RESUMO
Dysregulation of liver sinusoidal endothelial cell (LSEC) differentiation and function has been reported in alcohol-associated liver disease (ALD). Impaired nitric oxide (NO) production stimulates LSEC capillarization and dysfunction; however, the mechanism underlying NO production remains unclear. Here, we investigated the role of thioredoxin-interacting protein (TXNIP), an important regulator of redox homeostasis, in endothelial cell NO production and its subsequent effects on ALD progression. We found that hepatic TXNIP expression was upregulated in patients with ALD and in ethanol diet-fed mice with high expression in LSECs. Endothelial cell-specific Txnip deficiency (TxnipΔEC) in mice exacerbated alcohol-induced liver injury, inflammation, fibrosis, and hepatocellular carcinoma development. Deletion of Txnip in LSECs led to sinusoidal capillarization, downregulation of NO production, and increased release of proinflammatory cytokines and adhesion molecules, whereas TXNIP overexpression had the opposite effects. Mechanistically, TXNIP interacted with transforming growth factor ß-activated kinase 1 (TAK1) and subsequently suppressed the TAK1 pathway. Inhibition of TAK1 activation restored NO production and decreased the levels of proinflammatory cytokines, thereby, blocking liver injury and inflammation in TxnipΔEC mice. Our findings indicate that upregulated TXNIP expression in LSECs serves a protective role in ameliorating ALD. Enhancing TXNIP expression could, therefore, be a potential therapeutic approach for ALD.
Assuntos
Hepatopatias Alcoólicas , Óxido Nítrico , Animais , Humanos , Camundongos , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Citocinas/metabolismo , Células Endoteliais/metabolismo , Inflamação/metabolismo , Fígado/metabolismo , Cirrose Hepática/metabolismo , Hepatopatias Alcoólicas/genética , Hepatopatias Alcoólicas/metabolismo , Óxido Nítrico/metabolismoRESUMO
A male Korean raccoon dog of unknown age was rescued and placed at the Daejeon Wildlife Rescue Center, Korea. Physical examination revealed severe emaciation and dehydration, as well as thick crusts and alopecia over most of the body. During medical care, the animal died and was submitted for postmortem examination. Firm, brown-red lesions of various sizes were observed on the surface of the lungs. In cross-sections of the lungs, pulmonary vessels were thickened and dilated, with white irregular papillary luminal projections. Histologically, pulmonary blood vessels were severely hyperplastic, characterized by thickened dilated walls and fibrous papillary projections covered with a single layer of endothelial cells (ECs). Hyperplastic fibrous connective tissue was confirmed by Masson trichrome staining. The ECs expressed CD31. We diagnosed the lesion as intravascular papillary endothelial hyperplasia, a unique non-neoplastic reactive process that has not been reported previously in pulmonary vessels of canids, equids, or felids, to our knowledge.
Assuntos
Canidae , Células Endoteliais , Masculino , Animais , Hiperplasia/veterinária , Cães Guaxinins , Diagnóstico Diferencial , Pulmão , República da CoreiaRESUMO
BACKGROUND: Chronic obstructive pulmonary disease (COPD) refers to a lung disorder associated with symptoms of dyspnea, cough, and sputum production. Traditionally, Yijin-tang (YJT), a mixture of Pinellia ternate, Poria cocos, ginger, Chinese liquorice, and tangerine peel, has been prescribed for the treatment of respiratory system diseases caused by dampness phlegm. This experiment investigated the therapeutic effect of YJT in a mouse model of cigarette smoke (CS)- and lipopolysaccharide (LPS)-induced COPD. METHODS: COPD was induced by exposing mice to CS for 1 hour per day for 8 weeks, with intranasal delivery of LPS on weeks 1, 3, 5, and 7. YJT was administered at doses of 100 and 200 mg/kg 1 hour before CS exposure for the last 4 weeks. RESULTS: YJT significantly suppressed CS- and LPS-induced increases in inflammatory cell counts and reduced interleukin-1 beta (IL-1ß), IL-6, tumor necrosis factor-alpha (TNF-α), and monocyte chemoattractant protein-1 (MCP-1) levels in bronchoalveolar lavage fluid (BALF) and lung tissue. In addition, YJT not only decreased airway wall thickness, average alveolar intercept, and lung fibrosis, but it also suppressed the expression of matrix metallopeptidase (MMP)-7, MMP-9, and transforming growth factor-B (TGF-ß) and collagen deposition. Moreover, YJT suppressed phosphorylation of nuclear factor-kappa B (NF-κB) as well as expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). CONCLUSION: Collectively, our findings show that YJT attenuates respiratory inflammation and airway remodeling caused by CS and LPS exposure; therefore, therapeutic applications in COPD can be considered.
