Two full-length glutamate-gated chloride channel (GluCl) cDNAs, encoding GluCla3 and GluClb subunits, were cloned from ivermectin-susceptible (IVS) and -resistant (IVR) Cooperia oncophora adult worms. The IVS and IVR GluCla3 subunits differ at three amino acid positions, while the IVS and IVR GluClb subunits differ at two amino acid positions. The aim of this study was to determine whether mutations in the IVR subunits affect agonist sensitivity. The subunits were expressed singly and in combination in Xenopus laevis oocytes. Electrophysiological whole-cell voltage-clamp recordings showed that mutations in the IVR GluCla3 caused a modest but significant threefold loss of sensitivity to glutamate, the natural ligand for GluCl receptors. As well, a significant decrease in sensitivity to the anthelmintics ivermectin and moxidectin was observed in the IVR GluCla3 receptor.Mutations in the IVR GluClb subunit abolished glutamate sensitivity. Co-expressing the IVS GluCla3 and GluClb subunits resulted in heteromeric channels that were more sensitive to glutamate than the respective homomeric channels, demonstrating co-assembly of the subunits. In contrast, the heteromeric IVR channels were less sensitive to glutamate than the homomeric IVR GluCla3 channels. The heteromeric IVS channels were significantly more sensitive to glutamate than the heteromeric IVR channels. Of the three amino acids distinguishing the IVS and IVR GluCla3 subunits, only one of them, L256F, accounted for the differences in response between the IVS and IVR GluCla3 homomeric channels.
Human cytomegalovirus (CMV) is a major cause of nonhereditary adverse birth outcomes, including hearing and visual loss, neurologic deficits, and intrauterine growth retardation (IUGR), and may contribute to outcomes such as stillbirth and preterm delivery. However, the mechanisms by which CMV could cause adverse birth outcomes are not fully understood. This study reviewed proposed mechanisms underlying the role of CMV in stillbirth, preterm birth, and IUGR. Targeted literature searches were performed in PubMed and Embase to identify relevant articles. Several potential mechanisms were identified from in vitro studies in which laboratory-adapted and low-passage strains of CMV and various human placental models were used. Potential mechanisms identified included impairment of trophoblast progenitor stem cell differentiation and function, impairment of extravillous trophoblast invasiveness, dysregulation of Wnt signaling pathways in cytotrophoblasts, tumor necrosis factor-α mediated apoptosis of trophoblasts, CMV-induced cytokine changes in the placenta, inhibition of indoleamine 2,3-dioxygenase activity, and downregulation of trophoblast class I major histocompatibility complex molecules. Inherent challenges for the field remain in the identification of suitable in vivo animal models. Nonetheless, we believe that our review provides useful insights into the mechanisms by which CMV impairs placental development and function and how these changes could result in adverse birth outcomes.
The glutamate-gated chloride channels (GluCls) are members of the ligand-gated ion channel superfamily that are thought to be involved in the mode of action of ivermectin and mechanism of resistance. Using reverse-transcriptase PCR techniques, 2 full-length GluCl cDNAs, encoding GluClalpha3 and GluClbeta subunits, were cloned from Cooperia oncophora, a nematode parasite of cattle. The two sequences show a high degree of identity to similar subunits from other nematodes. The C. oncophora GluClalpha3 subunit is most closely related to the Haemonchus contortus GluClalpha3B subunit, while C. oncophora GluClbeta subunit shares high sequence identity with the H. contortus GluClbeta subunit. Using single-strand conformation polymorphism, the genetic variability of these two genes was analysed in an ivermectin-susceptible isolate and an ivermectin-resistant field isolate of C. oncophora. Statistical analysis suggested an association between the C. oncophora GluClalpha3 gene and ivermectin resistance. No such association was seen with the GluClbeta gene.
Two full-length beta-tubulin cDNAs, representing isotypes 1 and 2, were cloned from the cattle nematode Cooperia oncophora. The predicted protein sequences span 448 amino acids, and show a high degree of identity to beta-tubulins from other nematodes. While C. oncophora isotype 1 sequence had the highest identity to Haemonchus contortus isotype 1 and Teladorsagia circumcincta sequences (95% identity), the C. oncophora isotype 2 sequence was most similar to H. contortus isotype 2 and Trichostrongylus colubriformis (92% identity). Alignment of the two C. oncophora sequences with other trichostrongylid beta-tubulins deposited in GenBank showed a clear distinction between isotype 1 and 2 beta-tubulin classes. The two classes differed at 19 amino acid positions, most notably at the carboxy terminus. These isotype-defining residues were conserved among different trichostrongylid species within a class. Analysis of fragments of both genes revealed a high degree of genetic variability in coding and non-coding regions. However, all nucleotide differences detected in the coding region were silent, as they did not result in any amino acid substitution. Analysis of 2 groups of worms for the codon 200 polymorphism associated with benzimidazole resistance revealed a proportion of worms in 1 of the groups bearing a tyrosine at this position.
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