RESUMO
BACKGROUND AND AIM: Blood pressure is an independent predictor of target organ damage (TOD). Recent data from literature suggest that TOD can be present also in pre-hypertensive subjects, diagnosed with pressure monitoring (PM). Aim of this study is to clarify whether an augmentation of the carotid Intima-Media Thickness (cIMT) in office prehypertensives is a TOD associated to monitoring prehypertension (MP). PATIENTS AND METHODS: We have analyzed our database of individuals office normotensives showing an increase of cIMT. The ambulatory blood pressure monitoring (ABPM) of these was compared with those of office monitoring normotensives, matched by age and gender, antropometric characteristics, negative for familial hypertension and other risk factors (true normotensives, TN). RESULTS: We have selected 15 presumable prehypetensives (PP) and 8 TN subjects. The ABPM (ambulatory blood pressure monitoring) analysis confirmed that neither the PP nor TN showed systolic (S) and diastolic (D) BP within-day values above their day-night upper reference limits. However the statistical comparison between PP and TN revealed that the first group had a significant elevation of SBP and DBP Daily Mean Level (DML(SBP/DBP): 121 ± 2/81 ± 2 vs 112 ± 2/70 ± 2 mmHg, respectively, p = 0.007 and p = 0.002), confirming the MP diagnosis. CONCLUSIONS: These results demonstrate that cIMT increase in PP fulfill the criteria for MP diagnosis, suggesting that MP should be undertaken in all PP with altered cIMT, but larger prospective studies are needed.
Assuntos
Pressão Arterial , Determinação da Pressão Arterial , Doenças das Artérias Carótidas/diagnóstico por imagem , Artéria Carótida Primitiva/diagnóstico por imagem , Espessura Intima-Media Carotídea , Visita a Consultório Médico , Pré-Hipertensão/diagnóstico , Adulto , Determinação da Pressão Arterial/métodos , Monitorização Ambulatorial da Pressão Arterial , Distribuição de Qui-Quadrado , Feminino , Humanos , Masculino , Valor Preditivo dos Testes , Pré-Hipertensão/fisiopatologia , Estudos RetrospectivosRESUMO
Interaction of the p55 tumor necrosis factor receptor 1 (TNF-R1)-associated signal transducer TRADD with FADD signals apoptosis, whereas the TNF receptor-associated factor 2 protein (TRAF2) is required for activation of the nuclear transcription factor nuclear factor kappa B. TNF-induced activation of the stress-activated protein kinase (SAPK) was shown to occur through a noncytotoxic TRAF2-dependent pathway. TRAF2 was both sufficient and necessary for activation of SAPK by TNF-R1; conversely, expression of a dominant-negative FADD mutant, which blocks apoptosis, did not interfere with SAPK activation. Therefore, SAPK activation occurs through a pathway that is not required for TNF-R1-induced apoptosis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , MAP Quinase Quinase Quinase 1 , Proteínas Quinases Ativadas por Mitógeno , Proteínas/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Acetilcisteína/farmacologia , Apoptose , Proteínas de Transporte/metabolismo , Linhagem Celular , Dactinomicina/farmacologia , Ativação Enzimática , Proteína de Domínio de Morte Associada a Fas , Sequestradores de Radicais Livres/farmacologia , Células HeLa , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais , Fator 1 Associado a Receptor de TNF , Fator 2 Associado a Receptor de TNF , TransfecçãoRESUMO
Myogenic differentiation is a highly orchestrated multistep process controlled by extracellular growth factors that modulate largely unknown signals into the cell affecting the muscle-transcription program. P38MAPK-dependent signalling, as well as PI3K/Akt pathway, has a key role in the control of muscle gene expression at different stages during the myogenic process. P38MAPK affects the activities of transcription factors, such as MyoD and myogenin, and contributes, together with PI3K/Akt pathway, to control the early and late steps of myogenic differentiation. The aim of our work was to better define the role of PKR, a dsRNA-activated protein kinase, as potential component in the differentiation program of C2C12 murine myogenic cells and to correlate its activity with p38MAPK and PI3K/Akt myogenic regulatory pathways. Here, we demonstrate that PKR is an essential component of the muscle development machinery and forms a functional complex with p38MAPK and/or Akt, contributing to muscle differentiation of committed myogenic cells in vitro. Inhibition of endogenous PKR activity by a specific (si)RNA and a PKR dominant-negative interferes with the myogenic program of C2C12 cells, causing a delay in activation of myogenic specific genes and inducing the formation of thinner myofibers. In addition, the construction of three PKR mutants allowed us to demonstrate that both N and C-terminal regions of PKR are critical for the interaction with p38MAPK and Akt. The novel discovered complex permits PKR to timely regulate the inhibition/activation of p38MAPK and Akt, controlling in this way the different steps characterizing skeletal muscle differentiation.
