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1.
Nucleic Acids Res ; 52(11): 6543-6557, 2024 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-38752483

RESUMO

Pif1 helicase functions in both the nucleus and mitochondria. Pif1 tightly couples ATP hydrolysis, single-stranded DNA translocation, and duplex DNA unwinding. We investigated two Pif1 variants (F723A and T464A) that have each lost one site of interaction of the protein with the DNA substrate. Both variants exhibit minor reductions in affinity for DNA and ATP hydrolysis but have impaired DNA unwinding activity. However, these variants translocate on single-stranded DNA faster than the wildtype enzyme and can slide on the DNA substrate in an ATP-independent manner. This suggests they have lost their grip on the DNA, interfering with coupling ATP hydrolysis to translocation and unwinding. Yeast expressing these variants have increased gross chromosomal rearrangements, increased telomere length, and can overcome the lethality of dna2Δ, similar to phenotypes of yeast lacking Pif1. However, unlike pif1Δ mutants, they are viable on glycerol containing media and maintain similar mitochondrial DNA copy numbers as Pif1 wildtype. Overall, our data indicate that a tight grip of the trailing edge of the Pif1 enzyme on the DNA couples ATP hydrolysis to DNA translocation and DNA unwinding. This tight grip appears to be essential for the Pif1 nuclear functions tested but is dispensable for mitochondrial respiratory growth.


Assuntos
Núcleo Celular , DNA Helicases , DNA Mitocondrial , Mitocôndrias , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Núcleo Celular/metabolismo , DNA Helicases/metabolismo , DNA Helicases/genética , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/genética , Hidrólise , Mitocôndrias/metabolismo , Mitocôndrias/genética , Mitocôndrias/enzimologia , Mutação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
2.
Nucleic Acids Res ; 49(1): 416-431, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33313902

RESUMO

G-Quadruplexes are non-B form DNA structures present at regulatory regions in the genome, such as promoters of proto-oncogenes and telomeres. The prominence in such sites suggests G-quadruplexes serve an important regulatory role in the cell. Indeed, oxidized G-quadruplexes found at regulatory sites are regarded as epigenetic elements and are associated with an interlinking of DNA repair and transcription. PARP-1 binds damaged DNA and non-B form DNA, where it covalently modifies repair enzymes or chromatin-associated proteins respectively with poly(ADP-ribose) (PAR). PAR serves as a signal in regulation of transcription, chromatin remodeling, and DNA repair. PARP-1 is known to bind G-quadruplexes with stimulation of enzymatic activity. We show that PARP-1 binds several G-quadruplex structures with nanomolar affinities, but only a subset promote PARP-1 activity. The G-quadruplex forming sequence found in the proto-oncogene c-KIT promoter stimulates enzymatic activity of PARP-1. The loop-forming characteristics of the c-KIT G-quadruplex sequence regulate PARP-1 catalytic activity, whereas eliminating these loop features reduces PARP-1 activity. Oxidized G-quadruplexes that have been suggested to form unique, looped structures stimulate PARP-1 activity. Our results support a functional interaction between PARP-1 and G-quadruplexes. PARP-1 enzymatic activation by G-quadruplexes is dependent on the loop features and the presence of oxidative damage.


Assuntos
Quadruplex G , Poli(ADP-Ribose) Polimerase-1/metabolismo , Catálise , Dano ao DNA , Ativação Enzimática , Guanina/análogos & derivados , Guanina/química , Humanos , Oxirredução , Regiões Promotoras Genéticas , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato
3.
J Pharmacol Exp Ther ; 371(2): 278-289, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31439806

