RESUMO
Salicylates from plant sources have been used for centuries by different cultures to treat a variety of ailments such as inflammation, fever and pain. A chemical derivative of salicylic acid, aspirin, was synthesised and mass produced by the end of the 19th century and is one of the most widely used drugs in the world. Its cardioprotective properties are well established; however, recent evidence shows that it can also act as a chemopreventive agent. Its antithrombotic and anti-inflammatory actions occur through the inhibition of cyclooxygenases. The precise mechanisms leading to its anticancer effects are not clearly established, although multiple mechanisms affecting enzyme activity, transcription factors, cellular signalling and mitochondrial functions have been proposed. This review presents a brief account of the major COX-dependent and independent pathways described in connection with aspirin's anticancer effects. Aspirin's unique ability to acetylate biomolecules besides COX has not been thoroughly investigated nor have all the targets of its primary metabolite, salicylic acid been identified. Recent reports on the ability of aspirin to acetylate multiple cellular proteins warrant a comprehensive study to investigate the role of this posttranslational modification in its anticancer effects. In this review, we also raise the intriguing possibility that aspirin may interact and acetylate cellular molecules such as RNA, and metabolites such as CoA, leading to a change in their function. Research in this area will provide a greater understanding of the mechanisms of action of this drug.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Anticarcinógenos/farmacologia , Aspirina/farmacologia , Neoplasias/prevenção & controle , Humanos , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológicoRESUMO
Aspirin is a salicylate drug that is extensively used for its anti-inflammatory, antipyretic, analgesic and anti-thrombotic effects. More recently, it has been shown to decrease the incidence of cancers of epithelial origin. In most cases, aspirin is relatively safe. However, it does cause a host of adverse effects and toxicities, including gastrointestinal bleeding, ulcerations, nephrotoxicity and hypersensitivity reactions. Although the inhibition of cyclooxygenases by aspirin, which leads to its anti-inflammatory/analgesic properties, has been well studied, the mechanisms involved in its chemopreventive effects as well as some of its adverse effects are as yet ill-defined. Studies over the past decades suggest that, besides cyclooxygenases, aspirin acetylates other cellular proteins. These studies used radiolabeled 3H or 14C aspirin, the only approach used to date for the detection of proteins acetylated by aspirin. In a recent study using protein-specific anti-acetyl lysine antibodies and immunological methods, we demonstrated the ability of aspirin to acetylate the tumor suppressor protein p53. In this review, we present current research from the literature on the aspirin-induced acetylation of proteins. We also describe an immunological approach to detecting acetylated proteins in aspirin-treated cells, and demonstrate that multiple proteins are acetylated. Since post-translational modification of proteins, such as acetylation, may lead to the alteration of their function, it is possible that some of the hitherto unexplained beneficial or adverse effects of aspirin could occur as a result of these modifications. The identification of these novel acetylation targets of aspirin represents a new area for investigation.
RESUMO
This study assessed and compared the efficacy of culturally tailored behavioral interventions to increase use and acceptability of sexual barrier products among HIV-positive women in Zambia. It also sought to evaluate cultural preferences as facilitators or impediments to potential use of vaginal chemical barriers for sexual risk reduction within the Zambian context. Women (N=240), recruited from the University Teaching Hospital HIV Voluntary Counseling and Testing Center, were randomized into group or individual intervention arms. Participants attended a baseline assessment, three intervention sessions and follow up assessments at six and 12 months. All participants increased use and acceptability of female condoms and vaginal products and maintained male condom use at six and 12 months. Preliminary data indicated that group participants increased male condom use at six months and trial use and acceptability of female condoms and lubricants predicted their use in the group condition. Results support group interventions to increase sexual barrier use and acceptability in HIV-positive women within the Zambian context. From a public health standpoint, groups may represent a cost-effective and culturally congruent intervention.