Assuntos
Diferenciação Celular , Desenvolvimento Muscular , Fibras Musculares Esqueléticas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , eIF-2 Quinase/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Ativação Enzimática , Camundongos , Dados de Sequência Molecular , Fibras Musculares Esqueléticas/enzimologia , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fatores de Tempo , Transfecção , eIF-2 Quinase/genéticaRESUMO
The management of HBV or HCV has improved dramatically over the last decade with the development of new drugs. This paper provides a review of new available and developing treatment options for HBV and HCV associated liver diseases. In the closer future the most realistic therapeutical option for most of the patients with HBV and HCV infection will be combination and/or long-term usage of the new, stronger antiviral drugs, if they maintain good safety profiles, achieve low resistance rates and will be available at lower prices.
Assuntos
Antivirais/uso terapêutico , Hepatite B/tratamento farmacológico , Hepatite C/tratamento farmacológico , Vacinas contra Hepatite Viral/uso terapêutico , Antivirais/química , Antivirais/economia , Farmacorresistência Viral , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Humanos , Conformação Molecular , Estereoisomerismo , Vacinas contra Hepatite Viral/química , Vacinas contra Hepatite Viral/economiaRESUMO
Hepatocellular carcinoma (HCC) is a common cancer worldwide. A majority of HCC patients present with an unresectable or metastatic HCC and systemic therapy with cytotoxic agents provides marginal benefits. Some evidence suggests that certain natural phytochemical compounds used in conjunction with chemotherapeutic agents could enhance therapeutic efficacy by sensitizing cells to treatment.
Assuntos
Anticarcinógenos/uso terapêutico , Antineoplásicos Fitogênicos/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Resistência a Medicamentos , Sinergismo Farmacológico , Humanos , Resultado do TratamentoRESUMO
The hepatitis B virus (HBV) X protein (pX) is capable of activating transcription regulated by viral and cellular promoters containing binding sites for different transcription factors, including AP1. In this study we have analyzed the mechanisms of AP1 induction by pX. The hepatitis B virus transactivator was able to activate TRE (12-O-tetradecanoylphorbol-13-acetate response element)-directed transcription in different cell lines, including HepG2, HeLa, CV1, and PLC/PRF/5 cells. pX-induced AP1 activation in HepG2 cells was associated with an increase in the DNA-binding activity of c-Jun/c-Fos heterodimers, which was not dependent either on an increase in the overall amount of c-Fos and c-Jun proteins in the cells or on formation of dimers between pX and the two proteins, thus suggesting the involvement of posttranslational modifications of the transcription factor. The observation that the overexpression of c-Jun and c-Fos in the cells results in a strong augmentation of the effect of pX on TRE-directed transcription is additional evidence indicating the involvement of posttranscriptional modifications of c-Jun/c-Fos heterodimers. The increased AP1 binding observed in the presence of pX was unaffected by the protein kinase C inhibitors calphostin C and sphingosine and by the protein kinase A inhibitor HA1004, while it was almost completely blocked by staurosporine, a potent and nonspecific protein kinase inhibitor, suggesting that protein kinase C- and A-independent phosphorylation events might play a role in the phenomenon. The ability of pX also to increase TRE-directed transcription in cell lines in which AP1-binding activity is not increased (i.e., HeLa, CV1, and PLC/PRF/5 cells) suggests that pX can activate canonical TRE sites by different mechanisms as well.