MEF

Vitamin E (VE) deficiency results in pronounced muscle weakness and atrophy but the cell biological mechanism of pathology is unknown. We previously showed that VE supplementation promotes membrane repair in cultured cells and that oxidants potently inhibit repair. Here we provide three independent lines of evidence that VE is required for skeletal muscle myocyte plasma membrane repair in vivo. We also show that when another lipid-directed antioxidant, glutathione peroxidase 4 (Gpx4), is genetically deleted in mouse embryonic fibroblasts, repair fails catastrophically, unless cells are supplemented with VE. We conclude that lipid-directed antioxidant activity provided by VE, and possibly also Gpx4, is an essential component of the membrane repair mechanism in skeletal muscle. This work explains why VE is essential to muscle health and identifies VE as a requisite component of the plasma membrane repair mechanism in vivo.

Notch signaling involves proteolytic cleavage of the transmembrane Notch receptor after binding to its transmembrane ligands, Delta or Jagged; and the resultant soluble intracellular domain of Notch stimulates a cascade of transcriptional events. The Delta1 ligand also undergoes proteolytic cleavage upon Notch binding, resulting in the production of a free intracellular domain. We demonstrate that the expression of the intracellular domain of Delta1 results in a non‐proliferating senescent‐like cell phenotype which is dependent on the expression of the cell cycle inhibitor, p21, and is abolished by co‐expression of constitutively active Notch1. These data suggest a new intracellular role for Delta1.

Dans de nombreux secteurs industriels, on constate un besoin croissant d’inspecter des parties difficilement accessibles de structures. Il est donc important de mettre en place des moyens d’investigation non destructifs permettant de realiser ce genre de controles. Les ondes guidees repondent a cette problematique et sont grandement utilises pour le suivi de l’etat de sante des structures (barres, plaques, coques, cylindres, etc.). Ces ondes ultrasonores guidees peuvent par exemple etre generees et detectees par des pastilles piezoelectriques (PZT) colles sur la structure a inspecter et excitees en basse frequence sur leur mode de resonance radiale. La reception peut se faire soit avec une autre pastille collee a un autre endroit soit avec la meme pastille emettrice. Dans ce papier, nous montrons la faisabilite de la detection d’un defaut de corrosion (perte d’epaisseur) au moyen de ces ondes guidees sur une barre en acier ferritique de dimensions finies. Dans un premier temps, des ...

Exigencias de calidad, reduccion de costos y rapida respuesta al mercado requieren de maquinas herramientas con mayor y mejores prestaciones desde el punto de vista de su control. El conocimiento de la fuerza de mecanizado es de gran importancia tanto para la implementacion de tecnicas de control adaptivas como para el monitoreo del estado de la herramienta de corte (desgaste, rotura del filo, etc.). En este trabajo se presenta el diseno de una celda de carga para medir las fuerzas presentes en procesos de mecanizado, su fabricacion y resultados experimentales. Este dispositivo permite medir fuerzas estaticas a partir de la deformacion captada por galgas extensiometricas (strain-gauge) pegadas en la superficie de elementos deformables o resortes del tipo anillo octogonal. Las condiciones de aplicacion exigen el diseno de una celda de carga capaz de medir las tres componentes ortogonales de una fuerza orientada en el espacio. Ademas, su rigidez debe ser lo suficientemente alta para e...

To elucidate the function of the T-box transcription factor Tbx20 in mammalian development, we generated a graded loss-of-function series by transgenic RNA interference in entirely embryonic stem cell-derived mouse embryos. Complete Tbx20 knockdown resulted in defects in heart formation, including hypoplasia of the outflow tract and right ventricle,which derive from the anterior heart field (AHF), and decreased expression of Nkx2-5 and Mef2c, transcription factors required for AHF formation. A mild knockdown led to persistent truncus arteriosus (unseptated outflow tract) and hypoplastic right ventricle, entities similar to human congenital heart defects, and demonstrated a critical requirement for Tbx20 in valve formation. Finally, an intermediate knockdown revealed a role for Tbx20 in motoneuron development, specifically in the regulation of the transcription factors Isl2 and Hb9, which are important for terminal differentiation of motoneurons. Tbx20 could activate promoters/enhanc...

