Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 591
Filtrar
1.
Nat Prod Res ; 33(11): 1550-1555, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29334261

RESUMO

Two lignans including a new one, five flavonoids and five coumarins were isolated from the whole plant of Viola philippica (synonymised as Viola yedoensis Makino). The new compound was structurally determined as (7R,8S,8'S) -3,3'-dimethoxy- 4,4',9-trihydroxy- 7,9'-epoxy-8,8'-lignan 9-O-rutinoside by analysis of its NMR, MS and CD spectroscopic data. The known compounds were characterised by comparing their NMR and MS data with those reported. Among the known compounds, 5-hydroxy-4'-methoxyflavone-7-O- rutinoside, 6,7-di-O-ß-D- glucopyranosylesculetin, and 7R,8S-dihydrodehydrodiconiferyl alcohol 4-O-ß-D- glucopyranoside were isolated and identified from this genus for the first time. Of these compounds, 5-hydroxy-4'-methoxyflavone-7-O-rutinoside and (7R,8S,8'S) -3,3'-dimethoxy- 4,4',9-trihydroxy- 7,9'-epoxy-8,8'-lignan 9-O-rutinoside were potently active against α-glucosidase, while the two dimeric coumarins, 5, 5'-bi (6, 7-dihydroxycoumarin) and 6,6',7,7'-tetrahydroxy-5,8'-bicoumarin potently inhibited HCV protease.


Assuntos
Cumarínicos/farmacologia , Flavonoides/farmacologia , Inibidores de Glicosídeo Hidrolases/farmacologia , Hepatite C/enzimologia , Lignanas/farmacologia , Inibidores de Proteases/farmacologia , Viola/química , Dicroísmo Circular , Cumarínicos/química , Flavonoides/química , Inibidores de Glicosídeo Hidrolases/química , Lignanas/química , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Inibidores de Proteases/química
2.
Sci Rep ; 7(1): 16978, 2017 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-29208982

RESUMO

Sphingosine-1-phospate is a potent bioactive lipid metabolite that regulates cancer progression. Because sphingosine kinase 1 and sphingosine kinase 2 (SPHK 1/2) are both essential for sphingosine-1-phospate production, they could be a therapeutic target in various cancers. Peretinoin, an acyclic retinoid, inhibits post-therapeutic recurrence of hepatocellular carcinoma via unclear mechanisms. In this study, we assessed effects of peretinoin on SPHK expression and liver cancer development in vitro and in vivo. We examined effects of peretinoin on expression, enzymatic and promoter activity of SPHK1 in a human hepatoma cell line, Huh-7. We also investigated effects of SPHK1 on hepatocarcinogenesis induced by diethylnitrosamine using SPHK1 knockout mice. Peretinoin treatment of Huh-7 cells reduced mRNA levels, protein expression and enzymatic activity of SPHK1. Peretinoin reduced SPHK1 promoter activity; this effect of peretinoin was blocked by overexpression of Sp1, a transcription factor. Deletion of all Sp1 binding sites within the SPHK1 promoter region abolished SPHK1 promoter activity, suggesting that peretinoin reduced mRNA levels of SPHK1 via Sp1. Additionally, diethylnitrosamine-induced hepatoma was fewer and less frequent in SPHK1 knockout compared to wild-type mice. Our data showed crucial roles of SPHK1 in hepatocarcinogenesis and suggests that peretinoin prevents hepatocarcinogenesis by suppressing mRNA levels of SPHK1.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Retinoides/farmacologia , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Dietilnitrosamina/toxicidade , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Hepatite C/enzimologia , Hepatite C/genética , Humanos , Fígado/metabolismo , Cirrose Hepática/enzimologia , Cirrose Hepática/genética , Cirrose Hepática/virologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/genética , Camundongos Knockout , Camundongos Transgênicos , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Esfingolipídeos/genética , Esfingolipídeos/metabolismo
3.
Clin Chim Acta ; 475: 128-136, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29031454

