Ca2+ Regulates the Drosophila Stoned-A and Stoned-B Proteins Interaction with the C2B Domain of Synaptotagmin-1

The dicistronic Drosophila stoned gene is involved in exocytosis and/or endocytosis of synaptic vesicles. Mutations in either stonedA or stonedB cause a severe disruption of neurotransmission in fruit flies. Previous studies have shown that the coiled-coil domain of the Stoned-A and the µ-homology domain of the Stoned-B protein can interact with the C2B domain of Synaptotagmin-1. However, very little is known about the mechanism of interaction between the Stoned proteins and the C2B domain of Synaptotagmin-1. Here we report that these interactions are increased in the presence of Ca2+. The Ca2+-dependent interaction between the µ-homology domain of Stoned-B and C2B domain of Synaptotagmin-1 is affected by phospholipids. The C-terminal region of the C2B domain, including the tryptophan-containing motif, and the Ca2+ binding loop region that modulate the Ca2+-dependent oligomerization, regulates the binding of the Stoned-A and Stoned-B proteins to the C2B domain. Stoned-B, but not Stoned-A, interacts with the Ca2+-binding loop region of C2B domain. The results indicate that Ca2+-induced self-association of the C2B domain regulates the binding of both Stoned-A and Stoned-B proteins to Synaptotagmin-1. The Stoned proteins may regulate sustainable neurotransmission in vivo by binding to Ca2+-bound Synaptotagmin-1 associated synaptic vesicles

Post-Translational Modifications and Lipid Binding Profile of Insect Cell-Expressed Full-Length Mammalian Synaptotagmin 1

ABSTRACT: Synaptotagmin 1 (Syt1) is a Ca2+ sensor for SNARE-mediated, Ca2+-triggered synaptic vesicle fusion in neurons. It is composed of luminal, transmembrane, linker, and two Ca2+-binding (C2) domains. Here we describe expression and purification of full-length mammalian Syt1 in insect cells along with an extensive biochemical characterization of the purified protein. The expressed and purified protein is properly folded and has increased α-helical content compared to the C2AB fragment alone. Post-translational modifications of Syt1 were analyzed by mass spectrometry, revealing the same modifications of Syt1 that were previously described for Syt1 purified from brain extract or mammalian cell lines, along with a novel modification of Syt1, tyrosine nitration. A lipid binding screen with both full-length Syt1 and the C2AB fragments of Syt1 and Syt3 isoforms revealed new Syt1−lipid interactions. These results suggest a conserved lipid binding mechanism in which Ca2+-independent interactions are mediated via a lysine rich region of the C2B domain while Ca2+-dependent interactions are mediated via the Ca2+-binding loops

Murid Herpesvirus-4 Exploits Dendritic Cells to Infect B Cells

Dendritic cells (DCs) play a central role in initiating immune responses. Some persistent viruses infect DCs and can disrupt their functions in vitro. However, these viruses remain strongly immunogenic in vivo. Thus what role DC infection plays in the pathogenesis of persistent infections is unclear. Here we show that a persistent, B cell-tropic gamma-herpesvirus, Murid Herpesvirus-4 (MuHV-4), infects DCs early after host entry, before it establishes a substantial infection of B cells. DC-specific virus marking by cre-lox recombination revealed that a significant fraction of the virus latent in B cells had passed through a DC, and a virus attenuated for replication in DCs was impaired in B cell colonization. In vitro MuHV-4 dramatically altered the DC cytoskeleton, suggesting that it manipulates DC migration and shape in order to spread. MuHV-4 therefore uses DCs to colonize B cells

Origine et développement du combustible Uranium-Molybdène (U-Mo)

Historiquement, la plupart des réacteurs expérimentaux, en particulier les Réacteurs de Test et de Recherche, producteurs de hauts flux neutroniques, ont utilisé du combustible métallique à haut enrichissement en U235 (>90%). La politique actuelle est de plafonner l'enrichissement en U235 à 20% (objectif de non-prolifération). Après une première génération de combustible enrichi à 19.75% en U235 de type siliciure non retraitable, un consensus s'est dégagé au niveau international autour d'une nouvelle génération de combustible enrichi à 19.75% en U235, le combustible U-Mo (uranium - molybdène). Ce combustible permet de maintenir, voire d'améliorer, les performances actuelles des réacteurs tout en garantissant une fin de cycle acceptable (combustible retraitable). CEA, COGEMA, CERCA, FRAMATOME et TECHNICATOME ont regroupé leurs moyens techniques, financiers et leur savoir-faire pour développer en commun ce nouveau combustible U-Mo. Il est destiné aux réacteurs existants déjà convertis ou devant se convertir à l'uranium faiblement enrichi (en France, en Allemagne, au Japon, en Suède, ...) ainsi qu'aux nouveaux réacteurs tels que le RJH en France ou RRR en Australie. Ce programme de R& D démarré en 1999 est conduit en cohérence avec les travaux réalisés aux USA par l'Argonne National Laboratory. Compte tenu de la nécessité impérieuse pour les réacteurs de disposer en 2006 de ce nouveau combustible, seul à offrir une véritable solution de fin de cycle, l'objectif du groupe U-Mo français est de terminer la R& D et la qualification du combustible en 2005

Chimeric Theiler's virus with altered tropism for the central nervous system.

Theiler's virus is a neurotropic murine picornavirus which, depending on the strain, causes either an acute encephalitis or a persistent demyelinating disease. Following intracranial inoculation, the demyelinating strains infect sequentially the grey matter of the brain, the grey matter of the spinal cord, and finally the white matter of the spinal cord, where they persist and cause chronic demyelination. The neurovirulent strains cause a generally fatal encephalitis with lytic infection of neurons. The study of chimeric Theiler's viruses, obtained by recombining the genomes of demyelinating and neurovirulent strains, has shown that the viral capsid contains determinants for persistence and demyelination. In this article we describe the recombinant virus R5, in which the capsid protein VP1 and a small portion of protein 2A come from the neurovirulent GDVII strain and the rest of the genome comes from the persistent DA strain. The capsid of virus R5 also contains one mutation at amino acid 34 of VP3 (Asn-->His). Virus R5 does not persist in the central nervous system (CNS) of immunocompetent SJL/J or BALB/c mice. However, it replicates efficiently and persists in the CNS of BALB/c nu/nu mice, showing that its growth in the CNS is not impaired. In BALB/c nu/nu mice, whereas virus DA causes mortality with large amounts of viral antigens in the white matter of the spinal cord, virus R5 does not kill the animals, persists in the neurons of the grey matter of the brain, and never reaches the white matter of the spinal cord. This phenotype is due to the chimerism of the capsid and/or to the mutation in VP3. These results indicate that the capsid plays an important role in the characteristic migration of Theiler's virus within the CNS

A genetic approach to study the pathogenesis of Theiler's virus persistent infection.

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Thermal conductivity measurement of IRIS-TUM fuel plates (disperse U-8wt.%-Mo)

The thermal diffusivity of ground U-8wt.%-Mo powder in an aluminium matrix has been measured in the temperature range of 300°C to 400°C. By means of best estimates for the heat capacity and density of this complex compound a thermal conductivity of about 25 W/mK has been determined.JRC.E.3-Materials researc