RESUMEN
Application of the tetracycline-inducible gene expression system (Tet-On) in human induced pluripotent stem cells (hiPSCs) has become a fundamental transgenic tool owing to its regulatable gene expression. One of the major hurdles in hiPSC application is non-uniform transgene activation. Here, we report that the supplementation of reverse tetracycline transactivator (rtTA) in polyclonal hiPSCs populations can achieve the uniform transgene activation of Tet-On. Furthermore, the choice of antibiotic selection markers connected by an internal ribosomal entry site (IRES) can influence the expression of upstream transgenes. In particular, expression of the rtTA is more uniform in cell populations when linked to puromycin as compared to neomycin, obviating the need for sub-cloning or supplementation of rtTA. Finally, to expand the range of applications, we adopted our findings to tetracycline-inducible MyoD vector (Tet-MyoD). Our Tet-MyoD promises efficient, robust, and reproducible directed myogenic differentiation of hiPSCs.
RESUMEN
Muscular dystrophy is a disease characterized by progressive muscle weakness and degeneration. There are currently no available treatments for most muscular diseases, such as muscular dystrophy. Moreover, current therapeutics are focused on improving the quality of life of patients by relieving the symptoms or stress caused by the disease. Although the causative genes for many muscular diseases have been identified, the mechanisms underlying their pathogenesis remain unclear. Patient-derived induced pluripotent stem cells (iPSCs) have become a powerful tool for understanding the pathogenesis of intractable diseases, as well as for phenotype screening, which can serve as the basis for developing new drugs. However, it is necessary to develop an efficient and reproducible myogenic differentiation system. Previously, we reported a tetracycline-inducible MyoD overexpression model of myogenic differentiation using human iPSCs (hiPSCs). However, this model has certain disadvantages that limit its use in various applications, such as a drug screening. In this study, we developed an efficient and reproducible myogenic differentiation system by further modifying our previous protocol. The new protocol achieves efficient differentiation of feeder-free hiPSCs to myogenic cells via small-scale culture in six-well microplates to large-scale culture in 384-well microplates for high-throughput applications.
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Células Madre Pluripotentes Inducidas/citología , Diferenciación Celular/fisiología , Línea Celular , Células Cultivadas , Evaluación Preclínica de Medicamentos , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Desarrollo de Músculos/fisiología , Calidad de VidaRESUMEN
P-glycoprotein (P-gp; MDR1) recognizes and actively transports many structurally diverse compounds (hydrophobic neutral and cationic). We studied MDR1-mediated drug transport using a high-throughput (96-well) oocyte expression system. MDR1-expressing oocytes contained sufficient ATP levels to conduct fundamental efflux studies; the optimal experimental temperature was 25 degrees C. [(3)H]Vinblastine efflux by MDR1-expressing oocytes was detectable and afforded a K(m) of 145.5+/-25.4microM. [(3)H]Vinblastine (5.6+/-0.3microM) and [(3)H]digoxin (1.0+/-0.1microM) were individually injected into MDR1-expressing oocytes and their efflux monitored. Quinidine and verapamil, known MDR1 substrates/inhibitors, showed trans-inhibition on MDR1-mediated [(3)H]vinblastine and [(3)H]digoxin efflux. Conversely, doxorubicin demonstrated cis-inhibition without trans-inhibition on MDR1-mediated [(3)H]vinblastine efflux. The MDR1-expressing oocyte system offers researchers with an alternative in vitro method to screen compounds and may allow one to probe P-gp drug-drug and/or drug-inhibitor interactions.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Digoxina/metabolismo , Doxorrubicina/farmacología , Quinidina/farmacología , Verapamilo/farmacología , Vinblastina/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Transporte Biológico Activo , Relación Dosis-Respuesta a Droga , Doxorrubicina/metabolismo , Interacciones Farmacológicas , Femenino , Ensayos Analíticos de Alto Rendimiento , Humanos , Cinética , Microinyecciones , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Quinidina/metabolismo , Temperatura , Verapamilo/metabolismo , Xenopus laevisRESUMEN
Planarians are useful animals for regenerative and neuroscience research at the molecular level. Previously, we have reported the distribution and function of neurotransmitter-synthesizing neurons in the planarian central nervous system. In order to understand the neural projections and connections, it is important to understand the distribution of neurotransmitter receptors. In this study, we isolated a serotonin receptor gene and named it DjSER-7 (Dugesia japonica serotonin receptor type 7). DjSER-7-expressing cells were distributed in the planarian brain. According to electrophysiological analysis using Xenopus oocytes, current response was detected upon exposure to serotonin, but not other neurotransmitters in oocytes that were co-injected with mRNAs of both DjSER-7 and Galpha chimera B-2, which can interact with either Gq-, Gs- or Gi-coupled receptor. In contrast, current response was not detected after exposure to neurotransmitters in oocytes injected with only DjSER-7 mRNA. Our results indicated that DjSER-7 responds to serotonin, as indicated by electrophysiological analysis using Xenopus oocytes.
