RESUMEN
Limitation of cultured rat hepatoma cells for an essential amino acid results in a specific decrease in expression of several genes that are preferentially expressed in the liver, including the serum albumin and transthyretin genes. In the work presented here, we examined whether the coordinate repression of these genes is caused by decreased activity of one or more of the liver-enriched transcription factors, hepatocyte nuclear factor-1 (HNF-1), HNF-3, HNF-4 or C/EBP. To address this question, HepG2 human hepatoma cells were transiently transfected with luciferase reporter constructs containing multiple copies of individual transcription factor binding sites. Limitation for an essential amino acid resulted in specific repression of a construct in which luciferase expression was directed by HNF-1. A single HNF-1 binding site located adjacent to the TATA box plays a major role in transcription directed by the serum albumin promoter in transient transfection assays. Amino acid limitation of cells transfected with an albumin promoter/luciferase reporter construct resulted in specific repression of promoter activity. In addition, bacterial methylation or site-directed mutagenesis of the HNF-1 binding site in the albumin proximal promoter region eliminated the regulation of an albumin promoter-luciferase reporter construct under conditions of amino acid limitation. These results demonstrated that the HNF-1 binding site played a major role in regulation of the albumin promoter by amino acid availability. Deletion analysis of the albumin promoter confirmed regulation through the HNF-1 binding site and also identified a second amino acid regulatory element in the upstream region of the albumin promoter, which has been shown previously to contain a functional binding site for HNF-3. The repression of albumin promoter and HNF-1 reporter constructs in amino acid-limited cells occurred without a change in the DNA binding activity of HNF-1. Moreover, HNF-3 DNA binding activity was also not decreased in amino acid-limited cells. These results suggest that the regulation of transcription by amino acids occurs at the level of transcriptional activation by HNF-1 and HNF-3, rather than by alteration of the DNA binding activity of either factor.
Asunto(s)
Aminoácidos/deficiencia , Carcinoma Hepatocelular/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Aminoácidos/genética , Animales , Carcinoma Hepatocelular/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Factor Nuclear 1 del Hepatocito , Factor Nuclear 1-alfa del Hepatocito , Factor Nuclear 1-beta del Hepatocito , Humanos , Neoplasias Hepáticas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ratas , Células Tumorales CultivadasRESUMEN
Pseudorabies virus (PRV) early protein 0 (EP0) is a transactivator containing a RING finger domain. To assess the transactivation mechanism of PRV EP0, we performed the in vitro transcription by combining HeLa nuclear extract, purified recombinant EP0 and simple promoter constructs, and evaluated the results by primer extension. The data showed that EP0 could significantly activate the TATA-containing synthetic promoters. Moreover, EP0 activated transcription by stabilizing the formation of transcription initiation complex instead of enhancing the elongation rate. To further understand the role of EP0 on assembling the transcription initiation complex, we performed the pull-down assay using affinity precipitation of proteins from HeLa nuclear extracts and bacterially expressed glutathione-S-transferase EP0 RING finger fusion. The data showed that at least six nuclear proteins physically interacted with the EP0 RING finger. Overall, the transactivation of PRV EP0 is accomplished by enhancing the transcription initiation and is associated with at least six nuclear proteins.
