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1.
Acta Trop ; 210: 105621, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32659283

RESUMO

The carcinogenic liver fluke Opisthorchis viverrini causes several hepatobiliary diseases including a bile duct cancer-cholangiocarcinoma (CCA), which is a major public health problem in many countries in the Greater Mekong Sub-region. Praziquantel is the main drug against this parasite, however, reduced drug efficacy has been observed in some endemic areas. Therefore, alternative drugs are needed to prepare for praziquantel resistance in the future. The selenoprotein thioredoxin glutathione reductase (TGR) enzyme, which plays a crucial role in cellular redox balance of parasitic flatworms, has been shown as a potential drug target against these parasites. Hence, this study aimed to investigate the TGR of O. viverrini and assess its potential as a drug target. An open reading frame (ORF) that encodes O. viverrini TGR (Ov-TGR) was cloned from an O. viverrini cDNA library and the nucleotide were sequenced. The 1,812 nucleotides of the Ov-TGR full ORF encoded a polypeptide of 603 amino acid residues with a predicted molecular mass of 66 kDa. The putative amino acid sequence shared 55-96.8% similarities with TGRs from other helminths and mammals. Phylogenetic analysis revealed a close relationship of Ov-TGR with that of other trematodes. The ORF of Ov-TGR was inserted into pABC2 plasmid and transformed into Escherichia coli strain C321.ΔA to facilitate selenocysteine incorporation. The recombinant Ov-TGR (rOv-TGR-SEC) was expressed as a soluble protein and detected as a dimer form in the non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Its thioredoxin reductase (TrxR) and glutathione reductase (GR) activities were detected using DTNB, Trx and GSSG substrates with the Michaelis constant (Km) of 292.6 ± 52.3 µM, 8.09 ± 1.91 µM and 13.74 ± 1.2 µM, respectively. The TGR enzyme activities were effectively inhibited by a well-known inhibitor, auranofin in a dose-dependent manner. Moreover, auranofin expressed a lethal toxic effect on both newly excysted juveniles (NEJs) and adult worms of O. viverrini in vitro. Taken together, these results indicated that Ov-TGR is crucial for O. viverrini survival and maybe a potential target for the development of novel agents against opisthorschiasis.


Assuntos
Complexos Multienzimáticos/fisiologia , NADH NADPH Oxirredutases/fisiologia , Opisthorchis/enzimologia , Animais , Auranofina/farmacologia , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/genética , NADH NADPH Oxirredutases/antagonistas & inibidores , NADH NADPH Oxirredutases/genética , Fases de Leitura Aberta , Opisthorchis/efeitos dos fármacos , Filogenia
2.
Apoptosis ; 22(3): 463-474, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28012059

RESUMO

Anacardic acid is a medicinal phytochemical that inhibits proliferation of fungal as well as several types of cancer cells. It induces apoptotic cell death in various cell types, but very little is known about the mechanism involved in the process. Here, we used budding yeast Saccharomyces cerevisiae as a model to study the involvement of some key elements of apoptosis in the anacardic acid-induced cell death. Plasma membrane constriction, chromatin condensation, DNA degradation, and externalization of phosphatidylserine (PS) indicated that anacardic acid induces apoptotic cell death in S. cerevisiae. However, the exogenous addition of broad-spectrum caspase inhibitor Z-VAD-FMK or deletion of the yeast caspase Yca1 showed that the anacardic acid-induced cell death is caspase independent. Apoptosis-inducing factor (AIF1) deletion mutant was resistant to the anacardic acid-induced cell death, suggesting a key role of Aif1. Overexpression of Aif1 made cells highly susceptible to anacardic acid, further confirming that Aif1 mediates anacardic acid-induced apoptosis. Interestingly, instead of the increase in the intracellular reactive oxygen species (ROS) normally observed during apoptosis, anacardic acid caused a decrease in the intracellular ROS levels. Quantitative real-time PCR analysis showed downregulation of the BIR1 survivin mRNA expression during the anacardic acid-induced apoptosis.


Assuntos
Ácidos Anacárdicos/farmacologia , Apoptose/efeitos dos fármacos , NADH NADPH Oxirredutases/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/efeitos dos fármacos , Antioxidantes/farmacologia , Apoptose/fisiologia , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Metanossulfonato de Etila/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , NADH NADPH Oxirredutases/genética , RNA Fúngico/biossíntese , RNA Fúngico/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/genética
3.
Reprod Fertil Dev ; 29(7): 1447-1455, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27346638

RESUMO

GRIM-19 is associated with apoptosis, abnormal proliferation, immune tolerance and malignant transformation, and it also plays an important role in early embryonic development. Although the homologous deletion of GRIM-19 causes embryonic lethality in mice, the precise role of GRIM-19 in embryo implantation has not been elucidated. Here we show that GRIM-19 plays an important role in endometrial receptivity and embryo implantation. Day 1 to Day 6 pregnant mouse uteri were collected. Immunohistochemistry studies revealed the presence of GRIM-19 on the luminal epithelium and glandular epithelium throughout the implantation period in pregnant mice. The protein and mRNA levels of GRIM-19 were markedly decreased on Day 4 of pregnancy in pregnant mice, but there was no change in GRIM-19 levels in a group of pseudopregnant mice. Overexpression of GRIM-19 decreased the adhesion rate of RL95-2-BeWo co-cultured spheroids and increased apoptosis. Furthermore, STAT3 and IL-11 mRNA and protein levels were reduced by overexpressing GRIM-19, but protein and mRNA levels of TNF-α were increased. These findings indicate the involvement of GRIM-19 in the embryo implantation process by regulating adhesion, apoptosis and immune tolerance.


