RESUMEN
We identified three zebrafish mutants with defects in biliary development. One of these mutants, pekin (pn), also demonstrated generalized hypopigmentation and other defects, including disruption of retinal cell layers, lack of zymogen granules in the pancreas, and dilated Golgi in intestinal epithelial cells. Bile duct cells in pn demonstrated an accumulation of electron dense bodies. We determined that the causative defect in pn was a splice site mutation in the atp6ap2 gene that leads to an inframe stop codon. atp6ap2 encodes a subunit of the vacuolar H(+)-ATPase (V-H(+)-ATPase), which modulates pH in intracellular compartments. The Atp6ap2 subunit has also been shown to function as an intracellular renin receptor that stimulates fibrogenesis. Here we show that mutants and morphants involving other V-H(+)-ATPase subunits also demonstrated developmental biliary defects, but did not demonstrate the inhibition of fibrogenic genes observed in pn. The defects in pn are reminiscent of those we and others have observed in class C VPS (vacuolar protein sorting) family mutants and morphants, and we report here that knockdown of atp6ap2 and vps33b had an additive negative effect on biliary development. Our findings suggest that pathways which are important in modulating intracompartmental pH lead to defects in digestive organ development, and support previous studies demonstrating the importance of intracellular sorting pathways in biliary development.
Asunto(s)
Sistema Biliar/anomalías , Proteínas de la Membrana/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/anomalías , Animales , Sistema Biliar/enzimología , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Proteínas de la Membrana/genética , Mutación , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ATPasas de Translocación de Protón Vacuolares/genética , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
The antiviral factor CPSF6-358 interferes with the nuclear entry of human immunodeficiency virus type 1 (HIV-1). HIV-1 acquires resistance to CPSF6-358 through the N74D mutation of the capsid (CA), which alters its nuclear entry pathway. Here we show that compared to wild-type (WT) HIV-1, N74D HIV-1 is more sensitive to cyclosporine, has increased sensitivity to nevirapine, and is impaired in macrophage infection prior to reverse transcription. These phenotypes suggest a difference in the N74D reverse transcription complex that manifests early after infection and prior to interaction with the nuclear pore. Overall, our data indicate that N74D HIV-1 replication in transformed cells requires cyclophilin A but is dependent on other interactions in macrophages.
Asunto(s)
Proteínas de la Cápside/genética , Ciclofilina A/farmacología , VIH-1/genética , VIH-1/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/virología , Mutación , Transporte Activo de Núcleo Celular/efectos de los fármacos , Transporte Activo de Núcleo Celular/genética , Animales , Factores de Restricción Antivirales , Aotidae , Proteínas de la Cápside/metabolismo , Proteínas Portadoras/farmacología , División Celular , Línea Celular , VIH-1/efectos de los fármacos , Humanos , Pruebas de Sensibilidad Microbiana , ADN Polimerasa Dirigida por ARN/metabolismo , Inhibidores de la Transcriptasa Inversa/farmacología , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína LigasasRESUMEN
The total asymmetric synthesis of (+)- and (-)-clusianone and (+)- and (-)-clusianone methyl enol ether is reported. Asymmetric induction is achieved through the use of ACC alkylation, providing the key intermediates with an er of 99:1. The four synthetic compounds were evaluated for their anti-HIV activity. Both (+)- and (-)-clusianone displayed significant anti-HIV activity.