RESUMO

BACKGROUND: The ratio of serum γ-glutamyl transferase (GGT) to alanine aminotransferase (ALT) (GGT/ALT) is a marker for evaluating effects to antivirotic treatment and a helpful predictive factor for the prognosis of Child-Pugh A hepatocellular carcinoma (HCC) patients after surgery. The relationship between the incidence of postoperative acute kidney injury (AKI) and preoperative GGT/ALT is studied in hepatectomized hepatitis B- or C- associated HCC patients. METHODS: A total of 253 hepatitis B or C virus-related HCC patients undergoing hepatectomy between September 2012 and August 2016 at our hospital were included in the retrospective study. Serum ALT and GGT value were recorded, and the GGT/ALT was computed. AKI was defined that based on the "Kidney Disease Improving Global Outcomes (KDIGO) criteria". RESULTS: AKI was observed in 22 (8.7%) patients. Mean GGT/ALT of patients with AKI was significantly higher than in those without it (6.0 vs 2.1, P<0.001). Multivariate analysis revealed an increase in GGT/ALT as an independent risk factor for AKI in hepatitis B- or C- associated HCC patients, particularly in patients with Barcelona Clinic Liver Cancer (BCLC) stage 0 or A staged HCC (odds ratio (OR) 1.400, P<0.001). Multivariate analysis showed that ALT (OR 0.966, P=0.044) was somewhat inversely associated with the incidence of AKI in hepatitis B- or C- associated HCC patients. The best cutoff point of GGT/ALT was 2.92. Multivariate analysis showed that preoperative GGT/ALT ≥2.92 predicted poor prognosis of postoperative AKI in patients with HCC after hepatectomy (odds ratio 17.697, P<0.001). After propensity score matching, preoperative GGT/ALT ≥2.92 remained an independent risk factor for AKI in HCC patients (OR 13.947, P=0.003). CONCLUSIONS: The GGT/ALT of patients with AKI was significantly higher than those without it. Evaluation of GGT/ALT before surgery can be a helpful predictive tool for postoperative AKI in hepatitis B- or C- associated HCC patients undergoing hepatectomy, particularly in patients with BCLC stage 0 or A staged HCC. Hepatitis B- or C- associated HCC patients with low ALT especially within the normal range may have a high risk of AKI. However, the reason remains to be elucidated.


Assuntos
Lesão Renal Aguda/diagnóstico , Alanina Transaminase/sangue , Carcinoma Hepatocelular/enzimologia , Hepatite B/enzimologia , Hepatite C/enzimologia , Neoplasias Hepáticas/enzimologia , gama-Glutamiltransferase/sangue , Lesão Renal Aguda/enzimologia , Lesão Renal Aguda/etiologia , Lesão Renal Aguda/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Carcinoma Hepatocelular/complicações , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/cirurgia , Feminino , Hepatectomia/efeitos adversos , Hepatite B/complicações , Hepatite B/patologia , Hepatite B/cirurgia , Hepatite C/complicações , Hepatite C/patologia , Hepatite C/cirurgia , Humanos , Rim/enzimologia , Rim/patologia , Rim/cirurgia , Neoplasias Hepáticas/complicações , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/cirurgia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Período Pós-Operatório , Prognóstico , Estudos Retrospectivos , Fatores de Risco
4.
Sci Rep ; 7(1): 5876, 2017 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-28724915

RESUMO

HCV infection can decrease NAD+/NADH ratio, which could convert lipid metabolism to favor HCV replication. In hepatocytes, quinolinate phosphoribosyl transferase (QPRT) catabolizes quinolinic acid (QA) to nicotinic acid mononucleotide (NAMN) for de novo NAD synthesis. However, whether and how HCV modulates QPRT hence the lipogenesis is unknown. In this work, we found QPRT was reduced significantly in livers of patients or humanized C/OTg mice with persistent HCV infection. Mechanistic studies indicated that HCV NS3/4A promoted proteasomal degradation of QPRT through Smurf2, an E3 ubiquitin-protein ligase, in Huh7.5.1 cells. Furthermore, QPRT enzymatic activity involved in suppression of HCV replication in cells. Activation of QPRT with clofibrate (CLO) or addition of QPRT catabolite NAD both inhibited HCV replication in cells, probably through NAD+-dependent Sirt1 inhibition of cellular lipogenesis. More importantly, administration of CLO, a hypolipidemic drug used in clinics, could significantly reduce the viral load in HCV infected C/OTg mice. Take together, these results suggested that HCV infection triggered proteasomal degradation of QPRT and consequently reduced de novo NAD synthesis and lipogenesis, in favor of HCV replication. Hepatic QPRT thus likely served as a cellular factor that dampened productive HCV replication.


Assuntos
Hepacivirus/fisiologia , Hepatite C/enzimologia , Hepatite C/virologia , Pentosiltransferases/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Lipogênese , Camundongos , NAD/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Ubiquitina-Proteína Ligases/metabolismo , Proteínas não Estruturais Virais/metabolismo , Replicação Viral
5.
Clin Invest Med ; 40(2): E73-E80, 2017 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-28447580

RESUMO

PURPOSE: Monitoring of hepatitis C virus (HCV) treatment response is performed by serial HCV RNA measurements; however, this may not be useful for predicting treatment success or failure with oral direct-acting antiviral agent (DAA) therapies. Liver enzyme levels, which are elevated in chronic HCV and tend to decline on therapy, may serve as a more logistically and economically feasible alternative for monitoring treatment response. SOURCE: The Ottawa Hospital Viral Hepatitis Clinic patients (n=219), receiving interferon-free oral DAA treatments, were assessed for liver enzymes and HCV RNA levels at baseline, week 4 and ≥12 weeks post-treatment. Suppression cut points used for this analysis were ALT ≤ 40U L-1 and AST ≤ 30U L-1. The primary outcome was week 12 sustained virologic response (SVR). By our analysis, all indicators had strong PPV (>90%) but limited NPV (.