Assuntos
Implantação do Embrião/genética , Implantação do Embrião/fisiologia , Endométrio/fisiologia , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/fisiologia , Animais , Apoptose/genética , Apoptose/fisiologia , Adesão Celular/genética , Adesão Celular/fisiologia , Técnicas de Cocultura , Implantação do Embrião/imunologia , Endométrio/imunologia , Feminino , Tolerância Imunológica/genética , Tolerância Imunológica/fisiologia , Interleucina-11/genética , Interleucina-11/metabolismo , Camundongos , Gravidez , Pseudogravidez/genética , Pseudogravidez/fisiopatologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Esferoides Celulares , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
4.
J Biol Regul Homeost Agents ; 30(3): 649-653, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27655481

RESUMO

Cancer includes a number of related diseases due to abnormal cell proliferation that spreads to nearby tissues. Many compounds (physical, chemical and biological) have been used to try to halt this abnormal proliferation, but the therapeutic results are poor, due also to the side effects. It has been reported that ecto-nicotinamide adenine dinucleotide oxidase di-sulfide-thiol exchanger 2 (ENOX2), also known as tumor-associated nicotinamide adenine dinucleotide oxidase (tNOX), was found to be located on the cancer cell surface, essential for cancer cell growth. Capsaicin and other anti-oxidants are capable of inhibiting tNOX, causing apoptosis of cells, exerting anti-tumor activity. It is interesting that some authors reported that ENOX2 is present in the serum of cancer patients several years before the clinical symptoms of the tumor. However, this result has to be confirmed. In this article we discuss ENOX2 and its inhibition as a hope of improving cancer therapy.


Assuntos
NADH NADPH Oxirredutases/fisiologia , Neoplasias/tratamento farmacológico , Neoplasias/prevenção & controle , Anticarcinógenos/farmacologia , Anticarcinógenos/uso terapêutico , Antineoplásicos Fitogênicos/farmacologia , Antineoplásicos Fitogênicos/uso terapêutico , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais , Capsaicina/farmacologia , Capsaicina/uso terapêutico , Catequina/análogos & derivados , Catequina/farmacologia , Catequina/uso terapêutico , Regulação para Baixo/efeitos dos fármacos , Detecção Precoce de Câncer , Indução Enzimática/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Isoflavonas/farmacologia , Isoflavonas/uso terapêutico , NAD/fisiologia , NADH NADPH Oxirredutases/antagonistas & inibidores , NADH NADPH Oxirredutases/sangue , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/sangue , Proteínas de Neoplasias/fisiologia , Neoplasias/enzimologia
5.
J Immunol ; 197(6): 2069-78, 2016 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-27489284

RESUMO

Mast cells are among the principal effector cells in the pathogenesis of allergic asthma. In allergic reactions, allergen (Ag)-induced cross-linking of IgE bound to FcεRI on mast cells results in the production of vascular endothelial growth factor (VEGF), which is essential for the initiation and development of the allergic response. Despite the central role of VEGF in allergic asthma, the signaling events responsible for the production of VEGF remain unclear, particularly in Ag-stimulated mast cells. In the present study, we observed that blocking leukotriene B4 receptor 2 (BLT2) completely abrogated the production of VEGF in Ag-stimulated bone marrow-derived mast cells (BMMCs). The synthesis of BLT2 ligands (leukotriene B4 and 12(S)-hydroxyeicosatetraenoic acid) was also required for VEGF production, suggesting a mediating role of an autocrine BLT2 ligands-BLT2 axis in the production of VEGF in mast cells. The NADPH oxidase 1-reactive oxygen species-NF-κB cascade is downstream of BLT2 during Ag signaling to VEGF synthesis in mast cells. Furthermore, the level of VEGF synthesis in genetically mast cell-deficient Kit(W/Wv) mice was significantly lower than that in wild-type mice in the OVA-induced asthma model, suggesting that mast cells play a critical role in the synthesis of VEGF in OVA-induced allergic asthma. Importantly, VEGF production was restored to the levels observed in wild-type mice after adoptive transfer of normal BMMCs into Kit(W/Wv) mice but was not restored in BLT2(-/-) BMMC-reconstituted Kit(W/Wv) mice in the OVA-induced asthma model. Taken together, our results suggest that BLT2 expression in mast cells is essential for the production of VEGF in OVA-induced allergic asthma.


