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1.
Biochem Biophys Res Commun ; 490(3): 726-731, 2017 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-28642136

RESUMO

We tested the effectiveness of a novel 13-bp hepatitis B virus (HBV)-derived cis-acting element (CAE) (ACCTCGACAAGGC), called the DT2 CAE, in augmenting transgene expression delivered by plasmid vectors in eukaryotic cells. The addition of the DT2 CAE just upstream of the start codon of several different target proteins (luciferase, EGFP, LHB, HBsAg, and MIF) in DNA plasmid constructs enhanced their translation in a posttranscriptional manner, irrespective of cell type (cell lines or primary cells) or promoter (CMV or HBV preS1 promoters), suggesting its feasibility for enhanced protein production in eukaryotic cell systems. In conclusion, a novel HBV-derived DT2 CAE could be used effectively for enhanced protein production in eukaryotic cell culture systems.


Assuntos
Técnicas de Transferência de Genes , Vetores Genéticos/genética , Vírus da Hepatite B/genética , Plasmídeos/genética , Transgenes , Animais , Sequência de Bases , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Regiões Promotoras Genéticas , Biossíntese de Proteínas
2.
Mediators Inflamm ; 2017: 8123281, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28804222

RESUMO

Epithelial-mesenchymal transition (EMT) is a biological process that allows epithelial cells to assume a mesenchymal cell phenotype. EMT is considered as a therapeutic target for several persistent inflammatory airway diseases related to tissue remodeling. Herein, we investigated the role of endoplasmic reticulum (ER) stress and c-Src in TGF-ß1-induced EMT. A549 cells, primary nasal epithelial cells (PNECs), and inferior nasal turbinate organ cultures were exposed to 4-phenylbutylic acid (4PBA) or PP2 and then stimulated with TGF-ß1. We found that E-cadherin, vimentin, fibronectin, and α-SMA expression was increased in nasal polyps compared to inferior turbinates. TGF-ß1 increased the expression of EMT markers such as E-cadherin, fibronectin, vimentin, and α-SMA and ER stress markers (XBP-1s and GRP78), an effect that was blocked by PBA or PP2 treatment. 4-PBA and PP2 also blocked the effect of TGF-ß1 on migration of A549 cells and suppressed TGF-ß1-induced expression of EMT markers in PNECs and organ cultures of inferior turbinate. In conclusion, we demonstrated that 4PBA inhibits TGF-ß1-induced EMT via the c-Src pathway in A549 cells, PNECs, and inferior turbinate organ cultures. These results suggest an important role for ER stress and a diverse role for TGF-ß1 in upper airway chronic inflammatory disease such as CRS.


Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Genes src/fisiologia , Fator de Crescimento Transformador beta1/farmacologia , Células A549 , Movimento Celular/efeitos dos fármacos , Chaperona BiP do Retículo Endoplasmático , Genes src/genética , Humanos , Pólipos Nasais/metabolismo , Técnicas de Cultura de Órgãos , Transdução de Sinais/efeitos dos fármacos
3.
Biochem Biophys Res Commun ; 471(1): 156-62, 2016 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-26828270

RESUMO

GV1001, a synthetic peptide derived from human telomerase, has a range of diverse biological activities, including an antioxidant function. Here, we investigated the role of GV1001 in hepatitis C virus (HCV)-infected Huh7.5 (JFH-1) cells. We showed that GV1001 inhibited the production of ROS with decreased MAP kinase signaling. Interestingly, GV1001 lost its antioxidant activity as ROS levels decreased, resulting in a reduction in extracellular heat shock protein 90 (eHSP90) as low-density lipoprotein receptor-related protein 1 (LRP1) was blocked or knocked-down. GV1001 binds to eHSP90 and is delivered into the cell by endocytosis via LRP1. Endocytosed GV1001 finally suppressed ROS generation, presumably by hindering the interaction between eHSP90 and NADPH oxidase (NOX). Importantly, GV1001 suppressed HCV RNA replication in JFH-1 cells by inhibiting the binding of HSP90 to FKBP8, a member of the FK506-binding protein family. We also found that HSP90 expression was high in HCV-infected hepatocytes. Therefore, our data suggest that GV1001 may be a good therapeutic agent by controlling HCV RNA replication, as well as by preferentially targeting cells under conditions of oxidative stress.


Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Hepacivirus/fisiologia , Hepatócitos/virologia , Fragmentos de Peptídeos/administração & dosagem , RNA Viral/genética , Espécies Reativas de Oxigênio/metabolismo , Telomerase/administração & dosagem , Linhagem Celular , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/fisiologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética
4.
Mol Cancer ; 14: 23, 2015 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-25645622

RESUMO

BACKGROUND: The underlying mechanisms of carcinogenesis and gender disparity in hepatitis B virus (HBV)-induced hepatocellular carcinoma (HCC) remain unclear. Recently, we reported a novel HCC-related W4P/R mutation in the large surface protein (LHB) of HBV genotype C, which was found exclusively in male HCC patients. METHODS: LHB sequences from a carrier (wild type; WT) and W4P variant LHB sequence from an HCC patient were cloned and used to generate NIH3T3 and Huh7 cell lines. Cell proliferation and in vitro tumorigenicity were assessed by cell growth and transformation assays. Male and female nude mice were injected with the cells to determine in vivo tumorigenicity. To confirm the effect of estrogen in W4P-mediated tumorigenicity, male mice were injected with estrogen and challenged with W4P-expressing cells. The serum levels of different cytokines from the mouse model and patients were analyzed by ELISA. A critical role of interleukin (IL)-6 signaling in W4P-mediated tumorigenicity was tested by inhibition of Jak2. RESULTS: Although both WT and W4P variant LHBs enhanced cell proliferation by regulating the cell cycle and facilitated cell colony formation, the W4P variant demonstrated significantly higher activity. NIH3T3 cells expressing variant LHB, but not the WT, induced tumor in a nude mouse model. Tumor masses produced by variant LHB were significantly larger in male than female mice, and significantly reduced by estrogen. IL-6, but not tumor necrosis factor-α, was elevated in male mice harboring W4P-induced tumor, and was reduced by estrogen. IL-6 levels of HCC patients with the W4P variant were significantly higher than those of patients with WT LHB. W4P LHB induced higher production of IL-6 than WT LHB in cell lines, and the level was reduced by estrogen. The ability to reduce cell proliferation and colony formation of W4P LHB was hampered by inhibition of IL-6 signaling. CONCLUSIONS: This study suggests that the W4P mutation during the natural course of chronic hepatitis B infection may contribute to HCC development, particularly in male patients, in an IL-6-dependent manner.


Assuntos
Transformação Celular Viral , Variação Genética , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/genética , Animais , Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Ciclo Celular , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Estrogênios/farmacologia , Feminino , Expressão Gênica , Genótipo , Hepatite B/complicações , Hepatite B/virologia , Humanos , Interleucina-6/biossíntese , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Mutação , Células NIH 3T3 , Fatores Sexuais , Transdução de Sinais/efeitos dos fármacos , Carga Tumoral
5.
J Gen Virol ; 96(Pt 7): 1850-4, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25828947

RESUMO

Hepatitis B virus (HBV) infection is associated with a broad spectrum of clinical manifestations, including cirrhosis and hepatocellular carcinoma (HCC). Endoplasmic reticulum (ER) stress and subsequent oxidative stress have been implicated in liver carcinogenesis and disease progression with chronic inflammation. In our previous study, several mutations in the precore/core region of HBV genotype C were identified from 70 Korean chronic patients, and the mutations were associated with HCC and/or HBV e antigen serostatus. Here, we found that the naturally occurring mutations P5T/H/L of the HBV core antigen induced ER stress. The upregulation of ER stress resulted in higher reactive oxygen species production, intracellular calcium concentration, inflammatory cytokines as well as surface antigen production and apoptosis of cells. This study suggested that these mutations may contribute to the progression of liver disease in chronic patients.


Assuntos
Estresse do Retículo Endoplasmático , Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Vírus da Hepatite B/fisiologia , Interações Hospedeiro-Patógeno , Mutação de Sentido Incorreto , Espécies Reativas de Oxigênio/metabolismo , Apoptose , Genótipo , Antígenos do Núcleo do Vírus da Hepatite B/genética , Vírus da Hepatite B/classificação , Vírus da Hepatite B/genética , Vírus da Hepatite B/isolamento & purificação , Hepatite B Crônica/virologia , Humanos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , República da Coreia
6.
J Clin Microbiol ; 51(12): 3928-36, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24025913

RESUMO

The issue of hepatitis B virus (HBV) mutations possibly leading to a gender disparity in the progression of liver diseases has not been explored. We aimed to elucidate the relationships of the novel pre-S1 mutations, W4P/R, with the progression of liver diseases and male predominance in a South Korean chronic cohort by use of a molecular epidemiologic study. We developed a fluorescence resonance energy transfer (FRET)-based real-time PCR (RT-PCR) assay for the detection of the W4P/R mutations and applied it to 292 chronic HBV patients. The pre-S1 mutations from 247 (84.6%) of a total of 292 patients were detected by this assay. W4P/R mutants were found to be significantly related to severe liver diseases (hepatocellular carcinoma [HCC] and liver cirrhosis, 12.4% [19/153] of patients, versus chronic hepatitis and asymptomatic carriage, 1.1% [1/94] of patients) (P < 0.001). All of the W4P/R mutants were found in males only. The novel HBV pre-S1 mutations, W4P/R, may be associated with disease severity in male patients chronically infected with HBV genotype C. The W4P/R mutations may provide in part an explanation for the relatively high ratio of male to female incidence in HCC generation in South Korean chronic HBV patients.