Assuntos
Antivirais/administração & dosagem , Antivirais/uso terapêutico , Hepacivirus/efeitos dos fármacos , Hepacivirus/patogenicidade , Hepatite C/tratamento farmacológico , Hepatite C/enzimologia , Administração Oral , Adulto , Feminino , Hepatite C/patologia , Humanos , Fígado/enzimologia , Fígado/virologia , Masculino , Pessoa de Meia-Idade , RNA Viral/genética , Resultado do Tratamento
6.
Virology ; 507: 231-241, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28456022

RESUMO

Hepatitis C virus (HCV) subverts the cellular DEAD-box RNA helicase DDX6 to promote virus infection. Using polysome gradient analysis and the subgenomic HCV Renilla reporter replicon genome, we determined that DDX6 does not affect HCV translation. Rather expression of the subgenomic HCV Renilla luciferase reporter at late times, as well as labeling of newly synthesized viral RNA with 4-thiouridine showed that DDX6 modulates replication. Because DDX6 is an effector protein of the microRNA pathway, we also investigated its role in miR-122-directed HCV gene expression. Similar to sequestering miR-122, depletion of DDX6 modulated HCV RNA stability. Interestingly, miR-122-HCV RNA interaction assays with mutant HCV genomes sites and compensatory exogenous miR-122 showed that DDX6 affects the function of miR-122 at one particular binding site. We propose that DDX6 facilitates the miR-122 interaction with HCV 5' UTR, which is necessary for stabilizing the viral genome and the switch between translation and replication.


Assuntos
RNA Helicases DEAD-box/metabolismo , Hepacivirus/metabolismo , Hepatite C/enzimologia , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , RNA Viral/metabolismo , Regiões 5' não Traduzidas , Linhagem Celular Tumoral , RNA Helicases DEAD-box/genética , Hepacivirus/genética , Hepatite C/genética , Hepatite C/metabolismo , Hepatite C/virologia , Humanos , MicroRNAs/genética , Proteínas Proto-Oncogênicas/genética , RNA Viral/genética
7.
J Biol Chem ; 292(15): 6202-6212, 2017 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-28228479

RESUMO

Grazoprevir is a potent pan-genotype and macrocyclic inhibitor of hepatitis C virus (HCV) NS3/4A protease and was developed for treating chronic HCV infection. In HCV genotype (GT) 1a, grazoprevir maintains potent activity against a majority of NS3 resistance-associated amino acid substitutions, including the highly prevalent and naturally occurring Q80K polymorphism that impacts simeprevir, another NS3/4A protease inhibitor. The basis for an unexpected difference in the clinical impact of some NS3 substitutions was investigated. Phenotypic analysis of resistance-associated substitutions identified in NS3 from GT1a-infected patients who failed therapy with grazoprevir (in combination with elbasvir, an inhibitor of HCV NS5A protein) showed that positions 56, 156, and 168 in NS3 were most impactful because they diminished protein-inhibitor interactions. Although an amino acid substitution from aspartic acid to alanine at position 168 (D168A) reduced the potency of grazoprevir, its combination with R155K unexpectedly nullified this effect. Molecular dynamics and free-energy surface studies indicated that Asp-168 is important in anchoring Arg-155 for ligand binding but is not critical for Lys-155 because of the inherent flexibility of its side chain. Moreover, modeling studies supported a strong direct cation-heterocycle interaction between the Lys-155 side chain of the double substitution, R155K/D168A, and the lone pair on the quinoxaline in grazoprevir. This unique interaction provides a structural basis for grazoprevir's higher potency than simeprevir, an inhibitor to which the double substitution confers a significant reduction in potency. Our findings are consistent with the detection of R155K/D168A in NS3 from virologic failures treated with simeprevir but not grazoprevir.


