RESUMEN
Bcl-2 is a pro-survival member of Bcl-2 like superfamily, playing an important role in regulating the apoptotic process. In this study, the full-length Bcl-2 (EcBcl-2) was obtained, consisting of a 5'UTR of 290 bp, an ORF of 699 bp and a 3'UTR of 920 bp. EcBcl-2 gene encoded a polypeptide of 232 amino acids with an estimated molecular mass of 26.12 KDa and a predicted isoelectric point (pI) of 6.93. The deduced amino acid sequence analysis showed that EcBcl-2 consisted of the conserved residues and characteristic domains known to the critical functionality for Bcl-2. qRT-PCR analysis revealed that EcBcl-2 transcript was expressed in all the examined tissues, while the strongest expression level was observed in liver, followed by the expression in blood, gill, kidney, spleen, heart, intestine and muscle. The groupers challenged with V. alginolyticus showed a significant increase of EcBcl-2 mRNA in immune tissues. In addition, western blotting analysis confirmed that the up-regulation of EcBcl-2 protein expression was detected in liver. Subcellular localization analysis revealed that EcBcl-2 was localized in both nucleus and cytoplasm. Overexpression of EcBcl-2 can inhibit the LPS-induced apoptosis and activate the transcription activity of NF-κB and AP-1, while the deletion of BH1, BH2, BH3 or BH4 domain from EcBcl-2 can impede the signaling transduction. These results indicate that EcBcl-2 may play a regulatory role in the apoptotic process.
Asunto(s)
Lubina/genética , Linfoma de Células B/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Regiones no Traducidas 3'/genética , Regiones no Traducidas 5'/genética , Secuencia de Aminoácidos , Animales , Apoptosis/genética , Secuencia de Bases , Línea Celular Tumoral , Núcleo Celular/genética , Clonación Molecular/métodos , Citoplasma/genética , Proteínas de Peces/genética , Regulación de la Expresión Génica/genética , Células HeLa , Humanos , FN-kappa B/genética , ARN Mensajero/genética , Alineación de Secuencia , Factor de Transcripción AP-1/genética , Transcripción Genética/genética , Regulación hacia Arriba/genéticaRESUMEN
PTEN is a key tumor suppressor gene that can play a regulatory role in the cellular proliferation, survival and apoptosis. In this study, the full-length PTEN (EcPTEN) was obtained, containing a 5'UTR of 745 bp, an ORF of 1269 bp and a 3'UTR of 106 bp. The EcPTEN gene encoded a polypeptide of 422 amino acids with an estimated molecular mass of 49.14 KDa and a predicted isoelectric point (pI) of 6.34. The deduced amino acid sequence analysis showed that EcPTEN comprised the conserved residues and the characteristic domains known to the critical functionality of PTEN. qRT-PCR analysis revealed that EcPTEN mRNA was broadly expressed in all the examined tissues, while the highest expression level was observed in liver, followed by the expression in blood, kidney, spleen, heart, gill, muscle and intestine. The groupers challenged with Vibrio alginolyticus showed a sharp increase of EcPTEN mRNA expression in immune tissues. In addition, western blotting analysis confirmed that the up-regulation of EcPTEN protein expression was steadily induced in liver. Subcellular localization analysis indicated that EcPTEN was localized in both nucleus and cytoplasm. Overexpression of EcPTEN can activate the apoptotic cascade and abrogate NF-kB, AP-1, Stat3 and Myc promoter activity in Hela cells. These results indicated that EcPTEN harboring highly-conserved domains with a close sequence similarity to those of PTP superfamily may disrupt the mammalian signalings and play a regulatory role in the apoptotic process.