RESUMO

Doxorubicin is a risk factor for secondary lymphedema in cancer patients exposed to surgery or radiation. The risk is presumed to relate to its cytotoxicity. However, the present study provides initial evidence that doxorubicin directly inhibits lymph flow and this action appears distinct from its cytotoxic activity. We used real-time edge detection to track diameter changes in isolated rat mesenteric lymph vessels. Doxorubicin (0.5-20 µmol/l) progressively constricted lymph vessels and inhibited rhythmic contractions, reducing flow to 24.2% ± 7.7% of baseline. The inhibition of rhythmic contractions by doxorubicin paralleled a tonic rise in cytosolic Ca2+ concentration in lymphatic muscle cells, which was prevented by pharmacological antagonism of ryanodine receptors. Washout of doxorubicin partially restored lymph vessel contractions, implying a pharmacological effect. Subsequently, high-speed optical imaging was used to assess the effect of doxorubicin on rat mesenteric lymph flow in vivo. Superfusion of doxorubicin (0.05-10 µmol/l) maximally reduced volumetric lymph flow to 34% ± 11.6% of baseline. Likewise, doxorubicin (10 mg/kg) administered intravenously to establish clinically achievable plasma concentrations also maximally reduced volumetric lymph flow to 40.3% ± 6.0% of initial values. Our findings reveal that doxorubicin at plasma concentrations achieved during chemotherapy opens ryanodine receptors to induce "calcium leak" from the sarcoplasmic reticulum in lymphatic muscle cells and reduces lymph flow, an event linked to lymph vessel damage and the development of lymphedema. These results infer that pharmacological block of ryanodine receptors in lymphatic smooth muscle cells may mitigate secondary lymphedema in cancer patients subjected to doxorubicin chemotherapy. SIGNIFICANCE STATEMENT: Doxorubicin directly inhibits the rhythmic contractions of collecting lymph vessels and reduces lymph flow as a possible mechanism of secondary lymphedema, which is associated with the administration of anthracycline-based chemotherapy. The inhibitory effects of doxorubicin on rhythmic contractions and flow in isolated lymph vessels were prevented by pharmacological block of ryanodine receptors, thereby identifying the ryanodine receptor family of proteins as potential therapeutic targets for the development of new antilymphedema medications.


Assuntos
Doxorrubicina/farmacologia , Linfa/metabolismo , Vasos Linfáticos/metabolismo , Células Musculares/metabolismo , Contração Muscular/fisiologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Relação Dose-Resposta a Droga , Linfa/efeitos dos fármacos , Vasos Linfáticos/efeitos dos fármacos , Masculino , Células Musculares/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Ratos , Ratos Sprague-Dawley
4.
Bioorg Med Chem Lett ; 29(3): 430-434, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30578035

RESUMO

The hepatitis C virus (HCV) represents a substantial threat to human health worldwide. The virus expresses a dual-function protein, NS3 having both protease and RNA helicase activities that are essential for productive viral replication and sustained infections. While viral protease and polymerase inhibitors have shown great successes in treating chronic HCV infections, drugs that specifically target the helicase activity have not advanced. A robust and quantitative 96-well plate-based fluorescent DNA unwinding assay was used to screen a class of indole thio-barbituric acid (ITBA) analogs using the full-length, recombinant HCV NS3, and identified three naphthoyl-containing analogs that efficiently inhibited NS3 helicase activity in a dose-dependent manner, with observed IC50 values of 21-24 µM. Standard gel electrophoresis helicase assays using radiolabeled duplex DNA and RNA NS3 substrates confirmed the inhibition of NS3 unwinding activity. Subsequent anisotropy measurements demonstrated that the candidate compounds did not disrupt NS3 binding to nucleic acids. Additionally, the rate of ATP hydrolysis and the protease activity were also not affected by the inhibitors. Thus, these results indicate that the three ITBA analogs containing N-naphthoyl moieties are the foundation of a potential series of small molecules capable of inhibiting NS3 activity via a novel interaction with the helicase domain that prevents the productive unwinding of nucleic acid substrates, and may represent the basis for a new class of therapeutic agents with the potential to aid in the treatment and eradication of hepatitis C virus.


