RESUMEN
Ultrafast excited-state dynamics of the simplest nitrostilbenes, namely trans-4-nitrostilbene (t-NSB), was studied in solvents of various polarities with ultrafast broadband time-resolved fluorescence and transient absorption spectroscopies, and by quantum-chemical computations. The results revealed that the initially excited S1(ππ*) state deactivation dynamics is strongly influenced by the solvent polarity. Specifically, the t-NSB S1-state lifetime decreases by three orders of magnitude from â¼60 ps in high-polarity solvents to â¼60 fs in nonpolar solvents. The strong solvent-polarity dependence arises from the differences in dipole moments among the S1 and relevant states, including the major intersystem crossing (ISC) receiver triplet states, and therefore, the solvent polarity can modulate their relative energies and ISC rates. In nonpolar solvents, the sub-100 fs lifetime is due to a combination of efficient ISC and internal conversion. In medium-polarity solvents, the S1-state population decays via a competing ISC relaxation mechanism in a biphasic manner, and the ISC rates are found to obey the inverse energy gap law of the strong coupling case. In high-polarity solvents, the S1 state is stabilized to a much lower energy such that ISC becomes energetically infeasible, and the S1 state decays via barrier crossing along the torsion angle of the central ethylenic bond to the nonfluorescent perpendicular configuration. Regardless of the initial S1-state deactivation pathways in various solvents, the excited-state population is ultimately trapped in the metastable T1-state perpendicular configuration, at which a slower ISC occurs to bring the system to the ground state and bifurcate into either trans or cis form of NSB.
RESUMEN
Pompe disease is caused by autosomal recessive mutations in the acid alpha-glucosidase (GAA) gene, which encodes GAA. Although enzyme replacement therapy has recently improved patient survival greatly, the results in skeletal muscles and for advanced disease are still not satisfactory. Here, we report the derivation of Pompe disease-induced pluripotent stem cells (PomD-iPSCs) from two patients with different GAA mutations and their potential for pathogenesis modeling, drug testing and disease marker identification. PomD-iPSCs maintained pluripotent features and had low GAA activity and high glycogen content. Cardiomyocyte-like cells (CMLCs) differentiated from PomD-iPSCs recapitulated the hallmark Pompe disease pathophysiological phenotypes, including high levels of glycogen and multiple ultrastructural aberrances. Drug rescue assessment showed that exposure of PomD-iPSC-derived CMLCs to recombinant human GAA reversed the major pathologic phenotypes. Furthermore, l-carnitine treatment reduced defective cellular respiration in the diseased cells. By comparative transcriptome analysis, we identified glycogen metabolism, lysosome and mitochondria-related marker genes whose expression robustly correlated with the therapeutic effect of drug treatment in PomD-iPSC-derived CMLCs. Collectively, these results demonstrate that PomD-iPSCs are a promising in vitro disease model for the development of novel therapeutic strategies for Pompe disease.
Asunto(s)
Enfermedad del Almacenamiento de Glucógeno Tipo II/tratamiento farmacológico , Enfermedad del Almacenamiento de Glucógeno Tipo II/patología , Células Madre Pluripotentes Inducidas/patología , Modelos Biológicos , Adenina/análogos & derivados , Adenina/farmacología , Adenina/uso terapéutico , Animales , Secuencia de Bases , Biomarcadores/metabolismo , Carnitina/farmacología , Carnitina/uso terapéutico , Diferenciación Celular/efectos de los fármacos , Monitoreo de Drogas , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Enfermedad del Almacenamiento de Glucógeno Tipo II/fisiopatología , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Ratones , Datos de Secuencia Molecular , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Miocitos Cardíacos/ultraestructura , alfa-Glucosidasas/farmacología , alfa-Glucosidasas/uso terapéuticoRESUMEN
UNLABELLED: Alteration of cell surface proteolysis has been proposed to play a role in liver fibrosis, a grave complication of biliary atresia (BA). In this study we investigated the roles of hepatocyte growth factor activator inhibitor (HAI)-1 and -2 in the progression of BA. The expression levels of HAI-1 and -2 were significantly increased in BA livers compared with those in neonatal hepatitis and correlated with disease progression. In BA livers, HAI-1 and -2 were coexpressed in cells involved in ductular reactions. In other selective cholangiopathies, ductular cells positive for HAI-1 or HAI-2 also increased in number. Inflammatory cytokines, growth factors, and bile acids differentially up-regulated expression of HAI-1 and -2 transcripts in fetal liver cells and this induction could be antagonized by a cyclooxygenase-2 inhibitor. Conditioned media from cell lines stably overexpressing HAI-1 or HAI-2 enhanced the fibrogenic activity of portal fibroblasts and stellate cells, suggesting that both proteins might be involved in liver fibrosis. Because HAI-1 and -2 colocalized in ductular reactions sharing similar features to those observed during normal liver development, we sought to investigate the role of HAI-1 and -2 in cholangiopathies by exploring their functions in fetal liver cells. Knockdown of HAI-1 or HAI-2 promoted bidirectional differentiation of hepatoblast-derived cells. In addition, we showed that the hepatocyte growth factor activator, mitogen-activated protein kinase kinase 1, and phosphatidylinositol 3-kinase signaling pathways were involved in hepatic differentiation enhanced by HAI-2 knockdown. CONCLUSION: HAI-1 and -2 are overexpressed in the liver in cholangiopathies with ductular reactions and are possibly involved in liver fibrosis and hepatic differentiation; they could be investigated as disease markers and potential therapeutic targets.