Assuntos
Preservativos Femininos/estatística & dados numéricos , Infecções por HIV/prevenção & controle , Educação em Saúde/métodos , Educação Sexual/métodos , Comportamento Sexual/psicologia , Adulto , Preservativos/estatística & dados numéricos , Cultura , Feminino , Conhecimentos, Atitudes e Prática em Saúde , Humanos , Satisfação do Paciente , Comportamento de Redução do Risco , Espermicidas/uso terapêutico , ZâmbiaRESUMO
BACKGROUND: No trials of co-trimoxazole (trimethoprim-sulfamethoxazole) prophylaxis for HIV-infected adults or children have been done in areas with high levels of bacterial resistance to this antibiotic. We aimed to assess the efficacy of daily co-trimoxazole in such an area. METHODS: We did a double-blind randomised placebo-controlled trial in children aged 1-14 years with clinical features of HIV infection in Zambia. Primary outcomes were mortality and adverse events possibly related to treatment. Analysis was by intention to treat. FINDINGS: In October, 2003, the data and safety monitoring committee recommended early stopping of the trial. 541 children had been randomly assigned; seven were subsequently identified as HIV negative and excluded. After median follow-up of 19 months, 74 (28%) children in the co-trimoxazole group and 112 (42%) in the placebo group had died (hazard ratio [HR] 0.57 [95% CI 0.43-0.77], p=0.0002). This benefit applied in children followed up beyond 12 months (n=320, HR 0.48 [0.27-0.84], test for heterogeneity p=0.60) and across all ages (test for heterogeneity p=0.82) and baseline CD4 counts (test for heterogeneity p=0.36). 16 (6%) children in the co-trimoxazole group had grade 3 or 4 adverse events compared with 18 (7%) in the placebo group. These events included rash (one placebo), and a neutrophil count on one occasion less than 0.5x10(9)/L (16 [6%] co-trimoxazole vs seven [3%] placebo, p=0.06). Pneumocystis carinii was identified by immunofluorescence in only one (placebo) of 73 nasopharyngeal aspirates from children with pneumonia. INTERPRETATION: Our results suggest that children of all ages with clinical features of HIV infection should receive co-trimoxazole prophylaxis in resource-poor settings, irrespective of local resistance to this drug.
Assuntos
Infecções Oportunistas Relacionadas com a AIDS/prevenção & controle , Antibacterianos/uso terapêutico , Antibioticoprofilaxia , Combinação Trimetoprima e Sulfametoxazol/uso terapêutico , Antibacterianos/efeitos adversos , Criança , Pré-Escolar , Método Duplo-Cego , Feminino , Infecções por HIV/mortalidade , Hospitalização , Humanos , Lactente , Masculino , Taxa de Sobrevida , Combinação Trimetoprima e Sulfametoxazol/efeitos adversos , ZâmbiaRESUMO
OBJECTIVE AND DESIGN: Bacterial products as well as the host airway inflammatory responses contribute to the pathogenesis of Pseudomonas infections. We sought to determine if Pseudomonas elastase (PE) induces mitogen-activated protein (MAP) kinase activity in association with interleukin-8 (IL-8) production by alveolar epithelial cells. METHODS: We utilized Western blot analysis to detect phosphorylation of signaling intermediates and ELISA was used to measure IL-8 production. RESULTS: We found that PE induces phosphorylation of the extracellular signal-regulated (ERK1/2) proteins of the MAPK pathway in A549 epithelial cells. Similar results were obtained using primary cultures of rabbit alveolar type II epithelial cells. PE also enhanced IL-8 production, which was abolished in the presence of the ERK activation inhibitor U0126. CONCLUSIONS: We conclude that PE activates the ERK1/2 arm of the MAPK pathway and that activation of this pathway results in enhanced IL-8 production. The results demonstrate that PE may augment pulmonary inflammation via cellular signaling that regulates expression of IL-8.
Assuntos
Células Epiteliais/metabolismo , Interleucina-8/biossíntese , Proteínas Quinases Ativadas por Mitógeno/biossíntese , Elastase Pancreática/farmacologia , Pseudomonas aeruginosa/enzimologia , Alvéolos Pulmonares/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Western Blotting , Linhagem Celular , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Humanos , Fosforilação , Proteínas Tirosina Fosfatases/metabolismo , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/efeitos dos fármacos , Coelhos , Estimulação QuímicaRESUMO
We previously demonstrated that exposure of CCL39 lung fibroblasts to alpha-thrombin inhibits interleukin-6 (IL-6)-induced tyrosine phosphorylation of Stat3 (signal transducers and activators of transcription 3) via activation of mitogen-activated protein (MAP) kinase kinase 1 [Bhat et al. (1998) Arch. Biochem. Biophys. 350, 307-314]. In this study, using CCL39/MRC-5 cells, we investigated if additional signaling intermediates are involved in alpha-thrombin's inhibitory effects on IL-6-induced Stat3 signaling. We also determined if alpha-thrombin inhibits oncostatin M (OSM)-induced Stat3/Stat1, and interferon-gamma (IFN-gamma)-induced Stat1 tyrosine phosphorylation. We demonstrate that, although both IL-6 and OSM belong to the same cytokine family, alpha-thrombin inhibited only the IL-6-induced Stat3 tyrosine phosphorylation. The tyrosine phosphatase PTP1D coprecipitated with Stat3 from alpha-thrombin + IL-6, but not from alpha-thrombin + OSM-treated cells. Pretreatment of cells with a phosphatase inhibitor reversed the inhibitory actions of alpha-thrombin, suggesting a role for PTP1D in alpha-thrombin-mediated inhibition of IL-6-induced Stat3 signaling. Interestingly, alpha-thrombin failed to inhibit OSM- and IFN-gamma-induced Stat1 tyrosine phosphorylation. Cytokine-specific inhibition of the Stat3 signaling involving MAP kinase kinase 1 and PTP1D by alpha-thrombin may play an important role in regulation of gene expression.