RESUMO
BACKGROUND: Palmijihwanghwan (PJH) is a traditional medicine and eight constituents derived from PJH possess anti-inflammatory activities. However, the scientific evidence for its potential as a therapeutic agent for inflammatory lung disease has not yet been studied. In this study, we examined the protective effect of PJH in a mouse model of chronic obstructive pulmonary disease (COPD) induced by cigarette smoke (CS) with lipopolysaccharide (LPS). METHODS: Mice received CS exposure for 8 weeks and intranasal instillation of LPS on weeks 1, 3, 5 and 7. PJH (100 and 200 mg/kg) was administrated daily 1 h before CS treatment for the last 4 weeks. RESULTS: Compared with CS plus LPS-exposed mice, mice in the PJH-treated group showed significantly decreased inflammatory cells count and reduced inflammatory cytokines including interleukin-1 beta (IL-1ß), IL-6 and tumor necrosis factor alpha (TNF-α) levels in broncho-alveolar lavage fluid (BALF) and lung tissue. PJH also suppressed the phosphorylation of nuclear factor kappa B (NF-κB) and extracellular signal-regulated kinase1/2 (ERK1/2) caused by CS plus LPS exposure. Furthermore, CS plus LPS induced increases in matrix metallopeptidase (MMP)-7, MMP-9, and transforming growth factor-ß (TGF-ß) expression and collagen deposition that were inhibited in PJH-treated mice. CONCLUSIONS: This study demonstrates that PJH prevents respiratory inflammation and airway remodeling caused by CS with LPS exposure suggesting potential therapy for the treatment of COPD.
Assuntos
Anti-Inflamatórios/farmacologia , Medicina Tradicional Chinesa/métodos , Extratos Vegetais/farmacologia , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Animais , Modelos Animais de Doenças , Lipopolissacarídeos/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Doença Pulmonar Obstrutiva Crônica/etiologia , Poluição por Fumaça de Tabaco/efeitos adversosRESUMO
The outbreak of coronavirus (CoV) disease 2019 (COVID-19) caused by the severe acute respiratory syndrome CoV-2 (SARS-CoV-2) has turned into a pandemic. The enzyme 3C-like protease (3CLpro) is essential for the maturation of viral polyproteins in SARS-CoV-2 and is therefore regarded as a key drug target for treating the disease. To identify 3CLpro inhibitors that can suppress SARS-CoV-2 replication, we performed a virtual screening of 500,282 compounds in a Korean compound bank. We then subjected the top computational hits to inhibitory assays against 3CLpro in vitro, leading to the identification of a class of non-covalent inhibitors. Among these inhibitors, compound 7 showed an EC50 of 39.89 µM against SARS-CoV-2 and CC50 of 453.5 µM. This study provides candidates for the optimization of potent 3CLpro inhibitors showing antiviral effects against SARS-CoV-2.