Asunto(s)
Colestasis/patología , Hepatitis/patología , Cirrosis Hepática/patología , Glicoproteínas de Membrana/genética , Proteínas Inhibidoras de Proteinasas Secretoras/genética , Animales , Diferenciación Celular/fisiología , Línea Celular , Colestasis/fisiopatología , Femenino , Fibroblastos/citología , Células Estrelladas Hepáticas/citología , Hepatitis/congénito , Hepatitis/fisiopatología , Hepatocitos/citología , Humanos , Lactante , Recién Nacido , Cirrosis Hepática/congénito , Masculino , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas Inhibidoras de Proteinasas Secretoras/metabolismo , Ratas , Transducción de Señal/fisiología , Células Madre/citología , Células Madre/fisiologíaRESUMEN
Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein that is highly expressed in embryonic stem cells (ESCs) and its role in maintenance of pluripotency has been suggested previously. In epithelial cancer cells, activation of the EpCAM surface-to-nucleus signaling transduction pathway involves a number of membrane proteins. However, their role in somatic cell reprogramming is still unknown. Here we demonstrate that EpCAM and its associated protein, Cldn7, play a critical role in reprogramming. Quantitative RT-PCR analysis of Oct4, Sox2, Klf4, and c-Myc (OSKM) infected mouse embryonic fibroblasts (MEFs) indicated that EpCAM and Cldn7 were up-regulated during reprogramming. Analysis of numbers of alkaline phosphatase- and Nanog-positive clones, and the expression level of pluripotency-related genes demonstrated that inhibition of either EpCAM or Cldn7 expression resulted in impairment in reprogramming efficiency, whereas overexpression of EpCAM, EpCAM plus Cldn7, or EpCAM intercellular domain (EpICD) significantly enhanced reprogramming efficiency in MEFs. Furthermore, overexpression of EpCAM or EpICD significantly repressed the expression of p53 and p21 in the reprogramming MEFs, and both EpCAM and EpICD activated the promoter activity of Oct4. These observations suggest that EpCAM signaling may enhance reprogramming through up-regulation of Oct4 and possible suppression of the p53-p21 pathway. In vitro and in vivo characterization indicated that the EpCAM-reprogrammed iPSCs exhibited similar molecular and functional features to the mouse ESCs. In summary, our studies provide additional insight into the molecular mechanisms of reprogramming and suggest a more effective means of induced pluripotent stem cell generation.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Moléculas de Adhesión Celular/metabolismo , Reprogramación Celular , Células Madre Pluripotentes/metabolismo , Factores de Transcripción/metabolismo , Animales , Antígenos de Neoplasias/química , Moléculas de Adhesión Celular/química , Claudinas/metabolismo , Embrión de Mamíferos/citología , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Molécula de Adhesión Celular Epitelial , Fibroblastos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Factor 4 Similar a Kruppel , Ratones , Modelos Biológicos , Células Madre Pluripotentes/citología , Estructura Terciaria de Proteína , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The future clinical use of embryonic stem cell (ESC)-based hepatocyte replacement therapy depends on the development of an efficient procedure for differentiation of hepatocytes from ESCs. Here we report that a high density of human ESC-derived fibroblast-like cells (hESdFs) supported the efficient generation of hepatocyte-like cells with functional and mature hepatic phenotypes from primate ESCs and human induced pluripotent stem cells. Molecular and immunocytochemistry analyses revealed that hESdFs caused a rapid loss of pluripotency and induced a sequential endoderm-to-hepatocyte differentiation in the central area of ESC colonies. Knockdown experiments demonstrated that pluripotent stem cells were directed toward endodermal and hepatic lineages by FGF2 and activin A secreted from hESdFs. Furthermore, we found that the central region of ESC colonies was essential for the hepatic endoderm-specific differentiation, because its removal caused a complete disruption of endodermal differentiation. In conclusion, we describe a novel in vitro differentiation model and show that hESdF-secreted factors act in concert with regional features of ESC colonies to induce robust hepatic endoderm differentiation in primate pluripotent stem cells.