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1.
Food Res Int ; 157: 111295, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35761600

RESUMO

In this study, the radicals formed in camellia oil upon a heating process were identified and quantified using Electron Spin Resonance (ESR) spectroscopy coupled with spin-trapping technique. PBN and DMPO were served as spin traps. The total amounts of free radicals of heated camellia oil showed an increasing trend with the extension of heating time at 140 °C, 150 °C and 160 °C. In accordance with hyperfine splitting constants (aN, aH) â”€ the crucial parameter for identifying free radical species â”€ of free radical, it was definitely confirmed that alkyl, alkoxyl, oxygen-centered and DMPO oxidate free radicals were present in heated camellia oil. A free radical transition pathway was proposed that alkyl free radical is initially generated, then, alkyl peroxy free radical is subsequently generated in the presence of oxygen which eventually shifts into alkoxyl free radical by means of an intermediate formed from H-capture reaction of alkyl peroxy free radical.


Assuntos
Camellia , Temperatura Alta , Óxidos N-Cíclicos/química , Óxidos N-Cíclicos/metabolismo , Radicais Livres/química , Oxigênio , Marcadores de Spin
2.
Acta Crystallogr F Struct Biol Commun ; 76(Pt 8): 350-356, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32744246

RESUMO

CVT-313 is a potent CDK2 inhibitor that was identified by screening a purine-analogue library and is currently in preclinical studies. Since this molecule has the potential to be developed as a CDK2 inhibitor for cancer therapy, the potency of CVT-313 to bind and stabilize CDK2 was evaluated, together with its ability to inhibit aberrant cell proliferation. CVT-313 increased the melting temperature of CDK2 by 7°C in thermal stabilization studies, thus indicating its protein-stabilizing effect. CVT-313 inhibited the growth of human lung carcinoma cell line A549 in a dose-dependent manner, with an IC50 of 1.2 µM, which is in line with the reported biochemical potency of 0.5 µM. To support the further chemical modification of CVT-313 and to improve its biochemical and cellular potency, a crystal structure was elucidated in order to understand the molecular interaction of CVT-313 and CDK2. The crystal structure of CDK2 bound to CVT-313 was determined to a resolution of 1.74 Šand clearly demonstrated that CVT-313 binds in the ATP-binding pocket, interacting with Leu83, Asp86 and Asp145 directly, and the binding was further stabilized by a water-mediated interaction with Asn132. Based on the crystal structure, further modifications of CVT-313 are proposed to provide additional interactions with CDK2 in the active site, which may significantly increase the biochemical and cellular potency of CVT-313.


Assuntos
Trifosfato de Adenosina/química , Antineoplásicos/farmacologia , Quinase 2 Dependente de Ciclina/química , Inibidores de Proteínas Quinases/farmacologia , Purinas/farmacologia , Células A549 , Trifosfato de Adenosina/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Sítios de Ligação , Proliferação de Células/efeitos dos fármacos , Clonagem Molecular , Cristalografia por Raios X , Óxidos N-Cíclicos/química , Óxidos N-Cíclicos/metabolismo , Óxidos N-Cíclicos/farmacologia , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Relação Dose-Resposta a Droga , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Indolizinas/química , Indolizinas/metabolismo , Indolizinas/farmacologia , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Purinas/química , Purinas/metabolismo , Compostos de Piridínio/química , Compostos de Piridínio/metabolismo , Compostos de Piridínio/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Roscovitina/química , Roscovitina/metabolismo , Roscovitina/farmacologia
3.
Sci Rep ; 10(1): 12240, 2020 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-32699288

RESUMO

Tea plant often suffers from low temperature induced damage during its growth. How to improve the cold resistance of tea plant is an urgent problem to be solved. Nitric oxide (NO), γ-aminobutyric acid (GABA) and proline have been proved that can improve the cold resistance of tea plants, and signal transfer and biosynthesis link between them may enhance their function. NO is an important gas signal material in plant growth, but our understanding of the effects of NO on the GABA shunt, proline and NO biosynthesis are limited. In this study, the tea roots were treated with a NO donor (SNAP), NO scavenger (PTIO), and NO synthase inhibitor (L-NNA). SNAP could improve activities of arginine decarboxylase, ornithine decarboxylase, glutamate decarboxylase, GABA transaminase and Δ1-pyrroline-5-carboxylate synthetase and the expression level of related genes during the treatments. The contents of putrescine and spermidine under SNAP treatment were 45.3% and 37.3% higher compared to control at 24 h, and the spermine content under PTIO treatment were 57.6% lower compare to control at 12 h. Accumulation of proline of SNAP and L-NNA treatments was 52.2% and 43.2% higher than control at 48 h, indicating other pathway of NO biosynthesis in tea roots. In addition, the NO accelerated the consumption of GABA during cold storage. These facts indicate that NO enhanced the cold tolerance of tea, which might regulate the metabolism of the GABA shunt and of proline, associated with NO biosynthesis.