Assuntos
Proteínas de Ligação a DNA/antagonistas & inibidores , Interleucina-6/antagonistas & inibidores , Proteínas Tirosina Fosfatases/fisiologia , Transdução de Sinais , Trombina/farmacologia , Transativadores/antagonistas & inibidores , Animais , Linhagem Celular , Cricetinae , Proteínas de Ligação a DNA/metabolismo , Antagonismo de Drogas , Inibidores Enzimáticos/farmacologia , Humanos , Interferon gama/farmacologia , Interleucina-6/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Oncostatina M , Peptídeos/farmacologia , Fosforilação , Fosfotirosina/metabolismo , Testes de Precipitina , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Fator de Transcrição STAT1 , Fator de Transcrição STAT3 , Transdução de Sinais/efeitos dos fármacos , Transativadores/metabolismo , Vanadatos/farmacologiaRESUMO
OBJECTIVES: We investigated the ability of several human neutralizing monoclonal antibodies (mAbs), originally raised against human immunodeficiency virus (HIV) clade B isolates, to neutralize primary clade C isolates as single agents and in combination. STUDY DESIGN/METHODS: HIV clade C isolates from five different countries were tested for susceptibility to neutralization by anti-clade B mAbs in human peripheral blood mononuclear cells. Monoclonal antibody combinations were evaluated for possible synergy. RESULTS: All 20 primary HIV clade C isolates could be neutralized 97.5% to 100% by a quadruple combination of mAbs IgG1b12, 2G12, 2F5, and 4E10. These mAbs recognized conserved epitopes and were highly synergistic, resulting in strong cross-clade neutralization. CONCLUSIONS: In our previous experiment, a synergistic combination of human neutralizing mAbs protected all macaque neonates against oral challenge with a simian-human immunodeficiency virus encoding HIV env. Together, our data suggest that passive immunization with currently available anti-clade B mAbs could play a role in preventing HIV clade C transmission through breastfeeding.
Assuntos
Anticorpos Monoclonais/imunologia , Infecções por HIV/virologia , HIV-1/classificação , HIV-1/imunologia , Especificidade de Anticorpos , Reações Antígeno-Anticorpo , Sinergismo Farmacológico , Anticorpos Anti-HIV/imunologia , Humanos , Leucócitos Mononucleares/virologia , Testes de NeutralizaçãoRESUMO
Voluntary testing and counseling (VTC) for HIV/AIDS is now widely accepted as an effective HIV prevention and control strategy among heterosexual couples in sub-Saharan Africa. The most appropriate format and venue for VTC remains a topic of debate among clinicians and public health professionals. Our research done in Lusaka, Zambia, took a tripartite approach to exploring the most acceptable format and venue for VTC: a community survey of attitudes towards VTC, a pre- and postcounseling knowledge survey, and a pilot study of same-day VTC in urban antenatal care clinics. A community survey of 181 individuals was conducted in July-August 1996 based on a structured questionnaire. A pre- and post-VTC intervention knowledge survey was conducted during the same period among 82 couples attending the Zambia-UAB HIV Research Project (ZUHRP) HIV VTC center in Lusaka. Finally, same-day HIV VTC was pilot tested in six antenatal clinic locations during February-May 1997 and June-August 1998. The community survey revealed that 98% of participants support promotion of HIV VTC in the community and 83.8% prefer the same-day testing format. The knowledge survey revealed misconceptions about discordance within a couple and perinatal transmission of HIV. Pilot testing in antenatal clinics was well received, with 84% of pregnant women requesting testing and 25% having positive HIV serologies. Women with primary school or less education, those seeking antenatal care in local clinics, and those seen before the third trimester of pregnancy were more likely to request HIV testing. Testing and counseling for HIV were shown to be feasible and effective in the antenatal clinic setting. Implementation of same-day HIV VTC in antenatal clinics is an effective strategy to prevent vertical transmission and should be expanded to include couples to leverage a decrease in heterosexual transmission as well.
