RESUMO
BACKGROUND: Antiretrovirals have been associated with serious adverse drug reactions. Several factors have been suggested as independent risk factors for their development. Identification of these factors may help in prevention and management of the adverse drug reactions. OBJECTIVE: To describe the factors associated with adverse drug reactions, their management, and the clinical outcomes. DESIGN: A retrospective cohort study. SETTING: Kenyatta National Hospital, Comprehensive Care Centre. SUBJECTS: Adult patients receiving antiretrovirals from 2003 to 2006. MAIN OUTCOME MEASURES: The primary outcomes were the risk-factors, interventions and outcomes of documented adverse drug reaction after exposure to antiretrovirals. RESULTS: Systematic random sampling was used to pick 350 patients' files. The risk factors for experiencing at least one adverse drug reaction were: having a baseline CD4 count less than 123 (odds ratio [OR] = 1.82, 95% confidence interval [CI: 1.18 to 2.79; p = 0.006); treatment with antiretrovirals for more than 32 months (OR = 1.76, CI: 1.15 to 2.71; p = 0.010), using didanosine containing regimens (OR = 3.7, CI: 1.40 to 9.70; p = 0.008) or being on stavudine containing regimens (OR = 4.4, CI: 2.53 to 7.71; p = 0.001). The most common intervention was addition of a non-antiretroviral while 41% of events resulted in a change of anti-retroviral therapy. CONCLUSIONS: Current standard regimens in resource-limited countries are associated with an increased risk of adverse drug reactions. Almost half of adverse reactions are managed by addition of a non-anti-retroviral drug alone but 41% necessitated a change of anti-retrovirals.
Assuntos
Antirretrovirais/efeitos adversos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/diagnóstico , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/terapia , Infecções por HIV/tratamento farmacológico , Adulto , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/etiologia , Feminino , Infecções por HIV/complicações , Humanos , Quênia , Masculino , Avaliação de Processos e Resultados em Cuidados de Saúde , Estudos Retrospectivos , Fatores de RiscoRESUMO
BACKGROUND: Skin develops through a process of epidermal proliferation, maturation, and remodelling of the epidermis and dermis. This period also involves the maturation of the skin immune system, such that antigen applied though the skin of a neonatal mouse always results in immunosuppression, whereas in adults, immunity will occur. OBJECTIVES: Using proteomics, to identify proteins uniquely involved in the development of the skin and skin immune system. METHODS: Proteins were extracted from whole skin of mice aged 4 and 21 days, and separated using two-dimensional electrophoresis. RESULTS: Of the 25 proteins that were sequenced by peptide mass fingerprinting with matrix-assisted laser desorption/ionization-time of flight-mass spectrometry, three were known markers of keratinocyte differentiation and proliferation. These were cyclophilin A, epidermal fatty acid binding protein 5 and stefin A. Of interest were the two isoforms of stefin A, an intracellular protease inhibitor, found in neonatal skin. The strong expression of stefin A in neonates was confirmed by immunohistochemical analysis, suggesting an important role in the development of the epidermis. Additionally, Western blotting identified two larger isoforms in adult skin, revealing a change in the stefin A during development. CONCLUSIONS: We propose that stefin A is involved in development of the skin, that development of the skin and of immune function is linked, and that stefin A has an important function in neonatal skin and potentially the neonatal immune response.