Controlling cholesterol levels is a major challenge in human health, since hypercholesterolemia can lead to serious cardiovascular disease. Drugs that target carbohydrate metabolism can also modify lipid metabolism and hence cholesterol plasma levels. In this sense, dichloroacetate (DCA), a pyruvate dehydrogenase kinase (PDK) inhibitor, augments usage of the glycolysis-produced pyruvate in the mitochondria increasing oxidative phosphorylation (OXPHOS). In several animal models, DCA decreases plasma cholesterol and triglycerides. Thus, DCA was used in the 70 s to treat diabetes mellitus, hyperlipoproteinemia and hypercholesterolemia with satisfactory results. However, the mechanism of action remained unknown and we describe it here. DCA increases LDLR mRNA and protein levels as well as LDL intake in several cell lines, primary human hepatocytes and two different mouse models. This effect is mediated by transcriptional activation as evidenced by H3 acetylation on lysine 27 on the LDLR...

Targeting of myocyte enhancer binding factor 2 (MEF2) proteins to the nucleus depends on a C-terminal bipartite nuclear localization signal (NLS). By expression of green fluorescent protein (GFP)/MEF2 fusion proteins in transfected myoblasts, we show that MEF2C contains an additional 13 amino acids domain, located immediately upstream of the NLS, which contributes to its nuclear retention. We also show that the NLS present in MEF2 proteins is required for efficient nuclear localization of histone deacetylase 4 (HDAC4). In muscle cells, transfected HDAC4 is largely cytoplasmic or, to a lesser extent, pancellular. Co-transfection of either MEF2A or MEF2C causes HDAC4 to accumulate in the nucleus in association with MEF2. This effect strongly depends on MEF2 NLS; it also requires the specific interaction of HDAC4 with MEF2, since the isolated NLS is not sufficient for targeting HDAC4 to the nucleus and other nuclear proteins, such as NF-Y, cannot substitute MEF2. Therefore, we demonstr...

The future application of human embryonic stem cells (hESC) for therapeutic approaches requires the development of xeno-free culture conditions to prevent the potential transmission of animal pathogens or xenobiotic substances to hESC. An important component of the majority of hESC culture systems developed is the requirement for fibroblasts to serve as feeders. For this purpose, several studies have used human foreskin fibroblasts established under xeno-free conditions. In this study we report xeno-free establishment and maintenance of human embryonic fibroblasts (XHEF) and demonstrate their ability to support long-term self-renewal of hESC under xeno-free culture conditions, using a commercially available complete medium. Importantly, our culture conditions allow enzymatic passaging of hESC. In contrast, hESC cultured on human foreskin fibroblasts (XHFF) under the same conditions were poorly maintained and rapidly subject to differentiation. Our study clearly shows that the source...

The μE motifs of the immunoglobulin μ heavy-chain gene enhancer bind ubiquitously expressed proteins of the basic helix-loop-helix (bHLH) family. These elements work together with other, more tissue-restricted elements to produce B-cell-specific enhancer activity by presently undefined combinatorial mechanisms. We found that μE2 contributed to transcription activation in B cells only when the μE3 site was intact, providing the first evidence for functional interactions between bHLH proteins. In vitro assays showed that bHLH zipper proteins binding to μE3 enhanced Ets-1 binding to μA. One of the consequences of this protein-protein interaction was to facilitate binding of a second bHLH protein, E47, to the μE2 site, thereby generating a three-protein–DNA complex. Furthermore, transcriptional synergy between bHLH and bHLH zipper factors also required an intermediate ETS protein, which may bridge the transcription activation domains of the bHLH factors. Our observations define an unusu...

c‐Ski, originally identified as an oncogene product, induces myogenic differentiation in nonmyogenic fibroblasts through transcriptional activation of muscle regulatory factors. Although c‐Ski does not bind to DNA directly, it binds to DNA through interaction with Smad proteins and regulates signaling activities of transforming growth factor‐β (TGF‐β). In the present study, we show that c‐Ski activates the myogenin promoter independently of regulation of endogenous TGF‐β signaling. Expression of myogenin is regulated by a transcription factor complex containing proteins of the MyoD family and the myocyte enhancer factor 2 (MEF2) family. c‐Ski acts on the MyoD–MEF2 complex and modulates the activity of MyoD in myogenin promoter regulation. Interestingly, histone deacetylase (HDAC) inhibitors up‐regulated basal activity of transcription from a MyoD‐responsive reporter, although c‐Ski failed to further augment this transcription in the presence of HDAC inhibitors. c‐Ski is observed bot...