Asunto(s)
Lubina , Enfermedades de los Peces/genética , Proteínas de Peces/genética , Regulación de la Expresión Génica , Lipopolisacáridos/farmacología , Fosfohidrolasa PTEN/genética , Vibriosis/veterinaria , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , ADN Complementario/metabolismo , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/microbiología , Proteínas de Peces/química , Proteínas de Peces/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Fosfohidrolasa PTEN/química , Fosfohidrolasa PTEN/metabolismo , Filogenia , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de Secuencia/veterinaria , Distribución Tisular , Vibriosis/genética , Vibriosis/inmunología , Vibriosis/microbiología , Vibrio alginolyticus/fisiologíaRESUMEN
Bax is a pro-apoptotic member of Bcl-2 like superfamily, playing an important role in regulating the apoptosis. In this study, the full-length Bax (EcBax) was obtained, containing a 5'UTR of 64 bp, an ORF of 579 bp and a 3'UTR of 1021 bp. The EcBax gene encoded a polypeptide of 192 amino acids with an estimated molecular mass of 21.55 KDa and a predicted isoelectric point (pI) of 6.75. The deduced amino acid sequence analysis showed that EcBax comprised the conserved residues and the characteristic domains known to the critical function of Bax. qRT-PCR analysis revealed that EcBax mRNA was broadly expressed in all of the examined tissues, while the highest expression level was observed in blood, followed by the expression in liver, gill, spleen, kidney, heart, muscle and intestine. A sharp increase of EcBax expression was observed in the vibrio challenge group by comparing with those in the control. Subcellular localization analysis revealed that EcBax was predominantly localized in the cytoplasm. EcBax exerted a regulatory role in modulating the mitochondrial membrane potential, promoting the cytochrome c release, and then activating the downstream caspase signaling. Moreover, the overexpression of EcBax can decrease the cell viability and antagonize NF-kB, AP-1, Stat3 promoter activity in Hela cells. These results indicate that EcBax containing the conserved domain of pro-apoptotic member of Bcl-2 family may disrupt the mammalian signaling and play a regulative role in the apoptotic process.
Asunto(s)
Enfermedades de los Peces/metabolismo , Proteínas de Peces/genética , Perciformes/genética , Vibriosis/veterinaria , Vibrio alginolyticus/inmunología , Proteína X Asociada a bcl-2/genética , Secuencia de Aminoácidos , Animales , Apoptosis , Secuencia de Bases , Supervivencia Celular , Clonación Molecular , Secuencia Conservada , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/microbiología , Proteínas de Peces/metabolismo , Expresión Génica , Perfilación de la Expresión Génica , Células HeLa , Humanos , Inmunidad Innata , Lipopolisacáridos/farmacología , Hígado/inmunología , Hígado/metabolismo , Especificidad de Órganos , Perciformes/inmunología , Perciformes/metabolismo , Filogenia , Transporte de Proteínas , Bazo/inmunología , Bazo/metabolismo , Vibriosis/inmunología , Vibriosis/metabolismo , Vibriosis/microbiología , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Vibrio alginolyticus containing the highly toxic extracellular product is one of the most serious threats to grouper survival and its minimum lethal dose is approximately 500 CFU/g fish body weight in grouper. To study the toxic effects of V. alginolyticus on the immune system in teleost, Calmodulin (CaM), an important molecular indicator gene, was cloned from the orange-spotted grouper (Epinephelus coioides). The full-length Ec-CaM consisted of a 5'-UTR of 103 bp, an ORF of 450 bp and a 3'-UTR of 104 bp. The Ec-CaM gene encoded a protein of 149 amino acids with an estimated molecular mass of 16.4 kDa and a predicted isoelectric point of 3.93. The deduced amino acid sequence showed that Ec-CaM contained four highly conserved EF-hand domains known to be critical for the function of CaM. Ec-CaM was widely expressed and the highest expression level was observed in liver. Following V. alginolyticus challenge, a sharp increase level of respiratory burst activity and apoptosis ratio were observed. Further analyses of CaM expression and p53 expression in liver, kidney and spleen by qRT-PCR demonstrated that the up-regulated expression of CaM and p53 were observed in the vibrio challenge group. Western blotting analysis confirmed that the Ec-CaM protein was strongly induced in liver at 12 h post-injection, while a sharp increase of p53 protein expression was observed at 24 h post-injection. These results showed CaM expression serving as a potential molecular indicator may help to assess the toxicological effects of V. alginolyticus on the ROS generation and apoptotic process in grouper.
