Dynamin is functionally coupled to insulin granule exocytosis

The insulin granule integral membrane protein marker phogrin-green fluorescent protein was co-localized with insulin in Min6B1 beta-cell secretory granules but did not undergo plasma membrane translocation following glucose stimulation. Surprisingly, although expression of a dominant-interfering dynamin mutant (Dyn/K44A) inhibited transferrin receptor endocytosis, it had no effect on phogringreen fluorescent protein localization in the basal or secretagogue-stimulated state. By contrast, co-expression of Dyn/K44A with human growth hormone as an insulin secretory marker resulted in a marked inhibition of human growth hormone release by glucose, KCl, and a combination of multiple secretagogues. Moreover, serial pulse depolarization stimulated an increase in cell surface capacitance that was also blocked in cells expressing Dyn/K44A. Similarly, small interference RNA-mediated knockdown of dynamin resulted in marked inhibition of glucose-stimulated insulin secretion. Together, these data suggest the presence of a selective kiss and run mechanism of insulin release. Moreover, these data indicate a coupling between endocytosis and exocytosis in the regulation of beta-cell insulin secretion.     

The long journey to a Systems Biology of neuronal function

Computational neurobiology was born over half a century ago, and has since been consistently at the forefront of modelling in biology. The recent progress of computing power and distributed computing allows the building of models spanning several scales, from the synapse to the brain. Initially focused on electrical processes, the simulation of neuronal function now encompasses signalling pathways and ion diffusion. The flow of quantitative data generated by the "omics" approaches, alongside the progress of live imaging, allows the development of models that will also include gene regulatory networks, protein movements and cellular remodelling. A systems biology of brain functions and disorders can now be envisioned. As it did for the last half century, neuroscience can drive forward the field of systems biology.     

Techniques de recherche des polymorphismes génétiques

Sequencing the human genome has allowed the discovery of millions of DNA sequence variants. Sequence variations in human DNA are mainly present asSingle Nucleotide Polymorphisms (SNPs); this common form of variation is found about once every 1,000 bases in the human genome and 1.8 million SNPs have now been identified and located. The accessibility of databases of SNPs opens the possibility of studying the influence of these polymorphisms on disease risks as well as on drug responses. Numerous approaches have been set up for the identification of SNPs. In this review we describe the main techniques used for the identification of these polymorphisms. They rely on two major consequences of sequence variations: the apparition or the disappearance of restriction enzyme sites or the alteration of DNA strand hybridization due to the presence of a mismatch. Southern blotting and restriction endonucleases have allowed the development of the technique ofrestriction fragment length polymorphisms (RFLPs), now performed on PCR products. Several other approaches such as denaturing high-performance liquid chromatography or real-time PCR can detect allele differences upon re-hybridization and heteroduplex formation. However, DNA sequencing remains the obligate step for the positive identification of known or unknown SNPs. At last, the development of high-throughput methods allows a large increase in the rate of discovery of SNPs likely.     

Coxsackievirus entry across epithelial tight junctions requires occludin and the small GTPases Rab34 and Rab5

The major group B coxsackievirus (CVB) receptor is a component of the epithelial tight junction (TJ), a protein complex that regulates the selective passage of ions and molecules across the epithelium. CVB enters polarized epithelial cells from the TJ, causing a transient disruption of TJ integrity. Here we show that CVB does not induce major reorganization of the TJ, but stimulates the specific internalization of occludin-a TJ integral membrane component-within macropinosomes. Although occludin does not interact directly with virus, depletion of occludin prevents CVB entry into the cytoplasm and inhibits infection. Both occludin internalization and CVB entry require caveolin but not dynamin; both are blocked by inhibitors of macropinocytosis and require the activity of Rab34, Ras, and Rab5, GTPases known to regulate macropinocytosis. Thus, CVB entry depends on occludin and occurs by a process that combines aspects of caveolar endocytosis with features characteristic of macropinocytosis.     

High reinforcing efficacy of nicotine in non-human primates

Although tobacco appears highly addictive in humans, there has been persistent controversy about the ability of its psychoactive ingredient nicotine to induce self-administration behavior in laboratory animals, bringing into question nicotine's role in reinforcing tobacco smoking. Because of ethical difficulties in inducing nicotine dependence in na?ve human subjects, we explored reinforcing effects of nicotine in experimentally-naive non-human primates given access to nicotine for periods of time up to two years. Five squirrel monkeys with no experimental history were allowed to intravenously self-administer nicotine by pressing one of two levers. The number of presses on the active lever needed to obtain each injection was fixed (fixed-ratio schedule) or increased progressively with successive injections during the session (progressive-ratio schedule), allowing evaluation of both reinforcing and motivational effects of nicotine under conditions of increasing response cost. Over time, a progressive shift toward high rates of responding on the active lever, but not the inactive lever, developed. The monkeys' behavior was clearly directed toward nicotine self-administration, rather than presentation of environmental stimuli associated with nicotine injection. Both schedules of reinforcement revealed a high motivation to self-administer nicotine, with monkeys continuing to press the lever when up to 600 lever-presses were needed for each injection of nicotine. Thus, nicotine, by itself, in the absence of behavioral or drug-exposure history, is a robust and highly effective reinforcer of drug-taking behavior in a non-human primate model predictive of human behavior. This supports the use of nicotinic ligands for the treatment of smokers, and this novel preclinical model offers opportunities to test future medications for the treatment of nicotine dependence.     