Assuntos
Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/genética , Hepatite B Crônica/patologia , Hepatite B Crônica/virologia , Mutação de Sentido Incorreto , Precursores de Proteínas/genética , Adulto , Idoso , Sequência de Bases , DNA Viral/química , DNA Viral/genética , Progressão da Doença , Feminino , Genótipo , Vírus da Hepatite B/isolamento & purificação , Hepatite B Crônica/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular , Dados de Sequência Molecular , República da Coreia/epidemiologia , Medição de Risco , Análise de Sequência de DNA , Fatores Sexuais
7.
J Hepatol ; 56(1): 63-9, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21827734

RESUMO

BACKGROUND & AIMS: Hepatitis B virus (HBV) genotype C infection is associated with progression of hepatocellular carcinoma (HCC). Specific mutations of the HBV surface (S) gene have been reported to contribute to the development of HCC. In this study, novel nucleotide changes (sW182*) that result in a premature stop at codon 182 in the S gene of genotype C are investigated with regards to the development of HCC. METHODS: A multi-probe real time PCR that enables rapid and reliable detection of sW182* was developed and applied to 292 DNA samples from Korean patients with diverse chronic liver diseases. RESULTS: sW182* was detected in a total of 73 patients out of the 275 with positive amplification (26.5%). Its prevalence was significantly higher in patients with progressive forms of the disease (HCC and liver cirrhosis) than in patients with less severe forms of the disease (chronic hepatitis and carrier) [31.8% (56/176 patients) vs. 17.2% (17/99 patients); p=0.010]. In addition, an in vitro study using cell lines stable expressing the S protein with sW182* also strongly supported its relationship with HCC. CONCLUSIONS: In the present study, we demonstrate that the sW182* of HBV could provide an important contribution to the progression of liver diseases, through molecular epidemiologic and in vitro studies.


Assuntos
Códon sem Sentido , Genes Virais , Vírus da Hepatite B/genética , Hepatite B Crônica/complicações , Hepatite B Crônica/virologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Carcinoma Hepatocelular/etiologia , Linhagem Celular , Proliferação de Células , Primers do DNA/genética , DNA Viral/genética , Progressão da Doença , Expressão Gênica , Genes cdc , Genótipo , Células HEK293 , Vírus da Hepatite B/classificação , Vírus da Hepatite B/patogenicidade , Hepatite B Crônica/genética , Humanos , Neoplasias Hepáticas/etiologia , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , Homologia de Sequência do Ácido Nucleico , Proteínas Virais/genética
8.
J Virol ; 85(1): 123-32, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20962085

RESUMO

Several lines of evidence have suggested that some naturally occurring mutations of hepatitis B virus (HBV) play a critical role in hepatocellular carcinoma (HCC). Here, we describe a novel HCC-related pre-S2 mutation, F141L. To prove the relationship between the F141L mutation and HCC, molecular epidemiology studies using MboII PCR restriction analysis (PRA) were performed, and the molecular mechanism was investigated through construction of a stable hepatocyte cell line expressing the large surface HB protein (LHB) with the F141L mutation (F141L-LHB). Application of MboII PRA to samples from 241 Korean patients with chronic liver diseases of different clinical stages confirmed that F141L mutants were significantly related to HCC, even in comparison to liver cirrhosis (HCC, 26.3% of patients, or 26/99; liver cirrhosis, 3.8% of patients, or 2/52; P = 0.001). By studying stable cell lines, we found that F141L-LHBs could induce cell cycle progression by downregulating the p53 and p21 pathways and upregulating CDK4 and cyclin A. Furthermore, we found that in a colony-forming assay, the colony-forming rates in cell lines expressing F141L-LHBs were about twice as high as those of the wild type. In conclusion, our results suggest that F141L-LHBs may contribute importantly to the pathogenesis of HCC by inducing cell proliferation and transformation. So, the F141L mutation examined in this study could serve as a diagnostic marker for the prognosis of HCC.


