)
- Author
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Talesa, V, primary, Grauso, M, additional, Giovannini, E, additional, Rosi, G, additional, and Toutant, J P, additional
- Published
- 1995
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17. Four acetylcholinesterase genes in the nematode Caenorhabditis elegans
- Author
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Arpagaus, M., Combes, D., Culetto, E., Grauso, M., Fedon, Y., Romani, R., and Toutant, J.-P.
- Published
- 1998
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18. Functional genomics of ionotropic acetylcholine receptors in Caenorhabditis elegans and Drosophila melanogaster
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Db, Sattelle, Culetto E, Grauso M, Valérie RAYMOND, Cj, Franks, and Towers P
19. Acetylcholinesterase in tentacles of Octopus vulgaris (Cephalopoda). Histochemical localization and characterization of a specific high salt-soluble and heparin-soluble fraction of globular forms
- Author
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Talesa, V., Grauso, M., Giovannini, E., and Rosi, G.
- Published
- 1995
- Full Text
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20. A dimeric mutant of human pancreatic ribonuclease with selective cytotoxicity toward malignant cells
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Daniela Spalletti-Cernia, Giuseppe D'Alessio, Renata Piccoli, Michela Grauso, Jaroslav Cinátl, Carmen Monaco, Sonia Di Gaetano, Claudia De Lorenzo, Paolo Laccetti, Josef Matoušek, Piccoli, R., DI GAETANO, S., DE LORENZO, C., Grauso, M., Monaco, C., SPALLETTI CERNIA, D., Laccetti, Paolo, Cinatl, J., Matousek, J., D'Alessio, G., Piccoli, Renata, and Laccetti, P.
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Male ,Cell Survival ,RNase P ,Molecular Sequence Data ,Mutant ,Antineoplastic Agents ,Apoptosis ,3T3 cells ,Mice ,Ribonucleases ,Tumor Cells, Cultured ,medicine ,Animals ,Humans ,Cytotoxic T cell ,Amino Acid Sequence ,Thyroid Neoplasms ,Multidisciplinary ,Sequence Homology, Amino Acid ,biology ,Seminal Vesicles ,RNA ,Biological activity ,3T3 Cells ,Ribonuclease, Pancreatic ,Biological Sciences ,Molecular biology ,Recombinant Proteins ,Cell Transformation, Neoplastic ,medicine.anatomical_structure ,Mutagenesis, Site-Directed ,biology.protein ,Cattle ,Pancreatic ribonuclease ,Drug Screening Assays, Antitumor ,Dimerization ,Sequence Alignment - Abstract
Monomeric human pancreatic RNase, devoid of any biological activity other than its RNA degrading ability, was engineered into a dimeric protein with a cytotoxic action on mouse and human tumor cells, but lacking any appreciable toxicity on mouse and human normal cells. This dimeric variant of human pancreas RNase selectively sensitizes to apoptotic death cells derived from a human thyroid tumor. Because of its selectivity for tumor cells, and because of its human origin, this protein represents a potentially very attractive, novel tool for anticancer therapy.
- Published
- 1999
21. Dietary Bovine Lactoferrin Reduces the Deleterious Effects of Lipopolysaccharide Injection on Mice Intestine.
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Blais A, Takakura N, Grauso M, Puel-Artero C, Blachier F, and Lan A
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- Animals, Female, Mice, Cattle, Permeability, Inflammation prevention & control, Lactoferrin pharmacology, Lactoferrin administration & dosage, Lipopolysaccharides, Dietary Supplements, Tumor Necrosis Factor-alpha metabolism, Jejunum drug effects, Jejunum metabolism, Intestinal Mucosa metabolism, Intestinal Mucosa drug effects, Colon drug effects, Colon metabolism, Colon pathology
- Abstract
Background/objectives: Injection of lipopolysaccharides (LPS) in experimental models induces a systemic inflammatory response that is associated with deleterious effects on intestinal morphology and physiology. In this study, we have studied in female mice the effects of dietary supplementation with bovine lactoferrin (bLF) given before intraperitoneal injection of LPS on jejunum and colon., Methods: The first study evaluated the efficiency of different bLF and LPS concentrations to determine the optimal experimental conditions. For the second study mice were fed with 1% bLF before the LPS challenge (3 mg/kg body weight). Plasmatic markers of inflammation, intestinal morphology, permeability, and expression of genes related to epithelial differentiation, epithelial barrier function and intestinal inflammation in both small intestine and colon were evaluated., Results: bLF ingestion before the LPS challenge reduced the TNF-α circulating concentration, compared to control animals. This decrease in plasma TNF-α was correlated with improved intestinal permeability. The morphology of jejunal epithelium, which was affected by LPS challenge, was partly maintained by bLF. Measurement of the expression of genes encoding proteins involved in epithelial differentiation, intestinal inflammation, and epithelial barrier function suggests an overall protective effect of bLF against the adverse effects of LPS in the jejunum. In the colon, the effects of bLF ingestion on the subsequent LPS challenge, although protective, remain different when compared with those observed on jejunum., Conclusions: Taken together, our data indicate that bLF dietary supplementation does have a protective effect on the deleterious intestinal alterations induced by LPS systemic inflammation.
- Published
- 2024
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22. Identification of the major immune differences in severe asthmatic children according to their atopic dermatitis status.
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Lezmi G, Poirault C, Grauso M, Dietrich C, Adel-Patient K, and Leite-de-Moraes M
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- Humans, Child, Cytokines, Th17 Cells, Bronchoalveolar Lavage Fluid, Dermatitis, Atopic, Asthma
- Abstract
Severe asthma (SA) affects 2% to 5% of asthmatic children. Atopic dermatitis can affect up to 34% of children with SA (cwSA). Atopic dermatitis and asthma share common genetic and immunological features. However, not all children with SA suffer from AD, and it remains unclear whether the overall immune profiles of these children are similar. In this study, seventeen cwSA (9.8 [7.1-13.2] years; seven with and ten without AD) were enrolled. Bronchoalveolar lavage (BAL) and blood samples were collected from these patients. Seventy-three cytokines/chemokines and distinct immune T cell populations were evaluated in blood and BAL. We found that BAL and blood immune profiles of cwSA with and without AD were globally similar. However, specific differences were observed, namely lower frequency of Tc2, Th17 and IL-17-producing mucosal associated invariant T (MAIT-17) cells and higher CD8/CD4 ratio and IL-22 concentrations in BAL and of CCL19 concentrations in plasma from cwSA with AD. Further, in contrast with cwSA without AD, we found a positive correlation between a set of plasma cytokines and almost all cytokines in BAL in cwSA with AD. In conclusion, this study shows the major immune differences between cwSA with and without AD in BAL and blood suggesting that distinct endotypes may be implicated in the inflammatory responses observed in these pediatric patients., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)
- Published
- 2024
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23. Assessment of local and systemic signature of eosinophilic esophagitis (EoE) in children through multi-omics approaches.
