RESUMO
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.
Assuntos
Bass/genética , Linfoma de Células B/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas/genética , Sequência de Aminoácidos , Animais , Apoptose/genética , Sequência de Bases , Linhagem Celular Tumoral , Núcleo Celular/genética , Clonagem Molecular/métodos , Citoplasma/genética , Proteínas de Peixes/genética , Regulação da Expressão Gênica/genética , Células HeLa , Humanos , NF-kappa B/genética , RNA Mensageiro/genética , Alinhamento de Sequência , Fator de Transcrição AP-1/genética , Transcrição Gênica/genética , Regulação para Cima/genéticaRESUMO
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.
Assuntos
Bass , Doenças dos Peixes/genética , Proteínas de Peixes/genética , Regulação da Expressão Gênica , Lipopolissacarídeos/farmacologia , PTEN Fosfo-Hidrolase/genética , Vibrioses/veterinária , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Proteínas de Peixes/química , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , PTEN Fosfo-Hidrolase/química , PTEN Fosfo-Hidrolase/metabolismo , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência/veterinária , Distribuição Tecidual , Vibrioses/genética , Vibrioses/imunologia , Vibrioses/microbiologia , Vibrio alginolyticus/fisiologiaRESUMO
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.
Assuntos
Doenças dos Peixes/metabolismo , Proteínas de Peixes/genética , Perciformes/genética , Vibrioses/veterinária , Vibrio alginolyticus/imunologia , Proteína X Associada a bcl-2/genética , Sequência de Aminoácidos , Animais , Apoptose , Sequência de Bases , Sobrevivência Celular , Clonagem Molecular , Sequência Conservada , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Proteínas de Peixes/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Células HeLa , Humanos , Imunidade Inata , Lipopolissacarídeos/farmacologia , Fígado/imunologia , Fígado/metabolismo , Especificidade de Órgãos , Perciformes/imunologia , Perciformes/metabolismo , Filogenia , Transporte Proteico , Baço/imunologia , Baço/metabolismo , Vibrioses/imunologia , Vibrioses/metabolismo , Vibrioses/microbiologia , Proteína X Associada a bcl-2/metabolismoRESUMO
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.
Assuntos
Bass , Calmodulina/genética , Doenças dos Peixes/genética , Proteínas de Peixes/genética , Regulação da Expressão Gênica , Vibrioses/veterinária , Vibrio alginolyticus/fisiologia , Sequência de Aminoácidos , Animais , Apoptose , Sequência de Bases , Calmodulina/metabolismo , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Proteínas de Peixes/metabolismo , Perfilação da Expressão Gênica , Dados de Sequência Molecular , Especificidade de Órgãos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Explosão Respiratória , Alinhamento de Sequência , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Vibrioses/genética , Vibrioses/imunologia , Vibrioses/microbiologiaRESUMO
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.
Assuntos
Bass/metabolismo , Proteínas de Ligação a Ácido Graxo/metabolismo , Proteínas de Peixes/metabolismo , Estresse Fisiológico , Sequência de Aminoácidos , Animais , Antioxidantes/metabolismo , Sequência de Bases , Bass/genética , Células Sanguíneas/citologia , Células Sanguíneas/metabolismo , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Peixes/genética , Regulação da Expressão Gênica , Concentração de Íons de Hidrogênio , Mucosa Intestinal/metabolismo , Fígado/citologia , Fígado/metabolismo , Dados de Sequência Molecular , Fases de Leitura Aberta , Filogenia , Pichia/genética , Pichia/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Explosão Respiratória , Temperatura , Fatores de TempoRESUMO
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).