Assuntos
Inibidores Enzimáticos/farmacologia , Indóis/farmacologia , RNA Helicases/antagonistas & inibidores , Tiobarbitúricos/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Hepacivirus , Indóis/química , Estrutura Molecular , RNA Helicases/metabolismo , Relação Estrutura-Atividade , Tiobarbitúricos/química , Proteínas não Estruturais Virais/metabolismo
5.
J Biol Chem ; 291(34): 18041-57, 2016 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-27369081

RESUMO

Cells engage numerous signaling pathways in response to oxidative stress that together repair macromolecular damage or direct the cell toward apoptosis. As a result of DNA damage, mitochondrial DNA or nuclear DNA has been shown to enter the cytoplasm where it binds to "DNA sensors," which in turn initiate signaling cascades. Here we report data that support a novel signaling pathway in response to oxidative stress mediated by specific guanine-rich sequences that can fold into G-quadruplex DNA (G4DNA). In response to oxidative stress, we demonstrate that sequences capable of forming G4DNA appear at increasing levels in the cytoplasm and participate in assembly of stress granules. Identified proteins that bind to endogenous G4DNA in the cytoplasm are known to modulate mRNA translation and participate in stress granule formation. Consistent with these findings, stress granule formation is known to regulate mRNA translation during oxidative stress. We propose a signaling pathway whereby cells can rapidly respond to DNA damage caused by oxidative stress. Guanine-rich sequences that are excised from damaged genomic DNA are proposed to enter the cytoplasm where they can regulate translation through stress granule formation. This newly proposed role for G4DNA provides an additional molecular explanation for why such sequences are prevalent in the human genome.


Assuntos
Citoplasma/metabolismo , Grânulos Citoplasmáticos/metabolismo , Dano ao DNA , Quadruplex G , Estresse Oxidativo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Citoplasma/genética , Grânulos Citoplasmáticos/genética , Células HeLa , Humanos , RNA Mensageiro/genética
6.
J Neurochem ; 120(4): 598-610, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22117694

RESUMO

D-serine is an endogenous neurotransmitter that binds to the NMDA receptor, thereby increasing the affinity for glutamate, and the potential for excitotoxicity. The primary source of D-serine in vivo is enzymatic racemization by serine racemase (SR). Regulation of D-serine in vivo is poorly understood, but is thought to involve a combination of controlled production, synaptic reuptake by transporters, and intracellular degradation by D-amino acid oxidase (DAO). However, SR itself possesses a well-characterized eliminase activity, which effectively degrades D-serine as well. D-serine is increased two-fold in spinal cords of G93A Cu,Zn-superoxide dismutase (SOD1) mice--the standard model of amyotrophic lateral sclerosis (ALS). ALS mice with SR disruption show earlier symptom onset, but survive longer (progression phase is slowed), in an SR-dependent manner. Paradoxically, administration of D-serine to ALS mice dramatically lowers cord levels of D-serine, leading to changes in the onset and survival very similar to SR deletion. D-serine treatment also increases cord levels of the alanine-serine-cysteine transporter 1 (Asc-1). Although the mechanism by which SOD1 mutations increases D-serine is not known, these results strongly suggest that SR and D-serine are fundamentally involved in both the pre-symptomatic and progression phases of disease, and offer a direct link between mutant SOD1 and a glial-derived toxic mediator.


Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Modelos Animais de Doenças , Mutação , Racemases e Epimerases/fisiologia , Serina/fisiologia , Superóxido Dismutase/fisiologia , Esclerose Lateral Amiotrófica/enzimologia , Esclerose Lateral Amiotrófica/patologia , Animais , Progressão da Doença , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Microglia/enzimologia , Microglia/metabolismo , Microglia/patologia , Racemases e Epimerases/química , Racemases e Epimerases/deficiência , Serina/antagonistas & inibidores , Serina/biossíntese , Superóxido Dismutase/química , Superóxido Dismutase/genética , Superóxido Dismutase-1 , Regulação para Cima/genética
7.
Protein Sci ; 31(2): 407-421, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34761452

RESUMO

Helicases are molecular motors with many activities. They use the energy from ATP hydrolysis to unwind double-stranded nucleic acids while translocating on the single-stranded DNA. In addition to unwinding, many helicases are able to remove proteins from nucleic acids. Bacteriophage T4 Dda is able to displace a variety of DNA binding proteins and streptavidin bound to biotinylated oligonucleotides. We have identified a subdomain of Dda that when deleted, results in a protein variant that has nearly wild type activity for unwinding double-stranded DNA but exhibits greatly reduced streptavidin displacement activity. Interestingly, this domain has little effect on displacement of either gp32 or BamHI bound to DNA but does affect displacement of trp repressor from DNA. With this variant, we have identified residues which enhance displacement of some proteins from DNA.