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Adel-Patient K, Campeotto F, Grauso M, Guillon B, Moroldo M, Venot E, Dietrich C, Machavoine F, Castelli FA, Fenaille F, Molina TJ, Barbet P, Delacourt C, Leite-de-Moraes M, and Lezmi G
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- Humans, Child, Multiomics, Cytokines metabolism, Adaptive Immunity, Biomarkers, Eosinophilic Esophagitis
- Abstract
Background: Eosinophilic oesophagitis (EoE) is a chronic food allergic disorder limited to oesophageal mucosa whose pathogenesis is still only partially understood. Moreover, its diagnosis and follow-up need repeated endoscopies due to absence of non-invasive validated biomarkers. In the present study, we aimed to deeply describe local immunological and molecular components of EoE in well-phenotyped children, and to identify potential circulating EoE-biomarkers., Methods: Blood and oesophageal biopsies were collected simultaneously from French children with EoE (n=17) and from control subjects (n=15). Untargeted transcriptomics analysis was performed on mRNA extracted from biopsies using microarrays. In parallel, we performed a comprehensive analysis of immune components on both cellular and soluble extracts obtained from both biopsies and blood, using flow cytometry. Finally, we performed non-targeted plasma metabolomics using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS). Uni/multivariate supervised and non-supervised statistical analyses were then conducted to identify significant and discriminant components associated with EoE within local and/or systemic transcriptomics, immunologic and metabolomics datasets. As a proof of concept, we conducted multi-omics data integration to identify a plasmatic signature of EoE., Results: French children with EoE shared the same transcriptomic signature as US patients. Network visualization of differentially expressed (DE) genes highlighted the major dysregulation of innate and adaptive immune processes, but also of pathways involved in epithelial cells and barrier functions, and in perception of chemical stimuli. Immune analysis of biopsies highlighted EoE is associated with dysregulation of both type (T) 1, T2 and T3 innate and adaptive immunity, in a highly inflammatory milieu. Although an immune signature of EoE was found in blood, untargeted metabolomics more efficiently discriminated children with EoE from control subjects, with dysregulation of vitamin B6 and various amino acids metabolisms. Multi-blocks integration suggested that an EoE plasma signature may be identified by combining metabolomics and cytokines datasets., Conclusions: Our study strengthens the evidence that EoE results from alterations of the oesophageal epithelium associated with altered immune responses far beyond a simplistic T2 dysregulation. As a proof of concept, combining metabolomics and cytokines data may provide a set of potential plasma biomarkers for EoE diagnosis, which needs to be confirmed on a larger and independent cohort., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Adel-Patient, Campeotto, Grauso, Guillon, Moroldo, Venot, Dietrich, Machavoine, Castelli, Fenaille, Molina, Barbet, Delacourt, Leite-de-Moraes and Lezmi.)
- Published
- 2023
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24. Route of Sensitization to Peanut Influences Immune Cell Recruitment at Various Mucosal Sites in Mouse: An Integrative Analysis.
- Author
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Briard M, Guinot M, Grauso M, Guillon B, Hazebrouck S, Bernard H, Bouchaud G, Michel ML, and Adel-Patient K
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- Allergens, Animals, Disease Models, Animal, Immunity, Innate, Lymphocytes, Mice, Mice, Inbred BALB C, Arachis, Food Hypersensitivity
- Abstract
Symptom occurrence at the first ingestion suggests that food allergy may result from earlier sensitization via non-oral routes. We aimed to characterize the cellular populations recruited at various mucosal and immune sites after experimental sensitization though different routes. BALB/cJ mice were exposed to a major allergenic food (peanut) mixed with cholera toxin via the intra-gastric (i.g.), respiratory, cutaneous, or intra-peritoneal (i.p.) route. We assessed sensitization and elicitation of the allergic reaction and frequencies of T cells, innate lymphoid cells (ILC), and inflammatory and dendritic cells (DC) in broncho-alveolar lavages (BAL), lungs, skin, intestine, and various lymph nodes. All cellular data were analyzed through non-supervised and supervised uni/multivariate analysis. All exposure routes, except cutaneous, induced sensitization, but intestinal allergy was induced only in i.g.- and i.p.-exposed mice. Multivariate analysis of all cellular constituents did not discriminate i.g. from control mice. Conversely, respiratory-sensitized mice constituted a distinct cluster, characterized by high local inflammation and immune cells recruitment. Those mice also evidenced changes in ILC frequencies at distant site (intestine). Despite absence of sensitization, cutaneous-exposed mice evidenced comparable changes, albeit less intense. Our study highlights that the initial route of sensitization to a food allergen influences the nature of the immune responses at various mucosal sites. Interconnections of mucosal immune systems may participate in the complexity of clinical manifestations as well as in the atopic march.
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- 2022
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25. A Comprehensive Analysis of Immune Constituents in Blood and Bronchoalveolar Lavage Allows Identification of an Immune Signature of Severe Asthma in Children.
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Adel-Patient K, Grauso M, Abou-Taam R, Guillon B, Dietrich C, Machavoine F, Briard M, Garcelon N, Faour H, Neuraz A, Delacourt C, Molina TJ, Leite-de-Moraes M, and Lezmi G
- Subjects
- Child, Female, Humans, Male, Asthma blood, Asthma immunology, Biomarkers analysis, Bronchoalveolar Lavage Fluid immunology
- Abstract
Background: Targeted approaches may not account for the complexity of inflammation involved in children with severe asthma (SA), highlighting the need to consider more global analyses. We aimed to identify sets of immune constituents that distinguish children with SA from disease-control subjects through a comprehensive analysis of cells and immune constituents measured in bronchoalveolar lavage (BAL) and blood., Methods: Twenty children with SA and 10 age-matched control subjects with chronic respiratory disorders other than asthma were included. Paired blood and BAL samples were collected and analyzed for a large set of cellular (eosinophils, neutrophils, and subsets of lymphocytes and innate lymphoid cells) and soluble (chemokines, cytokines, and total antibodies) immune constituents. First, correlations of all immune constituents between BAL and blood and with demographic and clinical data were assessed (Spearman correlations). Then, all data were modelled using supervised multivariate analyses (partial least squares discriminant analysis, PLS-DA) to identify immune constituents that significantly discriminate between SA and control subjects. Univariate analyses were performed (Mann-Whitney tests) and then PLS-DA and univariate analyses were combined to identify the most discriminative and significant constituents., Results: Concentrations of soluble immune constituents poorly correlated between BAL and blood. Certain constituents correlated with age or body mass index and, in asthmatics, with clinical symptoms, such as the number of exacerbations in the previous year, asthma control test score, or forced expiratory volume. Multivariate supervised analysis allowed construction of a model capable of distinguishing children with SA from control subjects with 80% specificity and 100% sensitivity. All immune constituents contributed to the model but some, identified by variable-important-in-projection values > 1 and p < 0.1, contributed more strongly, including BAL Th1 and Th2 cells and eosinophilia, CCL26 (Eotaxin 3), IgA and IL-19 concentrations in blood. Blood concentrations of IL-26, CCL13, APRIL, and Pentraxin-3 may also help in the characterization of SA., Conclusions: The analysis of a large set of immune constituents may allow the identification of a biological immune signature of SA. Such an approach may provide new leads for delineating the pathogenesis of SA in children and identifying new targets for its diagnosis, prediction, and personalized treatment., Competing Interests: GL reports personal fees from novartis pharma, personal fees from Astra zeneca, personal fees from YSSUP research, during the conduct of the study; personal fees from DBV technologies, personal fees from Aimune therapeutics, outside the submitted work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Adel-Patient, Grauso, Abou-Taam, Guillon, Dietrich, Machavoine, Briard, Garcelon, Faour, Neuraz, Delacourt, Molina, Leite-de-Moraes and Lezmi.)
- Published
- 2021
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26. Immune signatures distinguish frequent from non-frequent exacerbators among children with severe asthma.
