RESUMEN
The M-CAT binding factor transcription enhancer factor 1 (TEF-1) has been implicated in the regulation of several cardiac and skeletal muscle genes. Previously, we identified an E-box-M-CAT hybrid (EM) motif that is responsible for the basal and cyclic AMP-inducible expression of the rat cardiac alpha-myosin heavy chain (alpha-MHC) gene in cardiac myocytes. In this study, we report that two factors, TEF-1 and a basic helix-loop-helix leucine zipper protein, Max, bind to the alpha-MHC EM motif. We also found that Max was a part of the cardiac troponin T M-CAT-TEF-1 complex even when the DNA template did not contain an apparent E-box binding site. In the protein-protein interaction assay, a stable association of Max with TEF-1 was observed when glutathione S-transferase (GST)-TEF-1 or GST-Max was used to pull down in vitro-translated Max or TEF-1, respectively. In addition, Max was coimmunoprecipitated with TEF-1, thus documenting an in vivo TEF-1-Max interaction. In the transient transcription assay, overexpression of either Max or TEF-1 resulted a mild activation of the alpha-MHC-chloramphenicol acetyltransferase (CAT) reporter gene at lower concentrations and repression of this gene at higher concentrations. However, when Max and TEF-1 expression plasmids were transfected together, the repression mediated by a single expression plasmid was alleviated and a three- to fourfold transactivation of the alpha-MHC-CAT reporter gene was observed. This effect was abolished once the EM motif in the promoter-reporter construct was mutated, thus suggesting that the synergistic transactivation function of the TEF-1-Max heterotypic complex is mediated through binding of the complex to the EM motif. These results demonstrate a novel association between Max and TEF-1 and indicate a positive cooperation between these two factors in alpha-MHC gene regulation.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Regulación del Desarrollo de la Expresión Génica , Secuencias Hélice-Asa-Hélice , Miocardio/metabolismo , Cadenas Pesadas de Miosina/genética , Miosinas/genética , Proteínas Nucleares , Regiones Promotoras Genéticas , Factores de Transcripción/fisiología , Animales , Secuencia de Bases , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Miocardio/citología , Unión Proteica , Ratas , Secuencias Reguladoras de Ácidos Nucleicos , Factores de Transcripción de Dominio TEA , Transcripción GenéticaRESUMEN
PURPOSE: To study pulsatile ocular blood flow (POBF) throughout pregnancy. METHODS: We enrolled twenty-seven healthy women in the first trimester of gestation, only ten of which were followed through the second trimester, and fourteen non pregnant healthy women. In each subject we measured POBF with the POBF pneumotonometer (OBF Ltd. UK), IOP, blood pressure (BP) and heart rate (HR). An unpaired Student t-test was used to compare pregnant women with non-pregnant women, and a two-tailed paired Student t-test was used to compare the same women in the first and second trimester of pregnancy. p <0.05 is considered statistically significant. RESULTS: Results are presented as means +/- SD. In the first trimester of pregnancy the age was 32 +/- 6, POBF 1516.4 +/- 382 ml/min, IOP 13 +/- 3 mmHg, BP 92 +/- 6 mmHg, and HR 86 +/- 14 beats/min. In the second trimester POBF was 1629.11 +/- 352.4 ml/min, intraocular pressure (IOP) 12 +/- 3 mmHg, BP 96 +/- 3 mmHg, and HR 93 +/- 10 beats/min. In the control group the age was 27 +/- 9, POBF 972.23 +/- 329.3 ml/min, BP 88 +/- 4.3 mmHg, and HR 80 +/- 14 beats/min. POBF increases during the first trimester (p = 0.00008). In the second trimester POBF was higher compared to the first trimester (p = 0.0008). Non significant differences were observed for the other parameters. CONCLUSIONS: The POBF increases throughout gestation. During pregnancy there is an increase in estrogen which induces endothelial-dependent vasodilatation in several tissues. The estrogen changes may influence POBF.