Assuntos
Síndrome da Imunodeficiência Adquirida/prevenção & controle , Infecções por HIV/prevenção & controle , Transmissão Vertical de Doenças Infecciosas/prevenção & controle , Programas de Rastreamento , Complicações Infecciosas na Gravidez/prevenção & controle , Cuidado Pré-Natal , Síndrome da Imunodeficiência Adquirida/diagnóstico , Síndrome da Imunodeficiência Adquirida/transmissão , Atitude Frente a Saúde , Controle de Doenças Transmissíveis/organização & administração , Escolaridade , Feminino , Infecções por HIV/diagnóstico , Infecções por HIV/transmissão , Conhecimentos, Atitudes e Prática em Saúde , Promoção da Saúde , Humanos , Recém-Nascido , Masculino , Projetos Piloto , Gravidez , População Urbana , ZâmbiaRESUMO
We previously demonstrated that exposure of CCL39 lung fibroblast cells to alpha-thrombin inhibits interleukin-6 (IL-6)-induced tyrosine phosphorylation of Stat3 (signal transducers and activators of transcription-3) protein via a mitogen-activated protein (MAP)-kinase dependent mechanism. In the present study, we investigated the mechanism of regulation of IL-6-induced signaling by transforming growth factor-beta (TGF-beta) and compared this to alpha-thrombin-mediated inhibition. We demonstrate that exposure of CCL39 cells to TGF-beta completely inhibits IL-6-induced Stat3 tyrosine phosphorylation and gp130 gene expression. However, in contrast to alpha-thrombin, TGF-beta-mediated inhibition did not require activation of the MAP kinase pathway. Also, unlike alpha-thrombin, TGF-beta-mediated inhibition requires synthesis of new proteins. Interestingly, TGF-beta and alpha-thrombin both inhibit IL-6-induced expression of gp130 mRNA levels. These results demonstrate that although the end effects are the same, alpha-thrombin and TGF-beta utilize distinct mechanisms to inhibit IL-6-induced Stat3 signaling.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Interleucina-6/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Trombina/farmacologia , Transativadores/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Animais , Northern Blotting , Linhagem Celular , Cricetinae , Cicloeximida/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Immunoblotting , Interleucina-6/metabolismo , Pulmão/citologia , Fosforilação/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Fator de Transcrição STAT3 , Transdução de Sinais/efeitos dos fármacos , Trombina/metabolismoRESUMO
The efficacy and safety of intramuscular artemotil (ARTECEF) was compared to intravenous quinine in African children with cerebral malaria. This prospective block randomized open-label study was conducted at two centers in Zambia. Subjects were children aged 0 to 10 years of age with cerebral malaria and a Blantyre Coma Score of 2 or less. Ninety two children were studied; 48 received artemotil and 44 quinine. No significant differences in survival, coma resolution time, neurologic sequelae, parasite clearance time, and fever resolution time were seen between the two regimens. Rates for negative malaria smears one month after therapy were similar in both groups. Artemotil was a well-tolerated drug in the 48 patients in this study. It appears to be at least therapeutically equivalent to quinine for the treatment of pediatric cerebral malaria. It has the advantage of being able to be given intramuscularly once daily for only five days.