Asunto(s)
Herpesvirus Suido 1 , Proteínas Recombinantes de Fusión/metabolismo , TATA Box , Transactivadores/metabolismo , Transcripción Genética , Proteínas Virales/metabolismo , Dedos de Zinc , Secuencia de Aminoácidos , Detergentes , Células HeLa , Humanos , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Sarcosina/análogos & derivados , Transactivadores/genética , Transactivadores/aislamiento & purificación , Proteínas Virales/genética , Proteínas Virales/aislamiento & purificaciónRESUMEN
OBJECTIVE: Human uncoupling proteins 2 and 3 (UCP2 and UCP3) are two mitochondrial proteins that are involved in the control of metabolism of fatty acid and possibly protect against oxidative damage. The aim of this study was to analyze genetic associations of four polymorphisms of the UCP2 and UCP3 genes with insulin, leptin concentration and obesity in Taiwan aborigines. RESEARCH METHODS: Four polymorphisms were compared in 324 obese (body mass index (BMI) > or =30 kg/m(2)) and overweight (30>BMI > or =25 kg/m(2)) subjects, and 114 normal weight subjects (BMI <25 kg/m(2)) in an aboriginal community of southern Taiwan. Anthropometric characteristics and fasting levels of insulin, leptin, triglycerides and cholesterol were measured. RESULTS: Before and after adjusting for age distribution, only the Val55 allele in exon 4 of the UCP2 gene increased the risk of overweight and obesity (adjusted odds ratio (OR)=2.02, P=0.004) in comparison with Ala55. UCP2 V55V is also associated with higher fasting insulin levels than A55V (P=0.01) and A55A (P=0.04) in the obese/overweight group. Using the COCAPHASE program of the UNPHASED software, haplotype analysis of three single nucleotide polymorphisms (A55V-G866A-C-55T) revealed that A-G-C (73% in obese subjects and 77% in controls) was the most common haplotype and that the haplotype V-A-T (13% in obese subjects and 5% in controls) was significantly increased in obese and overweight subjects (BMI > or =25 kg/m(2)) (OR=2.62, P<0.001). DISCUSSIONS: UCP2 A55V variant might predispose to obesity and Val55 allele to confer population-attributable risk for 9.5% of obese disorders and increase insulin concentrations. The V-A-T haplotype within UCP2-UCP3 gene cluster is also significantly associated with obesity in Paiwan aborigines.
Asunto(s)
Canales Iónicos/genética , Proteínas Mitocondriales/genética , Nativos de Hawái y Otras Islas del Pacífico/genética , Obesidad/genética , Anciano , Antropometría , Colesterol/sangre , Ayuno/sangre , Femenino , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Genotipo , Haplotipos , Humanos , Insulina/sangre , Leptina/sangre , Desequilibrio de Ligamiento , Masculino , Persona de Mediana Edad , Obesidad/sangre , Obesidad/etnología , Fenotipo , Polimorfismo de Nucleótido Simple , Taiwán/epidemiología , Triglicéridos/sangre , Proteína Desacopladora 2 , Proteína Desacopladora 3RESUMEN
BACKGROUND: Asthma is a multi-factorial disorder caused by complex interactions between genetic and environmental factors. IFN-gamma and IFN regulatory factor 1 (IRF-1) affect Th1/Th2 cytokine balance, and influence the differentiation of Th2 cells, which influence the development of asthma. OBJECTIVE: This study investigated CA repeats polymorphism of the IFN-gamma gene and GT repeats polymorphism of the IRF-1 gene, which may predispose individuals to asthma pathogenesis. METHODS: In the present study, we used the transmission/disequilibrium test (TDT) to investigate the relationship between asthma and the IFN-gamma and IRF-1 polymorphisms by studying 348 subjects composed of 232 parents and 116 asthmatic children. RESULTS: For global TDT test, IFN-gamma CA repeats and IRF-1 GT repeat polymorphisms showed a significant association with asthma in children (P=0.009 and 0.017, respectively). We demonstrated that 13 CA repeats (138 bp) of IFN-gamma gene and 11 GT repeats (306 bp) of IRF-1 gene are significantly preferentially transmitted to asthmatic children (T/NT=89/61, chi2=8.43, P<0.005 and T/NT=75/49, chi2=8.18, P<0.005, respectively). The offspring will have an increased risk of asthma when their parents transmit IFN-gamma 13 CA repeats (OR=1.83, P=0.009) and IRF1 11 GT repeats (OR=1.88, P=0.007) to them. But we observed that the IFN-gamma and IRF-1 polymorphisms are not associated with IgE concentrations. CONCLUSION: These findings provide strong evidence of which IFN-gamma CA repeat and IRF-1 GT repeat polymorphisms influence the risk of asthma for children in Taiwan.