Assuntos
Camellia sinensis/metabolismo , Óxido Nítrico/metabolismo , Raízes de Plantas/metabolismo , Poliaminas/metabolismo , Prolina/metabolismo , Chá/metabolismo , Ácido gama-Aminobutírico/metabolismo , Carboxiliases/metabolismo , Temperatura Baixa , Resposta ao Choque Frio/fisiologia , Óxidos N-Cíclicos/metabolismo , Glutamato Descarboxilase/metabolismo , Imidazóis/metabolismo , Doadores de Óxido Nítrico/metabolismo , Ornitina Descarboxilase/metabolismo , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Putrescina/metabolismo , S-Nitroso-N-Acetilpenicilamina/metabolismo , Espermidina/metabolismo , Espermina/metabolismo
4.
Antioxid Redox Signal ; 33(13): 883-902, 2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-32475148

RESUMO

Aims: REDOX signaling from reactive oxygen species (ROS) generated by the mitochondria (mitochondrial reactive oxygen species [mtROS]) has been implicated in cancer growth and survival. Here, we investigated the effect of 5-(4-methoxyphenyl)-3H-1,2-dithiole-3-thione (AOL), a recently characterized member of the new class of mtROS suppressors (S1QELs), on human lung adenocarcinoma proteome reprogramming, bioenergetics, and growth. Results: AOL reduced steady-state cellular ROS levels in human lung cancer cells without altering the catalytic activity of complex I. AOL treatment induced dose-dependent inhibition of lung cancer cell proliferation and triggered a reduction in tumor growth in vivo. Molecular investigations demonstrated that AOL reprogrammed the proteome of human lung cancer cells. In particular, AOL suppressed the determinants of the Warburg effect and increased the expression of the complex I subunit NDUFV1 which was also identified as AOL binding site using molecular modeling computer simulations. Comparison of the molecular changes induced by AOL and MitoTEMPO, an mtROS scavenger that is not an S1QEL, identified a core component of 217 proteins commonly altered by the two treatments, as well as drug-specific targets. Innovation: This study provides proof-of-concept data on the anticancer effect of AOL on mouse orthotopic human lung tumors. A unique dataset on proteomic reprogramming by AOL and MitoTEMPO is also provided. Lastly, our study revealed the repression of NDUFV1 by S1QEL AOL. Conclusion: Our findings demonstrate the preclinical anticancer properties of S1QEL AOL and delineate its mode of action on REDOX and cancer signaling.


Assuntos
Adenocarcinoma de Pulmão/etiologia , Adenocarcinoma de Pulmão/metabolismo , Mitocôndrias/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/patologia , Óxidos N-Cíclicos/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Humanos
5.
Carbohydr Polym ; 240: 116307, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32475577

RESUMO

Nanocomposite film of pullulan (PULL), tempo cellulose nanofibrils (TOCNs) and, montmorillonite clay (MMT) were prepared using a solution casting method with aqueous solutions. X-ray diffraction data revealed that exfoliated MMT nanoplatelets are distributed within the PULL/TOCNs/MMT film structure. Fourier-transform infrared results revealed that there might be interactions among the TOCNs, MMT and PULL matrix led to improved tensile strength, thermal stability, water barrier properties, and decrease moisture susceptibility while maintained reasonable transparency and biodegradability of the ternary PULL nanocomposites. These excellent properties of the nanocomposites clearly indicate towards a new strategy for developing high-performance PULL-based nanocomposites by using two different types of fillers with various geometric shapes and aspect ratio. This kind of ternary nanocomposite film can be broadly used in food packaging and protection as a green and biodegradable film.