Assuntos
Antivirais/farmacologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Inibidores de Proteases/farmacologia , SARS-CoV-2/enzimologia , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Antivirais/metabolismo , Chlorocebus aethiops , Proteases 3C de Coronavírus/metabolismo , Avaliação Pré-Clínica de Medicamentos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Inibidores de Proteases/metabolismo , Ligação Proteica , República da Coreia , Bibliotecas de Moléculas Pequenas/metabolismo , Células VeroRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19) pandemic. The virus still spreads globally through human-to-human transmission. Nevertheless, there are no specific treatments clinically approved. This study aimed to compare antiviral activity of gemcitabine and its analogue 2'-fluoro-2'-deoxycytidine (2FdC) against SARS-CoV-2 as well as cytotoxicity in vitro. Fluorescent image-based antiviral assays revealed that gemcitabine was highly potent, with a 50% effective concentration (EC50) of 1.2 µM, more active than the well-known nucleoside monophosphate remdesivir (EC50 = 35.4 µM). In contrast, 2FdC was marginally active (EC50 = 175.2 µM). For all three compounds, the 50% cytotoxic concentration (CC50) values were over 300 µM toward Vero CCL-81 cells. Western blot and quantitative reverse-transcription polymerase chain reaction analyses verified that gemcitabine blocked viral protein expression in virus-infected cells, not only Vero CCL-81 cells but also Calu-3 human lung epithelial cells in a dose-dependent manner. It was found that gemcitabine has a synergistic effect when combined with remdesivir. This report suggests that the difluoro group of gemcitabine is critical for the antiviral activity and that its combination with other evaluated antiviral drugs, such as remdesivir, could be a desirable option to treat SARS-CoV-2 infection.
Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Tratamento Farmacológico da COVID-19 , Desoxicitidina/análogos & derivados , SARS-CoV-2/efeitos dos fármacos , Monofosfato de Adenosina/farmacologia , Alanina/farmacologia , Animais , Antivirais/farmacologia , COVID-19/metabolismo , COVID-19/virologia , Linhagem Celular , Chlorocebus aethiops , Desoxicitidina/farmacologia , Quimioterapia Combinada , Humanos , Concentração Inibidora 50 , SARS-CoV-2/fisiologia , Células Vero , Replicação Viral/efeitos dos fármacos , GencitabinaRESUMO
Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), is considered as the most significant global public health crisis of the century. Several drug candidates have been suggested as potential therapeutic options for COVID-19, including remdesivir, currently the only authorized drug for use under an Emergency Use Authorization. However, there is only limited information regarding the safety profiles of the proposed drugs, in particular drug-induced cardiotoxicity. Here, we evaluated the antiviral activity and cardiotoxicity of remdesivir using cardiomyocytes-derived from human pluripotent stem cells (hPSC-CMs) as an alternative source of human primary cardiomyocytes (CMs). In this study, remdesivir exhibited up to 60-fold higher antiviral activity in hPSC-CMs compared to Vero E6 cells; however, it also induced moderate cardiotoxicity in these cells. To gain further insight into the drug-induced arrhythmogenic risk, we assessed QT interval prolongation and automaticity of remdesivir-treated hPSC-CMs using a multielectrode array (MEA). As a result, the data indicated a potential risk of QT prolongation when remdesivir is used at concentrations higher than the estimated peak plasma concentration. Therefore, we conclude that close monitoring of the electrocardiographic/QT interval should be advised in SARS-CoV-2-infected patients under remdesivir medication, in particular individuals with pre-existing heart conditions.
Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Antivirais/farmacologia , COVID-19/virologia , Miócitos Cardíacos/virologia , Células-Tronco Pluripotentes/citologia , SARS-CoV-2/efeitos dos fármacos , Monofosfato de Adenosina/farmacologia , Alanina/farmacologia , Amidas/farmacologia , Animais , Antimaláricos/farmacologia , COVID-19/complicações , Chlorocebus aethiops , Cloroquina/farmacologia , Eletrocardiografia , Citometria de Fluxo , Cardiopatias/complicações , Humanos , Hidroxicloroquina/farmacologia , Microscopia de Fluorescência , Miócitos Cardíacos/efeitos dos fármacos , Células-Tronco Pluripotentes/virologia , Pirazinas/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Vero , Ensaio de Placa Viral , Tratamento Farmacológico da COVID-19RESUMO
Dose-dependent lipid accumulation was induced by glucose in HepG2 cells. GlcN also exerted a promotory effect on lipid accumulation in HepG2 cells under normal glucose conditions (NG, 5 mM) and liver of normal fed zebrafish larvae. High glucose (HG, 25 mM)-induced lipid accumulation was suppressed by l-glutamine-d-fructose 6-phosphate amidotransferase inhibitors. ER stress inhibitors did not suppress HG or GlcN-mediated lipid accumulation. HG and GlcN stimulated protein expression, DNA binding and O-GlcNAcylation of carbohydrate-responsive element-binding protein (ChREBP). Furthermore, both HG and GlcN increased nuclear sterol regulatory element-binding protein-1 (SREBP-1) levels in HepG2 cells. In contrast to its stimulatory effect under NG, GlcN suppressed lipid accumulation in HepG2 cells under HG conditions. Similarly, GlcN suppressed lipid accumulation in livers of overfed zebrafish. In addition, GlcN activity on DNA binding and O-GlcNAcylation of ChREBP was stimulatory under NG and inhibitory under HG conditions. Moreover, GlcN enhanced ChREBP, SREBP-1c, ACC, FAS, L-PK and SCD-1 mRNA expression under NG but inhibited HG-induced upregulation in HepG2 cells. The O-GlcNAc transferase inhibitor, alloxan, reduced lipid accumulation by HG or GlcN while the O-GlcNAcase inhibitor, PUGNAc, enhanced lipid accumulation in HepG2 cells and liver of zebrafish larvae. GlcN-induced lipid accumulation was inhibited by the AMPK activator, AICAR. Phosphorylation of AMPK (p-AMPK) was suppressed by GlcN under NG while increased by GlcN under HG. PUGNAc downregulated p-AMPK while alloxan restored GlcN- or HG-induced p-AMPK inhibition. Our results collectively suggest that GlcN regulates lipogenesis by sensing the glucose or energy states of normal and excess fuel through AMPK modulation.
Assuntos
Glucosamina/metabolismo , Lipogênese/genética , N-Acetilglucosaminiltransferases/genética , Proteínas Quinases/genética , Proteínas de Peixe-Zebra/genética , Quinases Proteína-Quinases Ativadas por AMP , Acetilglucosamina/análogos & derivados , Acetilglucosamina/farmacologia , Aloxano/farmacologia , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Glucosamina/genética , Glucose/genética , Glucose/metabolismo , Células Hep G2 , Humanos , Lipídeos/genética , Fígado/metabolismo , N-Acetilglucosaminiltransferases/antagonistas & inibidores , Oximas/farmacologia , Fenilcarbamatos/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas Quinases/efeitos dos fármacos , Ribonucleotídeos/farmacologia , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/antagonistas & inibidoresRESUMO
The success rate of assisted reproductive technology is closely correlated with maternal age. Reproductive aging pathologies are frequently caused by impaired DNA repair, genomic instability, and mitochondrial dysfunction. Several reports have shown that resveratrol can prevent age-related diseases by improving mitochondrial function. Improved blastocyst development and mitochondrial output by dichloroacetic acid (DCA) supplementation were reported in aged mice. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has significant effects on implantation rates in women with previous miscarriages. Therefore, this study was conducted to observe how those compounds influence the developmental and the reproductive potential of aged oocytes. BDF1 female mice at 58-62 weeks old were used for this study. MII oocytes were fertilized and cultured in MRC media supplemented with or without resveratrol (0.5 µM), GM-CSF (2 ng/ml) or DCA (1.0 mM). The addition of resveratrol, GM-CSF or DCA tended to increase blastocyst development and pregnancy rates. Supplementation with resveratrol significantly increased the pregnancy and implantation rates (p < 0.05). Moreover, resveratrol decreased reactive oxygen species production and increased mitochondrial membrane potential. These results suggest that the addition of resveratrol can increase pregnancy outcomes in women of advanced maternal age.