Assuntos
Alérgenos/imunologia , Mastócitos/metabolismo , Receptores do Leucotrieno B4/fisiologia , Fator A de Crescimento do Endotélio Vascular/biossíntese , Animais , Interleucina-13/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , NADH NADPH Oxirredutases/fisiologia , NADPH Oxidase 1 , NF-kappa B/fisiologia , Ovalbumina/imunologia , Espécies Reativas de Oxigênio/metabolismo
6.
Oncotarget ; 7(27): 41404-41420, 2016 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-27167343

RESUMO

Aberrant STAT3 activation occurs in most human gastric cancers (GCs) and contributes to the malignant progression of GC, but mechanism(s) underlying aberrant STAT3 remain largely unknown. Here we demonstrated that the gene associated with retinoid interferon-induced mortality 19 (GRIM-19) was severely depressed or lost in GC and chronic atrophic gastritis (CAG) tissues and its loss contributed to GC tumorigenesis partly by activating STAT3 signaling. In primary human GC tissues, GRIM-19 was frequently depressed or lost and this loss correlated with advanced clinical stage, lymph node metastasis, H. pylori infection and poor overall survival of GC patients. In CAG tissues, GRIM-19 was progressively decreased along with its malignant transformation. Functionally, we indentified an oncogenic role of GRIM-19 loss in promoting GC tumorigenesis. Ectopic GRIM-19 expression suppressed GC tumor formation in vitro and in vivo by inducing cell cycle arrest and apoptosis. Moreover, we revealed that GRIM-19 inhibited STAT3 transcriptional activation and its downstream targets by reducing STAT3 nuclear distribution. Conversely, knockdown of GRIM-19 induced aberrant STAT3 activation and accelerated GC cell growth in vitro and in vivo, and this could be partly attenuated by the blockage of STAT3 activation. In addition, we observed subcellular redistributions of GRIM-19 characterized by peri-nuclear aggregates, non-mitochondria cytoplasmic distribution and nuclear invasion, which should be responsible for reduced STAT3 nuclear distribution. Our studies suggest that mitochondrial GRIM-19 could not only serve as an valuable prognostic biomarker for GC development, but also as a potential therapeutic target for STAT3-dependent carcinogenesis of GC.


Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Carcinogênese/genética , Transformação Celular Neoplásica/genética , Mitocôndrias/metabolismo , NADH NADPH Oxirredutases/fisiologia , Fator de Transcrição STAT3/genética , Neoplasias Gástricas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Mitocôndrias/efeitos dos fármacos , Terapia de Alvo Molecular , NADH NADPH Oxirredutases/metabolismo , Prognóstico , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/mortalidade , Neoplasias Gástricas/patologia
7.
Proc Natl Acad Sci U S A ; 113(12): E1757-66, 2016 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-26951670

RESUMO

The virulence of many pathogens depends upon their ability to cope with immune-generated nitric oxide (NO·). In Escherichia coli, the major NO· detoxification systems are Hmp, an NO· dioxygenase (NOD), and NorV, an NO· reductase (NOR). It is well established that Hmp is the dominant system under aerobic conditions, whereas NorV dominates anaerobic conditions; however, the quantitative contributions of these systems under the physiologically relevant microaerobic regime remain ill defined. Here, we investigated NO· detoxification in environments ranging from 0 to 50 µM O2, and discovered a regime in which E. coli NO· defenses were severely compromised, as well as conditions that exhibited oscillations in the concentration of NO·. Using an integrated computational and experimental approach, E. coli NO· detoxification was found to be extremely impaired at low O2 due to a combination of its inhibitory effects on NorV, Hmp, and translational activities, whereas oscillations were found to result from a kinetic competition for O2 between Hmp and respiratory cytochromes. Because at least 777 different bacterial species contain the genetic requirements of this stress response oscillator, we hypothesize that such oscillatory behavior could be a widespread phenomenon. In support of this hypothesis,Pseudomonas aeruginosa, whose respiratory and NO· response networks differ considerably from those of E. coli, was found to exhibit analogous oscillations in low O2 environments. This work provides insight into how bacterial NO· defenses function under the low O2 conditions that are likely to be encountered within host environments.


Assuntos
Escherichia coli/metabolismo , Óxido Nítrico/metabolismo , Aerobiose , Simulação por Computador , Di-Hidropteridina Redutase/fisiologia , Proteínas de Escherichia coli/fisiologia , Hemeproteínas/fisiologia , Interações Hospedeiro-Patógeno , Modelos Biológicos , NADH NADPH Oxirredutases/fisiologia , Oxirredutases/fisiologia , Oxigênio/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo , Especificidade da Espécie
8.
Exp Anim ; 65(3): 197-205, 2016 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-26876598

RESUMO

Accumulating evidence suggests that reactive oxygen species (ROS) generated by endogenous metabolic enzymes are involved in a variety of intracellular mechanisms. In particular, superoxide-generating NADPH oxidase (Nox) 1 is highly expressed in the colon and has been implicated in physiological and pathophysiological states of colon tissues. However, its role in tissue repair following colitis has not been fully elucidated. Our study using experimental colitis in mice showed that repair of the mucosal layer did not occur in Nox1-deficient mice following dextran sulfate sodium-induced colitis. This was accompanied by inhibition of proliferation, cell survival, migration, and terminal differentiation (generation of goblet cells) of crypt progenitor cells, as determined by histochemical analyses. Furthermore, Nox1 expression as well as ROS production in the colon crypt was increased during the repair process, and Nox1 deficiency suppressed these events. The results suggest that Nox1 promotes colon mucosal wound repair by sustaining the bioactivity of crypt progenitor cells and plays a crucial role in the epithelial restitution in the case of damage associated with colitis.


