Cardiac Muscarinic Receptor Overexpression in Sudden Infant Death Syndrome

BACKGROUND: Sudden infant death syndrome (SIDS) remains the leading cause of death among infants less than 1 year of age. Disturbed expression of some neurotransmitters and their receptors has been shown in the central nervous system of SIDS victims but no biological abnormality of the peripheral vago-cardiac system has been demonstrated to date. The present study aimed to seek vago-cardiac abnormalities in SIDS victims. The cardiac level of expression of muscarinic receptors, as well as acetylcholinesterase enzyme activity were investigated. METHODOLOGY/PRINCIPAL FINDINGS: Left ventricular samples and blood samples were obtained from autopsies of SIDS and children deceased from non cardiac causes. Binding experiments performed with [(3)H]NMS, a selective muscarinic ligand, in cardiac membrane preparations showed that the density of cardiac muscarinic receptors was increased as shown by a more than doubled B(max) value in SIDS (n = 9 SIDS versus 8 controls). On average, the erythrocyte acetylcholinesterase enzyme activity was also significantly increased (n = 9 SIDS versus 11 controls). CONCLUSIONS: In the present study, it has been shown for the first time that cardiac muscarinic receptor overexpression is associated with SIDS. The increase of acetylcholinesterase enzyme activity appears as a possible regulatory mechanism

Gene expression mapping of histone deacetylases and co-factors, and correlation with survival time and 1H-HRMAS metabolomic profile in human gliomas

Primary brain tumors are presently classified based on imaging and histopathological techniques, which remains unsatisfaying. We profiled here by quantitative real time PCR (qRT-PCR) the transcripts of eighteen histone deacetylases (HDACs) and a subset of transcriptional co-factors in non-tumoral brain samples from 15 patients operated for epilepsia and in brain tumor samples from 50 patients diagnosed with grade II oligodendrogliomas (ODII, n = 9), grade III oligodendrogliomas (ODIII, n = 22) and glioblastomas (GL, n = 19). Co-factor transcripts were significantly different in tumors as compared to non-tumoral samples and distinguished different molecular subgroups of brain tumors, regardless of tumor grade. Among all patients studied, the expression of HDAC1 and HDAC3 was inversely correlated with survival, whereas the expression of HDAC4, HDAC5, HDAC6, HDAC11 and SIRT1 was significantly and positively correlated with survival time of patients with gliomas. (1)H-HRMAS technology revealed metabolomically distinct groups according to the expression of HDAC1, HDAC4 and SIRT1, suggesting that these genes may play an important role in regulating brain tumorigenesis and cancer progression. Our study hence identified different molecular fingerprints for subgroups of histopathologically similar brain tumors that may enable the prediction of outcome based on the expression level of co-factor genes and could allow customization of treatment

Homozygosity for the V377I mutation in mevalonate kinase causes distinct clinical phenotypes in two sibs with hyperimmunoglobulinaemia D and periodic fever syndrome (HIDS).

OBJECTIVE: Mevalonate kinase (MVK) deficiency is a rare autosomal recessive auto-inflammatory disorder characterised by recurring episodes of fever associated with multiple non-specific inflammatory symptoms and caused by mutations in the MVK gene. The phenotypic spectrum is wide and depends mostly on the nature of the mutations. Hyperimmunoglobulinaemia D and periodic fever syndrome (HIDS) is a relatively mild presentation and predominantly associated with a c.1129G>A (p.V377I) mutation in the MVK gene. We report cases of two sisters homozygous for this mutation but exhibiting distinct (symptomatic vs asymptomatic) phenotypes. METHODS: Patient history was obtained; physical and clinical examination and laboratory tests were performed; lipopolysaccharide (LPS) response of peripheral blood mononuclear cells was quantified. RESULTS: Low MVK enzymatic activity is not necessarily associated with inflammatory symptoms. Increased inflammatory cytokine secretion in response to LPS is associated with symptomatic MVK deficiency. CONCLUSIONS: Individuals who are homozygous for the common p.V377I mutation in the MVK gene may not display the characteristic inflammatory episodes diagnostic of MKD and thus may be lost for correct and timely diagnosis.journal article20162016 03 07importedThis work was funded by the Laboratoire d'Excellence (LABEX) TRANSPLANEX [ANR-11-LABX-0070_TRANSPLANTEX]. Additional support was received from the Institut national de la santé et de la recherche médicale (INSERM), the University of Strasbourg (UNISTRA) and the Institut Universitaire de France (IUF)