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Adel-Patient K, Grauso M, Abou-Taam R, Guillon B, Dietrich C, Machavoine F, Garcelon N, Briard M, Faour H, Neuraz A, Delacourt C, Molina TJ, Leite-de-Moraes M, and Lezmi G
- Subjects
- Child, Disease Progression, Humans, Asthma diagnosis, Asthma epidemiology, Pulmonary Disease, Chronic Obstructive
- Published
- 2021
- Full Text
- View/download PDF
27. In vivo Dominant-Negative Effect of an SCN5A Brugada Syndrome Variant.
- Author
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Doisne N, Grauso M, Mougenot N, Clergue M, Souil C, Coulombe A, Guicheney P, and Neyroud N
- Abstract
Loss-of-function mutations in the cardiac Na
+ channel α-subunit Nav 1.5, encoded by SCN5A , cause Brugada syndrome (BrS), a hereditary disease characterized by sudden cardiac death due to ventricular fibrillation. We previously evidenced in vitro the dominant-negative effect of the BrS Nav 1.5-R104W variant, inducing retention of wild-type (WT) channels and leading to a drastic reduction of the resulting Na+ current ( INa ). To explore this dominant-negative effect in vivo , we created a murine model using adeno-associated viruses (AAVs)., Methods: Due to the large size of SCN5A , a dual AAV vector strategy was used combining viral DNA recombination and trans -splicing. Mice were injected with two AAV serotypes capsid 9: one packaging the cardiac specific troponin-T promoter, the 5' half of hSCN5A cDNA, a splicing donor site and a recombinogenic sequence; and another packaging the complementary recombinogenic sequence, a splicing acceptor site, the 3' half of hSCN5A cDNA fused to the gfp gene sequence, and the SV40 polyA signal. Eight weeks after AAV systemic injection in wild-type (WT) mice, echocardiography and ECG were recorded and mice were sacrificed. The full-length hSCN5A-gfp expression was assessed by western blot and immunohistochemistry in transduced heart tissues and the Na+ current was recorded by the patch-clamp technique in isolated adult GFP-expressing heart cells., Results: Almost 75% of the cardiomyocytes were transduced in hearts of mice injected with hNav 1.5 and ∼30% in hNav 1.5-R104W overexpressing tissues. In ventricular mice cardiomyocytes expressing R104W mutant channels, the endogenous INa was significantly decreased. Moreover, overexpression of R104W channels in normal hearts led to a decrease of total Nav 1.5 expression. The R104W mutant also induced a slight dilatation of mice left ventricles and a prolongation of RR interval and P-wave duration in transduced mice. Altogether, our results demonstrated an in vivo dominant-negative effect of defective R104W channels on endogenous ones., Conclusion: Using a trans -splicing and viral DNA recombination strategy to overexpress the Na+ channel in mouse hearts allowed us to demonstrate in vivo the dominant-negative effect of a BrS variant identified in the N-terminus of Nav 1.5., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Doisne, Grauso, Mougenot, Clergue, Souil, Coulombe, Guicheney and Neyroud.)- Published
- 2021
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28. Hyperosmolar environment and intestinal epithelial cells: impact on mitochondrial oxygen consumption, proliferation, and barrier function in vitro.
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Grauso M, Lan A, Andriamihaja M, Bouillaud F, and Blachier F
- Subjects
- Caco-2 Cells, Enterocytes physiology, Humans, Interleukin-8 metabolism, Intestinal Mucosa metabolism, Intestinal Mucosa physiopathology, Signal Transduction, Cell Proliferation, Enterocytes metabolism, Mitochondria metabolism, Osmolar Concentration, Oxygen Consumption
- Abstract
The aim of the present study was to elucidate the in vitro short-term (2-h) and longer-term (24-h) effects of hyperosmolar media (500 and 680 mOsm/L) on intestinal epithelial cells using the human colonocyte Caco-2 cell line model. We found that a hyperosmolar environment slowed down cell proliferation compared to normal osmolarity (336 mOsm/L) without inducing cell detachment or necrosis. This was associated with a transient reduction of cell mitochondrial oxygen consumption, increase in proton leak, and decrease in intracellular ATP content. The barrier function of Caco-2 monolayers was also transiently affected since increased paracellular apical-to-basal permeability and modified electrolyte permeability were measured, allowing partial equilibration of the trans-epithelial osmotic difference. In addition, hyperosmotic stress induced secretion of the pro-inflammatory cytokine IL-8. By measuring expression of genes involved in energy metabolism, tight junction forming, electrolyte permeability and intracellular signaling, different response patterns to hyperosmotic stress occurred depending on its intensity and duration. These data highlight the potential impact of increased luminal osmolarity on the intestinal epithelium renewal and barrier function and point out some cellular adaptive capacities towards luminal hyperosmolar environment.
- Published
- 2019
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29. Mucosal healing progression after acute colitis in mice.
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Vidal-Lletjós S, Andriamihaja M, Blais A, Grauso M, Lepage P, Davila AM, Gaudichon C, Leclerc M, Blachier F, and Lan A
- Subjects
- Animals, Cell Movement, Cell Proliferation, Colitis, Ulcerative chemically induced, Colitis, Ulcerative pathology, Colon cytology, Colon drug effects, Dextran Sulfate toxicity, Disease Models, Animal, Humans, Inflammation Mediators immunology, Inflammation Mediators metabolism, Intestinal Mucosa cytology, Intestinal Mucosa drug effects, Male, Mice, Mice, Inbred C57BL, Permeability, RNA, Ribosomal, 16S, Colitis, Ulcerative immunology, Colon pathology, Gastrointestinal Microbiome immunology, Intestinal Mucosa pathology, Regeneration immunology
- Abstract
Background: Mucosal healing has become a therapeutic goal to achieve stable remission in patients with inflammatory bowel diseases. To achieve this objective, overlapping actions of complex cellular processes, such as migration, proliferation, and differentiation, are required. These events are longitudinally and tightly controlled by numerous factors including a wide range of distinct regulatory proteins. However, the sequence of events associated with colon mucosal repair after colitis and the evolution of the luminal content characteristics during this process have been little studied., Aim: To document the evolution of colon mucosal characteristics during mucosal healing using a mouse model with chemically-induced colitis., Methods: C57BL/6 male mice were given 3.5% dextran sodium sulfate (DSS) in drinking water for 5 d. They were euthanized 2 (day 7), 5 (day 10), 8 (day 13), and 23 (day 28) d after DSS removal. The colonic luminal environment and epithelial repair processes during the inflammatory flare and colitis resolution were analyzed with reference to a non-DSS treated control group, euthanized at day 0. Epithelial repair events were assessed histo-morphologically in combination with functional permeability tests, expression of key inflammatory and repairing factors, and evaluation of colon mucosa-adherent microbiota composition by 16S rRNA sequencing., Results: The maximal intensity of colitis was concomitant with maximal alterations of intestinal barrier function and histological damage associated with goblet cell depletion in colon mucosa. It was recorded 2 d after termination of the DSS-treatment, followed by a progressive return to values similar to those of control mice. Although signs of colitis were severe (inflammatory cell infiltrate, crypt disarray, increased permeability) and associated with colonic luminal alterations (hyperosmolarity, dysbiosis, decrease in short-chain fatty acid content), epithelial healing processes were launched early during the inflammatory flare with increased gene expression of certain key epithelial repair modulators, including transforming growth factor-β, interleukin (Il)-15, Il-22, Il-33, and serum amyloid A. Whereas signs of inflammation progressively diminished, luminal colonic environment alterations and microscopic abnormalities of colon mucosa persisted long after colitis induction., Conclusion: This study shows that colon repair can be initiated in the context of inflamed mucosa associated with alterations of the luminal environment and highlights the longitudinal involvement of key modulators., Competing Interests: Conflict-of-interest statement: The authors have nothing to disclose.