Assuntos
Bacteriófago T4 , DNA Helicases , Proteínas Virais , Proteínas de Bactérias , Bacteriófago T4/enzimologia , DNA/química , DNA Helicases/química , DNA de Cadeia Simples/genética , Proteínas Repressoras , Estreptavidina/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo
8.
Enzymes ; 50: 335-367, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34861942

RESUMO

RNA viruses cause many routine illnesses, such as the common cold and the flu. Recently, more deadly diseases have emerged from this family of viruses. The hepatitis C virus has had a devastating impact worldwide. Despite the cures developed in the U.S. and Europe, economically disadvantaged countries remain afflicted by HCV infection due to the high cost of these medications. More recently, COVID-19 has swept across the world, killing millions and disrupting economies and lifestyles; the virus responsible for this pandemic is a coronavirus. Our understanding of HCV and SARS CoV-2 replication is still in its infancy. Helicases play a critical role in the replication, transcription and translation of viruses. These key enzymes need extensive study not only as an essential player in the viral lifecycle, but also as targets for antiviral therapeutics. In this review, we highlight the current knowledge for RNA helicases of high importance to human health.


Assuntos
COVID-19 , Replicação Viral , RNA Helicases DEAD-box , Hepacivirus/genética , Humanos , SARS-CoV-2
9.
Methods Mol Biol ; 2209: 53-72, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33201462

RESUMO

RNA helicases are responsible for virtually all of RNA metabolism. Viral and bacterial pathogens typically encode their own RNA helicases. Hence, this family of enzymes is increasingly recognized as potential targets for treatment of a variety of diseases. However, the conserved structural similarities among helicase families present an obstacle to the idea of developing specific inhibitors. In order to identify potential modulators of RNA helicase activity, rapid screening approaches are needed. This has been accomplished by optimizing and adapting standard helicase assays to function in high-throughput modalities. These optimized assays have enabled the application of rapid screening approaches to be applied toward discovering helicase inhibitors. This chapter provides detailed protocols for utilizing a medium to high-throughput approach for inhibitor discovery.


Assuntos
Ensaios Enzimáticos/métodos , Inibidores Enzimáticos/análise , RNA Helicases/antagonistas & inibidores , RNA/química , Fluorescência , Humanos
10.
Chem Commun (Camb) ; 57(60): 7445-7448, 2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34232232

RESUMO

G-quadruplex DNA interacts with the N-terminal intrinsically disordered domain of the DEAD-box helicase Ded1p, diminishing RNA unwinding activity but enhancing liquid-liquid phase separation of Ded1p in vitro and in cells. The data highlight multifaceted effects of quadruplex DNA on an enzyme with intrinsically disordered domains.


Assuntos
RNA Helicases DEAD-box/metabolismo , DNA/metabolismo , Quadruplex G , Proteínas de Saccharomyces cerevisiae/metabolismo , Citoplasma/química , Citoplasma/metabolismo , RNA Helicases DEAD-box/química , DNA/genética , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Transição de Fase , Domínios Proteicos , RNA/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química
12.
Mol Endocrinol ; 21(10): 2541-50, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17622585