Assuntos
Bentonita/química , Celulose/química , Óxidos N-Cíclicos/química , Glucanos/química , Nanocompostos/química , Nanofibras/química , Bentonita/metabolismo , Biodegradação Ambiental , Celulose/metabolismo , Óxidos N-Cíclicos/metabolismo , Embalagem de Alimentos , Glucanos/metabolismo , Microbiologia do Solo , Resistência à Tração , Água/química
6.
Int J Mol Sci ; 21(9)2020 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-32370303

RESUMO

Ferredoxin:NADP+ oxidoreductase from Plasmodium falciparum (PfFNR) catalyzes the NADPH-dependent reduction of ferredoxin (PfFd), which provides redox equivalents for the biosynthesis of isoprenoids and fatty acids in the apicoplast. Like other flavin-dependent electrontransferases, PfFNR is a potential source of free radicals of quinones and other redox cycling compounds. We report here a kinetic study of the reduction of quinones, nitroaromatic compounds and aromatic N-oxides by PfFNR. We show that all these groups of compounds are reduced in a single-electron pathway, their reactivity increasing with the increase in their single-electron reduction midpoint potential (E17). The reactivity of nitroaromatics is lower than that of quinones and aromatic N-oxides, which is in line with the differences in their electron self-exchange rate constants. Quinone reduction proceeds via a ping-pong mechanism. During the reoxidation of reduced FAD by quinones, the oxidation of FADH. to FAD is the possible rate-limiting step. The calculated electron transfer distances in the reaction of PfFNR with various electron acceptors are similar to those of Anabaena FNR, thus demonstrating their similar "intrinsic" reactivity. Ferredoxin stimulated quinone- and nitro-reductase reactions of PfFNR, evidently providing an additional reduction pathway via reduced PfFd. Based on the available data, PfFNR and possibly PfFd may play a central role in the reductive activation of quinones, nitroaromatics and aromatic N-oxides in P. falciparum, contributing to their antiplasmodial action.


Assuntos
Ferredoxina-NADP Redutase/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Xenobióticos/metabolismo , Apicoplastos/enzimologia , Biocatálise , Óxidos N-Cíclicos/química , Óxidos N-Cíclicos/metabolismo , Transporte de Elétrons , Ferredoxinas/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Cinética , Estrutura Molecular , NADP/metabolismo , Oxirredução , Quinonas/química , Quinonas/metabolismo , Especificidade por Substrato , Xenobióticos/química
7.
ACS Chem Biol ; 15(7): 1759-1764, 2020 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-32433863

RESUMO

While kinases have been attractive targets to combat many diseases, including cancer, selective kinase inhibition has been challenging, because of the high degree of structural homology in the active site, where many kinase inhibitors bind. We have previously discovered that 8-anilino-1-naphthalene sulfonic acid (ANS) binds an allosteric pocket in cyclin-dependent kinase 2 (Cdk2). Here, we detail the positive cooperativity between ANS and orthosteric Cdk2 inhibitors dinaciclib and roscovitine, which increase the affinity of ANS toward Cdk2 5-fold to 10-fold, and the relatively noncooperative effects of ATP. We observe these effects using a fluorescent binding assay and heteronuclear single quantum correlation nuclear magnetic resonance (HSQC NMR), where we noticed a shift from fast exchange to slow exchange upon ANS titration in the presence of roscovitine but not with an ATP mimic. The discovery of cooperative relationships between orthosteric and allosteric kinase inhibitors could further the development of selective kinase inhibitors in general.


Assuntos
Naftalenossulfonato de Anilina/química , Óxidos N-Cíclicos/química , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Indolizinas/química , Inibidores de Proteínas Quinases/química , Compostos de Piridínio/química , Roscovitina/química , Regulação Alostérica , Naftalenossulfonato de Anilina/metabolismo , Óxidos N-Cíclicos/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Sinergismo Farmacológico , Humanos , Indolizinas/metabolismo , Simulação de Acoplamento Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/metabolismo , Compostos de Piridínio/metabolismo , Roscovitina/metabolismo
8.
Antioxid Redox Signal ; 33(1): 1-19, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32103677

RESUMO

Aims: Mitochondrial stress and dysfunction within the intestinal epithelium are known to contribute to the pathogenesis of inflammatory bowel disease (IBD). However, the importance of mitophagy during intestinal inflammation remains poorly understood. The primary aim of this study was to investigate how the mitophagy protein BCL2/adenovirus E1B 19 kDa protein-interacting protein 3-like (BNIP3L/NIX) mitigates mitochondrial damage during intestinal inflammation in the hopes that these data will allow us to target mitochondrial health in the intestinal epithelium as an adjunct to immune-based treatment strategies. Results: In the intestinal epithelium of patients with ulcerative colitis, we found that NIX was upregulated and targeted to the mitochondria. We obtained similar findings in wild-type mice undergoing experimental colitis. An increase in NIX expression was found to depend on stabilization of hypoxia-inducible factor-1 alpha (HIF1α), which binds to the Nix promoter region. Using the reactive oxygen species (ROS) scavenger MitoTEMPO, we were able to attenuate disease and inhibit both HIF1α stabilization and subsequent NIX expression, suggesting that mitochondrially derived ROS are crucial to initiating the mitophagic response during intestinal inflammation. We subjected a global Nix-/- mouse to dextran sodium sulfate colitis and found that these mice developed worse disease. In addition, Nix-/- mice were found to exhibit increased mitochondrial mass, likely due to the inability to clear damaged or dysfunctional mitochondria. Innovation: These results demonstrate the importance of mitophagy within the intestinal epithelium during IBD pathogenesis. Conclusion: NIX-mediated mitophagy is required to maintain intestinal homeostasis during inflammation, highlighting the impact of mitochondrial damage on IBD progression.


