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“The vasopressin type 2 (V2R) receptor belongs to the class of G-protein coupled receptors. It is mainly expressed in the membrane of kidney tubules, Avapritinib where it is activated by the extracellular arginine vasopressin. In men, inactivating and activating mutations cause nephrogenic diabetes insipidus and the nephrogenic syndrome of inappropriate antidiuresis respectively. Like most GPCRs, V2R’s third intracellular loop (V2R-i3) is involved in the binding and activation of its major effector, the G alpha S protein.

We overexpressed the V2R(224-274) fragment corresponding to V2R-i3 as a fusion protein with thioredoxin A at the N-terminus and a hexahistidine tag between the two proteins. Recombinant V2R-i3 was designed to harbor N- and C-terminal cysteines, in

order to introduce a disulfide bond between N- and C-terminal extremities and hence reproduce the hairpin fold presumably present in the full-length receptor. The fusion protein was produced as inclusion bodies in Escherichia coli and purified by nickel affinity chromatography under denaturing conditions. After a refolding step, thioredoxin and hexahistidine tags were specifically cleaved with the tobacco etch virus protease. The hydrolysis yield, initially very low, increased up to 80% thanks to optimization of buffers and refolding methods. The cleaved fragment, V2(224-274), devoid of any tag, was then eluted with low imidazole concentrations in a second nickel affinity chromatography in denaturing conditions. The final yield was sufficient to prepare Bromosporine nmr a (15)N-(13)C labeled NMR sample suitable for triple resonance

experiments. We assigned all NMR resonances and confirmed the correct peptide sequence. As expected, the peptide forms a hairpin stabilized by a disulfide bond between its N- and C-terminal parts, thus mimicking its native structure in the full-length receptor. This study may provide a strategy for producing and studying the structure/function relationship of GPCR Urease fragments. (C) 2011 Elsevier Inc. All rights reserved.”
“This study conceptualized loneliness as a mediator in the relation between social engagement and depressive symptoms and explored gender differences in the mediation model. Various indices of social engagement were considered including living arrangement, social network, and activity participation.

Using data from 674 community-dwelling Korean American older adults, we first examined the mediation effect of loneliness in the relation between each of 3 indices of social engagement (not living alone, social network, and activity participation) and depressive symptoms. Subsequently, gender differences in the mediation model were examined.

As hypothesized, loneliness was found to mediate the relation between each of the indices of social engagement and depressive symptoms in both men and women.