Constitutive Overexpression of Muscarinic Receptors Leads to Vagal Hyperreactivity

BACKGROUND: Alterations in muscarinic receptor expression and acetylcholinesterase (AchE) activity have been observed in tissues from Sudden Infant Death Syndrome (SIDS). Vagal overactivity has been proposed as a possible cause of SIDS as well as of vasovagal syncopes. The aim of the present study was to seek whether muscarinic receptor overexpression may be the underlying mechanism of vagal hyperreactivity. Rabbits with marked vagal pauses following injection of phenylephrine were selected and crossed to obtain a vagal hyperreactive strain. The density of cardiac muscarinic receptors and acetylcholinesterase (AchE) gene expression were assessed. Blood markers of the observed cardiac abnormalities were also sought. METHODOLOGY/PRINCIPAL FINDINGS: Cardiac muscarinic M(2) and M(3) receptors were overexpressed in hyperreactive rabbits compared to control animals (2.3-fold and 2.5-fold, respectively) and the severity of the phenylephrine-induced bradycardia was correlated with their densities. A similar overexpression of M(2) receptors was observed in peripheral mononuclear white blood cells, suggesting that cardiac M(2) receptor expression can be inferred with high confidence from measurements in blood cells. Sequencing of the coding fragment of the M(2) receptor gene revealed a single nucleotide mutation in 83% of hyperreactive animals, possibly contributing for the transcript overexpression. Significant increases in AchE expression and activity were also assessed (AchE mRNA amplification ratio of 3.6 versus normal rabbits). This phenomenon might represent a compensatory consequence of muscarinic receptors overexpression. Alterations in M(2) receptor and AchE expression occurred between the 5th and the 7th week of age, a critical period also characterized by a higher mortality rate of hyperreactive rabbits (52% in H rabbits versus 13% in normal rabbits) and preceeded the appearance of functional disorders. CONCLUSIONS/SIGNIFICANCE: The results suggest that cardiac muscarinic receptor overexpression plays a critical role in the development of vagal hyperreactivity, whereas AchE hyperactivity appears as a compensatory consequence of it. Since similar vagal disorders were observed recently by us in SIDS, muscarinic receptor overexpression could become a marker of risk of vasovagal syncopes and SIDS

Génération de souris par inactivation fonctionnelle des gènes SIRT (Etude de la fonction du gène Rb dans la différenciation apipocytaire et le métabolisme énergétique)