Assuntos
Carcinoma Hepatocelular/virologia , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/genética , Hepatite B Crônica/virologia , Neoplasias Hepáticas/virologia , Mutação , Precursores de Proteínas/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transformação Celular Viral , Genótipo , Vírus da Hepatite B/classificação , Hepatite B Crônica/patologia , Hepatócitos/patologia , Hepatócitos/virologia , Humanos , Neoplasias Hepáticas/patologia , Risco
9.
J Med Virol ; 83(3): 405-11, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21264860

RESUMO

Hepatitis B virus (HBV) e antigen (HBeAg) seroconversion during chronic HBV infection is known to play an important role in disease progression and patient response to antiviral agents. The aim of the present study was to analyze gender disparity in distribution of major hydrophilic region (MHR) variants according to HBeAg serostatus. Prevalence of MHR variants from 68 Korean patients with chronic hepatitis (31 HBeAg-positive and 37 HBeAg-negative) was examined in terms of HBeAg serostatus and sex by direct sequencing analysis of the MHR. Gender disparity was observed in the distribution of MHR variants according to HBeAg serostatus. In male patients, the prevalence of MHR variants was significantly higher in HBeAg negative patients than in HBeAg positive patients [58.8% (10/17 patients) vs. 14.3% (3/21 patients), P=0.004]. However, the same was not true in female patients [55.0% (11/20 patients) vs. 60.0% (6/10 patients), P=1.000)]. In addition, 2 mutation types (L110I and G145A) related to HBeAg serostatus were found. In conclusion, HBeAg seroconversion in male chronic patients infected with genotype C could lead to mutations of MHR, major target to host immune response, which might in turn contribute to HBV persistence and immune evasion.


Assuntos
Antígenos E da Hepatite B/imunologia , Vírus da Hepatite B/genética , Vírus da Hepatite B/imunologia , Hepatite B Crônica/genética , Hepatite B Crônica/imunologia , Adolescente , Adulto , Idoso , Sequência de Aminoácidos , Feminino , Genótipo , Antígenos de Superfície da Hepatite B/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação , Alinhamento de Sequência , Fatores Sexuais , Adulto Jovem
10.
J Med Virol ; 83(1): 65-70, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21108340

RESUMO

Deletions and insertions in the hepatitis B virus (HBV) X region have been associated with severe forms of liver disease, including hepatocellular carcinoma (HCC). However, the molecular epidemiologic features of this virus have been described rarely. Deletions and insertions in the X region were determined by direct sequencing in a Korean cohort of 267 patients with different clinical statuses. Deletions and insertions were observed in two sets of six patients each (2.2%, 6/267). The prevalence of deletions or insertions was significantly higher in patients with severe liver disease, HCC, or cirrhosis of the liver (7.2%, 10/132) compared to patients who were carriers or had chronic hepatitis (1.5%, 2/135) (P = 0.017). All deletions in six strains were concentrated at the C terminal end of HBx, encompassing the 113th to 154th codons. A total of four novel types of insertions (PKLL, GM, FFN, and tt) were observed in six patients. Of particular interest, all six strains with insertions were accompanied by double mutations in the basal core promoter (BCP). In conclusion, these results suggest that deletions or insertions in the X region may contribute to disease progression in Korean patients with genotype C infection.


Assuntos
Vírus da Hepatite B/genética , Hepatite B Crônica/virologia , Mutagênese Insercional , Deleção de Sequência , Transativadores/genética , Adulto , Idoso , Povo Asiático , Carcinoma Hepatocelular/virologia , Portador Sadio/virologia , DNA Viral/química , DNA Viral/genética , Feminino , Vírus da Hepatite B/isolamento & purificação , Hepatite B Crônica/patologia , Humanos , Neoplasias Hepáticas/virologia , Masculino , Pessoa de Meia-Idade , República da Coreia , Análise de Sequência de DNA , Índice de Gravidade de Doença , Proteínas Virais Reguladoras e Acessórias
11.
Essays Biochem ; 65(6): 941-950, 2021 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-34156060

RESUMO

There has been little progress for several decades in modalities to treat cervical cancer. While the cervix is a hormone-sensitive tissue, physiologic roles of estrogen receptor α (ERα), progesterone receptor (PR), and their ligands in this tissue are poorly understood. It has hampered critical assessments of data in early epidemiologic and clinical studies for cervical cancer. Experimental evidence obtained from studies using mouse models has provided new insights into the molecular mechanism of ERα and PR in cervical cancer. In a mouse model expressing human papillomavirus (HPV) oncogenes, exogenous estrogen promotes cervical cancer through stromal ERα. In the same mouse model, genetic ablation of PR promotes cervical carcinogenesis without exogenous estrogen. Medroxyprogesterone acetate, a PR-activating drug, regresses cervical cancer in the mouse model. These results support that ERα and PR play opposite roles in cervical cancer. They further support that ERα inhibition and PR activation may be translated into valuable treatment for a subset of cervical cancers.