Assuntos
Ácido Dicloroacético/farmacologia , Técnicas de Cultura Embrionária/métodos , Desenvolvimento Embrionário/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Resveratrol/farmacologia , Animais , Antioxidantes/farmacologia , Meios de Cultura , Feminino , Idade Materna , Camundongos , Gravidez , Taxa de GravidezRESUMO
LiCl is widely prescribed for bipolar disorder but adversely associated with a higher incidence of increased body weight. Here, we investigated effects and underlying mechanisms of LiCl on lipid accumulation. LiCl induced dose-dependent lipid accumulation in HepG2 and RAW264.7 cells under normal as well as high glucose conditions. LiCl exposure additionally promoted lipid accumulation in livers of zebrafish. SB216763, a specific GSK-3ß inhibitor, did not affect lipid accumulation in HepG2 cells. Expression of key lipogenic enzymes, such as FAS and aP2, as well as SR-B1 were increased in RAW264.7 cells. LiCl enhanced FAS, ACC and SCD-1 mRNA levels while suppressing CPT-1 in HepG2 cells. LiCl stimulated DNA binding activities of SREBP-1c and ChREBP. LiCl activated AMPK phosphorylation but the AMPK inhibitor, AICAR, did not suppress LiCl-induced lipid accumulation in RAW264.7. LiCl, but not SB216763, induced a significant increase in ROS in RAW264.7 and HepG2 cells. NOX activity was dose-dependently enhanced by LiCl. Furthermore, NOX-1, NOX-2 and DUOX-1 mRNA levels were upregulated at an early stage of LiCl stimulation. LiCl-induced lipid accumulation was suppressed by the antioxidant, NAC, and inhibitors of NOX, DPI and APO. Phosphorylation and transcriptional activity of CREB were enhanced by LiCl. The cell-permeable cAMP analog, di-butyryl cAMP, not only promoted lipid accumulation itself but also LiCl-induced lipid accumulation in RAW264.7 cells. H-89, a PKA inhibitor, suppressed CREB activation, lipid accumulation and NOX activity in RAW264.7 cells. Our results indicate that LiCl stimulates lipid accumulation in hepatocyte and macrophage cells potentially through increased PKA-dependent ROS production.
Assuntos
Antimaníacos/efeitos adversos , Metabolismo dos Lipídeos/efeitos dos fármacos , Cloreto de Lítio/efeitos adversos , Espécies Reativas de Oxigênio/metabolismo , Aumento de Peso/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Transtorno Bipolar/tratamento farmacológico , Glicogênio Sintase Quinase 3 beta/metabolismo , Células Hep G2 , Humanos , Indóis/farmacologia , Isoquinolinas/farmacologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Maleimidas/farmacologia , Camundongos , Células RAW 264.7 , Sulfonamidas/farmacologia , Peixe-ZebraRESUMO
Dengue fever is one of the most important mosquito-borne viral infections in large parts of tropical and subtropical countries and is a significant public health concern and socioeconomic burden. There is an urgent need to develop antivirals that can effectively reduce dengue virus (DENV) replication and decrease viral load. Niclosamide, an antiparasitic drug approved for human use, has been recently identified as an effective antiviral agent against a number of pH-dependent viruses, including flaviviruses. Here, we reveal that neutralization of low-pH intracellular compartments by niclosamide affects multiple steps of the DENV infectious cycle. Specifically, niclosamide-induced endosomal neutralization not only prevents viral RNA replication but also affects the maturation of DENV particles, rendering them non-infectious. We found that niclosamide-induced endosomal neutralization prevented E glycoprotein conformational changes on the virion surface of flaviviruses, resulting in the release of non-infectious immature virus particles with uncleaved pr peptide from host cells. Collectively, our findings support the potential application of niclosamide as an antiviral agent against flavivirus infection and highlight a previously uncharacterized mechanism of action of the drug.