RESUMO

Routine consumption of alcohol at low doses is associated with decreased risk of acquiring type 2 diabetes, whereas chronic and excessive alcohol consumption increases the risk. Although there is good epidemiologic evidence for these biphasic effects, careful validation of these effects on insulin signaling has not been reported, nor have biological mechanisms underlying these biphasic effects been proposed. In this study, we provide evidence in rats that low-dose alcohol intake (4 g/kg x d) enhances hepatic insulin signaling by suppressing p55gamma (a phosphatidylinositol 3-kinase regulatory subunit isoform) at the posttranscriptional level, leading to the increased association of the phosphatidylinositol 3-kinase catalytic subunit (p110) with insulin receptor substrate-1 (P < 0.05) and subsequent activation of downstream effectors such as Akt, glycogen synthase kinase 3beta, and nuclear sterol regulatory element binding protein (SREBP)-1. These results, combined with our previous data (confirmed in the present study) demonstrating that ethanol intake at high doses (13 g/kg x d) disrupts hepatic insulin signaling by inducing TRB3, a mammalian homolog of Drosophila (tribbles-related protein 3) that prevented activation of downstream effectors (such as Akt, GSK3beta, and nSREBP-1), provide clear mechanistic validation of the biphasic effects of ethanol on insulin signaling. We also report that ethanol induction of TRB3 can be partially blocked (P < 0.01) by compounds (4-phenyl butyric acid and taurine-ursodeoxycholic acid) known to reduce endoplasmic reticulum stress. Thus, alcohol exerts biphasic actions on hepatic insulin signaling, such that low doses activate insulin signaling pathways associated with reduced p55gamma to increase nSREBP-1, whereas high doses of ethanol elevate TRB3 and suppress insulin signaling to decrease SREBP-1.


Assuntos
Etanol/administração & dosagem , Saúde , Insulina/metabolismo , Fígado/efeitos dos fármacos , Animais , Dieta , Relação Dose-Resposta a Droga , Etanol/urina , Humanos , Fígado/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Ratos , Transdução de Sinais/efeitos dos fármacos
13.
Biochim Biophys Acta ; 1760(7): 1088-95, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16624488

RESUMO

Chronic airway inflammation is a key feature of bronchial asthma. Leukotrienes are potent inflammatory mediators that play a role in the pathophysiology of asthma, and their levels are elevated in the airways in response to allergen challenge. We examined the anti-inflammatory effect of thymoquinone (TQ), the active principle in the volatile oil of Nigella sativa seeds, on leukotriene (LT) biosynthesis in a mouse model of allergic asthma. Mice sensitized and challenged with ovalbumin (OVA) antigen had an increased amounts of leukotriene B4 and C4, Th2 cytokines, and eosinophils in bronchoalveolar lavage (BAL) fluid. In addition, there was also a marked increase in lung tissue eosinophilia and goblet cell numbers. Administration of TQ before OVA challenge inhibited 5-lipoxygenase, the main enzyme in leukotriene biosynthesis, expression by lung cells and significantly reduced the levels of LTB4 and LTC4. This was accompanied by a marked decrease in Th2 cytokines and BAL fluid and lung tissue eosinophilia, all of which are characteristics of airway inflammation. These results demonstrate the anti-inflammatory effect of TQ in experimental asthma.


Assuntos
Asma/patologia , Benzoquinonas/química , Regulação para Baixo , Inflamação , Leucotrienos/biossíntese , Animais , Araquidonato 5-Lipoxigenase/metabolismo , Líquido da Lavagem Broncoalveolar , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Hipersensibilidade/patologia , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Nigella sativa/metabolismo
14.
Clin Chest Med ; 28(1): 23-42, vii, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17338926

RESUMO

The secondary role of pathology in the present clinical management of pulmonary hypertension (PH) reflects to some extent the limitations of the current understanding of the disease. Ample room exists for the diagnostic translation of the pathobiologic studies, with the goal of improving the diagnostic and prognostic power of the pathologic assessment of pulmonary vascular remodeling. This article seeks to show the complementarities of the pathology and pathobiology of PH.