Assuntos
Antimaláricos/uso terapêutico , Artemisininas , Malária Cerebral/tratamento farmacológico , Sesquiterpenos/uso terapêutico , Antimaláricos/administração & dosagem , Antimaláricos/efeitos adversos , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Injeções Intramusculares , Malária Cerebral/mortalidade , Masculino , Estudos Prospectivos , Quinina/efeitos adversos , Quinina/uso terapêutico , Sesquiterpenos/administração & dosagem , Sesquiterpenos/efeitos adversos , Taxa de Sobrevida , Zâmbia/epidemiologiaRESUMO
The Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway is stimulated by angiotensin II (Ang II) via the type 1 receptor after acute pressure overload in the heart. The purpose of this study was to determine whether activation of the JAK-STAT pathway by Ang II is dependent on G proteins. Ang II (100 nmol/L for 120 minutes) caused formation of sis-inducing factor (SIF) complexes and tyrosine phosphorylation of STAT proteins in neonatal rat ventricular myocytes. The percentage of change in Ang II-stimulated SIF induction was not affected by pertussis toxin (PTX) or GP antagonist-2A, compounds that inhibit activation of G(i) and G(o) proteins. In contrast, GP antagonist-2A, a peptide that selectively inhibits activation of G(q) proteins, completely abolished Ang II-stimulated SIF induction and STAT3 tyrosine phosphorylation. Pretreatment of cardiac myocytes with U73122, an inhibitor of phosphatidylinositol-specific phospholipase C (PLC) activity, decreased Ang II-stimulated SIF induction and STAT3 tyrosine phosphorylation in a dose-dependent manner. Chelation of intracellular Ca(2+) with BAPTA-AM did not alter Ang II-stimulated SIF induction. In contrast, pretreatment of cardiac myocytes with Ro-31-8220, a potent and specific inhibitor of protein kinase C (PKC), decreased Ang II-stimulated SIF induction in a dose-dependent manner. Ang II-stimulated SIF induction was abolished in cardiac myocytes after downregulation of PKC by treatment with PMA. From these data, we conclude that Ang II-stimulated SIF induction and STAT3 tyrosine phosphorylation is mediated by PTX-insensitive G proteins through a G(q)-PLC-PKC-mediated pathway in neonatal rat ventricular myocytes.
Assuntos
Angiotensina II/farmacologia , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação ao GTP/fisiologia , Miocárdio/metabolismo , Toxina Pertussis , Transdução de Sinais/efeitos dos fármacos , Fatores de Virulência de Bordetella/farmacologia , Animais , Animais Recém-Nascidos , Proteínas de Ligação a DNA/metabolismo , Miocárdio/citologia , Fosfatidilinositóis/farmacologia , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Transativadores/efeitos dos fármacos , Transativadores/metabolismo , Ativação Transcricional/efeitos dos fármacosRESUMO
Studies from our laboratory have shown that exposure of human lung epithelial cells to urokinase plasminogen activator (uPA) induces their proliferation. This effect of uPA is likely to occur via activation of signal transduction pathways. To elucidate uPA-induced signal transduction mechanisms, we exposed H-157 cells to uPA and determined the induced tyrosine phosphorylation profile of proteins. We demonstrate that, in these cells, uPA prominently induced tyrosine phosphorylation of a 78-kDa protein. This effect was observed as early as 30 min and was sustained for at least 24 h. Treatment of cells with agents that abrogate uPA receptor (uPAR) function, including neutralizing anti-uPAR antibody, phosphatidylinositol-specific phospholipase C, or a selective antagonist that blocks the association of uPA with uPAR (A5 compound), all failed to prevent uPA-induced tyrosine phosphorylation. B-428, an active site inhibitor of uPA activity, prevented the uPA effect. Treatment of cells with hepatocyte growth factor, vascular endothelial growth factor, or transforming growth factor-beta, all of which are known to be activated by a uPA-dependent pathway, did not stimulate tyrosine phosphorylation of the 78-kDa protein. uPA induced an increase in [(3)H]thymidine incorporation into DNA, and cell numbers were unaffected in the presence of A5. These results demonstrate that, in H-157 cells, uPA induces tyrosine phosphorylation of a 78-kDa protein via a proteolysis-dependent but uPAR-independent mechanism. This novel signaling pathway represents a putative mechanism by which uPA could influence epithelial cell proliferation.