Assuntos
Neoplasias do Colo do Útero , Animais , Colo do Útero , Modelos Animais de Doenças , Estrogênios/uso terapêutico , Feminino , Hormônios Esteroides Gonadais/uso terapêutico , Humanos , Camundongos , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/genética
12.
Viruses ; 13(3)2021 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-33800513

RESUMO

Pyruvate kinase M2 (PKM2) mainly catalyzes glycolysis, but it also exerts non-glycolytic functions in several cancers. While it has been shown to interact with the human papillomavirus 16 (HPV16) E7 oncoprotein, the functional significance of PKM2 in HPV-associated cervical cancer has been elusive. Here, we show that HPV16 E7 increased the expression of PKM2 in cervical cancer cells. TCGA data analyses revealed a higher level of PKM2 in HPV+ than HPV- cervical cancers and a worse prognosis for patients with high PKM2 expression. Functionally, we demonstrate that shRNA-mediated PKM2 knockdown decreased the proliferation of HPV+ SiHa cervical cancer cells. PKM2 knockdown also inhibited the E7-induced proliferation of cervical cancer cells. ML265 activating the pyruvate kinase function of PKM2 inhibited cell cycle progression and colony formation. ML265 treatments decreased phosphorylation of PKM2 at the Y105 position that has been associated with non-glycolytic functions. On the contrary, HPV16 E7 increased the PKM2 phosphorylation. Our results indicate that E7 increases PKM2 expression and activates a non-glycolytic function of PKM2 to promote cervical cancer cell proliferation.


Assuntos
Proteínas de Transporte/genética , Proliferação de Células/genética , Papillomavirus Humano 16/patogenicidade , Proteínas de Membrana/genética , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus/genética , Hormônios Tireóideos/genética , Neoplasias do Colo do Útero/virologia , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Feminino , Expressão Gênica , Papillomavirus Humano 16/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Fosforilação , Hormônios Tireóideos/metabolismo , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/genética , Proteínas de Ligação a Hormônio da Tireoide
13.
J Clin Microbiol ; 48(9): 3073-80, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20631094

RESUMO

We developed a multiprobe real-time PCR assay targeting hsp65 (HMPRT-PCR) to detect and identify mycobacterial isolates and isolates directly from sputum specimens. Primers and probes for HMPRT-PCR were designed on the basis of the hsp65 gene sequence, enabling the recognition of seven pathogenic mycobacteria, including Mycobacterium tuberculosis, M. avium, M. intracellulare, M. kansasii, M. abscessus, M. massiliense, and M. fortuitum. This technique was applied to 24 reference and 133 clinical isolates and differentiated between all strains with 100% sensitivity and specificity. Furthermore, this method was applied to sputum specimens from 117 consecutive smear-positive patients with smear results of from a trace to 3+. These results were then compared to those obtained using the rpoB PCR-restriction analysis method with samples from cultures of the same sputum specimens. The HMPRT-PCR method correctly identified the mycobacteria in 89 samples (76.0%, 89/117), and moreover, the sensitivity level was increased to 94.3% (50/53) for sputa with an acid-fast bacillus score equal to or greater than 2+. Our data suggest that this novel HMPRT-PCR method could be a promising approach for detecting pathogenic mycobacterial species from sputum samples and culture isolates routinely in a clinical setting.


Assuntos
Proteínas de Bactérias/genética , Técnicas Bacteriológicas/métodos , Chaperonina 60/genética , Mycobacterium/classificação , Mycobacterium/genética , Reação em Cadeia da Polimerase/métodos , Escarro/microbiologia , Tuberculose/diagnóstico , Primers do DNA/genética , RNA Polimerases Dirigidas por DNA/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mycobacterium/isolamento & purificação , Sondas de Oligonucleotídeos/genética , Sensibilidade e Especificidade , Tuberculose/microbiologia
14.
Front Immunol ; 11: 652, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32508804