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Sistema Nervioso Central/metabolismo , Genes de Helminto , Planarias/genética , Receptores Acoplados a Proteínas G/genética , Receptores de Serotonina/metabolismo , Serotonina/metabolismo , Xenopus laevis/metabolismo , Secuencia de Aminoácidos , Animales , Sistema Nervioso Central/citología , ADN Complementario , Hibridación in Situ , Datos de Secuencia Molecular , Oocitos/metabolismo , Planarias/metabolismo , ARN Mensajero/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Serotonina/genética , Xenopus laevis/genéticaRESUMEN
Lamin A and C proteins, encoded by the lamin A/C gene (LMNA), are inner nuclear membrane proteins predominantly expressed in terminally differentiated cells. Mutations in LMNA can cause various forms of cardiomyopathy with arrhythmia in an autosomal dominant manner. We collected and evaluated the clinical characteristics of unclassified familial cardiomyopathy with advanced AV block and sporadic cases with advanced AV block. Mutation in LMNA was directly screened using the cycle sequencing method in 5 probands of the familial cardiomyopathy and 60 sporadic cases with advanced AV block. In four of the five familial cases (80%), we identified four distinct mutations: two protein-truncation mutations, R225X and 815_818delinsCCAGAC, and two missense mutations, Y259H and R166P. No sporadic cases carried LMNA mutation. Left ventricular end-diastolic diameter (LVEDD) was slightly enlarged in LMNA mutant carriers (123.5 +/- 9.5%) as well as in non-carriers (125.1 +/- 13.3%), while left ventricular fractional shortening (LVFS) was preserved in LMNA mutant carriers (32.3 +/- 4.8%) and non-carriers (37.6 +/- 6.8%). In LMNA mutation carriers, the average age at onset of advanced AV block is significantly lower than that in non-carriers (43.7 +/- 9.5 vs. 65.3 +/- 13 yr., p < 0.01). Ventricular tachycardia, sudden death, and poor prognosis were observed in LMNA mutation carriers. LMNA mutation could cause familial cardiomyopathy with insignificant LV remodeling, early-age onset of advanced AV block, and lethal ventricular arrhythmia. Screening of LMNA mutation might be beneficial for risk stratification and clinical management of this type of unclassified familial cardiomyopathy.
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Arritmias Cardíacas/genética , Bloqueo Atrioventricular , Cardiomiopatías/genética , Lamina Tipo A/genética , Mutación , Adulto , Edad de Inicio , Anciano , Análisis Mutacional de ADN , Femenino , Humanos , Masculino , Persona de Mediana Edad , Membrana Nuclear/metabolismoRESUMEN
Terf/TRIM17 is a member of the TRIM family of proteins, which is characterized by the RING finger, B-box, and coiled-coil domains. In the present study, we found that terf interacts with TRIM44. Terf underwent ubiquitination in vitro in the presence of the E2 enzyme UbcH6; this suggests that terf exhibits E3 ubiquitin ligase activity. It was also found that terf was conjugated with polyubiquitin chains and stabilized by the proteasome inhibitor in mammalian cells; this suggested that terf rendered itself susceptible to proteasomal degradation through polyubiquitination. We also found that TRIM44 inhibited ubiquitination of terf, and thus stabilized the protein. The N-terminal region of TRIM44 contains a zinc-finger domain found in ubiquitin hydrolases (ZF UBP) and ubiquitin specific proteases (USPs). Thus, we proposed that TRIM44 may function as a new class of the "USP-like-TRIM" which regulates the activity of associated TRIM proteins.