Despite our understanding that medial smooth muscle hypertrophy is a central feature of vascular remodeling, the molecular pathways underlying this pathology are still not well understood. Work over the past decade has illustrated a potential role for the multifunctional calmodulin-dependent kinase CaMKII in smooth muscle cell contraction, growth, and migration. Here we demonstrate that CaMKII is enriched in vascular smooth muscle (VSM) and that CaMKII inhibition blocks ANG II-dependent VSM cell hypertrophy in vitro and in vivo. Specifically, systemic CaMKII inhibition with KN-93 prevented ANG II-mediated hypertension and medial hypertrophy in vivo. Adenoviral transduction with the CaMKII peptide inhibitor CaMKIIN abrogated ANG II-induced VSM hypertrophy in vitro, which was augmented by overexpression of CaMKII-δ2. Finally, we identify the downstream signaling components critical for ANG II- and CaMKII-mediated VSM hypertrophy. Specifically, we demonstrate that CaMKII induces VSM hy...

MEF2 and NKX2-5 transcription factors interact with each other in cardiogenesis and are necessary for normal heart formation. Despite evidence suggesting that these two transcription factors function synergistically and possibly through direct physical interactions, molecular mechanisms by which they interact are not clear. Here we determined the crystal structures of ternary complexes of MEF2 and NKX2-5 bound to myocardin enhancer DNA in two crystal forms. These crystal structures are the first example of human MADS-box/homeobox ternary complex structures involved in cardiogenesis. Our structures reveal two possible modes of interactions between MEF2 and NKX2-5: MEF2 and NKX bind to adjacent DNA sites to recognize DNA in cis; and MEF2 and NKX bind to different DNA strands to interact with each other in trans via a conserved protein-protein interface observed in both crystal forms. Disease related mutations are mapped to the observed protein-protein interface. Our structural studies provide a starting point to understand and further study the molecular mechanisms of the interactions between MEF2 and NKX2.5 and their roles in cardiogenesis.

The myocyte enhancer factor 2 (MEF2) family of transcription factors plays important roles in developmental processes and adaptive responses. Although MEF2 proteins are known to bind DNA in the nucleus to regulate specific gene expression, there are reports that show that MEF2 also functions in the cytoplasm. Previous structural studies of MEF2 focused exclusively on DNA-bound MEF2 with and without various corepressors or coactivators. While these studies have established a comprehensive structural model of DNA recognition and cofactor recruitment by MEF2, the structure of MEF2 not bound to DNA, which include cytoplasmic MEF2 and free MEF2 in the nucleus, is unknown. Here we determined the structure of the MADS-box/MEF2 domain of MEF2B without DNA nor cofactor. The Apo structure of MEF2B reveals a largely preformed DNA binding interface that may be important for recognizing the shape of DNA from the minor groove side. In addition, our structure also reveals that the C-terminal helix...

The mechanisms underlying dopamine neuron loss in Parkinson's disease (PD) are not clearly defined. Here, we delineate a pathway by which dopaminergic loss induced by 1-methyl-4-phenyl 1,2,3,6 tetrahydropyridine (MPTP) is controlled in vivo. We reported previously that calpains play a central required role in dopamine loss after MPTP treatment. Here, we provide evidence that the downstream effector pathway of calpains is through cyclin-dependent kinase 5 (cdk5)-mediated modulation of the transcription factor myocyte enhancer factor 2 (MEF2). We show that MPTP-induced conversion of the cdk5 activator p35 to a pathogenic p25 form is dependent on calpain activity in vivo. In addition, p35 deficiency attenuates MPTP-induced dopamine neuron loss and behavioral outcome. Moreover, MEF2 is phosphorylated on Ser444, an inactivating site, after MPTP treatment. This phosphorylation is dependent on both calpain and p35 activity, consistent with the model that calpain-mediated activation of...

