RESUMO
Mutations in genes encoding zinc cluster transcription factors (ZCFs) such as TAC1, MRR1, and UPC2 play a key role in Candida albicans azole antifungal resistance. Artificial activation of the ZCF Mrr2 has shown increased expression of the gene encoding the Cdr1 efflux pump and resistance to fluconazole. Amino acid substitutions in Mrr2 have recently been reported to contribute to fluconazole resistance in clinical isolates. In the present study, 57 C. albicans clinical isolates with elevated fluconazole MICs were examined for mutations in MRR2 and expression of CDR1 Mutations in MRR2 resulting in 15 amino acid substitutions were uniquely identified among resistant isolates, including 4 substitutions (S466L, A468G, S469T, T470N) previously reported to reduce fluconazole susceptibility. Three additional, novel amino acid substitutions (R45Q, A459T, V486M) were also discovered in fluconazole-resistant isolates. When introduced into a fluconazole-susceptible background, no change in fluconazole MIC or CDR1 expression was observed for any of the mutations found in this collection. However, introduction of an allele leading to artificial activation of Mrr2 increased resistance to fluconazole as well as CDR1 expression. Moreover, Mrr2 amino acid changes reported previously to have the strongest effect on fluconazole susceptibility and CDR1 expression also exhibited no differences in fluconazole susceptibility or CDR1 expression relative to the parent strain. While all known fluconazole resistance mechanisms are represented within this collection of clinical isolates and contribute to fluconazole resistance to different extents, mutations in MRR2 do not appear to alter CDR1 expression or contribute to resistance in any of these isolates.
Assuntos
Candida albicans/efeitos dos fármacos , Candida albicans/metabolismo , Fluconazol/farmacologia , Proteínas Fúngicas/metabolismo , Fatores de Transcrição/metabolismo , Azóis/farmacologia , Candida albicans/genética , Farmacorresistência Fúngica/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/genética , Testes de Sensibilidade Microbiana , Mutação/genética , Fatores de Transcrição/genéticaRESUMO
Glyceroacetonide-Oxyma [(2,2-dimethyl-1,3-dioxolan-4-yl)methyl 2-cyano-2-(hydroxyimino)acetate (1)] displayed remarkable physico-chemical properties as an additive for peptide-forming reactions. Although racemization-free amide-forming reactions have been established for N-urethane-protected α-amino acids with EDCI, 1, and NaHCO3 in water or DMF-water media, amide-forming reactions of N-acyl-protected α-amino acids and segment couplings of oligopeptides still require further development. Diethylphosphoryl-glyceroacetonide-oxyma (DPGOx 3) exhibits relative stability in aprotic solvents and is an effective coupling reagent for N-acyl-protected α-amino acids and oligo peptide segments. The conditions reported here is also effective in lactam-forming reactions. Unlike most of the reported coupling reagents, simple aqueous work-up procedures can remove the reagents and by-products generated in the reactions.