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Vírus da Hepatite B/metabolismo , Naftalenos , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Sulfonamidas , Transativadores/metabolismo , Ativação Transcricional , Alcaloides/farmacologia , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Núcleo Celular/metabolismo , Cloranfenicol O-Acetiltransferase/biossíntese , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Células HeLa , Humanos , Isoquinolinas/farmacologia , Cinética , Substâncias Macromoleculares , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/metabolismo , Fosforilação , Compostos Policíclicos/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-fos/biossíntese , Proteínas Proto-Oncogênicas c-jun/biossíntese , Esfingosina/farmacologia , Estaurosporina , Acetato de Tetradecanoilforbol/farmacologia , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Transfecção , Células Tumorais Cultivadas , Vaccinia virus/genética , Proteínas Virais Reguladoras e AcessóriasRESUMO
Chronic hepatitis C virus (HCV) infection is a leading cause of liver cirrhosis and hepatocellular carcinoma (HCC) worldwide. The HCV capside core is a multifunctional protein with regulatory functions that affects transcription and cell growth in vitro and in vivo. Here, we show that both HCV genotype 1a and 3 core proteins activate MEK1 and Erk1/2 MAP kinases and that the costitutive expression of the HCV core results in a high basal activity of Raf1 and MAP/kinase/kinase, as determined by endogenous Raf1 in vitro kinase assay and immunodetection of hyperphosphorylated Erk1 and Erk2 even after a serum starvation. Moreover, the activation of both Erk1/2 and the downstream transcription factor Elk-1 in response to the mitogenic stimulus EGF is significantly prolonged. The sustained response to EGF in cells expressing the HCV core occurs despite a normal induction of the MAP phosphatases MKP regulatory feedback and is likely due to the costitutive activation of Raf-1 activity. The ability of HCV core proteins to directly activate the MAP kinase cascade and to prolong its activity in response to mitogenic stimuli may contribute to the neoplastic transformation of HCV infected liver cells.
Assuntos
MAP Quinase Quinase Quinase 1 , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas do Core Viral/fisiologia , Ativação Enzimática , Genótipo , Hepacivirus/genética , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas c-raf/fisiologia , Proteínas do Core Viral/biossíntese , Proteínas do Core Viral/genéticaRESUMO
The mechanisms by which pX, the transactivator of the Hepatitis B Virus (HBV), exerts its effects on transcription of viral and cellular genes have not yet been fully clarified. While previous reports suggested the possibility of a direct interaction of pX, which lacks intrinsic DNA-binding activity, with components of the cellular transcription machinery, more recent investigations support the hypothesis that pX might activate cellular kinases involved in transcriptional regulation and growth control. We analysed the mechanisms of c-Jun transcription factor activation by pX and in particular the role of cellular proteins involved in the transduction of mitogenic signals (namely Ha-Ras and Raf-1). In both HeLa and undifferentiated F9 cells pX was able to increase the activity of exogenous transfected c-Jun but not of c-Jun proteins bearing mutations in the serine residues located in the amino-terminal transcriptional activation domain. We show by use of Ha-Ras and Raf-1 dominant negative mutants that both Ha-Ras and Raf-1 are required for pX-induced activation of c-Jun transcriptional activity. In addition we show that pX is able to cooperate with Raf-1 in c-Jun activation. Our results are consistent with the hypothesis that at least one site of action of pX is peripheral and is located upstream of the Ras genes products.