Assuntos
Western Blotting/métodos , Cistatinas/biossíntese , Proteômica/métodos , Pele/metabolismo , Animais , Western Blotting/normas , Cistatina A , Cistatinas/análise , Eletroforese em Gel Bidimensional , Feminino , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Mapeamento de Peptídeos , Pele/citologiaRESUMO
Hepatic genes crucial for carbohydrate and lipid homeostasis are regulated by insulin and glucose metabolism. However, the relative contributions of insulin and glucose to the regulation of metabolic gene expression are poorly defined in vivo. To address this issue, adenovirus-mediated hepatic overexpression of glucokinase was used to determine the effects of increased hepatic glucose metabolism on gene expression in fasted or ad libitum fed rats. In the fasted state, a 3 fold glucokinase overexpression was sufficient to mimic feeding-induced increases in pyruvate kinase and acetyl CoA carboxylase mRNA levels, demonstrating a primary role for glucose metabolism in the regulation of these genes in vivo. Conversely, glucokinase overexpression was unable to mimic feeding-induced alterations of fatty acid synthase, glucose-6-phosphate dehydrogenase, carnitine palmitoyl transferase I or PEPCK mRNAs, indicating insulin as the primary regulator of these genes. Interestingly, glucose-6-phosphatase mRNA was increased by glucokinase overexpression in both the fasted and fed states, providing evidence, under these conditions, for the dominance of glucose over insulin signaling for this gene in vivo. Importantly, glucokinase overexpression did not alter sterol regulatory element binding protein 1-c mRNA levels in vivo and glucose signaling did not alter the expression of this gene in primary hepatocytes. We conclude that a modest hepatic overexpression of glucokinase is sufficient to alter expression of metabolic genes without changing the expression of SREBP-1c.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/biossíntese , Proteínas de Ligação a DNA/biossíntese , Glucoquinase/biossíntese , Fígado/enzimologia , Fatores de Transcrição , Adenoviridae/genética , Animais , Carnitina O-Palmitoiltransferase/biossíntese , Células Cultivadas , Relação Dose-Resposta a Droga , Ácido Graxo Sintases/metabolismo , Regulação da Expressão Gênica , Glucoquinase/metabolismo , Glucose/metabolismo , Glucosefosfato Desidrogenase/biossíntese , Modelos Biológicos , Fosfoenolpiruvato Carboxiquinase (GTP)/biossíntese , Piruvato Quinase/metabolismo , RNA/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Proteína de Ligação a Elemento Regulador de Esterol 1 , Fatores de TempoRESUMO
Amplification of the MYCN oncogene in neuroblastoma is associated with poor prognosis. The amplified unit of DNA can be up to 1 Mb in size and so could contain additional genes which affect tumour phenotype. The neuroblastoma amplified gene (NAG) gene was initially located 400 kb telomeric to MYCN at 2p24 and reported to be co-amplified in 5/8 (63%) cell lines and 9/13 (70%) tumours. The sequence of a 4.5 kb transcript was proposed from the analysis of overlapping cDNA clones. However, our Northern blot hybridisation experiments indicate that the main RNA species expressed in neuroblastoma is 7-8 kb in size. We describe for the first time the cloning and sequencing of the 7.3 kb transcript of the NAG gene together with its precise genomic location and full exon structure. The 5' end of the gene is located 30 kb telomeric to DDX1, with the two genes lying in opposite orientations. The 52 exons of the 7.3 kb transcript cover 420 kb of genomic DNA. In vitro translation studies confirmed the protein coding potential of the transcript. Co-amplification of the entire NAG gene with MYCN was found in 1/6 (17%) neuroblastoma cell lines and 10/50 (20%) primary tumours. Previous studies had measured co-amplification of only the 5' end of the gene, nearest to MYCN. In this study, co-amplification of the NAG gene was found to be significantly associated with low disease stage in MYCN-amplified tumours (P=0.0063).