Asunto(s)
Asma/genética , Factor 1 Regulador del Interferón/genética , Interferón gamma/genética , Polimorfismo Genético/genética , Adulto , Asma/sangre , Niño , Salud de la Familia , Femenino , Frecuencia de los Genes/genética , Predisposición Genética a la Enfermedad/genética , Humanos , Inmunoglobulina E/sangre , Desequilibrio de Ligamiento/genética , Masculino , Fenotipo , TaiwánRESUMEN
Transcription of the serum albumin gene occurs almost exclusively in the liver and is controlled in part by a strong liver-specific promoter. The upstream region of the serum albumin gene promoter is highly conserved among species and is footprinted in vitro by a number of nuclear proteins. However, the role of the upstream promoter region in regulating transcription and the identity of the transcription factors that bind to this region have not been established. In the present study, deletion analysis of the rat serum albumin promoter in transiently transfected HepG2 cells demonstrated that elimination of the region between -207 and -153 bp caused a two-fold decrease in promoter activity (P<0.05). Additional analysis of the -207 to -124 bp promoter interval led to the identification of two potential binding sites for hepatocyte nuclear factor-3 (HNF-3) located at -168 to -157 bp (site X) and -145 to -134 bp (site Y). Electrophoretic mobility-shift assays performed with the HNF-3 X and Y sites demonstrated that both sites are capable of binding HNF-3alpha and HNF-3beta. Placement of a single copy of the HNF-3 X site upstream from a minimal promoter increased promoter activity by about four-fold in HepG2 cells, and the reporter construct containing this site could be transactivated if co-transfected with an HNF-3 expression construct. Furthermore, inactivation of the HNF-3 X site by site-directed mutagenesis within the context of the -261 bp albumin promoter construct resulted in a 40% decrease in transcription (P<0.05). These results indicate that the positive effect of the -207 to -153 bp promoter interval is attributable to the presence of the HNF-3 X site within this interval. Additional results obtained with transfected HepG2 cells suggest that the HNF-3 Y site plays a lesser role in activation of transcription than the X site.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Albúmina Sérica/genética , Factores de Transcripción , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Células COS , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Factor Nuclear 3-alfa del Hepatocito , Factor Nuclear 3-beta del Hepatocito , Humanos , Mutagénesis Sitio-Dirigida , Proteínas Nucleares/química , Proteínas Nucleares/genética , Oligonucleótidos Antisentido , Ratas , Eliminación de Secuencia , Activación Transcripcional , Células Tumorales CultivadasRESUMEN
The pseudorabies virus (PRV) DNase gene has previously been mapped within the PRV genome. To characterize further the enzymic properties of PRV DNase, this enzyme was expressed in Escherichia coli with the use of a pET expression vector. The protein was purified to homogeneity and assayed for nuclease activity in vitro. Recombinant PRV DNase exhibited an alkaline pH preference and an absolute requirement for Mg2+ ions that could not be replaced by Ca2+ and Na+ ions. Further studies showed that PRV DNase exhibited endonuclease, 5'-exonuclease and 3'-exonuclease activities in both single-stranded and double-stranded DNA. This activity occurred randomly and no significant base preference was demonstrated. The multiple biochemical activities of PRV DNase are similar to the activities of Neurospora crassa endo-exonuclease and E. coli RecBCD, two additional enzymes that are involved in recombination. Taken together, the similarity of action between N. crassa endo-exonuclease, E. coli RecBCD, and PRV DNase suggests that PRV DNase might have a role in the process of recombination that occurs during PRV infection.
Asunto(s)
Desoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/metabolismo , Herpesvirus Suido 1/enzimología , Catálisis , Proteínas de Unión al ADN/metabolismo , Endonucleasas/química , Exodesoxirribonucleasa V , Exonucleasas/química , Peso Molecular , Proteínas Recombinantes , Especificidad por Sustrato , Proteínas Virales/metabolismoRESUMEN
In order to investigate the mechanism of long-term immunity and the effect of protective immunity induced by DNA vaccination, we constructed the expression plasmid containing a pseudorabies virus (PRV) gD gene encoding an envelope glycoprotein. Intramuscular vaccination of mice with the plasmid DNA induced a strong antibody response which lasted for one year after final vaccination. An IgM to IgG class switch occurred, indicating helper T-lymphocyte activity. We further analyzed the persistence and expression of gD gene by polymerase chain reaction and reverse transcriptase polymerase chain reaction. The results showed that gD gene was present and expressed in the muscle cell up to one year after final booster injection. Furthermore, mice vaccinated with the plasmid DNA were protected against a subsequent lethal challenge with PRV. Therefore, the DNA vaccination does induce a protective immunity and long-term antibody response against PRV, which could be maintained by persistent expression of gD gene in muscle cells.
Asunto(s)
Formación de Anticuerpos/inmunología , Herpesvirus Suido 1/inmunología , Seudorrabia/inmunología , Vacunación , Vacunas de ADN/inmunología , Animales , Anticuerpos Antivirales/inmunología , Femenino , Expresión Génica/genética , Ingeniería Genética , Vectores Genéticos , Herpesvirus Suido 1/efectos de los fármacos , Inmunidad Activa , Ratones , Ratones Endogámicos BALB C , Seudorrabia/prevención & control , Proteínas Recombinantes/genética , Factores de Tiempo , Vacunas de ADN/administración & dosificación , Proteínas del Envoltorio Viral/genéticaRESUMEN
The pseudorabies virus (PRV) DNase gene has an open reading frame of 1476 nt, capable of coding a 492-residue protein. A previous study showed that PRV DNase is an alkaline exonuclease and endonuclease, exhibiting an Escherichia coli RecBCD-like catalytic function. To analyse its catalytic mechanism further, we constructed a set of clones truncated at the N-terminus or C-terminus of PRV DNase. The deleted mutants were expressed in E. coli with the use of pET expression vectors, then purified to homogeneity. Our results indicate that (1) the region spanning residues 274-492 exhibits a DNA-binding ability 7-fold that of the intact DNase; (2) the N-terminal 62 residues and the C-terminal 39 residues have important roles in 3'-exonuclease activity, and (3) residues 63-453 are responsible for 5'- and 3'-exonuclease activities. Further chemical modification of PRV DNase revealed that the inactivation of DNase by diethyl pyrocarbonate, which was reversible on treatment with hydroxylamine, seemed to be attributable solely to the modification of histidyl residues. Because the herpesviral DNases contained only one well-conserved histidine residue, site-directed mutagenesis was performed to replace His(371) with Ala. The mutant lost most of its nuclease activity; however, it still exhibited a wild-type level of DNA-binding ability. In summary, these results indicate that PRV DNase contains an independent DNA-binding domain and that His(371) is the active-site residue that has an essential role in PRV DNase activity.
Asunto(s)
ADN Viral/metabolismo , Desoxirribonucleasas/metabolismo , Herpesvirus Suido 1/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Catálisis , ADN Viral/genética , Desoxirribonucleasas/genética , Herpesvirus Suido 1/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión ProteicaRESUMEN
Prostaglandin J(2) (PGJ(2)) and its metabolites Delta(12)-PGJ(2) and 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) are naturally occurring derivatives of prostaglandin D(2) that have been suggested to exert antiinflammatory effects in vivo. 15d-PGJ(2) is a high-affinity ligand for the peroxisome proliferator-activated receptor gamma (PPARgamma) and has been demonstrated to inhibit the induction of inflammatory response genes, including inducible NO synthase and tumor necrosis factor alpha, in a PPARgamma-dependent manner. We report here that 15d-PGJ(2) potently inhibits NF-kappaB-dependent transcription by two additional PPARgamma-independent mechanisms. Several lines of evidence suggest that 15d-PGJ(2) directly inhibits NF-kappaB-dependent gene expression through covalent modifications of critical cysteine residues in IkappaB kinase and the DNA-binding domains of NF-kappaB subunits. These mechanisms act in combination to inhibit transactivation of the NF-kappaB target gene cyclooxygenase 2. Direct inhibition of NF-kappaB signaling by 15d-PGJ(2) may contribute to negative regulation of prostaglandin biosynthesis and inflammation, suggesting additional approaches to the development of antiinflammatory drugs.