Assuntos
Hipertensão Pulmonar/patologia , Apoptose/fisiologia , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/fisiologia , Proteínas Morfogenéticas Ósseas/fisiologia , Colágeno/análise , Células Endoteliais/patologia , Células Endoteliais/fisiologia , Endotélio/patologia , Fibroblastos/fisiologia , Produtos do Gene nef/fisiologia , Humanos , Hipertensão Pulmonar/fisiopatologia , Hipertrofia , Imuno-Histoquímica , Mediadores da Inflamação/fisiologia , Artéria Pulmonar/patologia , Resistência Vascular/fisiologia
15.
Immunol Lett ; 106(1): 72-81, 2006 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-16762422

RESUMO

Prostaglandins (PGs) are potent proinflammatory mediators generated through arachidonic acid metabolism by cyclooxygenase-1 and -2 (COX-1 and COX-2) in response to different stimuli and play an important role in modulating the inflammatory responses in a number of conditions, including allergic airway inflammation. Thymoquinone (TQ) is the main active constituent of the volatile oil extract of Nigella sativa seeds and has been reported to have anti-inflammatory properties. We examined the effect of TQ on the in vivo production of PGs and lung inflammation in a mouse model of allergic airway inflammation. Mice sensitized and challenged through the airways with ovalbumin (OVA) exhibited a significant increase in PGD2 and PGE2 production in the airways. The inflammatory response was characterized by an increase in the inflammatory cell numbers and Th2 cytokine levels in the bronchoalveolar lavage (BAL) fluid, lung airway eosinophilia and goblet cell hyperplasia, as well as the induction of COX-2 protein expression in the lung. Intraperitoneal injection of TQ for 5 days before the first OVA challenge attenuated airway inflammation as demonstrated by the significant decrease in Th2 cytokines, lung eosinophilia, and goblet cell hyperplasia. This attenuation of airway inflammation was concomitant to the inhibition of COX-2 protein expression and PGD2 production. However, TQ had a slight inhibitory effect on COX-1 expression and PGE2 production. These findings suggest that TQ has an anti-inflammatory effect during the allergic response in the lung through the inhibition of PGD2 synthesis and Th2-driven immune response.


Assuntos
Anti-Inflamatórios/uso terapêutico , Benzoquinonas/uso terapêutico , Hiper-Reatividade Brônquica/tratamento farmacológico , Hiper-Reatividade Brônquica/metabolismo , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/metabolismo , Prostaglandina D2/biossíntese , Alérgenos/imunologia , Animais , Hiper-Reatividade Brônquica/imunologia , Hiper-Reatividade Brônquica/patologia , Citocinas/biossíntese , Citocinas/genética , Dinoprostona/biossíntese , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/farmacologia , Células Th2/metabolismo
16.
Int Immunopharmacol ; 6(7): 1135-42, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16714217

RESUMO

Thymoquinone (TQ), the main active constituent of the volatile oil extracted from Nigella sativa's seeds, has been reported to have an anti-inflammatory and immune stimulatory effect on bronchial asthma and inflammation. However, little is known about the factors and mechanisms underlying these effects. In the present study, we examined the effect of TQ on airway inflammation in a mouse model of allergic asthma. Intraperitoneal injection of TQ before airway challenge of ovalbumin (OVA)-sensitized mice resulted in a marked decrease in lung eosinophilia and the elevated Th2 cytokines observed after airway challenge with OVA antigen; both in vivo, in the bronchoalveolar lavage (BAL) fluid and in vitro, following stimulation of lung cells with OVA. TQ also decreased the elevated serum levels of OVA-specific IgE and IgG1. Histological examination of lung tissue demonstrated that TQ significantly inhibited allergen-induced lung eosinophilic inflammation and mucus-producing goblet cells. While TQ showed a significant effect in inhibiting IL-4, IL-5 and IL-13 and some effect in inducing IFN-gamma production in the BAL fluid, it did show a slight effect on in vitro production of IL-4 by cultured lung cells stimulated with OVA antigen. These data suggest that TQ attenuates allergic airway inflammation by inhibiting Th2 cytokines and eosinophil infiltration into the airways; thus demonstrating its potential anti-inflammatory role during the allergic response in the lung.