Assuntos
Colite/patologia , Colite/fisiopatologia , Colo , Mucosa Intestinal/enzimologia , Mucosa Intestinal/fisiologia , NADH NADPH Oxirredutases/metabolismo , NADH NADPH Oxirredutases/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Regeneração , Animais , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Colite/induzido quimicamente , Sulfato de Dextrana , Expressão Gênica , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Camundongos , NADH NADPH Oxirredutases/genética , NADPH Oxidase 1
9.
J Pharmacol Sci ; 127(3): 370-6, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25837936

RESUMO

Although it has been reported that endotoxin-induced expression of Nox1 in the heart contributes to apoptosis in cardiomyocytes, functional role of Nox1 at the physiological expression level has not been elucidated. The aim of this study was to clarify the role of Nox1 under a hypoxic condition using wild-type (WT, Nox1(+/Y)) and Nox1-deficient (Nox1(-/Y)) mice. ECG recordings from anesthetized mice revealed that Nox1(-/Y) mice were more sensitive to hypoxia, resulting in bradycardia, compared to WT mice. Atrial and ventricular electrocardiograms recorded from Langendorff-perfused hearts revealed that hypoxic perfusion more rapidly decreased heart rate in Nox1(-/Y) hearts compared with WT hearts. Sinus node recovery times measured under a hypoxic condition were prolonged more markedly in the Nox1(-/Y) hearts. Sinoatrial node dysfunction of Nox1(-/Y) hearts during hypoxia was ameriolated by the pre-treatment with the Ca(2+) channel blocker nifedipine or the K(+) channel opener pinacidil. Spontaneous action potentials were recorded from enzymatically-isolated sinoatrial node (SAN) cells under a hypoxic condition. There was no significant difference in the elapsed times from the commencement of hypoxia to asystole between WT and Nox1(-/Y) SAN cells. These findings suggest that Nox1 may have a protective effect against hypoxia-induced SAN dysfunction.


Assuntos
Bradicardia/etiologia , Bradicardia/prevenção & controle , Hipóxia/complicações , NADH NADPH Oxirredutases/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Bradicardia/fisiopatologia , Bloqueadores dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/uso terapêutico , Modelos Animais de Doenças , Eletrocardiografia , Frequência Cardíaca , Hipóxia/fisiopatologia , Técnicas In Vitro , Moduladores de Transporte de Membrana/farmacologia , Moduladores de Transporte de Membrana/uso terapêutico , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , NADPH Oxidase 1 , Nifedipino/farmacologia , Nifedipino/uso terapêutico , Pinacidil/farmacologia , Pinacidil/uso terapêutico , Bloqueio Sinoatrial/etiologia , Bloqueio Sinoatrial/fisiopatologia , Bloqueio Sinoatrial/prevenção & controle , Nó Sinoatrial/citologia , Nó Sinoatrial/fisiopatologia
10.
Cardiovasc Res ; 106(1): 121-30, 2015 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-25661082

RESUMO

AIMS: Sphingosylphosphorylcholine (SPC) elicits vasoconstriction at micromolar concentrations. At lower concentrations (≤1 µmol/L), however, it does not constrict intrapulmonary arteries (IPAs), but strongly potentiates vasoreactivity. Our aim was to determine whether this also occurs in a systemic artery and to delineate the signalling pathway. METHODS AND RESULTS: Rat mesenteric arteries and IPAs mounted on a myograph were challenged with ∼25 mmol/L [K+] to induce a small vasoconstriction. SPC (1 µmol/L) dramatically potentiated this constriction in all arteries by ∼400%. The potentiation was greatly suppressed or abolished by inhibition of phospholipase C (PLC; U73122), PKCε (inhibitory peptide), Src (PP2), and NADPH oxidase (VAS2870), and also by Tempol (superoxide scavenger), but not by inhibition of Rho kinase (Y27632). Potentiation was lost in mesenteric arteries from p47(phox-/-), but not NOX2(-/-), mice. The intracellular superoxide generator LY83583 mimicked the effect of SPC. SPC elevated reactive oxygen species (ROS) in vascular smooth muscle cells, and this was blocked by PP2, VAS2870, and siRNA knockdown of PKCε. SPC (1 µmol/L) significantly reduced the EC50 for U46619-induced vasoconstriction, an action ablated by Tempol. In patch-clamped mesenteric artery cells, SPC (200 nmol/L) enhanced Ba2+ current through L-type Ca2+ channels, an action abolished by Tempol but mimicked by LY83583. CONCLUSION: Our results suggest that low concentrations of SPC activate a PLC-coupled and NOX1-mediated increase in ROS, with consequent enhancement of voltage-gated Ca2+ entry and thus vasoreactivity. We speculate that this pathway is not specific for SPC, but may also contribute to vasoconstriction elicited by other G-protein coupled receptor and PLC-coupled agonists.


Assuntos
Canais de Cálcio/efeitos dos fármacos , Artérias Mesentéricas/fisiologia , NADH NADPH Oxirredutases/fisiologia , Fosforilcolina/análogos & derivados , Artéria Pulmonar/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Esfingosina/análogos & derivados , Vasoconstrição/efeitos dos fármacos , Animais , Canais de Cálcio/fisiologia , Óxidos N-Cíclicos/farmacologia , Relação Dose-Resposta a Droga , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiologia , Artérias Mesentéricas/efeitos dos fármacos , Camundongos , Camundongos Knockout , Modelos Animais , NADPH Oxidase 1 , NADPH Oxidase 2 , NADPH Oxidases/deficiência , NADPH Oxidases/genética , NADPH Oxidases/farmacologia , NADPH Oxidases/fisiologia , Fosforilcolina/farmacologia , Proteína Quinase C-épsilon/farmacologia , Artéria Pulmonar/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Esfingosina/farmacologia , Marcadores de Spin , Fosfolipases Tipo C/farmacologia , Vasoconstrição/fisiologia
11.
Free Radic Biol Med ; 83: 21-30, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25701431

RESUMO

Increased oxidative stress and activation of protein kinase C (PKC) under hyperglycemia have been implicated in the development of diabetic nephropathy. Because reactive oxygen species derived from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, NOX1 accelerate the translocation of PKC isoforms, NOX1 is postulated to play a causative role in the development of diabetic nephropathy. Hyperglycemia was induced in wild-type and Nox1-deficient mice (KO) by two doses of streptozotocin injection. At 3 weeks after the induction of hyperglycemia, glomeruli and cortical tubules were isolated from kidneys. The mRNA level of Nox1 was significantly upregulated in the renal cortex at 3 weeks of hyperglycemia. Urinary albumin and expression of inflammatory or fibrotic mediators were similarly elevated in diabetic wild-type and KO; however, increases in glomerular volume and mesangial matrix area were attenuated in diabetic KO. Nox1 deficiency significantly reduced the levels of renal thiobarbituric acid-reacting substances and 8-hydroxydeoxyguanosine, membranous translocation of PKCα/ß, activity of PKC, and phosphorylation of p38 mitogen-activated protein kinase in the diabetic kidney. Furthermore, increased staining of senescence-associated ß-galactosidase in glomeruli and cortical tubules of diabetic mice was significantly suppressed in KO. Whereas the levels of cyclin-dependent kinase inhibitors, p16(INK4A) and p21(Cip1), were equivalent between the genotypes, increased levels of p27(Kip1) and γ-H2AX, a biomarker for DNA double-strand breaks, were significantly attenuated in isolated glomeruli and cortical tubules of diabetic KO. Taken together, NOX1 modulates the p38/p27(Kip1) signaling pathway by activating PKC and promotes premature senescence in early stage diabetic nephropathy.


Assuntos
Senescência Celular/fisiologia , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/patologia , Mesângio Glomerular/patologia , Hiperglicemia/patologia , NADH NADPH Oxirredutases/fisiologia , Proteína Quinase C/metabolismo , Animais , Western Blotting , Células Cultivadas , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/metabolismo , Nefropatias Diabéticas/induzido quimicamente , Nefropatias Diabéticas/metabolismo , Mesângio Glomerular/metabolismo , Hiperglicemia/induzido quimicamente , Hiperglicemia/metabolismo , Técnicas Imunoenzimáticas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidase 1 , Oxirredução , Estresse Oxidativo , Proteína Quinase C/genética , RNA Mensageiro/genética , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , beta-Galactosidase/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
12.
Hypertension ; 65(3): 561-8, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25489057

RESUMO

Null mutations in the p67(phox) subunit of nicotinamide adenine dinucleotide phosphate-oxidase confer protection from salt sensitivity on Dahl salt-sensitive rats. Here, we track the sequential changes in medullary blood flow (MBF), glomerular filtration rate (GFR), urinary protein, and mean arterial pressure in SSp67(phox) null rats and wild-type littermates during 21 days of 4.0% NaCl high-salt (HS) diet. Optical fibers were implanted in the renal medulla and MBF was measured in conscious rats by laser Doppler flowmetry. Separate groups of rats were prepared with femoral venous catheters and GFR was measured by the transcutaneous assessment of fluorescein isothiocyanate-sinistrin disappearance curves. Mean arterial blood pressure was measured by telemetry. In wild-type rats, HS caused a rapid reduction in MBF, which was significantly lower than control values by HS day-6. Reduced MBF was associated with a progressive increase in mean arterial pressure, averaging 170±5 mm Hg by HS salt day-21. A significant reduction in GFR was evident on day-14 HS, after the onset of hypertension and reduced MBF. In contrast, HS had no significant effect on MBF in SSp67(phox) null rats and the pressor response to sodium was blunted, averaging 150±3 mm Hg on day-21 HS. GFR was maintained throughout the study and proteinuria was reduced. In summary, when p67(phox) is not functional in the salt-sensitive rats, HS does not cause reduced MBF and salt-sensitive hypertension is attenuated, and consequently renal injury is reduced and GFR is maintained.


