However, in the presence

of uncoupling agent carbonyl cyanide4-(trifluoromethoxy)phenylhydrazone (FCCP) (1 mu mol/l), pre-treatment with 0.5 nmol/l E2 protected mitochondrial membrane potential and Protein Tyrosine Kinase inhibitor consequently increased Ca2+ influx (2.3-fold in NC and 3.1-fold in BS). At the same time, 0.5 nmol/l E2 by increasing the affinity of mitochondrial Na+/Ca2+ exchanger for Na+ inhibited mitochondrial Ca2+ efflux in NC and BS by about 40%. Also, the specific binding of physiological E2 concentrations (0.1-10 nmol/l) to isolated synaptosomal mitochondria was detected. Using membrane impermeable E2 bound to bovine serum albumin and selective inhibitor of mitochondrial Na+/Ca2+ exchanger, we obtained that E2′s action on mitochondrial Ca2+ efflux at least partially is due to the direct effects on the mitochondrial membrane and/or Na+/Ca2+

exchanger located in inner mitochondrial membrane. Our results implicate E2 as a modulator of Ca2+ concentration in mitochondrial matrix, and ultimately in the cytosol. Given the vital role of Ca2+ in regulation of total nerve cells activity, especially energy metabolism, neurotransmission and directing the cells toward survival or cell death, the effects on mitochondrial Ca2+ transport could be one of the important modes of E2 neuromodulatory action independent of the genome. (C) 2012 IBRO. Published by Elsevier Ltd. All rights reserved.”
“A double mutant of human purine nucleoside phosphorylase (hDM) with the amino acid

mutations Glu201Gln: Asn243Asp this website cleaves adenosine-based prodrugs to their corresponding cytotoxic drugs. When fused to an anti-tumor targeting component, hDM is targeted to tumor cells, where it effectively catalyzes phosphorolysis of the prodrug, 2-fluoro-20-deoxyadenosine Batimastat (F-dAdo) to the cytotoxic drug, 2-fluoroadenine (F-Ade). This cytotoxicity should be restricted only to the tumor microenvironment, because the endogenously expressed wild type enzyme cannot use adenosine-based prodrugs as substrates. To gain insight into the interaction of hDM with F-dAdo, we have determined the crystal structures of hDM with F-dAdo and F-Ade. The structures reveal that despite the two mutations, the overall fold of hDM is nearly identical to the wild type enzyme. Importantly, the residues Gln201 and Asp243 introduced by the mutation form hydrogen bond contacts with F-dAdo that result in its binding and catalysis. Comparison of substrate and product complexes suggest that the side chains of Gln201 and Asp243 as well as the purine base rotate during catalysis possibly facilitating cleavage of the glycosidic bond. The two structures suggest why hDM, unlike the wild-type enzyme, can utilize F-dAdo as substrate. More importantly, they provide a critical foundation for further optimization of cleavage of adenosine-based prodrugs, such as F-dAdo by mutants of human purine nucleoside phosphorylase.