Chez les mammifères, les protéines SIRT, appelées aussi sirtuines, appartiennent à la famille des histones désacétylases (HDAC) de classe III et ont été nommées ainsi pour leur homologie avec le gène de la levure Saccharomyces cerevisiae Sir2. Il est clairement établi que le gène Sir2 joue un rôle primordial dans l extension de la durée de vie de la levure induite par la restriction calorique et que des copies supplémentaires de ce gène retardent le vieillissement chez la levure. Contrairement aux HDAC de classe I et II, les protéines SIRT nécessitent un cofacteur, le NAD+, pour désacetyler les histones ainsi que d importants facteurs régulateurs de la transcription. Le chef de file des sirtuines, SIRT1, semble impliqué dans des pathologies variées telles le diabète, l obésité, l insuffisance cardiaque, le SIDA et les maladies neurodégénératives. Même si le rôle de SIRT1 n a pas encore été clairement établi in vivo, il apparaît indissociable de la régulation de différents processus biologiques allant de l apoptose à la différentiation adipocytaire et musculaire ainsi que dans le métabolisme glucidique en régulant la néoglucogenèse. La fonction des autres SIRT n est pas encore élucidée.Le sujet de ma thèse de doctorat est d essayer de comprendre les fonctions biologiques des gènes SIRT en les inactivant de façon spatio-temporelle par l utilisation de la stratégie du knock-out conditionnel (inactivation conditionnelle) et le système Cre/Lox. Cette technologie permet d inactiver de façon contrôlée le gène d intérêt dans un organe donné d une souris adulte afin d éviter les problèmes liés aux anomalies que l absence du gène pourrait provoquer au cours du développement de l animal lors d un knock-out classique . Pour parvenir à notre objectif, nous avons réalisé des constructions modifiées des différents gènes SIRT. La particularité de ces constructions consiste à introduire des séquences Lox autour du domaine du gène codant pour la partie catalytique de l enzyme. Les Lox sont des séquences nucléotidiques de 34 paires de bases qui sont excisées par l enzyme Cre-recombinase lors de l inactivation du gène. Les vecteurs contenant les gènes SIRT modifiés ont été électroporés dans des cellules souches embryonnaires (ES) de souris 129/Sv afin de s intégrer au génome par recombinaison homologue. Les clones positifs ont été injectés par la suite dans des blastocystes. Nous avons obtenu ainsi des souris transgéniques pour le gène d intérêt. Ces souris doivent par la suite être croisées avec des souris exprimant une protéine de fusion Cre-ERT sous le contrôle d un promoteur spécifique d un organe donné. La protéine Cre-ERT est une fusion entre la Cre-recombinase et le récepteur aux oestrogènes ayant une affinité élevée pour le ligand tamoxifène. En cas d injection de tamoxifène, il y a activation de la Cre qui induit l inactivation du gène d intérêt à un moment donné de la vie de la souris et permet d étudier ainsi les effets biologiques qui en résultent.A l heure actuelle, les constructions pour les gènes SIRT1-7 ont été réalisées. Nous disposons des souris transgéniques pour le gène SIRT2 floxé et les croisements sont en cours avec des souris transgéniques CMV-Cre-ERT2 (inactivation de SIRT2 dans tous les tissus) et Syn-Cre-ERT2 (inactivation de SIRT2 dans les cellules nerveuses). Les autres constructions ont été électroporées dans les cellules ES et sont en cours d analyse.Partie II: Etude de la fonction du gène Rb dans la différentiation adipocytaire et le métabolisme énergétique.Le gène du rétinoblastome Rb est le premier gène suppresseur de tumeur identifié et a été largement étudié. Son rôle a bien été démontré dans différents processus biologiques tels le contrôle du cycle cellulaire, l apoptose, la prolifération et la différenciation cellulaires. Récemment, des études in vitro ont montré que la protéine pRb avait un rôle important dans la différentiation adipocytaire et qu un déficit en pRb provoque un changement dans le programme de différentiation d adipocytes blancs de souris en adipocytes bruns. Dans notre étude, nous avons utilisé le système Cre-lox pour inactiver spécifiquement le gène Rb dans le tissu adipeux. Soumises à un régime riche en graisses, des souris déficientes en pRb restent maigres par rapport à leurs congénères qui voient leurs poids augmenter de manière significative. L analyse histologique et en microscopie électronique du tissu adipeux blanc (WAT) des souris déficientes en pRb montre une transformation d une partie du WAT en tissu adipeux brun (BAT) avec une augmentation significative du nombre de mitochondries suggérant une activité métabolique élevée. L analyse moléculaire a démontré une augmentation de l expression des gènes impliqués dans la dépense énergétique tels UCP-1, PGC-1a et Dio2. Ceci se traduit physiologiquement par une augmentation de la consommation en O2 et de la production de CO2 démontrant une élévation de la dépense énergétique chez les souris pRb-déficientes. Ces données confirment que l absence de pRb provoque la conversion d une partie du WAT en BAT, un tissu connu pour son rôle actif dans la dépense énergétique. Le gène Rb devient ainsi une cible thérapeutique potentielle dans le but d induire une perte de poids chez les patients obèses.In mammals, the sirtuin family of histone deacetylases (HDACs) family was named after their