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Proteínas Portadoras/metabolismo , Testículo/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Proteínas Portadoras/genética , Línea Celular , Humanos , Péptidos y Proteínas de Señalización Intracelular , Masculino , Ratones , Proteínas de Motivos Tripartitos , Técnicas del Sistema de Dos Híbridos , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
BACKGROUND: Protein-protein interactions (PPIs) are challenging but attractive targets for small chemical drugs. Whole PPIs, called the 'interactome', have been emerged in several organisms, including human, based on the recent development of high-throughput screening (HTS) technologies. Individual PPIs have been targeted by small drug-like chemicals (SDCs), however, interactome data have not been fully utilized for exploring drug targets due to the lack of comprehensive methodology for utilizing these data. Here we propose an integrative in silico approach for discovering candidates for drug-targetable PPIs in interactome data. RESULTS: Our novel in silico screening system comprises three independent assessment procedures: i) detection of protein domains responsible for PPIs, ii) finding SDC-binding pockets on protein surfaces, and iii) evaluating similarities in the assignment of Gene Ontology (GO) terms between specific partner proteins. We discovered six candidates for drug-targetable PPIs by applying our in silico approach to original human PPI data composed of 770 binary interactions produced by our HTS yeast two-hybrid (HTS-Y2H) assays. Among them, we further examined two candidates, RXRA/NRIP1 and CDK2/CDKN1A, with respect to their biological roles, PPI network around each candidate, and tertiary structures of the interacting domains. CONCLUSION: An integrative in silico approach for discovering candidates for drug-targetable PPIs was applied to original human PPIs data. The system excludes false positive interactions and selects reliable PPIs as drug targets. Its effectiveness was demonstrated by the discovery of the six promising candidate target PPIs. Inhibition or stabilization of the two interactions may have potential therapeutic effects against human diseases.
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Sistemas de Liberación de Medicamentos/métodos , Preparaciones Farmacéuticas/metabolismo , Mapeo de Interacción de Proteínas/métodos , Evaluación Preclínica de Medicamentos/métodos , Humanos , Preparaciones Farmacéuticas/química , Unión Proteica/fisiología , Estructura Secundaria de Proteína/fisiología , Tecnología Farmacéutica/métodosRESUMEN
The chloride-ion-pumping channel, halorhodopsin from Halobacterium sp. shark was detergent-solubilized and 3-D crystallized. Proteins were solubilized using the nonionic detergent n-octyl-beta-D-glucoside and were crystallized as thin-plate crystals with polyethylene glycol 4000 as a precipitant. The crystals belong to the space group P4(1)2(1)2 with unit-cell dimensions a=b=74.5 A and c=138.6 A. The diffraction pattern was slightly anisotropic. The best ordered crystal diffracted up to 3.3 A resolution along c axis with synchrotron radiation.
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Halobacterium/enzimología , Halorrodopsinas/química , Cristalización , Difracción de Rayos XRESUMEN
UNLABELLED: Conduction defect caused by lamin A/C gene mutation. INTRODUCTION: Mutations of lamin A/C gene (LMNA) cause dilated cardiomyopathy (DCM) with atrioventricular (AV) conduction defect, although the electrophysiological and histological profiles are not fully understood. METHODS AND RESULTS: We analyzed a large Japanese family (21 affected and 203 unaffected members) of DCM with AV block. The responsible LMNA mutation of IVS3-10A>G was novel and caused an aberrant splicing. The first clinical manifestation was low-grade AV block or atrial fibrillation (AF), which developed in affected members aged >or=30 years. We observed that the AV block progressed to third-degree within several years. The electrophysiological study of the four affected members revealed an impairment of intra-AV nodal conduction. Because of advanced AV block, pacemakers were implanted in 14 out of 21 affected members at the mean age of 44 years. Three affected members died suddenly and two affected members died of heart failure and/or ventricular tachycardia (VT) even after the pacemaker implantation. Postmortem examination showed conspicuous fibrofatty degeneration of the AV node. Endomyocardial biopsies showed remarkably deformed nuclei and substantial glycogen deposits in the subsarcolemma. CONCLUSION: The clinical phenotype in this family was characterized by (1) the first manifestation of the prolonged PQ interval or AF in adolescence, (2) progressive intra-AV nodal block to the third degree in several years, and (3) progressive heart failure after pacemaker implantation. Histological study revealed preferential degeneration at the AV node area and novel cellular damages in the working myocardium.