RESUMO

Previously, a telomerase-derived 16-mer peptide, GV1001, developed as an anticancer vaccine, was reported to exert antiviral effects on human immunodeficiency virus or hepatitis C virus in a heat shock protein-dependent manner. Here we investigated whether GV1001 exerts antiviral effects on hepatitis B virus (HBV) and elucidated its underlying mechanisms. GV1001 inhibited HBV replication and hepatitis B surface antigen (HBsAg) secretion in a dose-dependent manner, showing synergistic antiviral effects with nucleos(t)ide analogs (NAs) including entecavir and lamivudine. This peptide also inhibited viral cccDNA and pgRNA. The intravenous GV1001 treatment of transgenic mice had anti-HBV effects. Our mechanistic studies revealed that GV1001 suppresses HBV replication by inhibiting capsid formation via type I interferon-mediated induction of heme oxygenase-1 (HO-1). GV1001 promoted the mitochondrial DNA stress-mediated release of oxidized DNA into the cytosol, resulting in IFN-I-dependent anti-HBV effects via the STING-IRF3 axis. We found that the anti-HBV effect of GV1001 was due to its ability to penetrate into the cytosol via extracellular heat shock protein, leading to phagosomal escape-mediated mtDNA stress. We demonstrated that the cell-penetrating and cytosolic localization capacity of GV1001 results in antiviral effects on HBV infections via mtDNA stress-mediated IFN-I production. Thus, GV1001, a peptide proven to be safe for human use, may be an anti-HBV drug that can be synergistically used with nucleot(s)ide analog.


Assuntos
Antivirais/metabolismo , Dano ao DNA/imunologia , DNA Mitocondrial/genética , Vírus da Hepatite B/fisiologia , Hepatite B/imunologia , Interferon Tipo I/metabolismo , Fragmentos de Peptídeos/farmacologia , Telomerase/farmacologia , Animais , Sinergismo Farmacológico , Guanina/análogos & derivados , Guanina/farmacologia , Heme Oxigenase-1/metabolismo , Células Hep G2 , Hepatite B/tratamento farmacológico , Antígenos de Superfície da Hepatite B/metabolismo , Humanos , Fator Regulador 3 de Interferon/metabolismo , Lamivudina/farmacologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Fragmentos de Peptídeos/uso terapêutico , Fagossomos/metabolismo , Transdução de Sinais , Telomerase/uso terapêutico , Replicação Viral
15.
Int Forum Allergy Rhinol ; 10(5): 636-645, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32104972

RESUMO

BACKGROUND: All-trans retinoic acid (ATRA), a derivative of vitamin A, is known to have anti-fibrogenic effects and regulates cell proliferation and differentiation. Therefore, these abilities of ATRA may influence tissue remodeling in the upper airway. The aims of the present study were to investigate the effects of ATRA on the myofibroblast differentiation, extracellular matrix (ECM) production, cell migration, and collagen gel contraction and to determine the molecular mechanisms of ATRA in TGF-ß1-induced nasal polyp-derived fibroblasts (NPDFs). METHODS: NPDFs were isolated from nasal polyp. Cytotoxicity was evaluated by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide assay. TGF-ß1-induced fibroblasts were pretreated with ATRA. The expression levels of alpha-smooth muscle actin (α-SMA), collagen type 1, fibronectin, phospho-mitogen-activated protein kinase, and p-p50 (nuclear factor-kappaB [NF-κB]) were measured by Western blot analysis, real-time polymerase chain reaction, and/or immunofluorescence staining. Cell migration was analyzed with cell migration scratch assay and Transwell migration assay. Collagen contractile activity was measured using a collagen gel contraction assay. RESULTS: ATRA had no significant cytotoxic effect in NPDFs. Expression levels of α-SMA, collagen type 1, and fibronectin stimulated by TGF-ß1 were significantly downregulated in the ATRA-pretreated fibroblasts. TGF-ß1-induced cell migration and collagen gel contraction were significantly inhibited by ATRA pretreatment. ATRA also significantly inhibited phosphorylation of c-Jun N-terminal kinase (JNK), p38, and p50 in TGF-ß1-induced NPDFs, but did not inhibit phosphorylation of extracellular signal-related kinase (ERK). CONCLUSION: ATRA downregulated myofibroblast differentiation, ECM production, cell migration, and collagen gel contraction via p38, JNK-dependent NF-κB-signaling pathways in TGF-ß1-induced NPDFs. The findings suggest that ATRA could serve as a novel therapeutic agent to ameliorate nasal polyp development.


Assuntos
Matriz Extracelular/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , NF-kappa B/metabolismo , Pólipos Nasais/patologia , Fator de Crescimento Transformador beta1/farmacologia , Tretinoína/farmacologia , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Pólipos Nasais/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
16.
J Med Virol ; 80(8): 1337-43, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18551606

