The incubation temperature was set to 37 °C. As positive controls for cell surface exposure, strains JG137 and JG138, producing OmcAstrep and MtrCstrep, were chosen; as a control for OM integrity under the incubation conditions, the periplasmic c-type cytochrome MtrA containing an N-terminal strep-tag (MtrAstrep) was Stem Cell Compound Library manufacturer produced in an S. oneidensisΔmtrA background (JG50) (Schuetz et al., 2009). Cells were grown anaerobically overnight in minimal media with fumarate as an electron acceptor. At an OD578 nm of ∼0.2, 0.1 mM arabinose

was added to induce OM cytochrome and MtrA/MtrB production. After 4 h of production, cells were harvested and washed twice with mineral media without fumarate and lactate and then resuspended in HEPES buffer (100 mM, pH 7.5) containing 50 μM MgCl2 to obtain a final OD578 nm between 3 and 5. All further measurements were performed in independent duplicates in an anaerobic glove box. Specific reduction rates were obtained by normalization to the protein content of the cell suspension. Fifty microliters of the cell suspension was pipetted in a well of a microtiter plate. The assay was started through the addition of 150 μL of a solution containing 10 mM lactate and 10 mM ferric citrate. At different time points (0–30 min), the reaction was stopped by

the addition of 100 μL 3 M HCl. The Fe2+ concentration of the samples was determined using the ferrozine reagent (Viollier et al., 2000). The MFC setup used in this study features an anode www.selleckchem.com/products/Cyclopamine.html and cathode chamber with a working volume of 8 mL each, separated by a Nafion-117 membrane (Quintech, Göppingen; Kloke et al., 2010). A saturated calomel reference electrode (SCE) was Fossariinae separated from the anode compartment by another Nafion membrane. Electrodes were made of graphite felt cubes (Alpha Aesar, Karlsruhe) connected to platinum wires (0.1 mm; Chempur, Karlsruhe). The anode compartment was filled with 5.5 mL mineral media containing 50 mM lactate and 0.1 mM arabinose. Five hundred microliters of a cell suspension with an OD578 nm of 4 was added to start the experiment. All MFC

experiments were performed in duplicate and conducted at a constant temperature of 30 °C. The whole setup was connected to a potentiostat (Pine Instruments, Grove City). The standard measurement protocol consisted of two phases: after a conditioning period with a constant current flux over 5 h (0.3 μA cm−3), MFC cultures were subjected to a continuous increase in current density at a rate of 1.1 μA cm−3 h−1 over 45 h (current sweep phase). The anode compartment was continuously flushed with nitrogen gas to maintain anoxic conditions. Additional terminal electron acceptors were not added. A markerless multideletion mutant in all annotated OM cytochromes of S. oneidensis was constructed to generate a strain platform that allows for analysis of OM cytochrome activity without the potential detection of redundant activities from similar proteins.

“
“Research Group on Alcohol and Pharmacodependence (GRAP) – INSERM ERI 24 – SFR Cap Sante – Pharmacy School, Universite de Picardie Jules Verne, Amiens, France selleck kinase inhibitor Committee on the Neurobiology of Addictive Disorders, The Scripps Research Institute, La Jolla, CA, USA We previously found that the brain-derived neurotrophic factor (BDNF) in the dorsolateral striatum (DLS) is part of a homeostatic pathway that gates ethanol self-administration [Jeanblanc et al. (2009). J Neurosci, 29, 13494–13502)]. Specifically, we showed that moderate levels (10%) of ethanol consumption increase BDNF expression

within the DLS, and that direct infusion of BDNF into the DLS decreases operant self-administration of a 10% ethanol solution. BDNF binding to its receptor, TrkB, activates the mitogen-activated protein kinase (MAPK), phospholipase C-γ (PLC-γ) and phosphatidylinositol 3-kinase (PI3K) pathways. Thus, here, we set out to identify which of these intracellular pathway(s) plays a role in the regulation of ethanol consumption by BDNF. We found that inhibition of the MAPK,

but not PLC-γ or PI3K, activity blocks the BDNF-mediated reduction of ethanol consumption. As activation of the MAPK pathway leads to the initiation of transcription and/or translation events, we tested whether the BDNF-mediated reduction of ethanol self-administration requires de novo protein synthesis. We found that the inhibitory effect of BDNF on ethanol intake is see more blocked by the protein synthesis inhibitor cycloheximide. Together, our results show that BDNF attenuates ethanol drinking via activation of the MAPK pathway in a protein synthesis-dependent manner within the DLS. “
“In contrast to mammals, adult zebrafish recover locomotor functions after spinal cord injury (SCI), in part due to axonal regrowth and regeneration permissivity of the

central nervous system. Upregulation of major vault protein (MVP) expression after spinal cord injury in the brainstem of the adult zebrafish prompted us to probe for its contribution to recovery after SCI. MVP is a multifunctional protein expressed not only in many types of tumours but also in Tyrosine-protein kinase BLK the nervous system, where its importance for regeneration is, however, unclear. Using an established zebrafish SCI model, we found that MVP mRNA and protein expression levels were increased in ependymal cells in the spinal cord caudal to the lesion site at 6 and 11 days after SCI. Double immunolabelling showed that MVP was co-localised with Islet-1 or tyrosine hydroxylase around the central canal of the spinal cord in sham-injured control fish and injured fish 11 days after surgery. MVP co-localised with the neural stem cell marker nestin in ependymal cells after injury.