Assuntos
Proteínas de Neoplasias/genética , Neuroblastoma/genética , Sequência de Bases , Northern Blotting , Clonagem Molecular , Sequência Conservada/genética , DNA Complementar/química , DNA Complementar/genética , Éxons , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica , Genes/genética , Humanos , Íntrons , Dados de Sequência Molecular , Proteína Proto-Oncogênica N-Myc , Proteínas de Neoplasias/metabolismo , Estadiamento de Neoplasias , Neuroblastoma/patologia , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Regiões Promotoras Genéticas/genética , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Transcrição Gênica , Células Tumorais CultivadasRESUMO
Phosphoenolpyruvate carboxykinase (PEPCK) is the key enzyme in glyceroneogenesis, an important metabolic pathway that functions to restrain the release of non-esterified fatty acids (NEFAs) from adipocytes. The antidiabetic drugs known as thiazolidinediones (TZDs) are thought to achieve some of their benefits by lowering elevated plasma NEFAs. Moreover, peroxisome proliferator activated receptor gamma (PPARgamma) mediates the antidiabetic effects of TZDs, though many TZD responses appear to occur via PPARgamma-independent pathways. PPARgamma is required for adipocyte PEPCK expression, hence PEPCK could be a major target gene for the antidiabetic actions of TZDs. Here we used tissue culture and transfection assays to confirm that the TZD, rosiglitazone, stimulates PEPCK gene transcription specifically in adipocytes. We made the novel observation that this effect was by far the most rapid and robust among several other genes expressed in adipocytes. Adipocytes were transfected with a PEPCK/chloramphenicol acetyltransferase chimeric gene, in which either of the two previously discovered PPARgamma/retinoid X receptor alpha response elements, PCK2 and gAF1/PCK1, had been inactivated by mutagenesis. We demonstrate that PCK2 alone is a bona fide thiazolidinedione response element. We show also that the regulation of PEPCK by PPARs is cell-specific and isotype-specific since rosiglitazone induces PEPCK gene expression selectively in adipocytes, and PPARalpha- and PPARbeta-specific activators are inefficient. Hence, TZDs could lower plasma NEFAs via PPARgamma and PEPCK by enhancing adipocyte glyceroneogenesis.
Assuntos
Adipócitos/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Tiazóis/farmacologia , Tiazolidinedionas , Fatores de Transcrição/metabolismo , Células 3T3 , Adipócitos/enzimologia , Tecido Adiposo/citologia , Animais , Carcinoma Hepatocelular , Hipoglicemiantes/farmacocinética , Hipoglicemiantes/farmacologia , Masculino , Camundongos , Fosfoenolpiruvato Carboxiquinase (GTP)/efeitos dos fármacos , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , RNA Mensageiro/efeitos dos fármacos , Ratos , Ratos Wistar , Sequências Repetitivas de Ácido Nucleico , Rosiglitazona , Tiazóis/farmacocinética , Transcrição Gênica/efeitos dos fármacos , Transfecção , Células Tumorais CultivadasRESUMO
Fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11), an important gluconeogenic enzyme, catalyses the hydrolysis of fructose 1, 6-bisphosphate to fructose 6-phosphate and P(i). Enzyme activity is mainly regulated by the allosteric inhibitors fructose 2, 6-bisphosphate and AMP. Although some observations about hormonal regulation of the enzyme have been published, the FBPase promoter has not been studied in detail. Here we report an in vitro characterization of the FBPase promoter with respect to the elements that are required for basal promoter activity. Transient transfection of H4IIE rat hepatoma cells, combined with site-directed mutagenesis, demonstrated that an enhancer box, three GC-boxes and a nuclear factor kappaB (NF-kappaB)-binding element are important for hepatic FBPase promoter activity. These elements are found in the region located between -405 to +25 bp relative to the transcription start site. Electrophoretic-mobility-shift assays and supershift analysis confirmed that upstream stimulatory factor 1 (USF1)/USF2, specificity protein 1 (Sp1)/Sp3 and NF-kappaB respectively bind to these sites. The present study provides the basis for a more comprehensive screening for mutations in FBPase-deficient patients and for further studies of the transcriptional regulation of this gene.