Assuntos
Hepacivirus/enzimologia , Simulação de Dinâmica Molecular , Mutação de Sentido Incorreto , Quinoxalinas/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química , Substituição de Aminoácidos , Linhagem Celular Tumoral , Hepacivirus/genética , Hepatite C/tratamento farmacológico , Hepatite C/enzimologia , Hepatite C/genética , Humanos , Quinoxalinas/uso terapêutico , Simeprevir/química , Simeprevir/uso terapêutico , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
8.
Postepy Biochem ; 63(4): 261-268, 2017.
Artigo em Polonês | MEDLINE | ID: mdl-29374427

RESUMO

From the earliest times, medicine has focused on finding the most suitable and effective treatment for every patient. At present, a dynamic development of diagnostic methods and techniques for designing new drugs allows to create therapies for many diseases at the molecular level. Among the many drugs appearing on the medical market every year, special attention should be paid to those whose action is based on the inhibition of proteolytic enzyme activity. Protease inhibitors are a diverse group of biologically active molecules for which antiviral, antimicrobial, antifungal, antiparasitic or anticancer effects have been documented. Successes in the treatment of HIV infection, hepatitis C and influenza diseases certainly encourage researchers to look for new inhibitors that could be used in new therapies. This paper provides an overview of selected information on enzyme inhibitors, especially protease inhibitors, which are already registered medicines and substances that are promising candidates for medical use.


Assuntos
Inibidores de Proteases/farmacologia , Inibidores de Proteases/uso terapêutico , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Antivirais/farmacologia , Antivirais/uso terapêutico , Infecções por HIV/tratamento farmacológico , Infecções por HIV/enzimologia , Hepatite C/tratamento farmacológico , Hepatite C/enzimologia , Humanos , Influenza Humana/tratamento farmacológico , Influenza Humana/enzimologia
9.
J Med Dent Sci ; 63(2-3): 45-52, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27773912

RESUMO

Nucleic acid amplification test (NAT), which was introduced by the Japanese Red Cross Society in October 1999, began to be performed for screening of blood transfusion formulations in Japan in August 2014. In this study, the precision of immunological screenings of hepatitis B (HBsAg, HBcAb, and HBsAb), hepatitis C (HCVAb), and human immunodeficiency (HIVAb) virus antigens in donated blood were evaluated. In addition, the sensitivity of the alanine aminotransferase (ALT) test for detection of the hepatitis B and C viruses was re-evaluated. Immunological screenings showed high precision of detecting the viral antigens. In contrast, the ALT test showed much lower precision of detecting the presence of the hepatitis B and C viruses. Results of the NAT and immunological screenings revealed that ALT levels in donors were more strongly correlated with their levels of gammaglutamyltranspeptidase (γGTP) and body mass index (BMI), than with the results of NAT and immunological screening. Our study indicates that elevated level(s) of ALT, were more likely to be associated with lifestyles factors such as high intake of alcohol or obesity than with infection. Therefore, ALT may be excluded as surrogate markers of HBV, HCV, and HIV in donated blood.


Assuntos
Alanina Transaminase/sangue , Hepatite B/sangue , Hepatite C/sangue , Vírus de Hepatite/isolamento & purificação , Adulto , Biomarcadores/sangue , Feminino , Anticorpos Anti-Hepatite/sangue , Antígenos de Hepatite/sangue , Hepatite B/enzimologia , Hepatite B/imunologia , Hepatite B/virologia , Hepatite C/enzimologia , Hepatite C/imunologia , Hepatite C/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Técnicas de Amplificação de Ácido Nucleico , Curva ROC , Análise de Regressão
10.
ACS Infect Dis ; 2(11): 839-851, 2016 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-27676132

RESUMO

Domain II of the nonstructural protein 5 (NS5A) of the hepatitis C virus (HCV) is involved in intermolecular interactions with the viral RNA genome, the RNA-dependent RNA polymerase NS5B, and the host factor cyclophilin A (CypA). However, domain II of NS5A (NS5ADII) is largely disordered, which makes it difficult to characterize the protein-protein or protein-nucleic acid interfaces. Here we utilized a mass spectrometry-based protein footprinting approach in attempts to characterize regions forming contacts between NS5ADII and its binding partners. In particular, we compared surface topologies of lysine and arginine residues in the context of free and bound NS5ADII. These experiments have led to the identification of an RNA binding motif (305RSRKFPR311) in an arginine-rich region of NS5ADII. Furthermore, we show that K308 is indispensable for both RNA and NS5B binding, whereas W316, further downstream, is essential for protein-protein interactions with CypA and NS5B. Most importantly, NS5ADII binding to NS5B involves a region associated with RNA binding within NS5B. This interaction down-regulated RNA synthesis by NS5B, suggesting that NS5ADII modulates the activity of NS5B and potentially regulates HCV replication.