Assuntos
Vírus da Hepatite B/genética , Proteína Oncogênica p21(ras)/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Oncogênicas de Retroviridae/metabolismo , Transativadores/metabolismo , Ativação Transcricional , Linhagem Celular , Cloranfenicol O-Acetiltransferase/genética , Genes ras , Células HeLa , Humanos , Proteínas Oncogênicas v-raf , Transdução de Sinais , Proteínas Virais Reguladoras e AcessóriasRESUMO
CTLs- and lymphokine-induced apoptosis of infected hepatocytes during the course of chronic viral hepatitis is thought to be important for both disease termination and prevention of hepatocellular transformation. We therefore studied apoptosis induced by Fas (APO-1 or CD95)-a widely expressed cell surface receptor whose ligand is involved in lymphocyte cytotoxicity-in a set of human hepatoma cell lines. As normal hepatocytes, all of the human hepatoma cell lines tested do express detectable amounts of Fas on their surface. Nevertheless, only PLC/PRF/5 cells undergo apoptosis following treatment with anti-Fas. Systematic cloning and sequence analysis of the Fas cDNA did not show mutations in the Fas gene in any of the cells lines tested. However, due to alternative splicing, 5 to 10% of the Fas cDNAs are deleted of 63 internal nucleotides corresponding to the transmembrane domain, thus encoding for a soluble and secreted form of Fas (Fas delta TM), potentially able to neutralize anti-Fas or Fas-Ligand. Although we could not demonstrate a direct correlation between resistance of different hepatoma cell lines to Fas mediated death and endogenous expression of this transcript, we show that PLC/PRF 5 stable transfectants overexpressing Fas delta TM are less sensitive to anti-Fas than control cells. In three different cell lines, resistance to anti-Fas was overcome by treatment with the protein synthesis inhibitor cycloheximide. Although this could suggest the existence of short-lived repressors of the Fas-activated apoptotic signalling pathway(s), we show that translational inhibition is not required for the synergistic effect of cycloheximide to take place, and that resistant hepatoma cells can be sensitized to anti-Fas by subinhibitory concentrations of this protein synthesis inhibitor. Since cycloheximide is able to activate intracellular signalling independently on its effects on protein synthesis, we suggest that it might provide a costimulatory signal that cooperates with Fas in the induction of cell death and that, at least in the cells we tested, resistance to Fas is not an active process involving gene transcription and translation but only the consequence of an inadequate apoptotic stimulation.
Assuntos
Apoptose , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Receptor fas/fisiologia , Adulto , Sequência de Aminoácidos , Sequência de Bases , Cicloeximida/farmacologia , DNA Complementar/química , Humanos , Masculino , Dados de Sequência Molecular , Proteínas Repressoras/fisiologia , Células Tumorais CultivadasRESUMO
Withdrawal from the cell cycle of differentiating myocytes is regulated by the myogenic basic helix-loop-helix (bHLH) protein MyoD and the pocket proteins pRb, p107 and pRb2/p130. Downstream effectors of 'pocket' proteins are the components of the E2F family of transcription factors, which regulate the G1/S-phase transition. We analysed by EMSA the composition of E2F complexes in cycling, quiescent undifferentiated and differentiated C2C12 skeletal muscle cells. An E2F complex containing mainly E2F4 and pRb2/p130 (E2F-G0/G1 complex) appears when DNA synthesis arrests, replacing the cyclinA/cdk2 containing E2F complex of proliferating myoblasts (E2F-G1/S complex). Serum stimulation reinduces DNA synthesis and the re-appearance of E2F-G1/S complexes in quiescent myoblasts but not in differentiated C2C12 myotubes. In differentiating C2C12 cells, E2F complexes switch and DNA synthesis in response to serum are prevented when MyoD DNA binding activity and the cdks inhibitor MyoD downstream effector p21 are induced. Thus, during myogenic differentiation, formation of E2F4 and pRb2/p130 containing