Assuntos
Frutose-Bifosfatase/genética , Fígado/enzimologia , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Sítios de Ligação , Carcinoma Hepatocelular/metabolismo , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Eletroforese , Elementos Facilitadores Genéticos , Deleção de Genes , Genes Reporter , Humanos , Hidrólise , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , NF-kappa B/genética , NF-kappa B/metabolismo , Isoformas de Proteínas , Ratos , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp3 , Fatores de Transcrição/metabolismo , Transcrição Gênica , Transfecção , Células Tumorais Cultivadas , Fatores Estimuladores UpstreamRESUMO
Glucocorticoids stimulate gluconeogenesis by increasing the rate of transcription of genes that encode gluconeogenic enzymes such as phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase. Previous studies have shown that hepatic nuclear factor 3 (HNF3) is required as an accessory factor for several glucocorticoid-stimulated genes, including PEPCK. Here, we show that adenovirus-mediated expression of an HNF3beta protein with a deleted C-terminal transactivation domain (HNF3betaDeltaC) reduces the glucocorticoid-induced expression of the PEPCK and glucose-6-phosphatase genes in H4IIE hepatoma cells. Furthermore, expression of this truncated HNF3 protein results in a proportionate reduction of glucocorticoid-stimulated glucose production from lactate and pyruvate in these cells. The expression of HNF3betaDeltaN, in which the N-terminal transactivation domain is deleted, does not exhibit any of these effects. These results provide direct evidence that members of the HNF3 family are required for proper regulation of hepatic gluconeogenesis. Modulation of the function of the HNF3 family of proteins might be used to reduce the excessive hepatic production of glucose that is an important pathophysiologic feature of diabetes mellitus.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Gluconeogênese/fisiologia , Proteínas Nucleares/fisiologia , Fatores de Transcrição , Adenoviridae/genética , Sequência de Bases , Linhagem Celular , Cloranfenicol O-Acetiltransferase/genética , Primers do DNA , Proteínas de Ligação a DNA/genética , Fator 3-beta Nuclear de Hepatócito , Humanos , Proteínas Nucleares/genética , Recombinação Genética , Transfecção , Células Tumorais CultivadasRESUMO
Chicken ovalbumin upstream promoter-transcription factors (COUP-TFs), orphan members of the nuclear receptor superfamily, play a key role in the regulation of organogenesis, neurogenesis, and cellular differentiation during embryogenic development. COUP-TFs are also involved in the regulation of several genes that encode metabolic enzymes. Although COUP-TFs function as potent transcription repressors, there are at least three different molecular mechanisms of activation of gene expression by COUP-TFs. First, as we have previously shown, COUP-TF is required as an accessory factor for the complete induction of phosphoenolpyruvate carboxykinase gene transcription by glucocorticoids. This action is mediated by the binding of COUP-TF to the glucocorticoid accessory factor 1 (gAF1) and 3 (gAF3) elements in the phosphoenolpyruvate carboxykinase gene glucocorticoid response unit. In addition, COUP-TF1 binds to DNA elements in certain genes and transactivates directly. Finally, COUP-TF1 serves as a coactivator through DNA-bound hepatic nuclear factor 4. Here we show that the same region of COUP-TFI, located between amino acids 184 and 423, is involved in these three mechanisms of transactivation by COUP-TFI. Furthermore, we show that GRIP1 and SRC-1 potentiate the activity of COUP-TFI and that COUP-TFI associates with these coactivators in vivo using the same region required for transcription activation. Finally, overexpression of GRIP1 or SRC-1 does not convert COUP-TFI from a transcriptional repressor into a transcriptional activator in HeLa cells.
Assuntos
Proteínas de Ligação a DNA/genética , Fatores de Transcrição/genética , Ativação Transcricional , Animais , Fator I de Transcrição COUP , Galinhas , Proteínas de Ligação a DNA/metabolismo , Glucocorticoides/genética , Glucocorticoides/metabolismo , Células HeLa , Humanos , Ovalbumina/metabolismo , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
Glucocorticoids exert their effects on gene transcription through ubiquitous receptors that bind to regulatory sequences present in many genes. These glucocorticoid receptors are present in all cell types, yet glucocorticoid action is controlled in a tissue-specific way. One mechanism for this control relies on tissue-specific transcriptional activators that bind in the vicinity of the glucocorticoid receptor and are required for receptor action. We now describe a gene-specific and tissue-specific inhibitory mechanism through which glucocorticoid action is repressed by a tissue-restricted transcription factor, hepatocyte nuclear factor-6 (HNF-6). HNF-6 inhibits the glucocorticoid-induced stimulation of two genes coding for enzymes of liver glucose metabolism, namely 6-phosphofructo-2-kinase and phosphoenolpyruvate carboxykinase. Binding of HNF-6 to DNA is required for inhibition of glucocorticoid receptor activity. In vitro and in vivo experiments suggest that this inhibition is mediated by a direct HNF-6/glucocorticoid receptor interaction involving the amino-terminal domain of HNF-6 and the DNA-binding domain of the receptor. Thus, in addition to its known property of stimulating transcription of liver-expressed genes, HNF-6 can antagonize glucocorticoid-stimulated gene transcription.