Assuntos
Vesículas Citoplasmáticas/efeitos dos fármacos , Vírus da Dengue/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Espaço Intracelular/efeitos dos fármacos , Niclosamida/farmacologia , Animais , Antivirais/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Vesículas Citoplasmáticas/química , Vesículas Citoplasmáticas/virologia , Vírus da Dengue/genética , Vírus da Dengue/crescimento & desenvolvimento , Endossomos/química , Endossomos/virologia , Humanos , Concentração de Íons de Hidrogênio , Espaço Intracelular/química , Espaço Intracelular/virologia , Estágios do Ciclo de Vida/efeitos dos fármacos , Células Vero , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Vírion/efeitos dos fármacos , Vírion/genética , Vírion/crescimento & desenvolvimento , Replicação Viral/efeitos dos fármacos , Replicação Viral/genéticaRESUMO
Picornaviruses are non-enveloped viruses that represent a large family of positive-sense single-stranded RNA viruses including a number of causative agents of many human and animal diseases such as coxsackievirus B3 (CVB3) and rhinoviruses (HRV). In this study, we performed a high-throughput screening of a compound library composed of â¼6000 small molecules in search of potential picornavirus 3C protease (3Cpro) inhibitors. As results, we identified quinone analogues that effectively inhibited both CVB3 3Cpro and HRV 3Cpro with IC50 values in low micromolar range. Together with predicted binding modes of these compounds to the active site of the viral protease, it is implied that structural features of these non-peptidic inhibitors may act as useful scaffold for further anti-picornavirus drug design and development.
Assuntos
Antivirais/farmacologia , Benzoquinonas/farmacologia , Inibidores de Proteases/farmacologia , Rhinovirus/efeitos dos fármacos , Proteínas Virais/antagonistas & inibidores , Proteases Virais 3C , Antivirais/síntese química , Antivirais/química , Benzoquinonas/síntese química , Benzoquinonas/química , Cisteína Endopeptidases/metabolismo , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Estrutura Molecular , Inibidores de Proteases/síntese química , Inibidores de Proteases/química , Rhinovirus/enzimologia , Relação Estrutura-Atividade , Proteínas Virais/metabolismoRESUMO
The Middle East respiratory syndrome-coronavirus (MERS-CoV), first identified in Saudi Arabia, is an emerging zoonotic pathogen that causes severe acute respiratory illness in humans with a high fatality rate. Since its emergence, MERS-CoV continues to spread to countries outside of the Arabian Peninsula and gives rise to sporadic human infections following the entry of infected individuals to other countries, which can precipitate outbreaks similar to the one that occurred in South Korea in 2015. Current therapeutics against MERS-CoV infection have primarily been adapted from previous drugs used for the treatment of severe acute respiratory syndrome. In search of new potential drug candidates, we screened a library composed of 2334 clinically approved drugs and pharmacologically active compounds. The drug saracatinib, a potent inhibitor of Src-family of tyrosine kinases (SFK), was identified as an inhibitor of MERS-CoV replication in vitro. Our results suggest that saracatinib potently inhibits MERS-CoV at the early stages of the viral life cycle in Huh-7 cells, possibly through the suppression of SFK signaling pathways. Furthermore, saracatinib exhibited a synergistic effect with gemcitabine, an anticancer drug with antiviral activity against several RNA viruses. These data indicate that saracatinib alone or in combination with gemcitabine can provide a new therapeutic option for the treatment of MERS-CoV infection.