homology to the yeast Saccharomyces cerevisiae gene Sir2. In yeast, it has been shown that Sir2 mediates the effects of calorie restriction on the extension of lifespan and that high levels of Sir2 activity promote longevity. Like their yeast homologs, the mammalian sirtuins (SIRT1-7) are class III HDACs and require NAD+ as a cofactor to deacetylate substrates such as histones and transcription regulators. Through this activity, sirtuins are shown to regulate important biological processes ranging from apoptosis, adipocyte and muscle differentiation, and energy expenditure to gluconeogenesis. SIRT1, the most studied sirtuin, seems to be implicated in several pathologies such as diabetes, obesity, heart failure and neurodegenerative disorders.The aim of this Ph.D. thesis was to help understand the biological function of SIRT genes through their inactivation in mice in a spatial and temporal controlled manner, using the Cre/Lox technology. This system allows the controlled inactivation of a gene of interest in a given organ of an adult mouse to avoid abnormalities that could occur during the development when using a classical knock-out . We have generated genetically modified constructs for all SIRT genes by introducing 2 LoxP sequences flanking the catalytic domain of the enzyme. LoxP sites are sequences of 34 nucleotides that can be recognized and excised by the Cre-recombinase enzyme. Vectors containing the modified SIRT gene constructs were electroporated in embryonic stem cells (ES) of 129/Sv mice in order to be integrated in their genome by homologous recombination. Positif clones were then injected in blastocysts of C57BL/6J pseudopregnant mice. We obtained transgenic mice for the gene of interest. These mice will be crossed with mice expressing the Cre recombinase fused to a modified estrogen receptor with high affinity for the synthetic ligand tamoxifen, under the control of a cell specific promoter that targets Cre expression in a specific organ or cell type (promoter-Cre-ERT2). After tamoxifen injection, the Cre recombinase is activated and subsequently the SIRT gene of interest will be inactivated in a specific cell type (e.g. adipocytes), whilst its activity remains in other cells, allowing the study of the biological effects that result from such gene inactivation.At present, we have completed the constructs for all SIRT(1-7) genes. We obtained mice with a floxed SIRT2 allele that were crossed with a CMV-Cre-ERT2 and Synapsin-Cre-ERT2 transgenic mice to generate cohorts of double transgenic mice expressing the floxed SIRT2 allele and the Cre-ERT2 either in all cell types or specifically in neurons. Constructs for other SIRT genes have been electroporated in ES cells and the generation of mice is underway.PART 2: Phenogenomics of adipocyte-specific retinoblastoma deficient miceThe role of the tumor suppressor retinoblastoma protein (pRb) has been firmly established in the control of cell cycle, apoptosis, and differentiation. Recently, it was demonstrated that lack of pRb promotes a switch from white to brown adipocyte differentiation in vitro. We used the Cre-Lox system to specifically inactivate pRb in adult adipose tissue. Under a high-fat diet, pRb-deficient (pRbad-/-) mice failed to gain weight because of increased energy expenditure. This protection against weight gain was caused by the activation of mitochondrial activity in white and brown fat as evidenced by histologic, electron microscopic, and gene expression studies. Moreover, pRb(-/-) mouse embryonic fibroblasts displayed higher proliferation and apoptosis rates than pRb(+/+) mouse embryonic fibroblasts, which could contribute to the altered white adipose tissue morphology. Taken together, our data support a direct role of pRb in adipocyte cell fate determination in vivo and suggest that pRb could serve as a potential therapeutic target to trigger mitochondrial activation in white adipose tissue and brown adipose tissue, favoring an increase in energy expenditure and subsequent weight loss.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Metal partitioning in calcerous sediment of the Tafna river and its estuary (Algeria)

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Mechanisms of Angiotensin II-Dependent Progression to Heart Failure

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Sirtuins: the 'magnificent seven', function, metabolism and longevity

The sirtuin family of histone deacetylases (HDACs) was named after their homology to the Saccharomyces cerevisiae gene silent information regulator 2 (Sir2). In the yeast, Sir2 has been shown to mediate the effects of calorie restriction on the extension of life span and high levels of Sir2 activity promote longevity. Like their yeast homologs, the mammalian sirtuins (SIRT1-7) are class III HDACs and require NAD(+) as a cofactor to deacetylate substrates ranging from histones to transcriptional regulators. Through this activity, sirtuins are shown to regulate important biological processes ranging from apoptosis, adipocyte and muscle differentiation, and energy expenditure to gluconeogenesis. We review here the current knowledge regarding the role of sirtuins in metabolism, longevity, and discuss the possible therapeutic applications that could result from the understanding of their function in different organs and pathologies