Assuntos
Dexametasona/farmacologia , Glucocorticoides/farmacologia , Proteínas de Homeodomínio/metabolismo , Receptores de Glucocorticoides/fisiologia , Transativadores/metabolismo , Animais , Linhagem Celular , Dexametasona/antagonistas & inibidores , Genes Reporter , Glucocorticoides/antagonistas & inibidores , Fator 6 Nuclear de Hepatócito , Proteínas de Homeodomínio/genética , Humanos , Neoplasias Hepáticas Experimentais , Luciferases/genética , Regiões Promotoras Genéticas , Ratos , Receptores de Glucocorticoides/genética , Proteínas Recombinantes de Fusão/metabolismo , TATA Box , Transativadores/genética , Transfecção , Células Tumorais CultivadasRESUMO
The cyclic AMP response element (CRE) of the rat phosphoenolpyruvate carboxykinase (PEPCK) gene promoter is required for a complete glucocorticoid response. Proteins known to bind the PEPCK CRE include the CRE-binding protein (CREB) and members of the CCAAT/enhancer-binding protein (C/EBP) family. We took two different approaches to determine which of these proteins provides the accessory factor activity for the glucocorticoid response from the PEPCK CRE. The first strategy involved replacing the CRE of the PEPCK promoter/chloramphenicol acetyltransferase reporter plasmid (pPL32) with a consensus C/EBP-binding sequence. This construct, termed pDeltaCREC/EBP, binds C/EBPalpha and beta but not CREB, yet it confers a nearly complete glucocorticoid response when transiently transfected into H4IIE rat hepatoma cells. These results suggest that one of the C/EBP family members may be the accessory factor. The second strategy involved co-transfecting H4IIE cells with a pPL32 mutant, in which the CRE was replaced with a GAL4-binding sequence (pDeltaCREGAL4), and various GAL4 DNA-binding domain (DBD) fusion protein expression vectors. Although chimeric proteins consisting of the GAL4 DBD fused to either CREB or C/EBPalpha are able to confer an increase in basal transcription, they do not facilitate the glucocorticoid response. In contrast, a fusion protein consisting of the GAL4 DBD and amino acids 1-118 of C/EBPbeta provides a significant glucocorticoid response. Additional GAL4 fusion studies were done to map the minimal domain of C/EBPbeta needed for accessory factor activity to the glucocorticoid response. Chimeric proteins containing amino acid regions 1-84, 52-118, or 85-118 of C/EBPbeta fused to the GAL4 DBD do not mediate a glucocorticoid response. We conclude that the amino terminus of C/EBPbeta contains a multicomponent domain necessary to confer accessory factor activity to the glucocorticoid response from the CRE of the PEPCK gene promoter.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Glucocorticoides/metabolismo , Proteínas Nucleares/metabolismo , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Proteínas Estimuladoras de Ligação a CCAAT , Primers do DNA , Elementos Facilitadores Genéticos , Inibidores Enzimáticos/farmacologia , Fosfoenolpiruvato Carboxiquinase (ATP)/antagonistas & inibidores , Inibidores de Proteínas Quinases , Ratos , Células Tumorais CultivadasRESUMO
The phosphoenolpyruvate carboxykinase (PEPCK) gene promoter contains a glucocorticoid response unit (GRU) that includes three accessory factor-binding sites (AF1, AF2, and AF3), two glucocorticoid receptor-binding sites (GR1 and GR2), and a cAMP response element. All of these elements, and the proteins that bind to them, are required for a complete glucocorticoid response. The PEPCK promoter also contains a retinoic acid response unit (RARU) that consists of two retinoic acid response elements (RARE1 and RARE2) that bind retinoic acid receptor/9-cis-retinoic acid receptor heterodimers. The sequences of RARE1 and RARE2 coincide with those for AF1 and AF3, respectively. Thus, the PEPCK promoter can mediate different hormone responses through hormone response units that utilize common elements, but that bind different sets of proteins. We reasoned that each response might require a unique structural assembly and therefore tested how various arrangements of the PEPCK promoter affect the actions of either glucocorticoids or retinoic acid. The activation of the PEPCK gene in response to glucocorticoids requires a specific set of cis-acting elements that must be precisely positioned within the GRU. The distance between AF2 and GR1 is critical for the glucocorticoid response, and since the AF2 factor, HNF3, interacts with GR in vitro, this protein-protein interaction may be important for the glucocorticoid response. By contrast, the distance and orientation requirements of AF1 and AF3 with respect to GR1 are more flexible. In the case of the RARU, although the relative positions of RARE1 and RARE2 are important for the retinoic acid response, more tolerance for distance and stereospecific alignment between RARE1 and RARE2 is allowed. In addition, we show that the GRU and the RARU can act as a module, within a restricted region, in the context of the PEPCK promoter and in limited contexts of a heterologous promoter. These observations suggest that the structural requirements of the GRU and the RARU are different, and this may have important implications for how multiple hormonal signals are integrated through this promoter.