complexes is an early event, but not enough on its own to prevent the reactivation of DNA synthesis. Using a subclone of C3H10T1/2 mouse fibroblasts stably expressing Estrogen Receptor-MyoD (ER-MyoD) chimerae, we found that estrogen directed MyoD activation prevents the reassociation of cyclinA/cdk2 to the E2F4 containing complex following serum stimulation and this correlates with suppression of E2F activity and the inability of cells to re-enter the cell cycle. Our data indicate that, in differentiating myocytes, one mechanism through which MyoD induces permanent cell cycle arrest involves p21 upregulation and suppression of the proliferation-associated cdks-containing E2F complexes formation.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas de Transporte , Proteínas de Ciclo Celular , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Proteínas de Ligação a DNA , Músculo Esquelético/metabolismo , Proteína MyoD/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Quinase 2 Dependente de Ciclina , DNA/metabolismo , Fatores de Transcrição E2F , Fator de Transcrição E2F4 , Fase G1/fisiologia , Camundongos , Camundongos Endogâmicos C3H , Músculo Esquelético/citologia , Proteína MyoD/genética , Fase de Repouso do Ciclo Celular/fisiologia , Proteína 1 de Ligação ao Retinoblastoma , Fator de Transcrição DP1 , Regulação para CimaRESUMO
The hepatitis B virus (HBV) X protein (pX) stimulates transcription regulated by cis-acting elements that control many viral and cellular genes, including the c-myc and the c-fos proto-oncogenes. Using several c-fos promoter deletion mutants, we found the serum-responsive element (SRE) located at -315, the modified TPA-responsive element located at -296 (fos-AP-1 binding site, FAP) and the region spanning from nucleotide -220 to -120, which contains an NF1-like site and several stretches of sequence homologous to the AP-2 consensus binding sites, to be responsive to pX. pX does not modify the pattern of the retarded complexes bound to the SRE/FAP region which, in our system, appears to be occupied by SRE-binding factors. The activation of the SRE does not involve complex formation between SRE-binding factors and pX, it is not associated with an increase in serum response factor binding to the SRE and it does not determine changes in SRE mobility-shift pattern.
Assuntos
Genes fos , Vírus da Hepatite B/genética , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Transativadores/metabolismo , Ativação Transcricional , Animais , Sequência de Bases , Núcleo Celular/metabolismo , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Regulação Viral da Expressão Gênica , Células HeLa , Humanos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Plasmídeos , Proteínas Quinases/metabolismo , Mapeamento por Restrição , Transcrição Gênica , Transfecção , Células Vero , Proteínas Virais Reguladoras e AcessóriasRESUMO
Hepatitis C virus (HCV) infection display a very high rate of progression to chronicity and, like many other viruses causing persistent infections, it displays a tropism for the cells of the immune system. Peripheral blood mononuclear cells (PBMCs) from 21 HCV chronic carriers and long-term T cell clones derived from circulating or liver infiltrating T lymphocytes were tested by cDNA "nested" PCR for positive and negative strand HCV-RNA. The presence of HCV genomes in PBMCs is a frequent, although not constant, finding and can be accompanied by active viral replication, as suggested by the coexistence of negative strand HCV-RNA. Infected T cells are more represented in livers than in periphery, as indicated by comparing HCV-RNA detection in T cell clones isolated from both the compartments. Sequencing of viral genomes present in PBMCs and liver infiltrating lymphocytes showed that all the three major HCV genotypes present in our population of chronic carriers can infect lymphoid cells. Although each clonal population of T cells is infected by a single strain of HCV, in the same patient lymphoid cells can harbor different viral populations, different from those circulating at that moment in the serum.