Asunto(s)
Lubina , Calmodulina/genética , Enfermedades de los Peces/genética , Proteínas de Peces/genética , Regulación de la Expresión Génica , Vibriosis/veterinaria , Vibrio alginolyticus/fisiología , Secuencia de Aminoácidos , Animales , Apoptosis , Secuencia de Bases , Calmodulina/metabolismo , Clonación Molecular , ADN Complementario/genética , ADN Complementario/metabolismo , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/microbiología , Proteínas de Peces/metabolismo , Perfilación de la Expresión Génica , Datos de Secuencia Molecular , Especificidad de Órganos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Estallido Respiratorio , Alineación de Secuencia , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Vibriosis/genética , Vibriosis/inmunología , Vibriosis/microbiologíaRESUMEN
We have isolated and characterized a fatty acid binding protein from the liver of the orange-spotted grouper (Epinephelus coioides). Amino acid sequence similarity of the Ec-FABP (E. coioides-FABP) was highest to FABP10s isolated from the livers of catfish, chicken, salamander and iguana. The open-reading frame of the Ec-FABP codes for a protein of 14.0 kDa with a calculated isoelectric point of 8.5. We first expressed a FABP10 protein from orange-spotted grouper (E. coioides) in Pichia pastoris, and then characterized the antioxidative potential of our recombinant Ec-FABP by DCF fluorescence assay. RT-PCR assays showed that endogenous Ec-FABP mRNA is most strongly expressed in liver with the most abundance and intestine. Change in the groupers' blood cells respiratory burst activity was examined during and after exposure to the pH and temperature stress using flow cytometry. Further analysis of Ec-FABP gene expression in liver tissue by quantitative real-time PCR demonstrated that Ec-FABP transcript levels increased when the fish were exposed to both pH and temperature stress, but the time when its mRNA expression level peaked differed under these stresses. Western blot analyses confirmed that the Ec-FABP protein was strongly expressed in the liver after exposure to the pH and temperature stress. These results suggest that Ec-FABP expression is stimulated by pH and temperature stress and that it may play important roles in general adaptive and antioxidant responses.
Asunto(s)
Lubina/metabolismo , Proteínas de Unión a Ácidos Grasos/metabolismo , Proteínas de Peces/metabolismo , Estrés Fisiológico , Secuencia de Aminoácidos , Animales , Antioxidantes/metabolismo , Secuencia de Bases , Lubina/genética , Células Sanguíneas/citología , Células Sanguíneas/metabolismo , Clonación Molecular , ADN Complementario/genética , ADN Complementario/metabolismo , Proteínas de Unión a Ácidos Grasos/genética , Proteínas de Peces/genética , Regulación de la Expresión Génica , Concentración de Iones de Hidrógeno , Mucosa Intestinal/metabolismo , Hígado/citología , Hígado/metabolismo , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Filogenia , Pichia/genética , Pichia/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Estallido Respiratorio , Temperatura , Factores de TiempoRESUMEN
To search for potential antitumor drugs with potent efficiency and low toxicity, a novel 1,4,7-triazacyclodecane and its platinum (II) complex were synthesized. These compounds were characterized by elemental analysis, IR, 1H NMR, 13C NMR, MS spectra, thermoanalysis and conductivity measurement. Antitumor activity study indicated these compounds had strong antitumor activity in vitro to some extent. Inhibition of human liver tumor of CA was examined by antitumor rate and growth rate, complex C showed inhibition activity on transplanting-tumor growth of CA, 12 mg x kg(-1) was as potent as cisplatin, its ID50 was 853.6 mg x kg(-1).