L’objectif de ce travail est d’effectuer une campagne d’essais expérimentaux d’impact et de compression après impact sur chant de stratifiés composites afin d’établir les scénarios d’endommagements. Un dispositif d'essai au poids tombant a été utilisé afin de réaliser les impacts sur chant sur stratifiés avec différents drapages. Des coupes microscopiques, des radiographies aux rayons X et des analyses ultrasonores ont ensuite été effectuées afin de visualiser et de déterminer le scénario d’endommagement. Des essais de compression après impact ont également été réalisés. Les résultats des tests expérimentaux sont comparés avec un modèle numérique composé d'éléments d’interface pour décrire les fissures matricielles et d’éléments volumiques. Enfin, la prédiction numérique de la tenue résiduelle après impact permettra de diminuer les masses, d’éviter des essais coûteux, et donc de raccourcir la durée de développement.Low velocity / low energy edge impact and quasi-static exper...

En los ultimos anos se ha incrementado el interes mundial en la prediccion y evaluacion de distintos escenarios de accidente en los reactores nucleares de potencia, En particular, los producidos por la perdida parcial o total del refrigerante del nucleo del reactor (Loss Of Coolant Accident-LOCA) se encuentran entre los mas severos. En estas condiciones, el sistema registra una caida de presion y/o una disminucion marcada en la velocidad de circulacion del refrigerante, por lo que el calor acumulado en las barras combustibles debe ser disipado en condiciones de refrigeracion defectuosas. En este trabajo se ha desarrollado un modelo que permite analizar y cuantificar el comportamiento del fluido refrigerante para caracterizar la termo hidraulica del canal en funcion de la presion del sistema, la velocidad y el flujo del fluido. El modelo cuantifica, las fracciones de vapor y el flujo de calor critico (CHF) en el caso de altos flujos de calor con desprendimiento de la ebullicion nucle...

Transient elevations of cytosolic Ca 2+ are a common mechanism of cellular signaling. In striated muscle, the sarco(endo)plasmic reticulum Ca 2+ ATPase (SERCA) plays an important role in terminating Ca 2+ transients by returning cytosolic Ca 2+ to intracellular stores. Stored Ca 2+ can then be released again for subsequent signaling. We down-regulated SERCA2 gene expression in cultured cardiac myocytes by means of endogenous transcription of small interfering RNA encoded by an exogenous cDNA template. The cDNA template was delivered by adenovirus vector. Reduction of SERCA expression in all myocytes in culture was documented by immunochemistry, real-time RT-PCR, and determination of ATP-dependent Ca 2+ transport. The reduction of SERCA2 expression was associated with the up-regulation of transient receptor potential (TRP) channel proteins (TRPC4 and TRPC5) and Na + /Ca 2+ exchanger, indicating that intracellular store deficiency was compensated for by Ca 2+ fluxes through the plasma...

Emerging evidence suggests that myocyte enhancer factor 2 (MEF2) transcription factors act as effectors of neurogenesis in the brain, with MEF2C the predominant isoform in developing cerebrocortex. Here, we show that conditional knockout of Mef2c in nestin-expressing neural stem/progenitor cells (NSCs) impaired neuronal differentiation in vivo , resulting in aberrant compaction and smaller somal size. NSC proliferation and survival were not affected. Conditional null mice surviving to adulthood manifested more immature electrophysiological network properties and severe behavioral deficits reminiscent of Rett syndrome, an autism-related disorder. Our data support a crucial role for MEF2C in programming early neuronal differentiation and proper distribution within the layers of the neocortex.

Laminopathies are a wide and heterogeneous group of rare human diseases caused by mutations of the LMNA gene or related nuclear envelope genes. The variety of clinical phenotypes and the wide spectrum of histopathological changes among patients carrying an identical mutation in the LMNA gene make the prognostic process rather difficult, and classical genetic screens appear to have limited predictive value for disease development. The aim of this study was to evaluate whether a comprehensive profile of circulating cytokines may be a useful tool to differentiate and stratify disease subgroups, support clinical follow-ups and contribute to new therapeutic approaches. Serum levels of 51 pro- and anti-inflammatory molecules, including cytokines, chemokines and growth factors, were quantified by a Luminex multiple immune-assay in 53 patients with muscular laminopathy (Musc-LMNA), 10 with non-muscular laminopathy, 22 with other muscular disorders and in 35 healthy controls. Interleukin-17 ...