RESUMO

Few reports have detailed mutation frequencies and mutation patterns in the entire X region according to clinical status. The aims of this study were to elucidate the relationships between mutation patterns and their frequencies in the X region and clinical status in a Korean cohort and determine specific X mutation types, related closely with liver disease progression. All X mutations were determined by direct sequencing in 184 patients with different clinical features. Mutation rates in the X region in patients with more severe liver disease, hepatocellular carcinoma (HCC) (3.6%) or liver cirrhosis (4%) were always significantly higher than in patients with corresponding less severe forms, chronic hepatitis (2.9%) or asymptomatic carriers (2.1%), but no significant difference in mutation rates was found in terms of HBeAg serostatus. All five mutation types (V5M/L, P38S, H94Y, I127T/N, and K130M and V131I) affecting the six codons were found to be related significantly to clinical severity. Among these, two mutation types (V5M/L and K130M and V131I) were observed more frequently in HBeAg negative patients than in HBeAg positive patients. In conclusion, the results suggest that an accumulation of mutations in the X region contributes to disease progression in chronic patients, at least Korean patients with genotype C. Specific mutation types appears to be related more to severe liver diseases such as HCC or liver cirrhosis. In particular, a novel mutation type (V5M/L) discovered firstly during the present study was found to be associated significantly with HCC.


Assuntos
Vírus da Hepatite B/classificação , Vírus da Hepatite B/genética , Hepatite B Crônica/fisiopatologia , Mutação , Índice de Gravidade de Doença , Transativadores/genética , Adulto , Idoso , Sequência de Aminoácidos , Carcinoma Hepatocelular/epidemiologia , Carcinoma Hepatocelular/fisiopatologia , Carcinoma Hepatocelular/virologia , Feminino , Genótipo , Vírus da Hepatite B/patogenicidade , Hepatite B Crônica/epidemiologia , Hepatite B Crônica/virologia , Humanos , Coreia (Geográfico)/epidemiologia , Cirrose Hepática/epidemiologia , Cirrose Hepática/fisiopatologia , Cirrose Hepática/virologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Prevalência , Proteínas Virais Reguladoras e Acessórias
17.
Diagn Microbiol Infect Dis ; 62(2): 193-8, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18653300

RESUMO

Early differentiation of mycobacteria in sputa is crucial. This study was set to evaluate the usefulness of a newly developed duplex polymerase chain reaction (PCR) for hsp65 gene-based method in differentiating mycobacteria in sputum with a positive acid-fast bacilli (AFB) smear before culturing. One hundred forty-seven sputa with positive AFB smear were included for the analysis. Mycobacterial species were identified using a newly developed duplex PCR for hsp65 gene followed by a nested PCR-direct sequencing and the conventional colony-based method. Final decision of mycobacterial species were made based on 1) results of species identification based on mycobacterial colonies or 2) results of species identification of other sputa from the same patients and clinical findings. The duplex PCR-based method correctly identified 83.2% sputa from tuberculosis patients and 82.2% sputa from nontuberculous mycobacteria patients, whereas the colony-based method correctly identified 86.1% and 77.8%, respectively. Sensitivity and specificity of the colony-based method for Mycobacterium tuberculosis were 86.1% and 100%, respectively, whereas those of the duplex PCR-based method were 83.2% and 95.6%, respectively. The duplex PCR-based method, to differentiate mycobacterial species in sputa, produced comparable results as those of the colony-based identification method.


Assuntos
Proteínas de Bactérias/genética , Chaperoninas/genética , Mycobacterium/classificação , Reação em Cadeia da Polimerase/métodos , Escarro/microbiologia , Tuberculose Pulmonar/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas de Bactérias/metabolismo , Chaperonina 60 , Chaperoninas/metabolismo , DNA Bacteriano/análise , DNA Bacteriano/isolamento & purificação , Diagnóstico Diferencial , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mycobacterium/genética , Mycobacterium/isolamento & purificação , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Sensibilidade e Especificidade , Análise de Sequência de DNA , Especificidade da Espécie , Tuberculose Pulmonar/microbiologia
18.
World J Gastroenterol ; 24(10): 1084-1092, 2018 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-29563753

RESUMO

AIM: To study sex disparity in susceptibility to hepatocellular carcinoma (HCC), we created a transgenic mouse model that expressed the full hepatitis B virus (HBV) genome with the W4P mutation. METHODS: Transgenic mice were generated by transferring the pHY92-1.1x-HBV-full genome plasmid (genotype A2) into C57Bl/6N mice. We compared serum levels of hepatitis B surface antigen (HBsAg), interleukin (IL)-6, and the liver enzymes alanine aminotransferase (ALT) and aspartate transaminase (AST), as well as liver histopathological features in male and female transgenic (W4P TG) mice and in nontransgenic littermates of 10 mo of age. RESULTS: W4P TG males exhibited more pronounced hepatomegaly, significantly increased granule generation in liver tissue, elevated HBsAg expression in the liver and serum, and higher serum ALT and IL-6 levels compared to W4P TG females or littermate control groups. CONCLUSION: Together, our data indicate that the W4P mutation in preS1 may contribute to sex disparity in susceptibility to HCC by causing increased HBV virion replication and enhanced IL-6-mediated inflammation in male individuals. Additionally, our transgenic mouse model that expresses full HBV genome with the W4P mutation in preS1 could be effectively used for the studies of the progression of liver diseases, including HCC.