- Published
- 2019
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30. Dietary Protein Intake Level Modulates Mucosal Healing and Mucosa-Adherent Microbiota in Mouse Model of Colitis.
- Author
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Vidal-Lletjós S, Andriamihaja M, Blais A, Grauso M, Lepage P, Davila AM, Viel R, Gaudichon C, Leclerc M, Blachier F, and Lan A
- Subjects
- Animal Feed, Animals, Diet, Disease Models, Animal, Intestinal Mucosa drug effects, Intestinal Mucosa microbiology, Male, Mice, Mice, Inbred C57BL, Colitis chemically induced, Colitis drug therapy, Dietary Proteins administration & dosage, Dietary Proteins pharmacology, Gastrointestinal Microbiome drug effects, Intestinal Mucosa pathology
- Abstract
Mucosal healing after an inflammatory flare is associated with lasting clinical remission. The aim of the present work was to evaluate the impact of the amount of dietary protein on epithelial repair after an acute inflammatory episode. C57BL/6 DSS-treated mice received isocaloric diets with different levels of dietary protein: 14% (P14), 30% (P30) and 53% (P53) for 3 ( day 10 ), 6 ( day 13 ) and 21 ( day 28 ) days after the time of colitis maximal intensity. While the P53 diet worsened the DSS- induced inflammation both in intensity and duration, the P30 diet, when compared to the P14 diet, showed a beneficial effect during the epithelial repair process by accelerating inflammation resolution, reducing colonic permeability and increasing epithelial repair together with epithelial hyperproliferation. Dietary protein intake also impacted mucosa-adherent microbiota composition after inflammation since P30 fed mice showed increased colonization of butyrate-producing genera throughout the resolution phase. This study revealed that in our colitis model, the amount of protein in the diet modulated mucosal healing, with beneficial effects of a moderately high-protein diet, while very high-protein diet displayed deleterious effects on this process.
- Published
- 2019
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31. Proanthocyanidin-containing polyphenol extracts from fruits prevent the inhibitory effect of hydrogen sulfide on human colonocyte oxygen consumption.
- Author
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Andriamihaja M, Lan A, Beaumont M, Grauso M, Gotteland M, Pastene E, Cires MJ, Carrasco-Pozo C, Tomé D, and Blachier F
- Subjects
- Cell Line, Tumor, Colon cytology, Humans, Plant Extracts chemistry, Proanthocyanidins chemistry, Colon metabolism, Fruit chemistry, Hydrogen Sulfide metabolism, Oxygen Consumption drug effects, Plant Extracts pharmacology, Polyphenols pharmacology, Proanthocyanidins pharmacology
- Abstract
Hydrogen sulfide (H
2 S), a metabolic end product synthesized by the microbiota from L-cysteine, has been shown to act at low micromolar concentration as a mineral oxidative substrate in colonocytes while acting as an inhibitor of oxygen consumption at higher luminal concentrations (65 µM and above). From the previous works showing that polyphenols can bind volatile sulfur compounds, we hypothesized that different dietary proanthocyanidin-containing polyphenol (PACs) plant extracts might modulate the inhibitory effect of H2 S on colonocyte respiration. Using the model of human HT-29 Glc-/+ cell colonocytes, we show here that pre-incubation of 65 µM of the H2 S donor NaHS with the different polyphenol extracts markedly reduced the inhibitory effect of NaHS on colonocyte oxygen consumption. Our studies on HT-29 Glc-/+ cell respiration performed in the absence or the presence of PACs reveal rapid binding of H2 S with the sulfide-oxidizing unit and slower binding of H2 S to the cytochrome c oxidase (complex IV of the respiratory chain). Despite acute inhibition of colonocyte respiration, no measurable effect of NaHS on paracellular permeability was recorded after 24 h treatment using the Caco-2 colonocyte monolayer model. The results are discussed in the context of the binding of excessive bacterial metabolites by unabsorbed dietary compounds and of the capacity of colonocytes to adapt to changing luminal environment.- Published
- 2018
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32. Peroxisome proliferator-activated receptor gamma (PPARγ) regulates lactase expression and activity in the gut.
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Fumery M, Speca S, Langlois A, Davila AM, Dubuquoy C, Grauso M, Martin Mena A, Figeac M, Metzger D, Rousseaux C, Colombel JF, Dubuquoy L, Desreumaux P, and Bertin B
- Subjects
- Aniline Compounds pharmacology, Aniline Compounds therapeutic use, Animals, Caco-2 Cells, Chromatin Immunoprecipitation, Diet, Humans, Lactase genetics, Lactose metabolism, Lactose Intolerance drug therapy, Male, Mice, Mice, Inbred C57BL, Microscopy, Fluorescence, Mutagenesis, Site-Directed, PPAR gamma agonists, PPAR gamma genetics, Phenylpropionates pharmacology, Phenylpropionates therapeutic use, Promoter Regions, Genetic, Rats, Rats, Sprague-Dawley, Transcription, Genetic drug effects, Intestine, Small metabolism, Lactase metabolism, PPAR gamma metabolism
- Abstract
Lactase (LCT) deficiency affects approximately 75% of the world's adult population and may lead to lactose malabsorption and intolerance. Currently, the regulation of LCT gene expression remains poorly known. Peroxisome proliferator activator receptorγ (PPARγ) is a key player in carbohydrate metabolism. While the intestine is essential for carbohydrate digestion and absorption, the role of PPARγ in enterocyte metabolic functions has been poorly investigated. This study aims at characterizing PPARγ target genes involved in intestinal metabolic functions. In microarray analysis, the LCT gene was the most upregulated by PPARγ agonists in Caco-2 cells. We confirmed that PPARγ agonists were able to increase the expression and activity of LCT both in vitro and in vivo in the proximal small bowel of rodents. The functional response element activated by PPARγ was identified in the promoter of the human LCT gene. PPARγ modulation was able to improve symptoms induced by lactose-enriched diet in weaned rats. Our results demonstrate that PPARγ regulates LCT expression, and suggest that modulating intestinal PPARγ activity might constitute a new therapeutic strategy for lactose malabsorption., (© 2017 The Authors. Published under the terms of the CC BY 4.0 license.)
- Published
- 2017
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33. Changes in the Luminal Environment of the Colonic Epithelial Cells and Physiopathological Consequences.
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Blachier F, Beaumont M, Andriamihaja M, Davila AM, Lan A, Grauso M, Armand L, Benamouzig R, and Tomé D
- Subjects
- Animals, Energy Metabolism, Humans, Hydrogen-Ion Concentration, Metabolome, Cellular Microenvironment, Colon pathology, Epithelial Cells pathology
- Abstract
Evidence, mostly from experimental models, has accumulated, indicating that modifications of bacterial metabolite concentrations in the large intestine luminal content, notably after changes in the dietary composition, may have important beneficial or deleterious consequences for the colonic epithelial cell metabolism and physiology in terms of mitochondrial energy metabolism, reactive oxygen species production, gene expression, DNA integrity, proliferation, and viability. Recent data suggest that for some bacterial metabolites, like hydrogen sulfide and butyrate, the extent of their oxidation in colonocytes affects their capacity to modulate gene expression in these cells. Modifications of the luminal bacterial metabolite concentrations may, in addition, affect the colonic pH and osmolarity, which are known to affect colonocyte biology per se. Although the colonic epithelium appears able to face, up to some extent, changes in its luminal environment, notably by developing a metabolic adaptive response, some of these modifications may likely affect the homeostatic process of colonic epithelium renewal and the epithelial barrier function. The contribution of major changes in the colonocyte luminal environment in pathological processes, like mucosal inflammation, preneoplasia, and neoplasia, although suggested by several studies, remains to be precisely evaluated, particularly in a long-term perspective., (Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)
- Published
- 2017
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34. Epithelial response to a high-protein diet in rat colon.
