RESUMEN
Retinoic acid is a widely used factor in both mouse and human embryonic stem cells. It suppresses differentiation to mesoderm and enhances differentiation to ectoderm. Fibroblast growth factor 2 (FGF2) is widely used to induce differentiation to neurons in mice, yet in primates, including humans, it maintains embryonic stem cells in the undifferentiated state. In this study, we established an FGF2 low-dose-dependent embryonic stem cell line from cynomolgus monkeys and then analyzed neural differentiation in cultures supplemented with retinoic acid and FGF2. When only retinoic acid was added to culture, neurons differentiated from FGF2 low-dose-dependent embryonic stem cells. When both retinoic acid and FGF2 were added, neurons and astrocytes differentiated from the same embryonic stem cell line. Thus, retinoic acid promotes the differentiation from embryonic stem cells to neuroectoderm. Although FGF2 seems to promote self-renewal in stem cells, its effects on the differentiation of stem cells are influenced by the presence or absence of supplemental retinoic acid.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Células Madre Embrionarias/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Macaca fascicularis/fisiología , Placa Neural/efectos de los fármacos , Tretinoina/farmacología , Animales , Relación Dosis-Respuesta a Droga , Células Madre Embrionarias/fisiología , Cariotipificación , Microscopía Fluorescente , Placa Neural/fisiologíaRESUMEN
APJ is a G-protein-coupled receptor with seven transmembrane domains, and its endogenous ligand, apelin, was identified recently. They are highly expressed in the cardiovascular system, suggesting that APJ is important in the regulation of blood pressure. To investigate the physiological functions of APJ, we have generated mice lacking the gene encoding APJ. The base-line blood pressure of APJ-deficient mice is equivalent to that of wild-type mice in the steady state. The administration of apelin transiently decreased the blood pressure of wild-type mice and a hypertensive model animal, a spontaneously hypertensive rat. On the other hand, this hypotensive response to apelin was abolished in APJ-deficient mice. This apelin-induced response was inhibited by pretreatment with a nitric-oxide synthase inhibitor, and apelin-induced phosphorylation of endothelial nitric-oxide synthase in lung endothelial cells from APJ-deficient mice disappeared. In addition, APJ-deficient mice showed an increased vasopressor response to the most potent vasoconstrictor angiotensin II, and the base-line blood pressure of double mutant mice homozygous for both APJ and angiotensin-type 1a receptor was significantly elevated compared with that of angiotensin-type 1a receptor-deficient mice. These results demonstrate that APJ exerts the hypotensive effect in vivo and plays a counterregulatory role against the pressor action of angiotensin II.
Asunto(s)
Receptor de Angiotensina Tipo 1/química , Receptores Acoplados a Proteínas G/fisiología , Alelos , Angiotensina II/metabolismo , Animales , Receptores de Apelina , Presión Sanguínea , Northern Blotting , ADN Complementario/metabolismo , Endotelio/enzimología , Endotelio Vascular/metabolismo , Inhibidores Enzimáticos/farmacología , Homocigoto , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Modelos Genéticos , NG-Nitroarginina Metil Éster/metabolismo , Óxido Nítrico Sintasa/antagonistas & inhibidores , Fosforilación , Estructura Terciaria de Proteína , ARN/metabolismo , Ratas , Ratas Endogámicas WKY , Receptor de Angiotensina Tipo 1/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Recombinación Genética , Serina/química , Factores de TiempoRESUMEN
We previously identified a regulatory element at the 3'-downstream region of the human angiotensinogen (hANG) gene. Using this element as a probe by the Southwestern screening, we isolated a cDNA clone, encoding Finb, a transcriptional activator with multiple zinc finger domains. The N-terminal zinc finger domain of Finb bound to the GGATGG sequence within the regulatory element. Unexpectedly, Finb repressed transcription dependent on the regulatory element. Inspection of the 5'-flanking region in the hANG promoter identified the GGATGG-like elements, which prompted us to examine the effect of Finb on the hANG promoter activity. We also found the two Finb binding elements in the 5'-flanking region of the hANG gene by the gel shift assay, both of which were necessary for transcriptional repression of the hANG promoter. These findings suggest that Finb functions as a sequence-specific transcriptional repressor of the hANG gene.
Asunto(s)
Angiotensinógeno/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo/genética , Proteínas Represoras/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Transcripción Genética/genética , Dedos de Zinc , Secuencia de Bases , Línea Celular Tumoral , Clonación Molecular , ADN Complementario/genética , Proteínas de Unión al ADN/genética , Humanos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Unión Proteica , Proteínas Represoras/química , Elementos de Respuesta/genética , Factores de Transcripción/genéticaRESUMEN
Mammals respond to reduced food availability by becoming more wakeful and active, yet the central pathways regulating arousal and instinctual motor programs (such as food seeking) according to homeostatic need are not well understood. We demonstrate that hypothalamic orexin neurons monitor indicators of energy balance and mediate adaptive augmentation of arousal in response to fasting. Activity of isolated orexin neurons is inhibited by glucose and leptin and stimulated by ghrelin. Orexin expression of normal and ob/ob mice correlates negatively with changes in blood glucose, leptin, and food intake. Transgenic mice, in which orexin neurons are ablated, fail to respond to fasting with increased wakefulness and activity. These findings indicate that orexin neurons provide a crucial link between energy balance and arousal.