RESUMEN
Many recent studies have suggested that activation of the aryl hydrocarbon receptor (AhR) reduces immune responses, thus suppressing allergies and autoimmune diseases. In our continuing study on natural AhR agonists in foods, we examined the influence of 37 health food materials on the AhR using a reporter gene assay, and found that aqueous ethanol extracts of cassia seed and rosemary had particularly high AhR activity. To characterize the AhR-activating substances in these samples, the chemical constituents of the respective extracts were identified. From an active ethyl acetate fraction of the cassia seed extract, eight aromatic compounds were isolated. Among these compounds, aurantio-obtusin, an anthraquinone, elicited marked AhR activation. Chromatographic separation of an active ethyl acetate fraction of the rosemary extract gave nine compounds. Among these compounds, cirsimaritin induced AhR activity at 10-10² µM, and nepitrin and homoplantagenin, which are flavone glucosides, showed marked AhR activation at 10-10³ µM.
Asunto(s)
Cassia/química , Factores Inmunológicos/química , Ledum/química , Hojas de la Planta/química , Receptores de Hidrocarburo de Aril/agonistas , Semillas/química , Animales , Antraquinonas/química , Antraquinonas/aislamiento & purificación , Antraquinonas/farmacología , Línea Celular Tumoral , Flavonas/química , Flavonas/aislamiento & purificación , Flavonas/farmacología , Flavonoides/química , Flavonoides/aislamiento & purificación , Flavonoides/farmacología , Expresión Génica/efectos de los fármacos , Genes Reporteros , Glucósidos/química , Glucósidos/aislamiento & purificación , Glucósidos/farmacología , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Factores Inmunológicos/aislamiento & purificación , Factores Inmunológicos/farmacología , Luciferasas/genética , Luciferasas/metabolismo , Luteolina/química , Luteolina/aislamiento & purificación , Luteolina/farmacología , Ratones , Extractos Vegetales/química , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismo , Relación Estructura-ActividadAsunto(s)
Antineoplásicos Fitogénicos/farmacología , Cornaceae/química , Descubrimiento de Drogas , Neoplasias/tratamiento farmacológico , Hojas de la Planta/química , Sumoilación/efectos de los fármacos , Taninos/farmacología , Antineoplásicos Fitogénicos/aislamiento & purificación , Antineoplásicos Fitogénicos/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Taninos Hidrolizables/química , Taninos Hidrolizables/aislamiento & purificación , Taninos Hidrolizables/metabolismo , Taninos Hidrolizables/farmacología , Japón , Estructura Molecular , Concentración Osmolar , Extractos Vegetales/química , Extractos Vegetales/farmacología , Taninos/química , Taninos/aislamiento & purificación , Taninos/metabolismoRESUMEN
Conophylline, a vinca alkaloid extracted from the tropical plant Ervatamia microphylla, has been shown to induce the differentiation of insulin-producing beta-cells in cultured cells and in animals. However, its mechanism of action and the molecular target have remained unclear. Therefore, we prepared a fishing probe with conophylline to identify the target protein by using latex nano-beads, which are newly innovated tools for affinity-purification. With these conophylline-linked nano-beads, we found that conophylline directly interacted with ARL6IP. ARL6IP may thus be involved in the mechanism of cellular differentiation of beta-cells, and this probe should be useful to find other target proteins.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Hipoglucemiantes/química , Proteínas de la Membrana/química , Nanopartículas/química , Plantas Medicinales/química , Alcaloides de la Vinca/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Línea Celular Tumoral , Hipoglucemiantes/metabolismo , Hipoglucemiantes/farmacología , Células Secretoras de Insulina/citología , Proteínas de la Membrana/metabolismo , Hojas de la Planta/química , Ratas , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Alcaloides de la Vinca/metabolismo , Alcaloides de la Vinca/farmacologíaRESUMEN
In drug development research, the elucidation and understanding of the interactions between physiologically active substances and proteins that numerous genes produce is important. Currently, most commercially available drugs and physiologically active substances have been brought to market without knowledge of factors interacting with the drugs and the substances. Affinity purification is a useful and powerful technique employed to understand factors that are targeted by drugs and physiologically active substances. However, use of conventional matrices for affinity chromatography often causes a decrease in efficiency of affinity purification and, as a result, more practical matrices for affinity purification have been developed for application in drug discovery research. In this paper, we describe the development of high-performance affinity beads (SG beads and FG beads) that enable one-step affinity purification of drug targets and the elucidation of the mechanism of the action of the drugs. We also describe a chemical screening system using our affinity beads. We hope that utilization of the affinity beads will contribute to the progress of research in chemical biology.