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Beaumont M, Andriamihaja M, Armand L, Grauso M, Jaffrézic F, Laloë D, Moroldo M, Davila AM, Tomé D, Blachier F, and Lan A
- Subjects
- Animal Feed, Animals, Cluster Analysis, Epithelial Cells metabolism, Gene Expression Profiling, Gene Expression Regulation, Glutathione metabolism, Male, Rats, Signal Transduction, Transcriptome, Colon metabolism, Diet, Dietary Proteins metabolism, Intestinal Mucosa metabolism
- Abstract
Background: High-protein diets (HPD) alter the large intestine microbiota composition in association with a metabolic shift towards protein degradation. Some amino acid-derived metabolites produced by the colon bacteria are beneficial for the mucosa while others are deleterious at high concentrations. The aim of the present work was to define the colonic epithelial response to an HPD. Transcriptome profiling was performed on colonocytes of rats fed an HPD or an isocaloric normal-protein diet (NPD) for 2 weeks., Results: The HPD downregulated the expression of genes notably implicated in pathways related to cellular metabolism, NF-κB signaling, DNA repair, glutathione metabolism and cellular adhesion in colonocytes. In contrast, the HPD upregulated the expression of genes related to cell proliferation and chemical barrier function. These changes at the mRNA level in colonocytes were not associated with detrimental effects of the HPD on DNA integrity (comet assay), epithelium renewal (quantification of proliferation and apoptosis markers by immunohistochemistry and western blot) and colonic barrier integrity (Ussing chamber experiments)., Conclusion: The modifications of the luminal environment after an HPD were associated with maintenance of the colonic homeostasis that might be the result of adaptive processes in the epithelium related to the observed transcriptional regulations.
- Published
- 2017
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35. Detrimental effects for colonocytes of an increased exposure to luminal hydrogen sulfide: The adaptive response.
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Beaumont M, Andriamihaja M, Lan A, Khodorova N, Audebert M, Blouin JM, Grauso M, Lancha L, Benetti PH, Benamouzig R, Tomé D, Bouillaud F, Davila AM, and Blachier F
- Subjects
- Amino Acids metabolism, Animals, Colon microbiology, DNA Damage drug effects, Fermentation drug effects, Gastrointestinal Microbiome genetics, Gene Expression Regulation drug effects, Hypoxia-Inducible Factor 1, alpha Subunit biosynthesis, Interleukin-6 biosynthesis, Intestine, Large metabolism, Intestine, Large microbiology, Nitric Oxide metabolism, Nitric Oxide Synthase Type II biosynthesis, Oxygen Consumption drug effects, Rats, Sulfides pharmacology, Colon metabolism, Energy Metabolism, Gastrointestinal Microbiome drug effects, Hydrogen Sulfide metabolism
- Abstract
Protein fermentation by the gut microbiota releases in the large intestine lumen various amino-acid derived metabolites. Among them, hydrogen sulfide (H2S) in excess has been suspected to be detrimental for colonic epithelium energy metabolism and DNA integrity. The first objective of this study was to evaluate in rats the epithelial response to an increased exposure to H2S. Experiments from colonocyte incubation and intra-colonic instillation indicate that low millimolar concentrations of the sulfide donor NaHS reversibly inhibited colonocyte mitochondrial oxygen consumption and increased gene expression of hypoxia inducible factor 1α (Hif-1α) together with inflammation-related genes namely inducible nitric oxide synthase (iNos) and interleukin-6 (Il-6). Additionally, rat colonocyte H2S detoxification capacity was severely impaired in the presence of nitric oxide. Based on the γH2AX ICW technique, NaHS did not induce DNA damage in colonocytes. Since H2S is notably produced by the gut microbiota from sulfur containing amino acids, the second objective of the study was to investigate the effects of a high protein diet (HPD) on large intestine luminal sulfide content and on the expression of genes involved in H2S detoxification in colonocytes. We found that HPD markedly increased H2S content in the large intestine but the concomitant increase of the content mass maintained the luminal sulfide concentration. HPD also provoked an increase of sulfide quinone reductase (Sqr) gene expression in colonocytes, indicating an adaptive response to increased H2S bacterial production. In conclusion, low millimolar NaHS concentration severely affects colonocyte respiration in association with increased expression of genes associated with intestinal inflammation. Although HPD increases the sulfide content of the large intestine, the colonic adaptive responses to this modification limit the epithelial exposure to this deleterious bacterial metabolite., (Copyright © 2016 Elsevier Inc. All rights reserved.)
- Published
- 2016
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36. Bestrophin-encoded Ca²⁺-activated Cl⁻ channels underlie a current with properties similar to the native current in the moth Spodoptera littoralis olfactory receptor neurons.
- Author
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François A, Grauso M, Demondion E, Bozzolan F, Debernard S, and Lucas P
- Subjects
- Amino Acid Sequence, Animals, Arthropod Antennae innervation, Arthropod Antennae metabolism, Calcium physiology, Cells, Cultured, Chloride Channels antagonists & inhibitors, Chloride Channels genetics, Cloning, Molecular, Cricetinae, Flufenamic Acid pharmacology, Gene Expression, Gene Expression Regulation, Developmental, Humans, Insect Proteins antagonists & inhibitors, Insect Proteins genetics, Male, Membrane Potentials drug effects, Molecular Sequence Data, Niflumic Acid pharmacology, Nitrobenzoates pharmacology, Olfactory Receptor Neurons drug effects, Olfactory Receptor Neurons physiology, Organ Specificity, Patch-Clamp Techniques, Permeability, Primary Cell Culture, Sequence Analysis, DNA, Sequence Homology, Amino Acid, Spodoptera cytology, Spodoptera genetics, Spodoptera metabolism, Chloride Channels metabolism, Insect Proteins metabolism, Olfactory Receptor Neurons metabolism
- Abstract
Responses of insect olfactory receptor neurons (ORNs) involve an entry of Ca²⁺ through olfactory heterodimeric receptor complexes. In moths, the termination of ORN responses was found to strongly depend on the external Ca²⁺ concentration through the activation of unknown Ca²⁺-dependent Cl⁻ channels. We thus investigated the molecular identity of these Cl⁻ channels. There is compelling evidence that bestrophins form Cl⁻ channels when expressed in heterologous systems. Here we provide evidence that antennae of the moth Spodoptera littoralis express three transcripts encoding proteins with hallmarks of bestrophins. One of these transcripts, SlitBest1b, is expressed in ORNs. The heterologous expression of SlitBest1b protein in CHO-K1 cells yielded a Ca²⁺-activated Cl⁻ current that shares electrophysiological properties with the native Ca²⁺-activated Cl⁻ current of ORNs. Both currents are anionic, present similar dependence on the intracellular Ca²⁺ concentration, partly inactivate over time, have the same anion permeability sequence, the same sequence of inhibitory efficiency of blockers, the same almost linear I-V relationships and finally both currents do not depend on the cell volume. Therefore, our data suggest that SlitBest1b is a good candidate for being a molecular component of the olfactory Ca²⁺-activated Cl⁻ channel and is likely to constitute part of the insect olfactory transduction pathway. A different function (e.g. regulation of other proteins, maintenance of the anionic homeostasis in the sensillar lymph) and a different role (e.g. involvement in the olfactory system development) cannot be excluded however.