Assuntos
Proteínas de Neoplasias/metabolismo , Ativadores de Plasminogênio/farmacologia , Tirosina/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/farmacologia , Divisão Celular/fisiologia , Enzimas/metabolismo , Substâncias de Crescimento/fisiologia , Humanos , Membranas Intracelulares/metabolismo , Peso Molecular , Proteínas de Neoplasias/química , Fosforilação , Receptores de Superfície Celular/fisiologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Transdução de Sinais/fisiologia , Células Tumorais CultivadasRESUMO
To study human herpesvirus 8 (HHV-8) transmission between individuals and in populations, we developed a system for genetic fingerprinting of HHV-8 strains based on variation in the HHV-8 K1, glycoprotein B (gB), and glycoprotein H (gH) genes. Using this system, we sequenced nearly the entire K1 gene (840 bp); two segments of the gB gene (open reading frame 8), totaling 813 bp; and a 702-bp segment of the gH gene (open reading frame 22) from blood and tissue samples obtained from 40 human immunodeficiency virus-infected and noninfected individuals, including those with Kaposi's sarcoma, primary effusion lymphoma, or Castleman's disease. The specimen collection was assembled from individuals living in diverse geographical locations, including the United States, Australia, New Zealand, Uganda, and Zambia. As reported by others, K1 was the most variable gene, with up to 16% variation at the nucleotide sequence level and up to 32% variation at the amino acid sequence level. Despite this extensive sequence variation, the K1 amino acid sequence contained 14 conserved cysteine sites, suggesting a conserved tertiary structure. gB and gH sequences were highly conserved, in most cases differing by <0.6% in pairwise comparisons. K1 was the most useful gene for strain discrimination, but the other genes enabled the discrimination of strains with identical K1 sequences. Individuals from diverse geographic locations were infected with four different HHV-8 genotypes; strains did not strictly segregate by continent of origin. The majority of HHV-8 strains from the United States and Europe were relatively closely related, whereas some strains identified from Uganda and Australia were phylogenetically distant. Genotype I strains were the most common and were found on three continents. Identical sequences were found in specimens obtained from different body sites and at different times from the same individual.
Assuntos
Infecções Oportunistas Relacionadas com a AIDS/virologia , Infecções por Herpesviridae/virologia , Herpesvirus Humano 8/classificação , Linfoma Relacionado a AIDS/virologia , Sarcoma de Kaposi/virologia , África , Sequência de Aminoácidos , Ásia , Austrália , Impressões Digitais de DNA , DNA Viral/química , DNA Viral/genética , Genótipo , Infecções por HIV/virologia , Herpesvirus Humano 8/genética , Humanos , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNA , Estados Unidos , Proteínas do Envelope Viral/genética , Proteínas Virais/genéticaRESUMO
Exposure of primary human lung fibroblasts (HLF) to interleukin-6 (IL-6) rapidly induced Stat3 (signal transducers and activators of transcription 3) tyrosine phosphorylation. In these cells, alpha-thrombin did not induce tyrosine phosphorylation of Stat3; however, it potently induced its serine phosphorylation. Interestingly, a short pretreatment of cells with alpha-thrombin significantly inhibited IL-6-induced tyrosine phosphorylation of Stat3. The inhibition by alpha-thrombin was attenuated if cells were pretreated with U0126, a specific inhibitor of the mitogen-activated protein (MAP) kinase kinase 1 (MAPKK1). Exposure of HLF cells to IL-6 induced a twofold increase in gp130 mRNA levels; however, alpha-thrombin inhibited this IL-6-induced response almost to control levels. These results demonstrate, for the first time, that in HLF cells alpha-thrombin inhibits IL-6-induced Stat3 signaling via activation of MAPKK1 and that this cross-talk regulates IL-6-induced gp130 gene expression.
Assuntos
Antígenos CD/genética , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica/efeitos dos fármacos , Interleucina-6/farmacologia , Glicoproteínas de Membrana/genética , Quinases de Proteína Quinase Ativadas por Mitógeno , Transdução de Sinais/efeitos dos fármacos , Trombina/farmacologia , Transativadores/metabolismo , Western Blotting , Butadienos/farmacologia , Células Cultivadas , Receptor gp130 de Citocina , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Interleucina-6/antagonistas & inibidores , Pulmão/citologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , MAP Quinase Quinase 1 , Nitrilas/farmacologia , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Fosfotirosina/metabolismo , Testes de Precipitina , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , RNA Mensageiro/metabolismo , Fator de Transcrição STAT3 , Trombina/antagonistas & inibidoresAssuntos
Hipotermia/terapia , Incubadoras , Reaquecimento/métodos , Pele , Temperatura Corporal , Feminino , Humanos , Recém-Nascido , Masculino , MãesRESUMO
To examine the effect of iron chelation on mortality in cerebral malaria, we enrolled 352 children in a trial of deferoxamine in addition to standard quinine therapy at 2 centres in Zambia, one rural and one urban. Entrance criteria included age < 6 years, Plasmodium falciparum parasitaemia, normal cerebral spinal fluid, and unrousable coma. Deferoxamine (100 mg/kg/d infused for a total of 72 h) or placebo was added to a 7 d regimen of quinine that included a loading dose. Mortality overall was 18.3% (32/175) in the deferoxamine group and 10.7% (19/177) in the placebo group (adjusted odds ratio 1.8; 95% confidence interval 0.9-3.6; P = 0.074). At the rural study site, mortality was 15.4% (18/117) with deferoxamine compared to 12.7% (15/118) with placebo (P = 0.78, adjusted for covariates). At the urban site, mortality was 24.1% (14/58) with deferoxamine and 6.8% (4/59) with placebo (P = 0.061, adjusted for covariates). Among survivors, there was a non-significant trend to faster recovery from coma in the deferoxamine group (adjusted odds ratio 1.2; 95% confidence interval 0.97-1.6; P = 0.089). Hepatomegaly was significantly associated with higher mortality, while splenomegaly was associated with lower mortality. This study did not provide evidence for a beneficial effect on mortality in children with cerebral malaria when deferoxamine was added to quinine, given in a regimen that included a loading dose.
Assuntos
Antídotos/uso terapêutico , Antimaláricos/uso terapêutico , Desferroxamina/uso terapêutico , Quelantes de Ferro/uso terapêutico , Malária Cerebral/tratamento farmacológico , Malária Cerebral/mortalidade , Parasitemia/tratamento farmacológico , Parasitemia/mortalidade , Quinina/uso terapêutico , Criança , Pré-Escolar , Coma/tratamento farmacológico , Método Duplo-Cego , Quimioterapia Combinada , Feminino , Febre/tratamento farmacológico , Humanos , Lactente , Masculino , Estudos Prospectivos , Taxa de Sobrevida , Resultado do Tratamento , Zâmbia/epidemiologiaRESUMO
We recently demonstrated that, in rat aortic smooth muscle cells, alpha-thrombin stimulated Stat3/SIF-A (signal transducers and activators of transcription 3/sis-inducing factor-A) activity [G. J. Bhat et al. (1997) Hypertension 29(Pt. 2), 356-360]. In the present study, we observed that exposure of CCL39 cells (a Chinese hamster lung fibroblast cell line) to alpha-thrombin resulted in a time-dependent decrease in basal SIF-A activity. We hypothesized that the decrease in basal SIF-A was due to the initiation of an inhibitory pathway, following alpha-thrombin exposure. To test this hypothesis, we determined if alpha-thrombin would inhibit Stat3 and SIF-A activation by interleukin-6 (IL-6), leukemia inhibitory factor (LIF), and ciliary neurotrophic factor (CNTF). In support of this hypothesis, alpha-thrombin inhibited the Stat3/SIF-A response induced by all the above cytokines. The inhibition by alpha-thrombin was concentration dependent, was sensitive to hirudin, and was mimicked by the thrombin receptor agonist peptide. The inhibition did not require the activation of phorbol 12-myristate 13-acetate-sensitive isoforms of protein kinase C and was reversed by pretreatment with the mitogen-activated protein kinase kinase 1 (MAPKK1 or MEK1) inhibitor PD98059. Inhibitory cross talk between alpha-thrombin and IL-6 was also observed in MRC-5 cells, a fibroblast cell line derived from human lung tissue. Thus, we identify a novel alpha-thrombin inhibitory pathway which, acting through a MAPKK1-dependent mechanism, blocks IL-6-, LIF-, and CNTF-induced Stat3/SIF-A activation. This inhibitory cross talk may provide an important regulatory function to modulate gene transcription by these cytokines, during immune and inflammatory responses.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Inibidores do Crescimento/farmacologia , Interleucina-6/farmacologia , Linfocinas/farmacologia , Quinases de Proteína Quinase Ativadas por Mitógeno , Proteínas do Tecido Nervoso/farmacologia , Transdução de Sinais/efeitos dos fármacos , Trombina/farmacologia , Transativadores/metabolismo , Animais , Linhagem Celular , Fator Neurotrófico Ciliar , Cricetinae , Cricetulus , Citocinas/farmacologia , Ativação Enzimática , Flavonoides/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Fator Inibidor de Leucemia , MAP Quinase Quinase 1 , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Fator de Transcrição STAT3 , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
In cultured neonatal rat cardiac fibroblasts and CHO-K1 cells expressing angiotensin II (Ang II) type 1 receptors (AT1) (T3CHO/AT1A cell line), Ang II induced a delayed tyrosine phosphorylation of Stat3 (Signal Transducers and Activators of Transcription) with maximal activation at 2 h. This was in contrast to the rapid tyrosine phosphorylation (15-30 min) of Stat3 by the cytokine interleukin-6 (IL-6). Using T3CHO/AT1A cells, we tested the hypothesis that the delayed tyrosine phosphorylation of Stat3 by Ang II resulted from the induction of an inhibitory pathway (0-30 min) prior to activation (1-2 h). In support of this hypothesis, we observed that a short treatment of cells with Ang II transiently inhibited the IL-6-induced Stat3 tyrosine phosphorylation. The inhibitory effect of Ang II could be attenuated by exposing the cells to a specific inhibitor of MAP kinase kinase 1, PD98059. Such modulatory cross-talk between Ang II and IL-6 may have relevance in pathophysiological conditions such as cardiac hypertrophy, and in acute phase and inflammatory responses.