Assuntos
Taxa de Filtração Glomerular/efeitos dos fármacos , Hipertensão/prevenção & controle , Medula Renal/irrigação sanguínea , Mutação/genética , NADH NADPH Oxirredutases/deficiência , Fluxo Sanguíneo Regional/efeitos dos fármacos , Cloreto de Sódio na Dieta/farmacologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Creatinina/metabolismo , Modelos Animais de Doenças , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Masculino , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/fisiologia , Proteinúria/fisiopatologia , Proteinúria/prevenção & controle , Ratos , Ratos Endogâmicos Dahl , Ratos Mutantes , Espécies Reativas de Oxigênio/metabolismo , Fluxo Sanguíneo Regional/fisiologia , Cloreto de Sódio na Dieta/efeitos adversos
13.
Free Radic Biol Med ; 76: 173-84, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25151121

RESUMO

Nicotine contributes to the onset and progression of several pulmonary diseases. Among the various pathophysiological mechanisms triggered by nicotine, oxidative stress and cell death are reported in several cell types. We found that chronic exposure to nicotine (48h) induced NOX1-dependent oxidative stress and apoptosis in primary pulmonary cells. In murine (MLE-12) and human (BEAS-2B) lung epithelial cell lines, nicotine acted as a sensitizer to cell death and synergistically enhanced apoptosis when cells were concomitantly exposed to hyperoxia. The precise signaling pathway was investigated in MLE-12 cells in which NOX1 was abrogated by a specific inhibitor or stably silenced by shRNA. In the early phase of exposure (1h), nicotine mediated intracellular Ca(2+) fluxes and activation of protein kinase C, which in its turn activated NOX1, leading to cellular and mitochondrial oxidative stress. The latter triggered the intrinsic apoptotic machinery by modulating the expression of Bcl-2 and Bax. Overexpression of Bcl-2 completely prevented nicotine's detrimental effects, suggesting Bcl-2as a downstream key regulator in nicotine/NOX1-induced cell damage. These results suggest that NOX1 is a major contributor to the generation of intracellular oxidative stress induced by nicotine and might be an important molecule to target in nicotine-related lung pathologies.


Assuntos
Apoptose/efeitos dos fármacos , Células Epiteliais/patologia , Pulmão/patologia , NADH NADPH Oxirredutases/fisiologia , Nicotina/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Western Blotting , Cálcio/metabolismo , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , NADH NADPH Oxirredutases/antagonistas & inibidores , NADPH Oxidase 1 , Agonistas Nicotínicos/farmacologia , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismo
14.
PLoS One ; 9(7): e101669, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25014110

RESUMO

Ulcerative colitis (UC) is a chronic inflammatory bowel disease affecting the rectum which progressively extents. Its etiology remains unknown and the number of treatments available is limited. Studies of UC patients have identified an unbalanced endoplasmic reticulum (ER) stress in the non-inflamed colonic mucosa. Animal models with impaired ER stress are sensitive to intestinal inflammation, suggesting that an unbalanced ER stress could cause inflammation. However, there are no ER stress-regulating strategies proposed in the management of UC partly because of the lack of relevant preclinical model mimicking the disease. Here we generated the IL10/Nox1dKO mouse model which combines immune dysfunction (IL-10 deficiency) and abnormal epithelium (NADPH oxidase 1 (Nox1) deficiency) and spontaneously develops a UC-like phenotype with similar complications (colorectal cancer) than UC. Our data identified an unanticipated combined role of IL10 and Nox1 in the fine-tuning of ER stress responses in goblet cells. As in humans, the ER stress was unbalanced in mice with decreased eIF2α phosphorylation preceding inflammation. In IL10/Nox1dKO mice, salubrinal preserved eIF2α phosphorylation through inhibition of the regulatory subunit of the protein phosphatase 1 PP1R15A/GADD34 and prevented colitis. Thus, this new experimental model highlighted the central role of epithelial ER stress abnormalities in the development of colitis and defined the defective eIF2α pathway as a key pathophysiological target for UC. Therefore, specific regulators able to restore the defective eIF2α pathway could lead to the molecular remission needed to treat UC.


Assuntos
Colite Ulcerativa/etiologia , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático , Inflamação/etiologia , Interleucina-10/fisiologia , NADH NADPH Oxirredutases/fisiologia , Animais , Western Blotting , Estudos de Casos e Controles , Proliferação de Células , Células Cultivadas , Colite Ulcerativa/metabolismo , Colite Ulcerativa/patologia , Colo/imunologia , Colo/metabolismo , Colo/patologia , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Feminino , Citometria de Fluxo , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , Inflamação/metabolismo , Inflamação/patologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidase 1 , Fosforilação , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Resposta a Proteínas não Dobradas
15.
Cancer Res ; 74(1): 38-43, 2014 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-24247717