- Published
- 2012
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37. Calcium activates a chloride conductance likely involved in olfactory receptor neuron repolarization in the moth Spodoptera littoralis.
- Author
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Pézier A, Grauso M, Acquistapace A, Monsempes C, Rospars JP, and Lucas P
- Subjects
- Animals, Anions metabolism, Calcium pharmacology, Calcium Signaling drug effects, Calcium-Calmodulin-Dependent Protein Kinase Type 2 antagonists & inhibitors, Calmodulin pharmacology, Cell Culture Techniques methods, Chloride Channels antagonists & inhibitors, Enzyme Inhibitors pharmacology, Male, Membrane Potentials drug effects, Membrane Transport Modulators pharmacology, Olfactory Receptor Neurons drug effects, Permeability, Pheromones pharmacology, Protein Kinase C antagonists & inhibitors, Spodoptera drug effects, Spodoptera metabolism, Calcium physiology, Chloride Channels physiology, Membrane Potentials physiology, Olfactory Receptor Neurons physiology, Spodoptera physiology
- Abstract
The response of insect olfactory receptor neurons (ORNs) to odorants involves the opening of Ca(2+)-permeable channels, generating an increase in intracellular Ca(2+) concentration. Here, we studied the downstream effect of this Ca(2+) rise in cultured ORNs of the moth Spodoptera littoralis. Intracellular dialysis of Ca(2+) from the patch pipette in whole-cell patch-clamp configuration activated a conductance with a K(1/2) of 2.8 microm. Intracellular and extracellular anionic and cationic substitutions demonstrated that Cl(-) carries this current. The anion permeability sequence I(-) > NO(3)(-) > Br(-) > Cl(-) > CH(3)SO(3)(-) >> gluconate(-) of the Ca(2+)-activated Cl(-) channel suggests a weak electrical field pore of the channel. The Ca(2+)-activated current partly inactivated over time and did not depend on protein kinase C (PKC) and CaMKII activity or on calmodulin. Application of Cl(-) channel blockers, flufenamic acid, 5-nitro-2-(3-phenylpropylamino) benzoic acid, or niflumic acid reversibly blocked the Ca(2+)-activated current. In addition, lowering Cl(-) concentration in the sensillar lymph bathing the ORN outer dendrites caused a significant delay in pheromone response termination in vivo. The present work identifies a new Cl(-) conductance activated by Ca(2+) in insect ORNs presumably required for ORN repolarization.
- Published
- 2010
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38. The nicotinic acetylcholine receptor gene family of the malaria mosquito, Anopheles gambiae.
- Author
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Jones AK, Grauso M, and Sattelle DB
- Subjects
- Amino Acid Sequence, Animals, Chromosome Mapping, Conserved Sequence, Drosophila Proteins genetics, Gene Order, Genome, Molecular Sequence Data, Multigene Family, Protein Subunits genetics, RNA Editing, Receptors, Nicotinic metabolism, Transcription, Genetic, Alternative Splicing, Anopheles genetics, Phylogeny, Receptors, Nicotinic genetics
- Abstract
Nicotinic acetylcholine receptors (nAChRs) mediate fast cholinergic synaptic transmission in the insect nervous system and are targets of widely selling insecticides. We have identified the nAChR gene family from the genome of the malaria mosquito vector, Anopheles gambiae, to be the second complete insect nAChR gene family described following that of Drosophila melanogaster. Like Drosophila, Anopheles possesses 10 nAChR subunits with orthologous relationships evident between the two insects. Interestingly, the Anopheles orthologues of Dbeta2 and Dbeta3 possess the vicinal cysteines that define alpha subunits. As with Dalpha4 and Dalpha6, the Anopheles orthologues are alternatively spliced at equivalent exons. Reverse transcription-polymerase chain reaction analysis shows that RNA A-to-I editing sites conserved between Dalpha6 of Drosophila and alpha7-2 of the tobacco budworm, Heliothis virescens, are not shared with the equivalent nAChR subunit of Anopheles. Indeed, RNA-editing sites identified in functionally significant regions of Dbeta1, Dalpha5, and Dalpha6 are not conserved in the mosquito orthologues, indicating considerable divergence of RNA molecules targeted for editing within the insect order Diptera. These findings shed further light on the diversity of nAChR subunits and may present a useful basis for the development of improved malaria control agents by enhancing our understanding of a validated mosquito insecticide target.
- Published
- 2005
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39. Heavy metals modulate the activity of the purinergic P2X4 receptor.
- Author
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Coddou C, Lorca RA, Acuña-Castillo C, Grauso M, Rassendren F, and Huidobro-Toro JP
- Subjects
- Adenosine Triphosphate biosynthesis, Adenosine Triphosphate pharmacology, Animals, Binding Sites drug effects, Binding Sites genetics, Cysteine pharmacology, Dose-Response Relationship, Drug, Drug Synergism, Evoked Potentials drug effects, Evoked Potentials physiology, Extracellular Fluid chemistry, Histidine chemistry, Histidine pharmacology, Metals, Heavy antagonists & inhibitors, Metals, Heavy pharmacology, Mutation, Oocytes drug effects, Oocytes physiology, Purinergic P2 Receptor Antagonists, Receptors, Purinergic P2 genetics, Receptors, Purinergic P2X4, Toxicity Tests methods, Xenopus laevis, Receptors, Purinergic P2 drug effects
- Abstract
To further characterize the nature of the regulatory metal-binding sites of the rat P2X(4) receptor, several transition heavy metals were tested to examine their ability to mimic the facilitator action of zinc or the inhibitory action of copper. cDNA coding for the rat P2X(4) receptor was injected into Xenopus laevis oocytes; the two-electrode voltage-clamp technique was used to measure and quantify the ATP-evoked currents in the absence or presence of the metals. Cadmium facilitated the ATP-gated currents in a reversible and voltage-independent manner; maximal potentiation occurred within less than 1 min. Cadmium displaced leftward, in a concentration-dependent manner, the ATP concentration-response curve. In contrast, mercury reduced the ATP-gated currents in a reversible, time, and concentration manner. Maximal inhibition occurred after about 5 min of metal application. Cobalt also augmented the ATP-evoked currents, but its action was long lasting and did not reverse even after 45 min of metal washout. Other metals such as lead, nickel, manganese, silver, or gallium did not significantly alter the ATP-gated currents. The co-application of cadmium plus zinc or mercury plus copper caused additive effects. Mutation of H140 by alanine (H140A) augmented both the cadmium-induced facilitation and the mercury-induced inhibition. In contrast, the H241A mutant showed characteristics indistinguishable from the wild type. The H286A mutant showed a normal cadmium-induced potentiation, but an increased mercury inhibition. Out of the metals examined, only cadmium mimicked closely the action of zinc, evidencing commonalities. While mercury mimicked the action of copper, both metals apparently interact at distinct metal-binding sites. The present findings allow us to infer that heavy metals modulate the P2X(4) receptor by acting in at least three separate metal-binding sites.
- Published
- 2005
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40. Histidine 140 plays a key role in the inhibitory modulation of the P2X4 nucleotide receptor by copper but not zinc.