Assuntos
Antivirais/farmacologia , Benzodioxóis/farmacologia , Inibidores Enzimáticos/farmacologia , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Quinazolinas/farmacologia , Replicação Viral/efeitos dos fármacos , Linhagem Celular , Infecção Hospitalar , Replicação do DNA , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Descoberta de Drogas , Humanos , Bibliotecas de Moléculas Pequenas , Quinases da Família src/antagonistas & inibidores , GencitabinaRESUMO
We aimed to elucidate the effect of soluble factors secreted by glioma on microglial activation. Conditioned medium (CM) from glioma cells, CRT-MG and C6, significantly induced nitric oxide (NO) production and stimulated the mRNA expression of inducible NO synthase (iNOS), interleukin (IL)-1beta, IL-6, tumor necrosis factor-alpha (TNF-α) and cyclooxygenase 2 (COX-2) in BV2 cells. Glioma CM stimulated p38 mitogen-activated protein kinase (MAPK) phosphorylation, and a p38 MAPK inhibitor, SB203580, suppressed CM-induced NO production in BV2 cells. In addition, CM stimulated nuclear factor-kappaB (NF-κB) DNA binding and transcriptional activity, which was repressed by SB203580. Gliomas displayed higher mRNA expression and release of TNF-α and IL-1ß than primary astrocyte cells. Neutralization of TNF-α and IL-1ß in C6-CM using a neutralizing antibody inhibited NO/iNOS expression in BV-2 cells. These results indicate potential contribution of diffusible tumor-derived factors to regulate microglial activation and subsequent tumor microenvironment.
Assuntos
Meios de Cultivo Condicionados/química , Glioma/química , Interleucina-1beta/metabolismo , Microglia/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Animais , Animais Recém-Nascidos , Anticorpos/farmacologia , Linhagem Celular Tumoral , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Ciclo-Oxigenase 2/metabolismo , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Glioma/metabolismo , Glioma/patologia , Humanos , Imidazóis/farmacologia , Recém-Nascido , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Camundongos , Nitritos/metabolismo , Piridinas/farmacologia , Ratos , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Enteroviruses were identified and characterized from patients with aseptic meningitis and other enterovirusrelated diseases in Chungnam, Korea from 2005 to 2006. Enteroviruses were isolated from 79 of 519 cases (15.2%) in 2005, and 37 of 386 cases (9.6%) in 2006. Based on partial VP1 sequencing, a total of 116 enterovirus isolates were resolved into 13 types. Prevalent among the Chungnam isolates were echovirus 18 and coxsackievirus B5 in 2005, and echoviruses 5 and 25 in 2006. This is the first time echoviruses 5 and 18 have been identified in Korea since enterovirus surveillance began there in 1993. The temporal distribution of enterovirus epidemics in Chungnam showed a remarkable seasonal pattern, with cases occurring during most of the three months of the summer from June to August. The highest rate of enterovirus-positive cases occurred in patients less than 1 year of age. The ratio of male to female enterovirus-positive patients was approximately 1.8:1. Comparison of the VP1 amino acid sequences of the 15 coxsackievirus B5 isolates with reference strains revealed that all Chungnam isolates are substituted at positions 23 (V23I), 19 (S19G), 75 (Y75F), and 95 (N95S). Upon comparing the nine ECV5 isolates with foreign strains, it was found that only the Chungnam isolates, with the exception of Kor06-ECV5-239cn, have P at position 153 and F at position 146. The three ECV9 isolates from 2006 show alterations at amino acids 36, 148, and 154 outside of the BC-loop and at position 84 in the BC-loop, whereas the seven isolates from 2005 and the other ECV9 strains in the database only show the alteration at position 84 (D, I, N, S). The five ECV25 isolates have an S residue at position 134, whereas most of the foreign strains have an N residue.