Assuntos
Ciclofilina A/metabolismo , Hepacivirus/metabolismo , Hepatite C/enzimologia , Hepatite C/virologia , RNA Viral/metabolismo , Proteínas não Estruturais Virais/metabolismo , Motivos de Aminoácidos , Ciclofilina A/genética , Regulação Viral da Expressão Gênica , Hepacivirus/química , Hepacivirus/genética , Hepatite C/genética , Interações Hospedeiro-Patógeno , Humanos , Ligação Proteica , Domínios Proteicos , RNA Viral/genética , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética
11.
Mol Immunol ; 78: 48-56, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27588826

RESUMO

BACKGROUND: Mannan-binding lectin (MBL) - associated serine protease 2 (MASP-2) co-activates the lectin pathway of complement in response to several viral infections. The quality of this response partly depends on MASP2 gene polymorphisms, which modulate MASP-2 function and serum levels. In this study we investigated a possible role of MASP2 polymorphisms, MASP-2 serum levels and MBL-mediated complement activation in the susceptibility to HIV/AIDS and HBV/HCV coinfection. METHODS: A total of 178 HIV patients, 89 (50%) coinfected with HBV/HCV, 51.7% female, average age 40 (12-73) years, and 385 controls were evaluated. MASP-2 levels and MBL-driven complement activation were evaluated by enzyme-linked immunosorbent assay and 11 MASP2 polymorphisms from the promoter to the last exon were haplotyped using multiplex sequence-specific PCR. RESULTS: Genotype distribution was in Hardy-Weinberg equilibrium and differed between HIV+ patients and controls (P=0.030), irrespective of HBV or HCV coinfection. The p.126L variant, which was associated with MASP-2 levels <200ng/mL (OR=5.0 [95%CI=1.3-19.2] P=0.019), increased the susceptibility to HIV infection (OR=5.67 [95%CI=1.75-18.33], P=0.004) and to HIV+HBV+ status (OR=6.44 [95%CI=1.69-24.53, P=0.006). A similar association occurred with the ancient haplotype harboring this variant, AGCDV (OR=2.35 [95%CI=1.31-4.23], P=0.004). On the other hand, p.126L in addition to other variants associated with low MASP-2 levels-p.120G, p.377A and p.439H, presented a protective effect against AIDS (OR=0.25 [95%CI=0.08-0.80], P=0.020), independently of age, sex, hepatic function and viral load. MASP-2 serum levels were lower in HIV+ and HIV+HBV+ patients than in controls (P=0.0004). Among patients, MASP-2 levels were higher in patients with opportunistic diseases (P=0.001) and AIDS (P=0.004). MASP-2 levels correlated positively with MBL/MASP2-mediated C4 deposition (r=0.29, P=0.0002) and negatively with CD4+ cell counts (r=-0.21, P=0.018), being related to decreased CD4+ cell counts (OR=5.8 [95%CI=1.23-27.5, P=0.026). CONCLUSIONS: Genetically determined MASP-2 levels seem to have a two-edge effect in HIV and probably HCV/HBV coinfection, whereas low levels increase the susceptibility to infection, but on the other side protects against AIDS.


Assuntos
Infecções por HIV/genética , Serina Proteases Associadas a Proteína de Ligação a Manose/genética , Síndrome de Imunodeficiência Adquirida/enzimologia , Síndrome de Imunodeficiência Adquirida/genética , Síndrome de Imunodeficiência Adquirida/imunologia , Adolescente , Adulto , Idoso , Criança , Coinfecção/enzimologia , Coinfecção/genética , Coinfecção/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Predisposição Genética para Doença/genética , Genótipo , Infecções por HIV/enzimologia , Infecções por HIV/imunologia , Hepatite B/enzimologia , Hepatite B/genética , Hepatite B/imunologia , Hepatite C/enzimologia , Hepatite C/genética , Hepatite C/imunologia , Humanos , Masculino , Serina Proteases Associadas a Proteína de Ligação a Manose/imunologia , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Multiplex , Polimorfismo de Nucleotídeo Único , Adulto Jovem
13.
World J Gastroenterol ; 22(14): 3746-57, 2016 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-27076759

