RESUMEN
The Trypanosoma brucei aminopurine transporter P2/TbAT1 has long been implicated in the transport of, and resistance to, the diamidine and melaminophenyl arsenical classes of drugs that form the backbone of the pharmacopoeia against African trypanosomiasis. Genetic alterations including deletions and single nucleotide polymorphisms (SNPs) have been observed in numerous strains and clinical isolates. Here, we systematically investigate each reported mutation and assess their effects on transporter function after expression in a tbat1(-/-) T. brucei line. Out of a set of six reported SNPs from a reported 'resistance allele', none significantly impaired sensitivity to pentamidine, diminazene or melarsoprol, relative to the TbAT1-WT allele, although several combinations, and the deletion of the codon for residue F316, resulted in highly significant impairment. These combinations of SNPs, and ΔF316, also strongly impaired the uptake of [(3)H]-adenosine and [(3)H]-diminazene, identical to the tbat1(-/-) control. The TbAT1 protein model predicted that residues F19, D140 and F316 interact with the substrate of the transporter. Mutation of D140 to alanine resulted in an inactive transporter, whereas the mutation F19A produced a transporter with a slightly increased affinity for [(3)H]-diminazene but reduced the uptake rate. The results presented here validate earlier hypotheses of drug binding motifs for TbAT1.
Asunto(s)
Modelos Moleculares , Proteínas de Transporte de Nucleósidos/química , Proteínas de Transporte de Nucleósidos/genética , Tripanocidas/farmacología , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei brucei/genética , Alelos , Diminazeno/farmacología , Resistencia a Múltiples Medicamentos/genética , Cinética , Melarsoprol/farmacología , Mutación , Proteínas de Transporte de Nucleósidos/metabolismo , Pruebas de Sensibilidad Parasitaria , Pentamidina/farmacología , Polimorfismo de Nucleótido Simple , Dominios y Motivos de Interacción de Proteínas , Trypanosoma brucei brucei/químicaRESUMEN
Three clinical research projects are described that are relevant to pediatric hearing loss. The three projects fall into two distinct areas. The first area emphasizes clinical studies that track developmental outcomes in children with hearing loss; one project is specific to cochlear implants and the other to hearing aids. The second area addresses speech perception test development for very young children with hearing loss. Although these two lines of research are treated as separate areas, they begin to merge as new behavioral tests become useful in developing protocols for contemporary studies that address longitudinal follow-up of children with hearing loss.