Assuntos
Gastroenterite/etiologia , Proteínas de Membrana/genética , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Mitofagia/genética , Animais , Antioxidantes/farmacologia , Sítios de Ligação , Biomarcadores , Linhagem Celular Tumoral , Colite/etiologia , Colite/metabolismo , Colite/patologia , Óxidos N-Cíclicos/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Gastroenterite/metabolismo , Gastroenterite/patologia , Humanos , Hipóxia/genética , Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Doenças Inflamatórias Intestinais/etiologia , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Modelos Biológicos , Regiões Promotoras Genéticas , Ligação Proteica , Espécies Reativas de Oxigênio/metabolismo , Elementos de Resposta
9.
Neuropeptides ; 80: 102021, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32033788

RESUMO

Neuropeptides are involved in the regulation of the sympathetic activity and blood pressure in the paraventricular nucleus of the hypothalamus (PVN). The present study was designed to determine how alarin modulates the renal sympathetic nerve activity (RSNA), arterial blood pressure and mean arterial pressure (MAP) in the PVN, and whether superoxide anions regulate the effects of alarin in the PVN of spontaneously hypertensive rats (SHRs). Acute experiment was carried out with male Wistar-Kyoto rats (WKY) and SHRs under anesthesia. RSNA, systolic blood pressure (SBP), diastolic blood pressure (DBP), and MAP were measured. Alarin microinjection into the PVN increased RSNA (7.8 ± 1.8 vs. 14.8 ± 2.3%), SBP (5.9 ± 1.4 vs. 12.1 ± 1.6 mmHg), DBP (5.1 ± 0.8 vs. 10.0 ± 1.1 mmHg), and MAP (5.4 ± 1.2 vs. 10.7 ± 1.3 mmHg) in WKY rats and SHRs,. Alarin antagonist ala6-25 Cys decreased RSNA, SBP, DBP, and MAP in SHRs, and inhibited the effects of alarin. The alarin level was increased in the PVN of SHR compared to WKY rats. (29.7 ± 4.9 vs. 14.6 ± 2.4 pg/mg protein). PVN microinjection of superoxide anion scavengers tempol and tiron, or NAD(P)H oxidase inhibitor apocynin, decreased RSNA, SBP, DBP, and MAP in SHRs, and inhibited the effects of alarin, but the superoxide dismutase inhibitor diethyldithiocarbamic acid potentiated the effects of alarin. Superoxide anions and NAD(P)H oxidase activity levels in the PVN were increased by alarin, but decreased by alarin antagonist ala6-25 Cys. The alarin-induced increases in superoxide anions and NAD(P)H oxidase activity levels were abolished by pre-treatment with ala6-25 Cys. The results suggest that alarin in the PVN increases sympathetic outflow and blood pressure. The enhanced activity of endogenous alarin in the PVN contributes to sympathetic activation in hypertension, and the superoxide anion is involved in these alarin-mediated processes in the PVN.


Assuntos
Peptídeo Semelhante a Galanina/metabolismo , Hipertensão/tratamento farmacológico , Núcleo Hipotalâmico Paraventricular/efeitos dos fármacos , Superóxidos/farmacologia , Sistema Nervoso Simpático/efeitos dos fármacos , Acetofenonas/farmacologia , Animais , Pressão Arterial/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Óxidos N-Cíclicos/metabolismo , Frequência Cardíaca/efeitos dos fármacos , Hipertensão/induzido quimicamente , Masculino , Núcleo Hipotalâmico Paraventricular/metabolismo , Ratos Endogâmicos WKY , Marcadores de Spin , Superóxidos/metabolismo , Sistema Nervoso Simpático/fisiologia
10.
Regul Toxicol Pharmacol ; 111: 104540, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31759138