RESUMO

AIM: To elucidate the mechanism(s) by which S-adenosyl-L-methionine (SAM) decreases hepatitis C virus (HCV) expression. METHODS: We examined the effects of SAM on viral expression using an HCV subgenomic replicon cell culture system. Huh7 HCV-replicon cells were treated with 1 mmol/L SAM for different times (24-72 h), then total RNA and proteins were isolated. cDNA was synthesized and real time-PCR was achieved to quantify HCV-RNA, superoxide dismutase 1 and 2 (SOD-1, SOD-2) catalase, thioredoxin 1, methionine adenosyltransferase 1A and 2A (MAT1A, MAT2A) expression, and GAPDH and RPS18 as endogenous genes. Expression of cellular and viral protein was evaluated by western-blot analysis using antibodies vs HCV-NS5A, SOD-1, SOD-2, catalase, thioredoxin-1, MAT1A, MAT2A, GAPDH and actin. Total glutathione levels were measured at different times by Ellman's recycling method (0-24 h). Reactive oxidative species (ROS) levels were quantified by the dichlorofluorescein assay (0-48 h); Pyrrolidin dithiocarbamate (PDTC) was tested as an antioxidant control and H2O2 as a positive oxidant agent. RESULTS: SAM exposition decreased HCV-RNA levels 50%-70% compared to non-treated controls (24-72 h). SAM induced a synergic antiviral effect with standard IFN treatment but it was independent of IFN signaling. In addition, 1 mmol/L SAM exposition did not modify viral RNA stability, but it needs cellular translation machinery in order to decrease HCV expression. Total glutathione levels increased upon SAM treatment in HCV-replicon cells. Transcriptional antioxidant enzyme expression (SOD-1, SOD-2 and thioredoxin-1) was increased at different times but interestingly, there was no significant change in ROS levels upon SAM treatment, contrary to what was detected with PDTC treatment, where an average 40% reduction was observed in exposed cells. There was a turnover from MAT1A/MAT2A, since MAT1A expression was increased (2.5 fold-times at 48 h) and MAT2A was diminished (from 24 h) upon SAM treatment at both the transcriptional and translational level. CONCLUSION: A likely mechanism(s) by which SAM diminish HCV expression could involve modulating antioxidant enzymes, restoring biosynthesis of glutathione and switching MAT1/MAT2 turnover in HCV expressing cells.


Assuntos
Antioxidantes/metabolismo , Antivirais/farmacologia , Glutationa/biossíntese , Hepacivirus/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Hepatócitos/efeitos dos fármacos , Metionina Adenosiltransferase/metabolismo , S-Adenosilmetionina/farmacologia , Replicação Viral/efeitos dos fármacos , Linhagem Celular , Relação Dose-Resposta a Droga , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hepacivirus/genética , Hepacivirus/crescimento & desenvolvimento , Hepatite C/enzimologia , Hepatite C/genética , Hepatócitos/enzimologia , Interações Hospedeiro-Patógeno , Humanos , Metionina Adenosiltransferase/genética , Estresse Oxidativo/efeitos dos fármacos , RNA Viral/biossíntese , Fatores de Tempo , Transfecção
14.
Virology ; 490: 99-108, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26855332

RESUMO

Hepatitis C virus (HCV) activates PI3K-Akt signaling to enhance entry and replication. Here, we found that this pathway also increased HCV translation. Knocking down the three Akt isoforms significantly decreased, whereas ectopic expression increased HCV translation. HCV translation upregulation by Akt required their kinase activities because Akt kinase-dead mutants downregulated HCV translation; and was dependent on PI3K activity since it was sensitive to PI3K inhibitor wortmannin. The viral 3'UTR was not involved in translation upregulation by Akt. HCV NS5A increased Akt phosphorylation/activity and HCV translation in the absence of the viral 3'UTR. Sterol regulatory element-binding proteins (SREBPs) were the downstream effectors of the PI3K-Akt pathway in regulating HCV translation because Akt1 and Akt2 activated both SREBP-1 and SREBP-2, whereas Akt3 upregulated SREBP-1. Knocking down SREBPs significantly decreased, while ectopic expression of SREBPs increased HCV translation. Taken together, we showed that the PI3K-Akt signaling pathway positively regulates HCV translation through SREBPs.


Assuntos
Hepacivirus/genética , Hepatite C/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Viral/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Hepacivirus/metabolismo , Hepatite C/enzimologia , Hepatite C/genética , Hepatite C/virologia , Interações Hospedeiro-Patógeno , Humanos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , RNA Viral/metabolismo , Transdução de Sinais , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Regulação para Cima
15.
Cell Microbiol ; 18(8): 1121-33, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-26814617

RESUMO

GBF1 is a host factor required for hepatitis C virus (HCV) replication. GBF1 functions as a guanine nucleotide exchange factor for G-proteins of the Arf family, which regulate membrane dynamics in the early secretory pathway and the metabolism of cytoplasmic lipid droplets. Here we established that the Arf-guanine nucleotide exchange factor activity of GBF1 is critical for its function in HCV replication, indicating that it promotes viral replication by activating one or more Arf family members. Arf involvement was confirmed with the use of two dominant negative Arf1 mutants. However, siRNA-mediated depletion of Arf1, Arf3 (class I Arfs), Arf4 or Arf5 (class II Arfs), which potentially interact with GBF1, did not significantly inhibit HCV infection. In contrast, the simultaneous depletion of both Arf4 and Arf5, but not of any other Arf pair, imposed a significant inhibition of HCV infection. Interestingly, the simultaneous depletion of both Arf4 and Arf5 had no impact on the activity of the secretory pathway and induced a compaction of the Golgi and an accumulation of lipid droplets. A similar phenotype of lipid droplet accumulation was also observed when GBF1 was inhibited by brefeldin A. In contrast, the simultaneous depletion of both Arf1 and Arf4 resulted in secretion inhibition and Golgi scattering, two actions reminiscent of GBF1 inhibition. We conclude that GBF1 could regulate different metabolic pathways through the activation of different pairs of Arf proteins.