RESUMO

ENOX1 is a highly conserved NADH oxidase that helps to regulate intracellular nicotinamide adenine dinucleotide levels in many cell types, including endothelial cells. Pharmacologic and RNA interference (RNAi)-mediated suppression of ENOX1 impairs surrogate markers of tumor angiogenesis/vasculogenesis, providing support for the concept that ENOX1 represents an antiangiogenic druggable target. However, direct genetic evidence that demonstrates a role for ENOX1 in vascular development is lacking. In this study, we exploited a zebrafish embryonic model of development to address this question. Whole-mount in situ hybridization coupled with immunofluorescence performed on zebrafish embryos demonstrate that enox1 message and translated protein are expressed in most tissues, and its expression is enriched in blood vessels and heart. Morpholino-mediated suppression of Enox1 in Tg(fli1-eGFP) and Tg(flk1-eGFP) zebrafish embryos significantly impairs the development of vasculature and blood circulation. Using in vivo multiphoton microscopy, we show that morpholino-mediated knockdown of enox1 increases NADH levels, consistent with loss of enzyme. VJ115 is a small-molecule inhibitor of Enox1's oxidase activity shown to increase intracellular NADH in endothelial cells; we used VJ115 to determine if the oxidase activity was crucial for vascular development. We found that VJ115 suppressed vasculogenesis in Tg(fli1-eGFP) embryos and impaired circulation. Previously, it was shown that suppression of ENOX1 radiosensitizes proliferating tumor vasculature, a consequence of enhanced endothelial cell apoptosis. Thus, our current findings, coupled with previous research, support the hypothesis that ENOX1 represents a potential cancer therapy target, one that combines molecular targeting with cytotoxic sensitization.


Assuntos
Endotélio Vascular/embriologia , Endotélio Vascular/crescimento & desenvolvimento , Complexos Multienzimáticos/fisiologia , NADH NADPH Oxirredutases/fisiologia , Animais , Animais Geneticamente Modificados , Endotélio Vascular/enzimologia , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Neovascularização Fisiológica/fisiologia , Peixe-Zebra
17.
Circulation ; 127(18): 1888-902, 2013 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-23564668

RESUMO

BACKGROUND: In diabetes mellitus, vascular complications such as atherosclerosis are a major cause of death. The key underlying pathomechanisms are unclear. However, hyperglycemic oxidative stress derived from NADPH oxidase (Nox), the only known dedicated enzyme to generate reactive oxygen species appears to play a role. Here we identify the Nox1 isoform as playing a key and pharmacologically targetable role in the accelerated development of diabetic atherosclerosis. METHODS AND RESULTS: Human aortic endothelial cells exposed to hyperglycemic conditions showed increased expression of Nox1, oxidative stress, and proinflammatory markers in a Nox1-siRNA reversible manner. Similarly, the specific Nox inhibitor, GKT137831, prevented oxidative stress in response to hyperglycemia in human aortic endothelial cells. To examine these observations in vivo, we investigated the role of Nox1 on plaque development in apolipoprotein E-deficient mice 10 weeks after induction of diabetes mellitus. Deletion of Nox1, but not Nox4, had a profound antiatherosclerotic effect correlating with reduced reactive oxygen species formation, attenuation of chemokine expression, vascular adhesion of leukocytes, macrophage infiltration, and reduced expression of proinflammatory and profibrotic markers. Similarly, treatment of diabetic apolipoprotein E-deficient mice with GKT137831 attenuated atherosclerosis development. CONCLUSIONS: These studies identify a major pathological role for Nox1 and suggest that Nox1-dependent oxidative stress is a promising target for diabetic vasculopathies, including atherosclerosis.


Assuntos
Aterosclerose/enzimologia , Aterosclerose/etiologia , Diabetes Mellitus Experimental/enzimologia , NADH NADPH Oxirredutases/fisiologia , NADPH Oxidases/fisiologia , Animais , Aterosclerose/patologia , Células Cultivadas , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/patologia , Células Endoteliais/enzimologia , Células Endoteliais/patologia , Humanos , Mediadores da Inflamação/fisiologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , NADPH Oxidase 1 , Técnicas de Cultura de Órgãos , Isoformas de Proteínas/fisiologia , Espécies Reativas de Oxigênio/metabolismo
18.
Acta Crystallogr D Biol Crystallogr ; 69(Pt 3): 324-32, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23519407

RESUMO

The synthesis of the dipeptide antibiotic bacilysin involves the sequential action of multiple enzymes in the bac operon. YwfH (also referred to as BacG) catalyzes the stereoselective reduction of dihydro-hydroxyphenylpyruvate (H2HPP) to tetrahydro-hydroxyphenylpyruvate (H4HPP) in this biosynthetic pathway. YwfH is an NADPH-dependent reductase that facilitates the conjugate addition of a hydride at the C4 olefin terminus of H2HPP. Here, the structure of YwfH is described at three conformational steps: the apo form, an apo-like conformation and the NADPH complex. YwfH is structurally similar to other characterized short-chain dehydrogenase/reductases despite having marginal sequence similarity. The structures of YwfH in different conformational states provide a rationale for the ping-pong reaction mechanism. The identification and role of the residues in the catalytic tetrad (Lys113-Tyr117-Ser155-Asn158) in proton transfer were examined by mutational analysis. Together, the structures and biochemical features revealed synchronized conformational changes that facilitate cofactor specificity and catalysis of H4HPP formation en route to tetrahydrotyrosine synthesis.


Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Tirosina/biossíntese , Apoproteínas/química , Apoproteínas/genética , Apoproteínas/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Domínio Catalítico/genética , Cristalografia por Raios X , Ácido Graxo Sintases/química , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/fisiologia , NADH NADPH Oxirredutases/química , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/fisiologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Ligação Proteica/genética , Conformação Proteica , Especificidade por Substrato/genética , Tirosina/análogos & derivados , Tirosina/química
19.
Arterioscler Thromb Vasc Biol ; 32(12): 2966-73, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23087362

RESUMO

OBJECTIVE: Although the matricellular protein thrombospondin-1 (TSP1) is highly expressed in the vessel wall in response to injury, its pathophysiological role in the development of vascular disease is poorly understood. This study was designed to test the hypothesis that TSP1 stimulates reactive oxygen species production in vascular smooth muscle cells and induces vascular dysfunction by promoting oxidative stress. METHODS AND RESULTS: Nanomolar concentrations of TSP1 found in human vascular disease robustly stimulated superoxide (O(2)(•-)) levels in vascular smooth muscle cells at both cellular and tissue level as measured by cytochrome c and electron paramagnetic resonance. A peptide mimicking the C terminus of TSP1 known to specifically bind CD47 recapitulated this response. Transcriptional knockdown of CD47 and a monoclonal inhibitory CD47 antibody abrogated TSP1-triggered O(2)(•-) in vitro and ex vivo. TSP1 treatment of vascular smooth muscle cells activated phospholipase C and protein kinase C, resulting in phosphorylation of the NADPH oxidase organizer subunit p47(phox) and subsequent Nox1 activation, leading to impairment of arterial vasodilatation ex vivo. Further, we observed that blockade of CD47 and NADPH oxidase 1 gene silencing in vivo in rats improves TSP1-induced impairment of tissue blood flow after ischemia reperfusion. CONCLUSIONS: Our data suggest a highly regulated process of reactive oxygen species stimulation and blood flow regulation promoted through a direct TSP1/CD47-mediated activation of Nox1. This is the first report, to our knowledge, of a matricellular protein acting as a ligand for NADPH oxidase activation and through specific engagement of integrin-associated protein CD47.


Assuntos
Antígeno CD47/fisiologia , Músculo Liso Vascular/fisiologia , NADH NADPH Oxirredutases/fisiologia , Fluxo Sanguíneo Regional/fisiologia , Trombospondina 1/fisiologia , Animais , Antígeno CD47/genética , Inativação Gênica , Masculino , Camundongos , Músculo Liso Vascular/efeitos dos fármacos , NADH NADPH Oxirredutases/genética , NADPH Oxidase 1 , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Proteína Quinase C/fisiologia , Ratos , Fluxo Sanguíneo Regional/efeitos dos fármacos , Transdução de Sinais/fisiologia , Superóxido Dismutase/fisiologia , Superóxido Dismutase-1 , Trombospondina 1/farmacologia , Fosfolipases Tipo C/fisiologia
20.
Biochim Biophys Acta ; 1820(11): 1744-52, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22846226

RESUMO

BACKGROUND: Tumor-associated NADH oxidase (tNOX; ENOX2) is a growth-related protein expressed in transformed cells. High concentrations of numerous chemotherapeutic agents have shown to inhibit tNOX activity and protein levels leading to a reduction in cell growth while little is known for the effects of low concentrations of chemotherapeutic agents on tNOX expression. METHODS: Effects of chemotherapeutic agents on cell function were evaluated with traditional in vitro assays and the xCELLigence System. Western blot analyses were used to study protein expression profiles of the epithelial-to-mesenchymal transition. RESULTS: We showed that doxorubicin treatment transiently up-regulates tNOX expression in human lung carcinoma A549 cells in association with enhanced cell migration. Similar results were observed in tamoxifen-exposed A549 cells. Furthermore, protein marker analyses revealed that the enhanced migration induced by tamoxifen was correlated with epithelial-to-mesenchymal transition, as evidenced by down-regulation of epithelial markers and up-regulation of mesenchymal markers. Importantly, tNOX overexpression enhanced cell migration, confirming the essential role of tNOX in cell migration. CONCLUSIONS: Based on these findings, we conclude that doxorubicin and tamoxifen induce a transient up-regulation of tNOX expression, leading to enhanced cell migration and EMT. GENERAL SIGNIFICANCE: These findings establish an essential role for tNOX in cell migration and survival and may provide a rational framework for the further development of tNOX inhibitors as a novel class of antitumor agents.


Assuntos
Antineoplásicos/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , NADH NADPH Oxirredutases/fisiologia , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Humanos , Camundongos , NADH NADPH Oxirredutases/antagonistas & inibidores , NADH NADPH Oxirredutases/genética , Estresse Oxidativo , Tamoxifeno/farmacologia , Regulação para Cima
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