Assuntos
Glucocorticoides/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Regiões Promotoras Genéticas , Elementos de Resposta/genética , Fatores de Transcrição , Tretinoína/metabolismo , Animais , Sítios de Ligação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Glucocorticoides/farmacologia , Fator 3-alfa Nuclear de Hepatócito , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Ratos , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tretinoína/farmacologiaRESUMO
Phosphoenolpyruvate carboxykinase (PEPCK) is a rate-controlling enzyme in hepatic gluconeogenesis, and it therefore plays a central role in glucose homeostasis. The rate of transcription of the PEPCK gene is increased by glucagon (via cAMP) and glucocorticoids and is inhibited by insulin. Under certain circumstances glucose also decreases PEPCK gene expression, but the mechanism of this effect is poorly understood. The glucose-mediated stimulation of a number of glycolytic and lipogenic genes requires the expression of glucokinase (GK) and increased glucose metabolism. HL1C rat hepatoma cells are a stably transfected line of H4IIE rat hepatoma cells that express a PEPCK promoter-chloramphenicol acetyltransferase fusion gene that is regulated in the same manner as the endogenous PEPCK gene. These cells do not express GK and do not normally exhibit a response of either the endogenous PEPCK gene, or of the trans-gene, to glucose. A recombinant adenovirus that directs the expression of glucokinase (AdCMV-GK) was used to increase glucose metabolism in HL1C cells to test whether increased glucose flux is also required for the repression of PEPCK gene expression. In AdCMV-GK-treated cells glucose strongly inhibits hormone-activated transcription of the endogenous PEPCK gene and of the expressed fusion gene. The glucose effect on PEPCK gene promoter activity is blocked by 5 mM mannoheptulose, a specific inhibitor of GK activity. The glucose analog, 2-deoxyglucose mimics the glucose response, but this effect does not require GK expression. 3-O-methylglucose is ineffective. Glucose exerts its effect on the PEPCK gene within 4 h, at physiologic concentrations, and with an EC50 of 6.5 mM, which approximates the Km of glucokinase. The effects of glucose and insulin on PEPCK gene expression are additive, but only at suboptimal concentrations of both agents. The results of these studies demonstrate that, by inhibiting PEPCK gene transcription, glucose participates in a feedback control loop that governs its production from gluconeogenesis.
Assuntos
Glucose/metabolismo , Glucose/farmacologia , Insulina/fisiologia , Neoplasias Hepáticas Experimentais/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/biossíntese , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Transcrição Gênica , Animais , Células Cultivadas , Cloranfenicol O-Acetiltransferase , Citomegalovirus , Repressão Enzimática , Vetores Genéticos , Glucoquinase/biossíntese , Glucoquinase/genética , Glicólise , Homeostase , Insulina/farmacologia , Cinética , Regiões Promotoras Genéticas , Ratos , Proteínas Recombinantes de Fusão/biossíntese , TransfecçãoRESUMO
Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the rate-limiting step of gluconeogenesis. The activity of this enzyme is controlled by several hormones, including glucocorticoids, glucagon, retinoic acid, and insulin, that principally affect the rate of transcription of the PEPCK gene. Glucocorticoids induce PEPCK gene transcription through a complex glucocorticoid response unit that consists of, from 5' to 3', accessory factor elements AF1 and AF2; two noncanonical glucocorticoid receptor-binding sites, GR1 and GR2; a third accessory factor element, AF3; and a cAMP-response element, CRE. A complete glucocorticoid response is dependent on the presence of both GR-binding sites, all three accessory elements, and the CRE. In this study we assess the relative roles of GR1 and GR2 in the context of the glucocorticoid response unit and use a combination of binding and function assays to compare GR1 and GR2 to glucocorticoid response elements (GREs) that conform closely to the consensus sequence. The relative binding affinity of GR follows the order: consensus GRE >> GR1 > GR2. Mutations that disrupt the binding of GR to GR1 result in a major reduction of the glucocorticoid response, whereas similar mutations of GR2 have a much smaller effect. Unlike the simple consensus GRE, neither GR1 nor GR2 mediate a glucocorticoid response through a heterologous promoter. The accessory elements appear to have different functional roles. AF2 is still needed for a maximal glucocorticoid response when GR1 is converted to a high-affinity GR-binding element, but AF1 and AF3 are not required.
Assuntos
Glucocorticoides/fisiologia , Fosfoenolpiruvato Carboxilase/genética , Receptores de Glucocorticoides/fisiologia , Animais , Ligação Competitiva/genética , Carcinoma Hepatocelular , Sequência Consenso , Glucocorticoides/metabolismo , Mutagênese Sítio-Dirigida , Fosfoenolpiruvato Carboxilase/metabolismo , Ligação Proteica/genética , Ratos , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Células Tumorais CultivadasRESUMO
The phosphoenolpyruvate carboxykinase (PEPCK) gene promoter contains a glucocorticoid response unit (GRU) that includes, as a linear array, two accessory factor binding sites (AF1 and AF2) and two glucocorticoid receptor binding sites. All of these elements are required for a complete glucocorticoid response. AF1 and AF2 also partially account for the response of the PEPCK gene to retinoic acid and insulin, respectively. A second retinoic acid response element was recently located just downstream of the GRU. In this study we show that mutation of the 3' half-site of this element results in a 60% reduction of the glucocorticoid response of PEPCK promoter-chloramphenicol acetyltransferase (CAT) fusion constructs in transient transfection assays, thus the half-site is now termed AF3. A variety of assays were used to show that chicken ovalbumin upstream promoter transcription factor (COUP-TF) binds specifically to AF3 and that upstream stimulatory factor (USF) binds to an E-box motif located 2 base pairs downstream of AF3. Mutations of AF3 that diminish binding of COUP-TF reduce the glucocorticoid response, but mutation of the USF binding site has no effect. The functional roles of AF1, AF2, and AF3 in the glucocorticoid response were explored using constructs that contained combinations of mutations in all three elements. All three elements are required for a maximal glucocorticoid response, and mutation of any two abolish the response.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Regiões Promotoras Genéticas , Receptores de Glucocorticoides/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Bases , Fator I de Transcrição COUP , Sequência Consenso , Metilação , Dados de Sequência MolecularRESUMO
OBJECTIVES: This study examined the extent to which four broad areas of concern associated with prescribing are perceived by general practitioners (GPs): their sense of burden in providing health care, their views on financial constraints and incentives, the use of a prescription to cope with clinical workload and their perception of demanding patients. A secondary aim was to relate these concerns to actual measures of prescribing behaviour using PACT data. METHOD: A questionnaire covering the four themes was sent to 386 GPs. Using factor analysis, new measures were constructed to reflect the GPs' perception of the four areas of concern. RESULTS: A total of 228 GPs (59%) completed the questionnaire. Results indicated a high level of concern among GPs regarding current pressures that could affect prescribing. Only the respondents' concern about the possible adverse effects of financial pressures upon medical decisions was related to prescribing: those concerned about financial pressures prescribed less generically (P < 0.0005), had higher practice costs compared with the Family Health Services Authority average (P = 0.002) and issued more prescriptions overall (P = 0.007). CONCLUSION: There is a continuing need to monitor and evaluate the effect of recent changes in primary care for their effect upon prescribing behaviour.