Assuntos
Fator 1 de Ribosilação do ADP/fisiologia , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Hepacivirus/fisiologia , Hepatite C/virologia , Replicação Viral , Linhagem Celular Tumoral , Hepatite C/enzimologia , Interações Hospedeiro-Patógeno , Humanos , Gotículas Lipídicas , Domínios Proteicos , Transporte Proteico , Via Secretória
16.
HIV Med ; 17(1): 62-7, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26122981

RESUMO

OBJECTIVES: The aim of the study was to establish the risk of liver toxicity in HIV/hepatitis C virus (HCV)-coinfected patients receiving etravirine, according to the degree of liver fibrosis. METHODS: A prospective cohort study of 211 HIV-infected patients initiating an etravirine-containing regimen was carried out. HCV coinfection was defined as a positive HCV RNA test, and baseline liver fibrosis was assessed by transient elastography. Hepatotoxicity was defined as clinical symptoms, or an aspartate aminotransferase (AST) or alanine aminotransferase (ALT) value > 5-fold higher than the upper limit of normal if baseline values were normal, or 3.5-fold higher if values were altered at baseline. RESULTS: Overall, 145 patients (69%) were HCV coinfected, with a lower nadir (165 versus 220 cells/µL, respectively; p = 0.03) and baseline (374 versus 498 cells/µL, respectively; p = 0.04) CD4 count than monoinfected patients. Etravirine was mainly used with two nucleoside reverse transcriptase inhibitors (129; 61%) or with a boosted protease inhibitor (PI) (28%), with no significant differences according to HCV serostatus. Transient elastography in 117 patients (81%) showed a median (range) stiffness value of 8.25 (3.5-69) kPa, with fibrosis stage 1 in 43 patients (37%) and fibrosis stage 4 in 28 patients (24%). During an accumulated follow-up time of 449.3 patient-years (median 548 days), only one patient with advanced fibrosis (50.8 kPa) had grade 3-4 liver toxicity (0.7%). Transaminases changed slightly, with no significant differences compared with baseline fibrosis, and nine and six patients had grade 1 and 2 transaminase increases, respectively. Also, HCV coinfection was not associated with a higher risk of discontinuation (25% discontinued versus 21% of monoinfected patients; p = 0.39, log-rank test) or virological failure (8% versus 12%, respectively; p = 0.4). CONCLUSIONS: Our data suggest that etravirine is a safe option for HIV/HCV-coinfected patients, including those with significant liver fibrosis.


Assuntos
Antirretrovirais/administração & dosagem , Coinfecção/tratamento farmacológico , Infecções por HIV/tratamento farmacológico , Hepatite C/tratamento farmacológico , Cirrose Hepática/epidemiologia , Piridazinas/administração & dosagem , Adulto , Idoso , Alanina Transaminase/metabolismo , Antirretrovirais/efeitos adversos , Aspartato Aminotransferases/metabolismo , Coinfecção/enzimologia , Feminino , Infecções por HIV/enzimologia , Hepatite C/enzimologia , Humanos , Cirrose Hepática/enzimologia , Cirrose Hepática/patologia , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Piridazinas/efeitos adversos , Resultado do Tratamento
17.
J Clin Lab Anal ; 30(3): 200-3, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-25689690

RESUMO

BACKGROUND: CD4(+) cell count, the common HIV infection screening test, is costly and unable to differentiate HIV monoinfection from its concurrent infection with hepatitis B or C virus. We aimed to ascertain diagnostic value of serum adenosine deaminase (ADA) activity as a useful tool to differentiate HIV mono- and co-infection. METHODS: Blood samples were collected from 30 HIV-HBV and 30 HIV-HCV coinfected patients, 33 HIV positive subjects, and 72 controls. CD4(+) cell count, serum total ADA (tADA), and ADA1, and ADA2 isoenzyme activities were determined and their sensitivity and specificity were computed. RESULTS: tADA and ADA2 activities were significantly higher and CD4(+) counts were markedly lower in all patients compared with controls. Strong inverse agreements between CD4(+) cell counts and both tADA and ADA2 activities were observed. Serum tADA and ADA1 activities showed the highest specificity and the highest sensitivity, respectively, for differentiating HIV monoinfection from HIV-HBV and HIV-HCV coinfections. CONCLUSIONS: We showed strong agreement and correlation between CD4(+) cell count and ADA enzyme activity. Based on high ADA sensitivity and specificity, it is concluded that determination of ADA activity might be a novel diagnostic tool to distinguish of HIV monoinfection from its coinfection with HBV or HCV.


