To that end we have successfully expressed the hydrosoluble ecto-

To that end we have successfully expressed the hydrosoluble ecto-domain of bovine CD38 (bCD38; residues 32-278), and corresponding glycosylation mutants, in the methylotrophic

yeast Pichia pastoris. The secreted proteins were purified to homogeneity by affinity chromatography on immobilized Cibacron blue. We found by site-directed mutagenesis and mass spectrometry that bCD38 was a monoglycosylated P5091 protein at Asn-201. The expression yield of the deglycosylated mutants was not significantly affected, indicating that glycosylation at Asn-201 was not required for a proper processing and secretion of this protein by A pastoris. Significant alterations in the kinetic parameters of NAD(+) were observed for the deglycosylated mutants. The thermo-stability of the recombinant enzyme was also influenced by mutation at position 201. Interestingly both parameters were dependent on the nature of the mutant and a stable deglycosylated mutant N201D of bCD38 could be produced that can be further used for structural studies. (C) 2009 Elsevier Inc. All rights reserved.”
“Recent in vivo studies have shown that erythropoietin (EPO) offers strong protection against brain edema. However, the intracellular and molecular mechanisms

behind this beneficial check details effect have not been specified. The aim of this study was to determine whether human erythropoietin (rhEPO) reduces the astrocytic swelling created by oxygen-glucose deprivation followed by reoxygenation (OGD/Reox) in vitro and whether this effect can be mediated through the modulation of aquaporin4 (AQP4) expression in the plasma

membrane (PM) and phosphorylation of the mitogen-activated protein kinase pathway (MAPK) pathway. Our results showed that OGD/Reox produced increase in cell volume, morphological swelling, and mitochondrial swelling. These changes were associated with the up-regulation of Adenylyl cyclase AQP4 in PM and the over-activation of MAPK. Silencing AQP4 expression using small interfering ribonucleic acid for AQP4 was found to block astrocytic swelling. Inhibition of the over-activation of MAPK mitigated the PM AQP4 overabundance and cellular swelling. As expected, treatment with rhEPO significantly reduced the OGD/Reox-induced increase in cell volume, morphological swelling, and mitochondrial swelling as well as the up-regulation of AQP4 in PM. In addition, cultures treated with the neutralizing anti-EPO antibody worsened the PM AQP4 abundance and cellular swelling, abolishing the protective effects mediated by rhEPO treatment. Furthermore, the over-activation of these MAPK after OGD/Reox was attenuated by rhEPO treatment significantly. In conclusion, our data strongly suggest that rhEPO can protect astrocytes from swelling caused by ischemia and reperfusion-like injury.

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