Assuntos
Infecções por Enterovirus/epidemiologia , Enterovirus/genética , Enterovirus/patogenicidade , Meningite Asséptica/virologia , Sequência de Aminoácidos , Animais , Proteínas do Capsídeo/genética , Linhagem Celular Tumoral , Chlorocebus aethiops , Primers do DNA , Feminino , Febre/epidemiologia , Febre/virologia , Amplificação de Genes , Genes Virais/genética , Humanos , Coreia (Geográfico)/epidemiologia , Masculino , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sepse/epidemiologia , Sepse/virologia , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/genética , Vômito/epidemiologia , Vômito/virologiaRESUMO
Activity of the taurine transporter (TAUT) is regulated by signal transduction in response to diverse stimuli including tumor promoters such as phobol ester. Regulation of the transcription rate of TAUT appears to play an important role in exerting biological roles of taurine in mammalian tissues in adverse environments. Although cDNA of human TAUT has been cloned and sequenced in placenta, thyroid cells, and retinal pigment epithelial cells, the promoter region of TAUT has never been reported. In order to clone the upstream region of the human TAUT promoter, we have compared TAUT cDNA sequences with the entire human genome sequence. Polymerase chain reaction (PCR) was performed from genomic DNA prepared from a SK-Hep-1 cell line for the amplification of the TAUT promoter region including the partial exon (150 bp) and the 5' untranslated region (UTR, 380 bp). The PCR product of the promoter region, which was 1800 bp long, was ligated into the pGEM-T vector, and sequenced. The 5' flanking region of the TAUT promoter was analysed for the identification of enhancer and regulation motifs. Surprisingly we found the consensus TPA responsive element (TGAGTCAG) which is responsible for gene regulation by the protein kinase C (PKC)-mediated signal transduction pathway. The well known fact that proto-oncogene AP1 (cFos/cJun heterodimer or cJun/cJun homodimer) binds to TRE implies that TAUT expression might be closely linked to tumor promotion. Since AP1 activity is also tightly regulated in nerve cells, AP1-regulated TAUT transcription might be an important step in nerve cell function. Furthermore, the TFIID binding site, cap signal for transcription initiation, PEA3 motif, heat shock factor binding motif, and many other motifs were found in the TAUT promoter region, and require characterization.
Assuntos
Proteínas de Transporte/genética , Glicoproteínas de Membrana/genética , Proteínas de Membrana Transportadoras , Regiões Promotoras Genéticas , Sequência de Bases , Proteínas de Transporte/classificação , Clonagem Molecular , Primers do DNA , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Glicoproteínas de Membrana/classificação , Proto-Oncogene Mas , Células Tumorais CultivadasRESUMO
Although taurine transporter (TAUT) activity has been known to be regulated by diverse intracellular and extracellular factors involved in the signal transduction pathway, such as protein kinase C, intracellular Ca concentration, and glucocorticoids, little is known concerning the underlying mechanisms. Evidence suggests that such stimulation-mediated changes in TAUT activity in mammalian cells are partly achieved through the modulation of TAUT transcription activity. In order to better understand the regulation of TAUT transcription activity and subsequently the role of taurine in the signal transduction pathway, we have cloned and sequenced the 5' flanking region of the human TAUT gene, and characterized the TAUT promoter region in human cells. For these reasons, the TAUT luciferase reporter vector was constructed using the 5' flanking region of the TAUT gene (1800 bp). The TAUT luciferase reporter vector was then transfected into SiHa cells, and luciferase activity was measured. The construct containing its own promoter of TAUT (pGL3 b TAU31) showed a 10 fold higher luciferase activity compared to the value found in the empty vector (pGL3 b). This implies a functional transcription of the homologous TAUT promoter. Similar results were obtained with the exon deleted construct [pGL3 b TAU31(-e)]. We also constructed the TAUT luciferase reporter gene (pGL3 pro TAU13) using a heterologous promoter. About 2.5 fold higher luciferase activity was observed in cells transfected with this construct containing the heterologous promoter compared to the value found in the control vector (PGL3 pro).