Assuntos
Adenosina Desaminase/sangue , Coinfecção/diagnóstico , Infecções por HIV/sangue , Infecções por HIV/diagnóstico , Hepatite B/diagnóstico , Hepatite C/diagnóstico , Adulto , Coinfecção/sangue , Coinfecção/enzimologia , Feminino , Infecções por HIV/enzimologia , Hepatite B/sangue , Hepatite B/enzimologia , Hepatite C/sangue , Hepatite C/enzimologia , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
19.
Clin Lab ; 62(11): 2155-2159, 2016 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-28164655

RESUMO

BACKGROUND: Hepatistis C virus (HCV) affects approximately 170 million people, and it is the leading cause of the chronic liver disease. The destruction of liver cells is reflected by an increase of different enzyme activities in the serum. These enzymes include alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), which play a significant role in the metabolism of many biological substances and exist mainly in the liver. In this study we investigated the activity of alcohol dehydrogenase and its isoenzymes and the total activity of ALDH in the sera of patients with hepatitis C. METHODS: Serum samples were taken for routine biochemical investigations from 50 patients with hepatitis C and from 50 healthy subjects. The activity of class I and II ADH isoenzymes and ALDH activity were measured by spectrofluorometric methods. For the measurement of total ADH activity and activity of class III and IV isoenzymes, the photometric methods were used. RESULTS: The analysis of our results shows a statistically significant increase in the activity of ADH I and ADH II (2.5-fold and 2-fold, respectively). Activities of both classes of alcohol dehydrogenase isoenzymes have good correlation with alanine and aspartate aminotransferase. The observed increase in total alcohol dehydrogenase activity was not very high but confirmed the elevation of class I and II isoenzyme activity. CONCLUSIONS: We can state that the activity of class I and II alcohol dehydrogenase isoenzymes in the sera of patients with hepatitis C is increased and it seems to be caused by the release of these isoenzymes from damaged liver cells.


Assuntos
Álcool Desidrogenase/sangue , Aldeído Desidrogenase/sangue , Hepatite C/sangue , Fígado/enzimologia , Adulto , Idoso , Bilirrubina/sangue , Biomarcadores/sangue , Estudos de Casos e Controles , Feminino , Hepatite C/diagnóstico , Hepatite C/enzimologia , Humanos , Isoenzimas , Fígado/patologia , Masculino , Pessoa de Meia-Idade , Fotometria , Espectrometria de Fluorescência , Regulação para Cima , Adulto Jovem
20.
Cell Immunol ; 300: 18-25, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26632272

RESUMO

Myeloperoxidase (MPO) is an important enzyme in the front-line protection against microorganisms. In peripheral blood, it is accepted that MPO is only produced by myeloid-lineage cells. Thus, MPO presence is unexpected in lymphocytes. We showed recently that B1-lymphocytes from mice have MPO. Here, we showed that subsets of human peripheral B, CD4(+) and CD8(+) T lymphocytes express MPO. The content of MPO in lymphocytes was very low compared to neutrophils/monocytes with a preferential distribution in the nucleus and perinuclear region. Also, we performed a MPO mRNA expression analysis from human blood cells derived from microarray raw data publicly available, showing that MPO is modulated in infectious disease. MPO was increased in CD4(+) T lymphocytes from HIV chronic infection and in CD8(+) T lymphocytes from HCV-positive patients. Our study points out MPO as a multifunctional protein due to its subcellular localization and expression modulation in lymphocytes indicating alternative unknown functions for MPO in lymphocytes.


Assuntos
Linfócitos B/enzimologia , Linfócitos T CD4-Positivos/enzimologia , Linfócitos T CD8-Positivos/enzimologia , Peroxidase/biossíntese , Linfócitos B/imunologia , Western Blotting , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Separação Celular , Citometria de Fluxo , Infecções por HIV/enzimologia , Infecções por HIV/imunologia , Hepatite C/enzimologia , Hepatite C/imunologia , Humanos , Imunofenotipagem , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Análise de Sequência com Séries de Oligonucleotídeos , Peroxidase/imunologia , Reação em Cadeia da Polimerase em Tempo Real
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA