Mammalian P2X receptors contain 10 conserved cysteine residues in their ectodomains, which form five disulfide bonds (SS1-5). Here, we analyzed the relevance of these disulfide pairs in rat P2X4 receptor function by replacing one or both cysteines with alanine or threonine, expressing receptors in HEK293 cells and studying their responsiveness to ATP in the absence and presence of ivermectin, an allostenic modulator of these channels. Response to ATP was not altered when both cysteines forming the SS3 bond (C132-C159) were replaced with threonines. Replacem ent of SS1 (C116-C165), SS2 (C126-C149) and SS4 (C217-C227), but not SS5 (C261-C270), cysteine pairs with threonines resulted in de creased sensitivity to ATP and faster deactivation times. The maximum current amplitude was reduced in SS2, SS4 and SS5 double mutants and could be partially rescued by ivermectin in SS2 and SS5 double mutants. This response pattern was also observed in numerous single residue mutants, but receptor function was not affected when the 217 cysteine was replaced with threonine or arginine or when the 261 cysteine was replaced with alanine. These results suggest that the SS1, SS2 and SS4 bonds contribute substantially to the structure of the ligand binding pocket, while the SS5 bond located towards the transmembrane domain contributes to receptor gating., M. B. Rokic, V. Tvrdoňová, V. Vávra, M. Jindřichová, T. Obšil, S. S. Stojilkovic, H. Zemková., and Obsahuje bibliografii
Resistance to anthelmintic medication of equid strongyles is a worldwide phenomenon and for this reason systematic investigations of resistant parasite populations are necessary. The purpose of the present study was to investigate the presence and distribution of equid strongyles resistant to the anthelmintics used in Romania, as well as the pre-treatment and post-treatment prevalence of species of strongylid nematodes. The Faecal Egg Count Reduction Test was performed between 2010 and 2013 on a total number of 588 horses and 23 donkeys from 26 locations (subgroups). Animals of the first group (I) consisting of subgroups no. 1-11 were treated with Albendazole (ABZ), those of the second group (II) consisting of subgroups no. 12-23 with Fenbendazole (FBZ), while Ivermectin (IVM) was used on animals of the third group (III) consisting of subgroups no. 24-26. Resistant strongyles have been found in 82% (average lower limit of the 95% confidence interval, LCL95%, was 65) of the total equids from the group treated with ABZ. In the group of horses treated with FBZ, resistant strongyles were identified in 75% of the subgroups (LCL95% = 44). No resistant strongyles have been identified in IVM-treated horse groups (LCL95% = 98). The pre-treatment prevalence of the species of the Strongylinae Müller, 1780 was 22%, whereas that with nematodes of the subfamily Cyathostominae Molin, 1861 78%. Post-treatment reduction of strongyline nematodes was observed (5%), which demonstrates the sensitivity of large strongyles to common anthelmintics. The post-treatment prevalence of cyathostomes was of 95%, which proves their resistance, especially to ABZ- and FBZ-based anthelmintics., Mihai Cernea, Romeo T. Cristina, Laura C. Ştefănuţ, Luís M. Madeira de Carvalho, Marian A. Taulescu, Vasile Cozma., and Obsahuje bibliografii