“Background/Aims: The Janus kinase 3 JAK3 participates in the signaling of immune cells. Lack of JAK3 triggers inflammatory bowel disease, which in turn has been shown to affect intestinal activity of the epithelial Na+ channel ENaC and thus colonic sodium absorption. At least in theory, inflammatory bowel disease in JAK3-deficient mice could lead to intestinal salt loss compromizing extracellular volume maintenance and blood pressure regulation. The
present study thus explored whether JAK3 deficiency impacts on colonic ENaC activity, fecal Na+ exretion, blood pressure and extracellular fluid volume regulation. Methods: Experiments were performed in gene-targeted mice lacking functional JAK3 (jak3(-/-)) and in wild type mice (jak3(+/+)). Colonic ENaC activity was estimated from find more amiloride-sensitive current in Ussing chamber experiments, fecal, serum and urinary Na+ concentration by flame photometry, blood pressure by the tail cuff method and serum aldosterone levels by immunoassay. Results: The amiloride (50 mu M)-induced deflection of the transepithelial potential difference was significantly lower and fecal Na+ excretion significantly higher in jak3(-/-) mice than in jak3(+/+) mice. Moreover, systolic IACS-10759 mouse arterial blood pressure was significantly lower and serum aldosterone concentration significantly higher
in jak3(-/-) mice than in jak3(+/+) mice. Both, absolute and fractional renal Na+ excretion were significantly lower in jak3(-/-) mice than in jak3(+/+) mice. Conclusions: JAK3 deficiency leads to impairment of colonic ENaC activity with intestinal Na+ loss, decrease of blood pressure, increased aldosterone release and subsequent
stimulation of renal tubular Na+ reabsorption. Copyright (C) 2013 S. Karger AG, Basel”
“The well-established method for high-throughput construction of an expression system of the yeast Saccharomyces cerevisiae uses homologous recombination between an expression plasmid and a target gene (with homologous regions of the plasmid on both ends added by PCR). This method has been widely used for membrane proteins using plasmids containing GFP, and has been successfully used to investigate the cellular localization and solubilization conditions of the proteins. Although the methanol-utilizing yeast Pichia pastoris is Flucloronide known as an excellent expression host, a method for high-throughput construction of an expression system like that in S. cerevisiae has not been reported. In this study, we have attempted to construct expression systems via homologous recombination in P. pastoris. The insertion of genes into a plasmid could be easily checked by colony-PCR. Expression systems for seven membrane proteins of medaka fish (Oryzias latipes) and yeast (S. cerevisiae) were constructed, and the expression of proteins was analyzed by fluorescence spectra, fluorescence microscopy, and SDS-PAGE (in-gel fluorescence detection). (c) 2010 Elsevier Inc. All rights reserved.