Asunto(s)
Cromatografía de Afinidad/métodos , Descubrimiento de Drogas/métodos , Microesferas , Unión Competitiva , Evaluación Preclínica de Medicamentos/métodos , Óxido Ferrosoférrico/química , Nanopartículas/química , Ácidos Polimetacrílicos/química , Unión ProteicaRESUMEN
6-((2-(Dimethylamino)ethyl)amino)-3-hydroxy-7H-indeno(2,1-c)-quinolin-7-one dihydrochloride (TAS-103) is a quinoline derivative that displays antitumor activity in murine and human tumor models. TAS-103 has been reported to be a potent topoisomerase II poison. However, other studies have indicated that cellular susceptibility to TAS-103 is not correlated with topoisomerase II expression. Because the direct target of TAS-103 remained unclear, we searched for a TAS-103 binding protein using high-performance affinity latex beads. We obtained a component of the signal recognition particle (SRP) as a TAS-103 binding protein. This component is a 54-kDa subunit (SRP54) of SRP, which mediates the proper delivery of secretory proteins in cells. We fractioned 293T cell lysates using gel-filtration chromatography and performed a coimmunoprecipitation assay using 293T cells expressing FLAG-tagged SRP54. The results revealed that TAS-103 disrupts SRP complex formation and reduces the amount of SRP14 and SRP19. TAS-103 treatment and RNAi-mediated knockdown of SRP54 or SRP14 promoted accumulation of the exogenously expressed chimeric protein interleukin-6-FLAG inside cells. In conclusion, we identified signal recognition particle as a target of TAS-103 by using affinity latex beads. This provides new insights into the mechanism underlying the effects of chemotherapies comprising TAS-103 and demonstrates the usefulness of the affinity beads.
Asunto(s)
Aminoquinolinas/farmacología , Proteínas Portadoras/metabolismo , Cromatografía de Afinidad/métodos , Indenos/farmacología , Microesferas , Preparaciones Farmacéuticas/metabolismo , Partícula de Reconocimiento de Señal/química , Extractos Celulares/química , Línea Celular , Núcleo Celular/química , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Células HeLa , Humanos , Interleucina-6/metabolismo , Riñón/citología , Peso Molecular , Interferencia de ARN , Coloración y EtiquetadoAsunto(s)
Marcadores de Afinidad , Microesferas , Nanopartículas , Proteínas/aislamiento & purificación , Antimetabolitos Antineoplásicos/administración & dosificación , Antimetabolitos Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Citarabina/administración & dosificación , Desoxicitidina Quinasa/fisiología , Evaluación Preclínica de Medicamentos , Humanos , Leucemia/tratamiento farmacológico , Metotrexato/administración & dosificación , Metotrexato/farmacología , Mitocondrias/metabolismo , Ácidos Polimetacrílicos , Porfirinas/metabolismoRESUMEN
Although the idea of homogeneous electrochemical immunoassay using antibody and an electroactive modified antigen as a probe looks to be very useful for high-throughput drug screening, there have been few reports. One reason for this is the difficulty experienced making an electroactive probe, because the introduction of electroactive compounds to antigens often interferes with the antigen-antibody interaction. To apply a homogeneous electrochemical assay to drug screening, we have designed new probes referring to the information of immobilization on beads which could identify the drug receptor. FK506 (also called Tacrolimus), immunosuppressive agent is modified with ferrocene derivatives as an electron mediator between glucose oxidase and an electrode, at a non-obstructing part. One of the probes still indicated the electrochemical activity as a mediator and had the specific binding capability for FKBP12 (FK506 binding protein). The current decrease in response to the additional FKBP12, detected with constant voltage amperometry using the probe, was observed within 5 min. Then, free FK506 as a leader drug, rapamycin and cyclosporine A as unknown drugs were used as a model for drug screening. Since the order of response currents at the same concentration of each drug reflected their binding constants, it was shown that binding capacity of an unknown drug candidate could be estimated by comparison of response currents between the leader drug and the unknown drug candidate. Thus, this glucose oxidase assisted homogeneous electrochemical drug-receptor binding assay has been proved to be a useful tool for drug screening.
