RESUMEN
Viral promoters are widely utilized in commercial and customized vectors to drive expression of genes of interest including reporter, effector and transfection control, because of their high transcription efficiency in a variety of primary and transformed cell lines. However, we observed altered rate of transcription for these promoters under conditions such as presence of an effector protein. These variations in viral promoter driven expressions can potentially lead to incorrect conclusion, especially in comparative and quantitative experiments. We found significantly reduced viral promoter activity in cells overexpressing tumor suppressor protein p53, whereas markedly induced transcription in cells overexpressing MAP/ERK kinase kinase 1 (Mekk 1). Using deletion constructs generated from the CMV promoter, we found the transcription reduction by p53 is possibly mediated through the TATA motif present in proximal CMV promoter. The activation of the CMV promoter by Mekk 1, on the other hand, is attributed to the proximal CRE binding site in the promoter. These findings may be of interest to investigators who use CMV (or other viral) promoter driven vectors for either comparative or quantitative gene expression, or effect on promoter activity.
Asunto(s)
Citomegalovirus/genética , Sistema de Señalización de MAP Quinasas , TATA Box , Activación Transcripcional , Proteína p53 Supresora de Tumor/metabolismo , Secuencia de Bases , Sitios de Unión , Secuencia de Consenso , Citomegalovirus/metabolismo , Activación Enzimática , Vectores Genéticos , Células HEK293 , Humanos , Quinasa 1 de Quinasa de Quinasa MAP/genética , Quinasa 1 de Quinasa de Quinasa MAP/metabolismo , Datos de Secuencia Molecular , Mapeo de Interacción de Proteínas , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismo , Proteína p53 Supresora de Tumor/genéticaRESUMEN
BACKGROUND: The Dt/V obtained by using ionic dialysance (D) as a surrogate for urea clearance (K) is a well-validated adjunct measure of hemodialysis adequacy, with a variable level of correlation with urea-based Kt/V. However, this correlation has not been examined based on patients' body size and ultrafiltration (UF) volume during the dialysis session. METHODS: Simultaneous evaluations of online Dt/V and single-pool variable-volume urea Kt/V were made. Patients were categorized into three subgroups based on their weight (<60, 60-80 and ≥80 kg), body mass index (<25, 25-30 and >30 kg/m2) and UF volume (<1.5, 1.5-3 and >3 L). The correlation between Dt/V and Kt/V was evaluated for the entire cohort per dialysis session in each subgroup. RESULTS: Mean Kt/V was greater than the mean Dt/V (1.72 versus 1.50, P < 0.001), with an overall correlation r value of 0.602. This correlation was stronger in the medium weight group versus lower and higher weights. The correlation between Dt/V and Kt/V was inversely related to the UF volume (r = 0.698, 0.621 and 0.558 for those with UF volume of <1.5, 1.5-3.0 and >3 L, respectively). A total of 99.3% of patients with Dt/V of >1.2 also had Kt/V >1.2 and 9.5% of those with Dt/V <1.2 had their Kt/V <1.2. CONCLUSIONS: There is a moderate degree of correlation between Dt/V and Kt/V in African-American hemodialysis patients, which is impacted by body size and UF volume. A Dt/V of >1.2 strongly predicts adequate dialysis as defined by Kt/V of >1.2.