Early energy deficit in Huntington disease: identification of a plasma biomarker traceable during disease progression

Huntington disease (HD) is a fatal neurodegenerative disorder, with no effective treatment. The pathogenic mechanisms underlying HD has not been elucidated, but weight loss, associated with chorea and cognitive decline, is a characteristic feature of the disease that is accessible to investigation. We, therefore, performed a multiparametric study exploring body weight and the mechanisms of its loss in 32 presymptomatic carriers and HD patients in the early stages of the disease, compared to 21 controls. We combined this study with a multivariate statistical analysis of plasma components quantified by proton nuclear magnetic resonance ((1)H NMR) spectroscopy. We report evidence of an early hypermetabolic state in HD. Weight loss was observed in the HD group even in presymptomatic carriers, although their caloric intake was higher than that of controls. Inflammatory processes and primary hormonal dysfunction were excluded. (1)H NMR spectroscopy on plasma did, however, distinguish HD patients at different stages of the disease and presymptomatic carriers from controls. This distinction was attributable to low levels of the branched chain amino acids (BCAA), valine, leucine and isoleucine. BCAA levels were correlated with weight loss and, importantly, with disease progression and abnormal triplet repeat expansion size in the HD1 gene. Levels of IGF1, which is regulated by BCAA, were also significantly lower in the HD group. Therefore, early weight loss in HD is associated with a systemic metabolic defect, and BCAA levels may be used as a biomarker, indicative of disease onset and early progression. The decreased plasma levels of BCAA may correspond to a critical need for Krebs cycle energy substrates in the brain that increased metabolism in the periphery is trying to provide.     

The murine gammaherpesvirus-68 gp150 acts as an immunogenic decoy to limit virion neutralization

Herpesviruses maintain long-term infectivity without marked antigenic variation. They must therefore evade neutralization by other means. Immune sera block murine gammaherpesvirus-68 (MHV-68) infection of fibroblasts, but fail to block and even enhance its infection of IgG Fc receptor-bearing cells, suggesting that the antibody response to infection is actually poor at ablating virion infectivity completely. Here we analyzed this effect further by quantitating the glycoprotein-specific antibody response of MHV-68 carrier mice. Gp150 was much the commonest glycoprotein target and played a predominant role in driving Fc receptor-dependent infection: when gp150-specific antibodies were boosted, Fc receptor-dependent infection increased; and when gp150-specific antibodies were removed, Fc receptor-dependent infection was largely lost. Neither gp150-specific monoclonal antibodies nor gp150-specific polyclonal sera gave significant virion neutralization. Gp150 therefore acts as an immunogenic decoy, distorting the MHV-68-specific antibody response to promote Fc receptor-dependent infection and so compromise virion neutralization. This immune evasion mechanism may be common to many non-essential herpesvirus glycoproteins.     

Noncompaction of the ventricular myocardium is associated with a de novo mutation in the beta-myosin heavy chain gene

Noncompaction of the ventricular myocardium (NVM) is the morphological hallmark of a rare familial or sporadic unclassified heart disease of heterogeneous origin. NVM results presumably from a congenital developmental error and has been traced back to single point mutations in various genes. The objective of this study was to determine the underlying genetic defect in a large German family suffering from NVM. Twenty four family members were clinically assessed using advanced imaging techniques. For molecular characterization, a genome-wide linkage analysis was undertaken and the disease locus was mapped to chromosome 14ptel-14q12. Subsequently, two genes of the disease interval, MYH6 and MYH7 (encoding the alpha- and beta-myosin heavy chain, respectively) were sequenced, leading to the identification of a previously unknown de novo missense mutation, c.842G>C, in the gene MYH7. The mutation affects a highly conserved amino acid in the myosin subfragment-1 (R281T). In silico simulations suggest that the mutation R281T prevents the formation of a salt bridge between residues R281 and D325, thereby destabilizing the myosin head. The mutation was exclusively present in morphologically affected family members. A few members of the family displayed NVM in combination with other heart defects, such as dislocation of the tricuspid valve (Ebstein's anomaly, EA) and atrial septal defect (ASD). A high degree of clinical variability was observed, ranging from the absence of symptoms in childhood to cardiac death in the third decade of life. The data presented in this report provide first evidence that a mutation in a sarcomeric protein can cause noncompaction of the ventricular myocardium.     

Phenotypic evolution from genetic polymorphisms in a radial network architecture

Background  

The genetic architecture of a quantitative trait influences the phenotypic response to natural or artificial selection. One of the main objectives of genetic mapping studies is to identify the genetic factors underlying complex traits and understand how they contribute to phenotypic expression. Presently, we are good at identifying and locating individual loci with large effects, but there is a void in describing more complex genetic architectures. Although large networks of connected genes have been reported, there is an almost complete lack of information on how polymorphisms in these networks contribute to phenotypic variation and change. To date, most of our understanding comes from theoretical, model-based studies, and it remains difficult to assess how realistic their conclusions are as they lack empirical support.