RNA polymerase II (RNAPII) requires a set of general transcription factors ) TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH ) to initiate transcription from a gene promoter in vitro. General transcription factors have been isolated from Saccharomyces cerevisiae, rat, human and Drosophila, and their corresponding cDNAs have been cloned. In this report, we describe a reconstituted in vitro transcription system that consists of the following preparations of factors from the yeast Schizosaccharomyces pombe: affinity-purified RNAPII, TFIIH, and recombinant TBP, TFIIB, TFIIE and TFIIF. We show that this system can support basal transcription from the adenovirus major late promoter when purified RNAPII is used and activated transcription when the RNAPII holoenzyme (RNAPII plus the Mediator proteins) is included in the reaction. In contrast, the TATA binding protein-associated factors had no effect on transcriptional activation in our Sc. pombe system. These results indicate that Sc. pombe uses the same set of general transcription factors as other eukaryotes and that the Mediator is involved in activated transcription.The ability to achieve basal levels of transcription from protein-encoding genes in eukaryotes requires RNA polymerase II (RNAPII) and a set of additional proteins called general transcription factors (GTFs). The GTFs have been purified to homogeneity from HeLa cells, rat liver, Drosophila and the yeast Saccharomyces cerevisiae, and have been named TFIIA, TFIID, TFIIE, TFIIF, TFIIB and TFIIH [1]. The cDNAs that encode these factors have been isolated, and their amino acid sequences show a high degree of evolutionary conservation. These findings indicate that the transcriptional machinery is highly conserved among eukaryotes.An in vitro transcription assay that consists of purified RNAPII and recombinant GTFs can carry out basal transcription but cannot respond to gene-specific transcriptional activators. Activated transcription requires additional multiprotein complexes named coactivators. The main coactivators required for activated transcription in in vitro systems are the TFIID complex and the Mediator [2]. Recent work suggests that the TFIID complex, which contains the TATA binding protein (TBP) and other TBPassociated factors (TAFs), plays an important role in facilitating activation by gene-specific transcription factors as wells as in recognition of the TATA box and other core promoter sequences (necessary for both basal and activated transcription) [3]. Mediator is a large multiprotein complex that is brought to promoters by DNA-bound, gene-specific transcriptional regulatory proteins and helps these proteins to communicate with factors bound to the core promoter. Mediator is required for transcription in vivo and for optimal levels of both basal and activated transcription in vitro in nuclear extracts from human cells [4,5]. Components of both the TFIID complex and Mediator are conserved from yeast to humans.The yeast Schizosaccharomyces pombe can be genetically manipulated and has served as an exce...
A growing number of surgical patients present to the operating room with implantable cardioverter defibrillators (ICD). Peri-operative care of these patients dictates that ICD function be suspended for many surgical procedures to avoid inappropriate, and possibly harmful, ICD therapy triggered by electromagnetic interference (EMI). An alternative to reprogramming the ICD is the use of a magnet to temporarily suspend its function. However, this approach is not without complications. We report a case where magnet use failed to inhibit ICD sensing of EMI, and a shock was delivered to the patient. Measures to decrease EMI, controversies regarding magnet use, and expert recommendations are discussed.
218Determinación del polimorfismo de CYP2C9*2 y su relación con la farmacocinética de acenocumarol en voluntarios sanos. Resumen:Antecedentes: La mayoría de los pacientes que reciben tratamientos con anticoagulantes orales por periodos prolongados presentan variabilidad en la respuesta. El acenocumarol es el anticoagulante oral más prescrito en nuestro país, es biotransformado principalmente por CYP2C9 e investigaciones recientes demuestran que la variante CYP2C9*2 es una de las responsables de la variabilidad de respuesta a acenocumarol.Objetivo: Determinar las diferencias en los pará-metros farmacocinéticos de acenocumarol en voluntarios que presentan la variante alélica CYP2C9*2.Métodos: Se estudiaron 24 voluntarios sanos.La detección de genotipos se realizó mediante PCR-RFLP y los parámetros farmacocinéticos se obtuvieron mediante la concentración plasmática de acenocumarol usando un método validado para UPLC-MS/MS.Resultados: Del total de 24 voluntarios,19 tenían el genotipo CYP2C9*1/*1 (wt/wt), 4 tenían genotipo CYP2C9*1/*2 (heterocigoto) y 1 voluntario tenía genotipo de CYP2C9*2/*2 (homocigoto recesivo). Los parámetros farmacocinéticos del acenocumarol no fueron significativamente diferentes entre los individuos con genotipo CYP2C9*2 y CYP2C9*1. Sin embargo, la farmacocinética de acenocumarol del individuo CYP2C9*2/*2 mostró diferencias relevantes con respecto a la observada en el grupo CYP2C9*1/*1 (tmáx aumentó 1,4 veces, ke disminuyó 1,8 veces y t1/2 aumentó 1,7 veces).Conclusión: La farmacocinética de acenocumarol en el individuo con el genotipo CYP2C9*2/*2 refleja una potencial relevancia de este polimorfismo en el tratamiento con acenocumarol. Aim: to determine pharmacokinetics parameters of acenocumarol in volunteers exhibiting the CYP2C9*2 polymorphic variant. Methods:Genotype detection was performed using PCR-RFLP and pharmacokinetics parameters were obtained from the acenocumarol concentrations, using a UPLC-MS/MS validated method. The project was approved by the institutional Ethics Committee of the University of Chile's Faculty of Medicine.Results: 19 out of 24 volunteers had the CYP2C9*1/*1 genotype, 4 the CYP2C9*1/*2 genotype (heterozygous) and 1 subject had the CYP2C9*2/*2 genotype (recessive homozygous). No statistically significant differences between acenocumarol pharmacokinetics parameters of CYP2C9*2 compared to those with normal variant, CYP2C9*1were observed.. However, a single individual with the CYP2C9*2/*2 genotype showed different pharmacokinetics parameters: tmáx and t1/2 were increased 1.4 and 1.7 times, respectively, and kc was 1.8 times lower compared to the group with the CYP2C9*1/*1 genotype. Conclusion:There are clear differences in genotype-dependent acenocoumarol pharmacokinetics in individuals with the CYP2C9*2/*2 genotype, reflecting a potential relevance of this polymorphism in anticoagulation with acenocumarol.Keywords: Pharmacogenetics, CYP2C9*2, anticoagulants, acenocoumarol.Determinación del polimorfismo de CYP2C9*2 y su relación con la farmacocinética de acenocum...
Changes in bioavailability of anticonvulsant drugs such as topiramate may cause loss of or worsened seizure control. Thus, the purpose of this study was to evaluate, in a double-blind crossover design, the bioavailability between two oral formulations of topiramate in healthy volunteers after a single dose. The protocol, approved by the Institutional Committee of Ethics, consisted of administration of 1 tablet of 100 mg of topiramate of each formulation (Toprel™ and Topamax™), to 20 healthy volunteers after a 12 h overnight fast, using an open, two-period, randomized, crossover and double-blind design. Thus, the plasma concentrations (Cp) of topiramate were measured at predetermined intervals of time, from 0 to 24 h, using a validated UPLC-MS/MS method. Based on plasma concentration-time profiles we obtained the following pharmacokinetic parameters:
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