- Author
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Coddou C, Morales B, González J, Grauso M, Gordillo F, Bull P, Rassendren F, and Huidobro-Toro JP
- Subjects
- Adenosine Triphosphate metabolism, Allosteric Site, Animals, Binding Sites, Cell Line, Copper chemistry, Copper pharmacology, Cysteine chemistry, DNA, Complementary metabolism, Dose-Response Relationship, Drug, Humans, Metals pharmacology, Mutagenesis, Site-Directed, Mutation, Oocytes metabolism, Patch-Clamp Techniques, Rats, Receptors, Purinergic P2X4, Time Factors, Transfection, Xenopus laevis, Zinc chemistry, Copper metabolism, Histidine chemistry, Receptors, Purinergic P2 chemistry, Zinc metabolism
- Abstract
To elucidate the role of extracellular histidines in the modulation of the rat P2X4 receptor by trace metals, we generated single, double, and triple histidine mutants for residues 140, 241, and 286, replacing them with alanines. cDNAs for the wild-type and receptor mutants were expressed in Xenopus laevis oocytes and in human embryonic kidney 293 cells and examined by the two electrode and patch clamp techniques, respectively. Whereas copper inhibited concentration-dependently the ATP-gated currents in the wild-type and in the single or double H241A and H286A receptor mutants, all receptors containing H140A were insensitive to copper in both cell systems. The characteristic bell-shaped concentration-response curve of zinc observed in the wild-type receptor became sigmoid in both oocytes and human embryonic kidney cells expressing the H140A mutant; in these mutants, the zinc potentiation was 2.5-4-fold larger than in the wild-type. Results with the H140T and H140R mutants further support the importance of a histidine residue at this position. We conclude that His-140 is critical for the action of copper, indicating that this histidine residue, but not His-241 or His-286, forms part of the inhibitory allosteric metal-binding site of the P2X4 receptor, which is distinct from the putative zinc facilitator binding site.
- Published
- 2003
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41. Functional genomics of ionotropic acetylcholine receptors in Caenorhabditis elegans and Drosophila melanogaster.
- Author
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Sattelle DB, Culetto E, Grauso M, Raymond V, Franks CJ, and Towers P
- Subjects
- Animals, Caenorhabditis elegans Proteins chemistry, Caenorhabditis elegans Proteins genetics, Drosophila Proteins chemistry, Drosophila Proteins genetics, Genomics, Models, Molecular, Phylogeny, Protein Conformation, Protein Subunits, Receptors, Cholinergic physiology, Caenorhabditis elegans genetics, Drosophila melanogaster genetics, Receptors, Cholinergic chemistry, Receptors, Cholinergic genetics
- Abstract
Genetics, genomics and electrophysiology are transforming our understanding of the nicotinic acetylcholine receptors (nAChRs). Caenorhabditis elegans contains the largest known family of nAChR subunit genes (27 members), while Drosophila melanogaster contains an exclusively neuronal nAChR gene family (10 members). In C. elegans, several genetic screens have enabled the identification of nAChR subunits, along with novel proteins that act upstream and downstream of functional nAChRs. The C. elegans genome project has identified many new candidate nAChR subunits and the calculated electrostatic potential energy profiles for the M2 channel-lining regions predict considerable functional diversity. The respective roles of subunits are under investigation using forward and reverse genetics. Electrophysiological and reporter gene studies have demonstrated roles for particular subunits in levamisole-sensitive muscle nAChRs and a role for nAChRs in pharyngeal pumping. Recombinant homomeric and heteromeric C. elegans nAChRs have been expressed in Xenopus laevis oocytes. In D. melanogaster, three new nAChR a subunits have been cloned, one of which shows multiple variant transcripts arising from alternative splicing and A-to-I pre-mRNA editing. Thus, studies on the genetic model organisms C. elegans and D. melanogaster have revealed different routes to generating molecular and functional diversity in the nAChR gene family and are providing new insights into the in vivo functions of individual family members.
- Published
- 2002
42. Neonicotinoids: insecticides acting on insect nicotinic acetylcholine receptors.
- Author
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Matsuda K, Buckingham SD, Kleier D, Rauh JJ, Grauso M, and Sattelle DB
- Subjects
- Animals, Humans, Insecticides chemistry, Receptors, Nicotinic chemistry, Structure-Activity Relationship, Cholinergic Agents pharmacology, Insecticides pharmacology, Receptors, Nicotinic drug effects
- Abstract
Imidacloprid is increasingly used worldwide as an insecticide. It is an agonist at nicotinic acetylcholine receptors (nAChRs) and shows selective toxicity for insects over vertebrates. Recent studies using binding assays, molecular biology and electrophysiology suggest that both alpha- and non-alpha-subunits of nAChRs contribute to interactions of these receptors with imidacloprid. Electrostatic interactions of the nitroimine group and bridgehead nitrogen in imidacloprid with particular nAChR amino acid residues are likely to have key roles in determining the selective toxicity of imidacloprid. Chemical calculation of atomic charges of the insecticide molecule and a site-directed mutagenesis study support this hypothesis.
- Published
- 2001
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43. Four genes encode acetylcholinesterases in the nematodes Caenorhabditis elegans and Caenorhabditis briggsae. cDNA sequences, genomic structures, mutations and in vivo expression.
- Author
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Combes D, Fedon Y, Grauso M, Toutant JP, and Arpagaus M
- Subjects
- Acetylcholinesterase chemistry, Acetylcholinesterase metabolism, Amino Acid Sequence, Animals, Binding Sites, Caenorhabditis elegans enzymology, Caenorhabditis elegans genetics, Cholinesterase Inhibitors pharmacology, Cloning, Molecular, Dimerization, Gallamine Triethiodide pharmacology, Genes, Helminth genetics, Inhibitory Concentration 50, Isoenzymes chemistry, Isoenzymes genetics, Isoenzymes metabolism, Molecular Sequence Data, Physical Chromosome Mapping, Propidium pharmacology, Protein Structure, Quaternary, RNA, Messenger analysis, RNA, Messenger genetics, Sequence Alignment, Sequence Deletion, Substrate Specificity, Trans-Splicing genetics, Acetylcholinesterase genetics, Caenorhabditis enzymology, Caenorhabditis genetics, Exons genetics, Introns genetics, Mutation genetics
- Abstract
We report the full coding sequences and the genomic organization of the four genes encoding acetylcholinesterase (AChE) in Caenorhabditis elegans and Caenorhabditis briggsae, in relation to the properties of the encoded enzymes. ace-1 and ace-2, located on chromosome X and I, respectively, encode two AChEs (ACE-1 and ACE-2) that present 35% identity. The C-terminal end of ACE-1 is homologous to the C terminus of T subunits of vertebrate AChEs. ACE-1 oligomerizes into amphiphilic tetramers. ACE-2 has a hydrophobic C terminus of H type. It associates into glycolipid-anchored dimers. In C. elegans and C. briggsae, ace-3 and ace-4 are organized in tandem on chromosome II, with only 356 nt and 369 nt, respectively, between the stop codon of ace-4 (upstream gene) and the ATG of ace-3. ace-3 produces only 5 % of the total AChE activity. It encodes an H subunit that associates into dimers of glycolipid-anchored catalytic subunits, which are highly resistant to the usual AChE inhibitors, and which hydrolyze butyrylthiocholine faster than acetylthiocholine. ACE-4 is closer to ACE-3 (54 % identity) than to ACE-1 or ACE-2. The usual sequence FGESAG surrounding the active serine residue in cholinesterases is changed to FGQSAG in ace-4. ACE-4 was not detected by our current biochemical methods, although the gene is transcribed in vivo. However the level of ace-4 mRNAs is far lower than those of ace-1, ace-2 and ace-3. The ace-2, ace-3 and ace-4 transcripts were found to be trans-spliced by both SL1 and SL2, although these genes are not included in typical operons. The molecular bases of null mutations g72 (ace-2), p1304 and dc2 (ace-3) have been identified., (Copyright 2000 Academic Press.)