Assuntos
Prescrições de Medicamentos , Tratamento Farmacológico , Medicina de Família e Comunidade , Padrões de Prática Médica , Adulto , Idoso , Antibacterianos/uso terapêutico , Atitude Frente a Saúde , Tomada de Decisões , Custos de Medicamentos , Prescrições de Medicamentos/economia , Prescrições de Medicamentos/estatística & dados numéricos , Tratamento Farmacológico/economia , Tratamento Farmacológico/estatística & dados numéricos , Análise Fatorial , Feminino , Humanos , Funções Verossimilhança , Masculino , Pessoa de Meia-Idade , Relações Médico-Paciente , Padrões de Prática Médica/economia , Fatores Socioeconômicos , Estatísticas não ParamétricasRESUMO
A previously characterized retinoic acid response element (RARE1) in the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter confers approximately 50% of the response of this gene to retinoic acid (RA). Transient transfection experiments were performed using constructs containing progressive 5' deletions of the PEPCK promoter to locate other elements that contribute to the RA response. A second RARE (RARE2) was located between -402 and -306. Methylation interference and mobility gel shift assays indicated that RAR/RXR bound specifically to a segment of DNA located between -337 and -321. This region contains consensus and degenerate half-sites for receptor binding separated by 5 bp. Mutations in either half-site selectively decreased the RA response and diminished RAR/RXR binding in mobility gel shift assays. When both RARE1 and RARE2 were mutated, 80% of the RA response was lost. Finally, RARE2 conferred a RA response in a heterologous promoter context. We conclude that RAR/RXR binds to RARE2, and that this DR5-type element is a major contributor to the response of the PEPCK gene to RA.
Assuntos
Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Tretinoína/farmacologia , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , DNA/genética , DNA/metabolismo , Dados de Sequência Molecular , Ratos , Receptores do Ácido Retinoico/metabolismo , Receptores X de Retinoides , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
Functional retinoic acid response elements (RAREs) have been described wherein the direct repeats are separated by 1, 2 or 5 bp (termed DR1, DR2 and DR5 respectively). We have previously shown that retinoic acid receptor/retinoid X receptor (RAR/RXR) binds a DR1 RARE within the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter and is the trans-acting complex that mediates the retinoic acid (RA) response. However, the mechanism of trans-activation is unknown. The consequences of RAR/RXR binding to the PEPCK RARE were examined using a circular permutation analysis as a first step to explore the possible role of DNA conformational changes in the RA response. The RAR/RXR heterodimer produced a distortion angle of 78 degrees. The DNA distortion was shown to be at the centre of the PEPCK RARE; RA did not affect the severity of the distortion angle or the location of the distortion centre. Monomers and homodimers of RAR also distorted the DNA, but to a lesser extent than did RAR/RXR. The results of a phasing analysis demonstrated that RAR/RXR heterodimers did not induce a static DNA bend, in either the presence or the absence of RA. A cyclization kinetics assay was employed to show that RAR/RXR binding affected DNA ring closure in a phase-sensitive, RA-insensitive, manner. Taken together, these observations support the idea that RAR/RXR heterodimers distort the structure of the PEPCK RARE, at least in part, by altering DNA flexibility. The conformational change in the PEPCK RARE upon RAR/RXR binding has implications for how RAR/RXR heterodimers recognize various RARE structures.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Regiões Promotoras Genéticas , Receptores do Ácido Retinoico/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Primers do DNA , Escherichia coli , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Plasmídeos , Reação em Cadeia da Polimerase , Receptores do Ácido Retinoico/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Receptor alfa de Ácido Retinoico , Receptores X de Retinoides , Retinoides/metabolismo , Fatores de Transcrição/biossínteseRESUMO
Advertisers are increasingly using symbols to circumvent logical argument when trying to persuade people (the "targets" of the advertisement) to make choices that are not strictly rational. Symbols can convey covert meanings and awaken or exploit subconscious feelings, such as a desire for power or a fear of doing harm. Some of the ways in which pharmaceutical advertisements use these techniques are examined: advertising by contagion; adding to our worries; polarity of choices; teasers; idealisation. Rational prescribing should be based on logic, but advertisements do not depend on logical arguments for their most powerful effects: the advertisers may subvert us by appealing to our unconscious desires.