Asunto(s)
Técnicas Biosensibles/instrumentación , Evaluación Preclínica de Medicamentos/instrumentación , Electroquímica/instrumentación , Ensayo de Inmunoadsorción Enzimática/instrumentación , Glucosa Oxidasa/química , Proteínas de Unión a Tacrolimus/análisis , Tacrolimus/análisis , Técnicas Biosensibles/métodos , Evaluación Preclínica de Medicamentos/métodos , Electroquímica/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Diseño de Equipo , Análisis de Falla de Equipo , Glucosa Oxidasa/análisis , Inmunosupresores/análisis , Inmunosupresores/química , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Tacrolimus/química , Proteínas de Unión a Tacrolimus/químicaRESUMEN
Muscle cell differentiation, or myogenesis, is a well-characterized process and involves the expression of specific sets of genes in an orderly manner. A prerequisite for myogenesis is the exit from the cell cycle, which is associated with the up-regulation of the tumor suppressor Rb. In this study, we set to investigate the regulatory mechanism of the Rb promoter that allows adequate up-regulation in differentiating myoblasts. We report that Rb expression is regulated by the transcription factors GABP, HCF-1 and YY1. Before induction of differentiation, Rb is expressed at a low level and GABP and YY1 are both present on the promoter. YY1, which exerts an inhibitory effect on Rb expression, is removed from the promoter as cells advance through myogenesis and translocates from the nucleus to the cytoplasm. On the other hand, upon induction of differentiation, the GABP cofactor HCF-1 is recruited to and coactivates the promoter with GABP. RNAi-mediated knock-down of HCF-1 results in inhibition of Rb up-regulation as well as myotube formation. These results indicate that the Rb promoter is subject to regulation by positive and negative factors and that this intricate activation mechanism is critical to allow the accurate Rb gene up-regulation observed during myogenesis.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Desarrollo de Músculos/fisiología , Regiones Promotoras Genéticas/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Animales , Diferenciación Celular , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Factores de Unión al ADN Específico de las Células Eritroides , Factor de Transcripción de la Proteína de Unión a GA , Factor C1 de la Célula Huésped , Humanos , Ratones , ARN Interferente Pequeño/farmacología , Proteína de Retinoblastoma , Factores de Transcripción/antagonistas & inhibidores , Activación Transcripcional , Factor de Transcripción YY1RESUMEN
A20 is an immediate-early NF-kappaB target gene. Prior to NF-kappaB stimulation, the A20 promoter is bound by the polymerase II machinery to allow rapid transcription activation. Here we show that the basal A20 transcription is repressed at the level of elongation in a promoter-specific fashion. Immunodepletion in vitro and RNA interference in cultured cells suggest that the basal elongation inhibition is conferred by DRB sensitivity-inducing factor (DSIF). We have identified a negative upstream promoter element called ELIE that controls DSIF activity. Remarkably, following NF-kappaB stimulation, inhibition of the A20 promoter by DSIF persists, but it is now regulated by NF-kappaB rather than ELIE. Similar regulation by DSIF is shown for another NF-kappaB-responsive gene, the IkappaBalpha gene. These findings reveal an intimate and dynamic relationship between DSIF inhibition of elongation and promoter-bound transcription factors. The potential significance of the differential regulation of DSIF activity by cis-acting elements is discussed.
Asunto(s)
FN-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Proteínas/genética , Proteínas Represoras , Secuencia de Bases , ADN Complementario/genética , Proteínas de Unión al ADN , Humanos , Proteínas I-kappa B/genética , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intracelular , Células Jurkat , Modelos Biológicos , Datos de Secuencia Molecular , Inhibidor NF-kappaB alfa , Proteínas Nucleares/genética , Interferencia de ARN , Factores de Transcripción , Transcripción Genética/efectos de los fármacos , Factores de Elongación Transcripcional , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
The transcription factor hGABP/E4TF1 is a heterotetrameric complex composed of two DNA-binding subunits (hGABP alpha/E4TF1-60) and two transactivating subunits (hGABP beta/E4TF1-53). In order to understand the molecular mechanism of transcriptional regulation by hGABP, we searched for proteins that interact with the non-DNA-binding subunit, hGABP beta, using yeast two-hybrid screening. We identified a human cDNA encoding a protein related to YAF-2 (YY1-associated factor 2), which was previously isolated as an interacting partner of the Ying-Yang-1 (YY1) transcription factor. Reflecting this similarity, both YAF-2 and this novel protein (named YEAF1 for YY1- and E4TF1/hGABP-associated factor-1) interacted with hGABP beta and YY1 in vitro and in vivo, indicating that YEAF1 and YAF-2 constitute a cofactor family for these two structurally distinct transcription factors. By using yeast three-hybrid assay, we demonstrated that hGABP beta and YY1 formed a complex only in the presence of YEAF1, indicating that YEAF1 is a bridging factor of these two transcription factors. These cofactors are functionally different in that YAF-2 positively regulates the transcriptional activity of hGABP but YEAF1 negatively regulates this activity. Also, YAF-2 mRNA is highly expressed in skeletal muscle, whereas YEAF1 mRNA is highly expressed in placenta. We speculate that the transcriptional activity of hGABP is in part regulated by the expression levels of these tissue-specific cofactors. These results provide a novel mechanism of transcriptional regulation by functionally distinct cofactor family members.