Assuntos
Angiotensina II/farmacologia , Proteínas de Ligação a DNA/fisiologia , Interleucina-6/farmacologia , Quinases de Proteína Quinase Ativadas por Mitógeno , Transdução de Sinais , Transativadores/fisiologia , Animais , Humanos , MAP Quinase Quinase 1 , Fosforilação , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Tirosina Quinases/fisiologia , Ratos , Fator de Transcrição STAT3 , Tirosina/metabolismoRESUMO
In rat neonatal cardiac fibroblasts and CHO-K1 cells expressing angiotensin type 1 receptors, angiotensin II (AII) rapidly caused a time dependent reduction in the SDS-polyacrylamide gel electrophoretic mobility of Stat3 (Signal Transducer and Activator of Transcription). This was concentration dependent and detected at a low/physiological concentration of AII (1 nM), with initial effect observed as early as 2 min; and maximal at 5 min. The rapid stimulation of Stat3 mobility retardation by AII, paralleled the rapid activation of MAP kinases (mitogen-activated protein kinases), and both were sensitive to the MAP kinase kinase 1 inhibitor, PD98059. Immunoprecipitation of Stat3 from [32P] labeled cells demonstrated a 4-fold increase in Stat3 phosphorylation in response to AII, and phosphoamino acid analysis indicated that phosphorylation occurred on serine residues. Angiotensin II-induced rapid phosphorylation of Stat3 was also sensitive to the MAP kinase kinase 1 inhibitor, PD98059. Treatment of immunoprecipitated Stat3 from AII-treated cells with protein phosphatase- PP-2A, reversed the AII-induced retardation of Stat3 mobility. These results demonstrate that AII rapidly induces Stat3 serine phosphorylation through a MAP kinase kinase 1 dependent pathway. Rapid stimulation of Stat3 serine phosphorylation by AII may have implications in the modulation of its transcriptional activity and gene expression.
Assuntos
Angiotensina II/farmacologia , Proteínas de Ligação a DNA/metabolismo , Miocárdio/metabolismo , Receptores de Angiotensina/fisiologia , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Angiotensina I , Animais , Animais Recém-Nascidos , Células CHO , Cricetinae , Proteínas de Ligação a DNA/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Fibroblastos/metabolismo , Flavonoides/farmacologia , Cinética , Quinases de Proteína Quinase Ativadas por Mitógeno , Fosfoproteínas Fosfatases , Fosforilação , Inibidores de Proteínas Quinases , Ratos , Receptores de Angiotensina/biossíntese , Proteínas Recombinantes/metabolismo , Fator de Transcrição STAT3 , Serina , Transativadores/isolamento & purificação , TransfecçãoRESUMO
Exposure of rat aortic vascular smooth muscle cells to alpha-thrombin resulted in the appearance of sis-inducing factor-A (SIF-A)-like DNA binding activity. This response to alpha-thrombin was delayed (detectable at 1 hour) compared with the rapid activation (15 to 30 minutes) by platelet-derived growth factor and the cytokine interleukin-6. alpha-Thrombin-induced SIF-A was sensitive to treatment with the tyrosine kinase inhibitor genistein. The thrombin inhibitor hirudin prevented the alpha-thrombin-mediated SIF-A induction. Cycloheximide had no effect on the ability of alpha-thrombin to induce SIF-A, suggesting that induction does not require new protein synthesis. alpha-Thrombin-induced SIF-A could be resolved into two additional subcomplexes termed SIF-A, and SIF-As. Antibodies against Stat3 reacted with alpha-thrombin-induced SIF-Af, suggesting that Stat3 or a related protein is present in this subcomplex. Induction of SIF-A DNA binding activity may contribute to alpha-thrombin-mediated cellular responses, including wound healing, cell proliferation, and inflammation in the vasculature.