Assuntos
Linfócitos T CD4-Positivos/virologia , Hepacivirus/genética , Hepatite C/virologia , Leucócitos Mononucleares/virologia , RNA Viral/sangue , Adulto , Sequência de Bases , Linfócitos T CD4-Positivos/citologia , Linhagem Celular , Células Clonais , DNA Viral/genética , Feminino , Hepacivirus/classificação , Hepacivirus/isolamento & purificação , Hepacivirus/metabolismo , Hepatite B/sangue , Hepatite B/virologia , Hepatite C/sangue , Humanos , Leucócitos Mononucleares/citologia , Fígado/citologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência MolecularRESUMO
Increasing evidence suggests that angiotensin II may act as a growth factor for several muscle cell types. Angiotensin II stimulation activates many immediate early response genes like c-Fos, c-Jun, c-Myc and Egr-1 in both vascular smooth muscle cells and cardiomyocytes, independently of whether a hyperplastic or hypertrophic response is taking place. In this study we report that angiotensin II significantly stimulates AP1-driven transcription in mouse skeletal muscle cells C2C12 stably transfected with a TRE-tk-CAT plasmid in a dose-dependent manner (peak stimulation at 10(-5) M of angiotensin II). Moreover, angiotensin II increases the binding of the AP1 complex to its DNA target in both quiescent C2C12 myoblasts and in differentiated C2C12 myotubes. Most of the TRE-bound complexes in both unstimulated and angiotensin II-treated cells consist of c-jun/c-fos heterodimers. Using a set of different protein kinase inhibitors, including HA1004, H7, tyrphostin, genistein and staurosporine, we could demonstrate that the angiotensin II-induced AP1 binding increase is not mediated by the cAMP-dependent pathway and that protein kinase C and tyrosine kinases are involved. Treatment of C2C12 cells with H2O2 induces a dose-dependent increase in c-jun/c-fos heterodimer binding, specifically reverted by the cysteine derivative and glutathione precursor N-acetyl-L-cysteine (NAC). The observation that the induction by angiotensin II of both the AP1 DNA binding activity and DNA synthesis in quiescent C2C12 myoblasts is abolished by NAC strongly suggests a role for reactive oxygen intermediates (ROIs) in the intracellular transduction of angiotensin II signals for immediate early gene induction and for cell proliferation.
Assuntos
Angiotensina II/farmacologia , Expressão Gênica/efeitos dos fármacos , Músculo Esquelético/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Transdução de Sinais/fisiologia , Animais , Antioxidantes/farmacologia , Sequência de Bases , Diferenciação Celular , Linhagem Celular , Genes Precoces/efeitos dos fármacos , Coração/efeitos dos fármacos , Camundongos , Dados de Sequência Molecular , Músculo Esquelético/citologia , Músculo Esquelético/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Miocárdio/metabolismo , Oligodesoxirribonucleotídeos , Inibidores de Proteínas Quinases , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-fos/biossíntese , Proteínas Proto-Oncogênicas c-jun/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição AP-1/metabolismo , TransfecçãoRESUMO
We have constructed two expression vectors in order to study the action of the HBV 17 Kd X protein on the c-fos and c-myc promoters. The results show that the promoters contain multiple elements that respond to X protein, suggesting involvement of multiple transcription factors. The exact mechanism of the interaction remains elusive, but our data allow speculation about the factors that may be influenced.
Assuntos
Genes fos/genética , Genes myc/genética , Vírus da Hepatite B/genética , Fatores de Transcrição/genética , Ativação Transcricional , Cloranfenicol O-Acetiltransferase/genética , Análise Mutacional de DNA , Vetores Genéticos/genética , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de FusãoRESUMO
Clinical and experimental evidence suggests the possible existence of one or more extrahepatic sites of HCV infection. In order to demonstrate the "in vivo" infection of lymphoid cells by HCV, we applied a nested PCR to total cytoplasmic RNA extracted from fresh or cultured peripheral blood mononuclear cells (PBMCs) of HCV chronically infected patients, using primers derived from the highly conserved 5' untranslated region of the HCV genome. The presence of virions in PBMCs occurs frequently, if not always, and is often accompanied by active viral replication. Moreover, the appearance of replicative intermediates after stimulation of cellular growth with mitogens suggests that latent genomes could undergo replication upon cellular activation and/or proliferation.