Assuntos
Anti-Inflamatórios/farmacologia , Benzoquinonas/farmacologia , Pneumonia/tratamento farmacológico , Hipersensibilidade Respiratória/tratamento farmacológico , Alérgenos/farmacologia , Animais , Líquido da Lavagem Broncoalveolar/química , Citocinas/imunologia , Modelos Animais de Doenças , Feminino , Células Caliciformes/efeitos dos fármacos , Células Caliciformes/imunologia , Células Caliciformes/patologia , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Contagem de Leucócitos , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/farmacologia , Células Th2/imunologia
17.
Free Radic Biol Med ; 39(8): 1089-98, 2005 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-16198235

RESUMO

We report herein the novel observation that alterations in oxidant/antioxidant balance are evident and cause vascular dysfunction in aortae of prediabetic nonobese-diabetic mice (NOD). We found that nitrotyrosine, a biochemical marker of oxidant stress, was higher in the NOD aortae when compared to age-matched non-autoimmune BALB/c controls or the diabetes-resistant NOD congenic strain, NOD.Lc7. The oxidant stress was localized to the intimal and medial layers, and endothelium-dependent relaxation to acetylcholine was decreased in isolated aortic rings from NOD mice. Inhibition of nitric oxide synthesis caused an endothelium-dependent contraction, and treatment with either a selective thromboxane A2/prostaglandin H2 receptor antagonist or a non-isozyme-specific cyclooxygenase inhibitor reversed this effect. Aortic rings from NOD.Lc7 did not display the paradoxical vasoconstriction. Furthermore, the vascular dysfunction was caused by oxidative stress, as treatment with a superoxide dismutase mimetic in vivo or with native antioxidant enzymes ex vivo inhibited the tissue oxidant stress and restored endothelium-dependent relaxation. Endothelial function was also restored by the inhibitors of NAD(P)H oxidase, diphenylene iodonium or apocynin. Our studies indicate that an oxidant stress that occurs prior to the onset of diabetes in this mouse model contributes to endothelial dysfunction independently of overt diabetes.


Assuntos
Diabetes Mellitus Tipo 1/fisiopatologia , Endotélio Vascular/fisiopatologia , Estresse Oxidativo , Estado Pré-Diabético/fisiopatologia , Prostaglandinas/metabolismo , Vasodilatação , Acetofenonas/farmacologia , Acetilcolina/farmacologia , Animais , Aorta/química , Aorta/efeitos dos fármacos , Aorta/fisiopatologia , Inibidores de Ciclo-Oxigenase/farmacologia , Diabetes Mellitus Tipo 1/metabolismo , Endotélio Vascular/química , Endotélio Vascular/efeitos dos fármacos , Homeostase , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/metabolismo , Óxido Nítrico/metabolismo , Oniocompostos/farmacologia , Oxirredução , Estado Pré-Diabético/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Receptores de Tromboxano A2 e Prostaglandina H2/antagonistas & inibidores , Receptores de Tromboxano A2 e Prostaglandina H2/metabolismo , Tirosina/análogos & derivados , Tirosina/análise , Tirosina/metabolismo
18.
Free Radic Biol Med ; 37(6): 869-80, 2004 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-15706661

RESUMO

Regulation of the basal manganese superoxide dismutase (SOD2) promoter depends on the transcriptional activity of the Sp family of transcription factors. Here we report that reduced expression in the presence of Tat is independent of induction with Tumor necrosis factor alpha and that Tat affects the interaction of Sp1 and Sp3 with the basal promoter. Footprinting and electrophoretic mobility shift assay (EMSA) analyses with extracts from HeLa cells showed that Sp1/Sp3 complexes populate the proximal SOD2 promoter, and that Tat leads to an increase in the binding activity of Sp3. In Drosophila S2 cells, both Sp1 and Sp3 activated the basal SOD2 promoter (88.1 +/- 39.4 fold vs. 10.3 +/- 3.5 fold, respectively), demonstrating a positive, yet lower transcriptional regulatory function for Sp3. Additionally, the inability of Sp3 to synergistically affect promoter activity indicates an efficient competition of Sp3 with Sp1 for the multiple Sp binding sites in the SOD2 basal promoter. Tat potentiated both Sp1 and Sp3 activation of the promoter in S2 cells, though the activity of Sp3 was still lower than that of Sp1. Thus, the consequence of a shift by Tat to increased Sp3-containing complexes on the basal SOD2 promoter is decreased SOD2 expression. Together, our studies demonstrate the functional importance of the interaction of Sp1, Sp3, and Tat, revealing a possible mechanism for the attenuation of basal manganese superoxide dismutase expression.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação Viral da Expressão Gênica , Produtos do Gene tat/genética , Produtos do Gene tat/fisiologia , HIV-1/genética , Fator de Transcrição Sp1/metabolismo , Superóxido Dismutase/biossíntese , Fatores de Transcrição/metabolismo , Acetilcisteína/química , Animais , Sítios de Ligação , Northern Blotting , Western Blotting , Diferenciação Celular , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Drosophila , Radicais Livres , Células HeLa , Humanos , Luciferases/metabolismo , Modelos Químicos , Estresse Oxidativo , Plasmídeos/metabolismo , Ligação Proteica , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp3 , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Fatores de Transcrição/genética , Transfecção , Produtos do Gene tat do Vírus da Imunodeficiência Humana
19.
Antioxid Redox Signal ; 20(10): 1655-70, 2014 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-23641945

