RESUMEN
Aggregated ß-amyloid peptides (Aß) are neurotoxic and responsible for neuronal death both in vitro and in vivo. From the structural point of view, Aß self-aggregation involves a conformational change in the peptide. Here, we investigated the relationship between conformational changes and amino acid residues of Aß(40). Urea unfolding in combination with NMR spectroscopy was applied to probe the stabilization of Aß(40) conformation. L17 and F19 residues were found more sensitive to environmental changes than the other residues. Replacement of these two residues with alanine could stabilize the conformation of Aß(40). Further analysis indicated that the Aß(40)(L17A/F19A) mutant could diminish the aggregation and reduce the neurotoxicity. These results suggest that L17 and F19 are the critical residues responsible for conformational changes which may trigger neurotoxic cascade of Aß(40).
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/toxicidad , Neuronas/efectos de los fármacos , Fragmentos de Péptidos/química , Fragmentos de Péptidos/toxicidad , Alanina/química , Alanina/genética , Sustitución de Aminoácidos , Péptidos beta-Amiloides/genética , Animales , Supervivencia Celular , Humanos , Leucina/química , Leucina/genética , Mutación , Resonancia Magnética Nuclear Biomolecular , Células PC12 , Fragmentos de Péptidos/genética , Fenilalanina/química , Fenilalanina/genética , Conformación Proteica , Ratas , Urea/químicaRESUMEN
Among 234 isolates comprising 26 different Candida species colonizing the oropharynx of 181 (54.3% of 399 surveyed) HIV-infected outpatients, 27 (11.7%) were fluconazole resistant. Antibacterial treatment was associated with increased rates of yeast colonization, while antiretroviral therapy and pneumococcal vaccination protected patients from yeast colonization.
Asunto(s)
Candida/aislamiento & purificación , Portador Sano , Infecciones por VIH , Orofaringe/microbiología , Adulto , Antifúngicos/farmacología , Candida/efectos de los fármacos , Portador Sano/epidemiología , Portador Sano/microbiología , Estudios de Cohortes , Farmacorresistencia Fúngica , Femenino , Fluconazol/farmacología , Infecciones por VIH/complicaciones , Infecciones por VIH/epidemiología , Infecciones por VIH/microbiología , Humanos , Modelos Logísticos , Masculino , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Pacientes Ambulatorios , Factores de Riesgo , TaiwánRESUMEN
We previously identified a novel centrosomal protein CPAP, which carries a 112-residue motif that is essential for microtubule destabilization. In this report, we define both the microtubule (MT) binding and destabilizing domains in human CPAP and analyze the mutations that affect its MT-destabilizing activity. Analysis of a series of CPAP truncated proteins showed that the MT-binding domain (MBD; residues 423-607) of CPAP is located next to its MT-destabilizing domain (MDD; residues 311-422). Site-specific mutagenesis revealed that the mutations that either disrupt the alpha-helical structure (Y341P, I346P, L348P, and triple-P) or alter the charge property (KR377EE) of the MDD significantly affect its MT-destabilizing ability. The activity for binding to a tubulin heterodimer was also significantly reduced in KR377EE mutant. Furthermore, we have analyzed the putative function of Drosophila d-SAS-4, a distant relative of human CPAP, which shares a conserved approximately 20-aa sequence with the MDD of CPAP. Our results show that mutations in this conserved sequence also eliminate d-SAS-4's MT-destabilizing activity, suggesting that d-SAS-4 and CPAP may play similar roles within cells.
Asunto(s)
Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Secuencia de Aminoácidos , Animales , Bovinos , Línea Celular Tumoral , Análisis Mutacional de ADN , Dimerización , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Microtúbulos/efectos de los fármacos , Datos de Secuencia Molecular , Proteínas Mutantes/metabolismo , Paclitaxel/farmacología , Péptidos/metabolismo , Fenotipo , Unión Proteica/efectos de los fármacos , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Tubulina (Proteína)/metabolismoRESUMEN
The ERG3 gene in Candida albicans was identified as a gene whose mRNA level was higher in the cph1/cph1 efg1/efg1 double mutant than in the wild-type cells. Further study showed that Efg1, but not Cph1, negatively regulated ERG3. Mutations in EFG1 consistently increased the susceptibility of the cells to antifungal agents.
Asunto(s)
Candida albicans/efectos de los fármacos , Proteínas de Unión al ADN/fisiología , Proteínas Fúngicas/fisiología , Regulación Fúngica de la Expresión Génica , Oxidorreductasas/genética , Factores de Transcripción/fisiología , Candida albicans/genética , Candida albicans/patogenicidad , Farmacorresistencia Fúngica , VirulenciaRESUMEN
Overexpression of CDR1, an efflux pump, is one of the major mechanisms contributing to drug resistance in Candida albicans. CDR1 p-lacZ was constructed and transformed into a Saccharomyces cerevisiae strain so that the lacZ gene could be used as the reporter to monitor the activity of the CDR1 promoter. Overexpression of CaNDT80, the C. albicans homolog of S. cerevisiae NDT80, increases the beta-galactosidase activity of the CDR1 p-lacZ construct in S. cerevisiae. Furthermore, mutations in CaNDT80 abolish the induction of CDR1 expression by antifungal agents in C. albicans. Consistently, the Candt80/Candt80 mutant is also more susceptible to antifungal drugs than the wild-type strain. Thus, the gene for CaNdt80 may be the first gene among the regulatory factors involved in drug resistance in C. albicans whose function has been identified.