Assuntos
Hepatite C/microbiologia , Hepatite Crônica/microbiologia , Leucócitos/microbiologia , RNA Viral/biossíntese , Adulto , Sequência de Bases , Portador Sadio , Células Cultivadas , DNA Viral , Feminino , Hepacivirus/genética , Hepacivirus/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Viral/análise , Replicação ViralRESUMO
It has previously been shown that the hepatitis B virus (HBV) X gene product, HBx, transactivates homologous and heterologous transcriptional regulatory sequences of viruses, including the human immunodeficiency virus type 1 (HIV1) long terminal repeat (LTR), and various cellular genes in vitro. To evaluate the transactivating function of HBx in vivo, we generated transgenic mice carrying the X open reading frame under the control of the human antithrombin III (ATIII) gene regulatory sequences. These mice express the 16 Kd HBx protein in the liver, as demonstrated by immunoprecipitation studies. Crossbreeding of HBx mice with transgenics carrying either the chloramphenicol acetyl transferase (CAT) bacterial or the lacZ reporter gene driven by the HIV1-LTR allowed us to demonstrate, for the first time, the in vivo transactivating function of HBx protein.
Assuntos
HIV-1/genética , Vírus da Hepatite B/genética , Sequências Repetitivas de Ácido Nucleico/genética , Transativadores/genética , Ativação Transcricional , Animais , Antitrombina III/genética , Cloranfenicol O-Acetiltransferase/genética , Regulação Viral da Expressão Gênica , Humanos , Camundongos , Camundongos Transgênicos , Fases de Leitura Aberta , Proteínas Virais Reguladoras e Acessórias , beta-Galactosidase/genéticaRESUMO
Testing for hepatitis C virus by ELISA requires confirmation by recombinant immunoblot assay (RIBA). The first-generation RIBA uses the same antigen as used in the ELISA and one further antigen. A second-generation RIBA in which two further antigens are present, detects positivity that is not found by either the ELISA or the original RIBA. Consequently, although it is adequate to test ELISA positive sera with the first-generation RIBA, the second-generation assay is recommended for confirming negativity.
Assuntos
Western Blotting/métodos , Anticorpos Anti-Hepatite/isolamento & purificação , Hepatite C/diagnóstico , Hepatite Crônica/imunologia , Kit de Reagentes para Diagnóstico , Ensaio de Imunoadsorção Enzimática , Humanos , Sensibilidade e EspecificidadeRESUMO
In order to investigate the transactivational function of HBV truncated preS/S proteins we have constructed two sets of plasmids and have tested their transactivational potential on the c-myc regulatory sequences and the TPA-responsive element. We found that preS/S proteins only become transactivationally active when truncated at the carboxy terminal end. Furthermore, using immunofluorescence microscopy we determined that the proteins are located exclusively in the cytoplasm, apparently ruling out DNA binding and activation of factors in the nucleus.
Assuntos
Genes myc/genética , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/genética , Sequências Reguladoras de Ácido Nucleico/genética , Ativação Transcricional , Cloranfenicol O-Acetiltransferase/genética , Análise Mutacional de DNA , Genes myc/efeitos dos fármacos , Antígenos de Superfície da Hepatite B/isolamento & purificação , Antígenos de Superfície da Hepatite B/farmacologia , Precursores de Proteínas/isolamento & purificação , Precursores de Proteínas/farmacologia , Proteínas Recombinantes de Fusão/genética , Sequências Reguladoras de Ácido Nucleico/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Many studies have shown that hepatitis B virus infection may also occur in hepatitis B surface antigen-negative patients. This occult infection has been identified both in patients with cryptogenic liver disease and in patients with hepatitis C virus-related chronic hepatitis, and much evidence suggests that it may be a risk factor of hepatocellular carcinoma development. However several aspects of this occult infection remain unclear such as its prevalence and the factor(s) involved in the lack of circulating hepatitis B surface antigen. Moreover, it is uncertain whether the occult hepatitis B virus infection may contribute to chronic liver damage, considering that it is usually associated with a suppressed viral replication. Evidence and hypotheses concerning this fascinating field of bio-medical research are reviewed.