Assuntos
Carcinoma Hepatocelular/virologia , Suscetibilidade a Doenças/virologia , Vírus da Hepatite B/genética , Hepatite B Crônica/virologia , Neoplasias Hepáticas/virologia , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/patologia , Suscetibilidade a Doenças/sangue , Feminino , Genoma Viral/genética , Antígenos de Superfície da Hepatite B/sangue , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/isolamento & purificação , Hepatite B Crônica/sangue , Hepatite B Crônica/patologia , Humanos , Interleucina-6/sangue , Fígado/enzimologia , Fígado/patologia , Fígado/virologia , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Precursores de Proteínas/sangue , Precursores de Proteínas/genética , Fatores Sexuais , Carga Viral
19.
Am J Rhinol Allergy ; 32(4): 228-235, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29676176

RESUMO

Purpose Methyl-CpG-binding protein 2 (MeCP2), known as a transcriptional regulator, has been suggested to play an important role in myofibroblast differentiation in the lung. The purpose of this study was to investigate the role of MeCP2 in transforming growth factor (TGF)- ß1-induced myofibroblast differentiation and extracellular matrix (ECM) production in nasal polyp-derived fibroblasts (NPDFs). Methods To identify the expression of MeCP2 in nasal polyp tissues, immunohistochemistry staining and Western blot were performed. TGF- ß1-induced NPDFs were treated with 5-azacytidine, a DNA methylation inhibitor, and the expression levels of α-SMA and fibronectin were determined by semiquantitative reverse transcription polymerase chain reaction, immunofluorescent staining, and Western blotting. The total soluble collagen was analyzed by the Sircol collagen assay. MeCP2 silenced by MeCP2-specific small interference ( si) RNA was verified by Western blot. Results The expression levels of MeCP2 increased in nasal polyp tissues compared to normal inferior turbinate tissues. 5-Azacytidine significantly inhibited the expression of α-SMA and fibronectin mRNA in a dose-dependent manner. In addition, 5-azacytidine suppressed collagen production and the expression of MeCP2 in the same manner. The expression levels of a-SMA and collagen production were significantly blocked by MeCP2 silencing in TGF- ß1-induced NPDFs. Conclusions Our data suggest that MeCP2 plays an essential role in TGF- ß1-induced myofibroblast differentiation and ECM production in NPDFs.


Assuntos
Matriz Extracelular/metabolismo , Fibroblastos/fisiologia , Proteína 2 de Ligação a Metil-CpG/metabolismo , Miofibroblastos/fisiologia , Pólipos Nasais/imunologia , Actinas/genética , Actinas/metabolismo , Adulto , Azacitidina/farmacologia , Diferenciação Celular , Células Cultivadas , Colágeno/genética , Colágeno/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Proteína 2 de Ligação a Metil-CpG/genética , RNA Interferente Pequeno/genética , Fator de Crescimento Transformador beta1/metabolismo , Conchas Nasais/citologia
20.
Oncol Rep ; 17(1): 81-7, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17143482

RESUMO

The multifunctional G-protein-coupled metabotropic glutamate receptor (mGluR) family comprises eight subtypes, some of which participate in tumorigenesis. The purpose of this study was to evaluate mGluR5 expression in oral squamous cell carcinoma (SCC) tissues and oral cancer cell lines. We also investigated the prognostic significance of mGluR5 and its functional importance in the migration, invasion, and adhesion of oral cancer cells. We evaluated the expression of mGluR5 in samples from 131 oral SCC patients and in several oral cancer cell lines by immunohistochemistry and RT-PCR. We observed varying levels of mGluR5 in human oral SCC tissues and cancer cell lines. There was a significant association between strong mGluR5 immunoreactivity and overall survival (P=0.0109). The functional significance of the expression of mGluR5 in oral cancer cells was then investigated in HSC3 oral tongue cancer cells. An mGluR5 agonist, DHPG increased tumor cell migration, invasion, and adhesion in HSC3 cells (P<0.05). This was reversed by the mGluR5 antagonist MPEP. Our results strongly suggest that mGluR5 is a new prognostic marker and contributes to tumor cell migration and invasion in oral cancer.


Assuntos
Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Receptores de Glutamato Metabotrópico/biossíntese , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Feminino , Humanos , Imuno-Histoquímica , Células KB , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Prognóstico , Receptor de Glutamato Metabotrópico 5 , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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