- Published
- 2000
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44. A dimeric mutant of human pancreatic ribonuclease with selective cytotoxicity toward malignant cells.
- Author
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Piccoli R, Di Gaetano S, De Lorenzo C, Grauso M, Monaco C, Spalletti-Cernia D, Laccetti P, Cinátl J, Matousek J, and D'Alessio G
- Subjects
- 3T3 Cells, Amino Acid Sequence, Animals, Apoptosis drug effects, Cattle, Cell Transformation, Neoplastic, Dimerization, Drug Screening Assays, Antitumor, Humans, Male, Mice, Molecular Sequence Data, Mutagenesis, Site-Directed, Recombinant Proteins biosynthesis, Recombinant Proteins chemistry, Recombinant Proteins toxicity, Ribonuclease, Pancreatic chemistry, Ribonuclease, Pancreatic genetics, Ribonucleases chemistry, Seminal Vesicles enzymology, Sequence Alignment, Sequence Homology, Amino Acid, Thyroid Neoplasms, Tumor Cells, Cultured, Antineoplastic Agents toxicity, Cell Survival drug effects, Ribonuclease, Pancreatic toxicity
- Abstract
Monomeric human pancreatic RNase, devoid of any biological activity other than its RNA degrading ability, was engineered into a dimeric protein with a cytotoxic action on mouse and human tumor cells, but lacking any appreciable toxicity on mouse and human normal cells. This dimeric variant of human pancreas RNase selectively sensitizes to apoptotic death cells derived from a human thyroid tumor. Because of its selectivity for tumor cells, and because of its human origin, this protein represents a potentially very attractive, novel tool for anticancer therapy.
- Published
- 1999
- Full Text
- View/download PDF
45. Molecular cloning and expression of a full-length cDNA encoding acetylcholinesterase in optic lobes of the squid Loligo opalescens: a new member of the cholinesterase family resistant to diisopropyl fluorophosphate.
- Author
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Talesa V, Grauso M, Arpagaus M, Giovannini E, Romani R, and Rosi G
- Subjects
- Acetylcholinesterase genetics, Acetylcholinesterase isolation & purification, Amino Acid Sequence, Animals, Base Sequence, Brain enzymology, COS Cells, Centrifugation, Density Gradient, Cloning, Molecular, DNA, Complementary genetics, Drug Resistance, Electrophoresis, Polyacrylamide Gel, Kinetics, Molecular Sequence Data, Polymerase Chain Reaction, Type C Phospholipases chemistry, Acetylcholinesterase biosynthesis, Brain drug effects, Cholinesterase Inhibitors pharmacology, DNA, Complementary biosynthesis, Decapodiformes metabolism, Isoflurophate pharmacology
- Abstract
Acetylcholinesterase cDNA was cloned by screening a library from Loligo opalescens optic lobes; cDNA sequence analysis revealed an open reading frame coding for a protein of 610 amino acids that showed 20-41% amino acid identity with the acetylcholinesterases studied so far. The characteristic structure of cholinesterase (the choline binding site, the catalytic triad, and six cysteines that form three intrachain disulfide bonds) was conserved in the protein. The heterologous expression of acetylcholinesterase in COS cells gave a recovery of acetylcholinesterase activity 20-fold higher than in controls. The enzyme, partially purified by affinity chromatography, showed molecular and kinetic features indistinguishable from those of acetylcholinesterase expressed in vivo, which displays a high catalytic efficiency. Both enzymes are true acetylcholinesterase corresponding to phosphatidylinositol-anchored G2a dimers of class I, with a marked substrate specificity for acetylthiocholine. The deduced amino acid sequence may explain some particular kinetic characteristics of Loligo acetylcholinesterase, because the presence of a polar amino acid residue (S313) instead of a nonpolar one [F(288) in Torpedo] in the acyl pocket of the active site could justify the high substrate specificity of the enzyme, the absence of hydrolysis with butyrylthiocholine, and the poor inhibition by the organophosphate diisopropyl fluorophosphate.
- Published
- 1999
- Full Text
- View/download PDF
46. Existence of four acetylcholinesterase genes in the nematodes Caenorhabditis elegans and Caenorhabditis briggsae.
- Author
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Grauso M, Culetto E, Combes D, Fedon Y, Toutant JP, and Arpagaus M
- Subjects
- Amino Acid Sequence, Animals, Caenorhabditis elegans genetics, Chromosome Mapping, Cloning, Molecular, Molecular Sequence Data, Phylogeny, Polymerase Chain Reaction, Sequence Homology, Amino Acid, Acetylcholinesterase genetics, Caenorhabditis genetics
- Abstract
Three genes, ace-1, ace-2 and ace-3, respectively located on chromosomes X, I and II, were reported to encode acetylcholinesterases (AChEs) of classes A, B and C in the nematode Caenorhabditis elegans. We have previously cloned and sequenced ace-1 in the two related species C. elegans and C. briggsae. We report here partial sequences of ace-2 (encoding class B) and of two other ace sequences located in close proximity on chromosome II in C. elegans and C. briggsae. These two sequences are provisionally named ace-x and ace-y, because it is not possible at the moment to establish which of these two genes corresponds to ace-3. Ace-x and ace-y are transcribed in vivo as shown by RT-PCR and they are likely to be included in a single operon.
- Published
- 1998
- Full Text
- View/download PDF
47. Sequence comparison of ACE-1, the gene encoding acetylcholinesterase of class A, in the two nematodes Caenorhabditis elegans and Caenorhabditis briggsae.
- Author
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Grauso M, Culetto E, Berge JB, Toutant JP, and Arpagaus M
- Subjects
- Acetylcholinesterase chemistry, Amino Acid Sequence, Animals, Base Sequence, Blotting, Southern, Cloning, Molecular, Codon genetics, DNA Primers, Introns, Molecular Sequence Data, Polymerase Chain Reaction, Sequence Homology, Amino Acid, Acetylcholinesterase genetics, Caenorhabditis enzymology, Caenorhabditis genetics, Caenorhabditis elegans enzymology, Caenorhabditis elegans genetics, Genes, Helminth
- Abstract
The ace-1 gene, which encodes acetylcholinesterase of class A, has been cloned and sequenced in C. briggsae and compared to its homologue in C. elegans. Both genes present an open reading frame of 1860 nucleotides. The percentages of identity are 80% and 95% at the nucleotide and aminoacid levels respectively. All residues characteristic of an acetylcholinesterase are found in conserved positions in C. briggsae ACE-1. The deduced C-terminus is hydrophilic, thus resembling the catalytic peptide T of vertebrate cholinesterases. Codon usage in both ace-1 genes appears to be lowly biased. This may indicate that these genes are lowly expressed. The splicing sites of the eight introns of ace-1 in C. elegans are conserved in C. briggsae, but introns are shorter in C. briggsae. No homology was found between intronic sequences in both species, except for the consensus border sequences.
- Published
- 1996
- Full Text
- View/download PDF
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