RESUMO

SIGNIFICANCE: Respiring mitochondria are a significant site for reactions involving reactive oxygen and nitrogen species that contribute to irreversible cellular, structural, and functional damage leading to multiple pathological conditions. Manganese superoxide dismutase (MnSOD) is a critical component of the antioxidant system tasked with protecting the oxidant-sensitive mitochondrial compartment from oxidative stress. Since global knockout of MnSOD results in significant cardiac and neuronal damage leading to early postnatal lethality, this approach has limited use for studying the mechanisms of oxidant stress and the development of disease in specific tissues lacking MnSOD. To circumvent this problem, a number of investigators have employed the Cre/loxP system to precisely knockout MnSOD in individual tissues. RECENT ADVANCES: Multiple tissue and organ-specific Cre-expressing mice have been generated, which greatly enhance the specificity of MnSOD knockout in tissues and organ systems that were once difficult, if not impossible to study. CRITICAL ISSUES: Evaluating the contribution of MnSOD deficiency to oxidant-mediated mitochondrial damage requires careful consideration of the promoter system used for creating the tissue-specific knockout animal, in addition to the collection and interpretation of multiple indices of oxidative stress and damage. FUTURE DIRECTIONS: Expanded use of well-characterized tissue-specific promoter elements and inducible systems to drive the Cre/loxP recombinational events will lead to a spectrum of MnSOD tissue knockout models, and a clearer understanding of the role of MnSOD in preventing mitochondrial dysfunction in human disease.


Assuntos
Estresse Oxidativo , Superóxido Dismutase/genética , Animais , Expressão Gênica , Técnicas de Inativação de Genes , Engenharia Genética , Humanos , Integrases/genética , Especificidade de Órgãos , Superóxido Dismutase/metabolismo
20.
Neurol Res Int ; 2012: 625245, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23029613

RESUMO

In mammalian systems, D-serine is perhaps the most biologically active D-amino acid described to date. D-serine is a coagonist at the NMDA-receptor, and receptor activation is dependent on D-serine binding. Because D-serine binding dramatically increases receptor affinity for glutamate, it can produce excitotoxicity without any change in glutamate per se. D-serine is twofold higher in the spinal cords of mSOD1 (G93A) ALS mice, and the deletion of serine racemase (SR), the enzyme that produces D-serine, results in an earlier onset of symptoms, but with a much slower rate of disease progression. Localization studies within the brain suggest that mSOD1 and subsequent glial activation could contribute to the alterations in SR and D-serine seen in ALS. By also degrading both D-serine and L-serine, SR appears to be a prime bidirectional regulator of free serine levels in vivo. Therefore, accurate and reproducible measurements of D-serine are critical to understanding its regulation by SR. Several methods for measuring D-serine have been employed, and significant issues related to validation and standardization remain unresolved. Further insights into the intracellular transport and tissue-specific compartmentalization of D-serine within the CNS will aid in the understanding of the role of D-serine in the pathogenesis of ALS.

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