RESUMO

BIA 10-2474 is a novel fatty acid amide hydrolase (FAAH) inhibitor developed for the treatment of medical conditions which would benefit from enhanced levels of endogenous anandamide (AEA) such as pain disorders. During a Phase I clinical trial one subject died after receiving BIA 10-2474 and four other subjects displayed neurological signs. As part of series of papers presenting all the toxicology data available prior to the clinical trial, we report here the preclinical toxicology studies examining once-a-day oral administration of BIA 10-2474 to male and female Wistar rats. These included a 14-day dose range finding (150, 200 and 250 mg/kg/day), a 4-week study (30, 90 and 150 mg/kg/day) and 13- and 26-week studies (both at 10, 30 and 90 mg/kg/day). The 13- and 26-week studies also included a 4-week recovery arm and a toxicokinetic arm for the parent compound, BIA 10-2474, and the two major metabolites (BIA 10-2445 and BIA 10-2583) were also measured in the 26-week study. At 150 mg/kg and below, all animals survived the scheduled treatment periods although neurological side-effects (abnormal or stiff gait, dragging of fore- or hind-limbs) were seen at 150 mg/kg in both the dose-range finding and 4-week studies. At 90 mg/kg/day, even up to 26-weeks treatment, no clinical signs were seen apart from some decreases in body weight gain. A number of consistent hematological and biochemical changes were noted which were considered related to treatment with BIA 10-2474. Morphologically, in the 4-week study, except for a slight gliosis in the hippocampus of one female at 150 mg/kg, no CNS histopathology was observed; hippocampus gliosis was not observed in subsequent studies. In the 13-week study axonal swelling was present in the medulla oblongata in about half the animals at 90 mg/kg/day and this increased to nearly all the rats at 90 mg/kg/day in the 26-week study. Additional signs seen only in the 26-week study at 90 mg/kg/day included axonal swelling of the fasiculus gracilis and vacuolar changes in the medulla oblongata and ventral commissure of the 3rd ventricle. Other findings included vacuolar degeneration in the ganglia of the GI tract, salivary glands, prostate gland, uterus, and parathyroid glands. The pituitary gland showed edema and mitotic figures in the pars nervosa. These observations outside the CNS were seen in most rats at 90 and 150 mg/kg/day independent of study duration. At 30 mg/kg/day, most of these observations were only seen in isolated cases except for the vacuolar degeneration in GI tract ganglia, which was absent at this dose after 4 weeks treatment but was present in almost all rats at 13 and 26 weeks. Hepatocellular hypertrophy and nephropathy were seen across all studies and the extent of these changes was similar in the 13- and 26-week studies. Most findings resolved after the 4-week recovery periods except for the axonal swelling seen in the medulla oblongata and spinal cord. BIA 10-2474 exposure was markedly higher than the exposure to either metabolite, BIA 10-2445 (19- to 192-fold) and BIA 10-2583 (63- to 526-fold). Exposure to metabolites differed between sexes with higher concentrations of BIA 10-2445 in females compared to males, but the inverse for BIA 10-2583. Although a No Observed Adverse Effect Level (NOAEL) of 30 mg/kg/day was concluded following the 4-week study, the histopathological findings at that dose in the 13- and 26-week studies resulted in the NOAEL being determined to be 10 mg/kg/day.


Assuntos
Óxidos N-Cíclicos/administração & dosagem , Óxidos N-Cíclicos/toxicidade , Bulbo/efeitos dos fármacos , Piridinas/administração & dosagem , Piridinas/toxicidade , Medula Espinal/efeitos dos fármacos , Testes de Toxicidade , Administração Oral , Animais , Óxidos N-Cíclicos/metabolismo , Feminino , Masculino , Bulbo/patologia , Nível de Efeito Adverso não Observado , Piridinas/metabolismo , Ratos , Ratos Wistar , Medula Espinal/patologia
11.
Sci Rep ; 9(1): 19837, 2019 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-31882663

RESUMO

Medical treatment utilizing non-thermal plasma is based on the production of reactive oxygen species (ROS) and their interactions with biomatters. On the basis of empirical data from practices, plasma treatment has been planned with regard to the setup of a plasma generator's parameters, including gas combination, gas-flow rate, and applied voltage. In this study, we quantitated plasma treatment in terms of the plasma dose on the target matter, which can be contrasted with the radiation dose to targets under radiation exposure. We measured the OH radical production in cell culture medium and intracellular ROS production from plasma treatment in comparison with those from X-ray exposure. The clonogenic cell deaths from plasma and X-ray exposures were also compared. In plasma treatment, the clonogenic cell death was better predicted by intracellular ROS production rather than by medium OH production.


Assuntos
Óxidos N-Cíclicos/metabolismo , Gases em Plasma/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Raios X , Animais , Linhagem Celular , Óxidos N-Cíclicos/química , Relação Dose-Resposta à Radiação , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Humanos , Camundongos , Exposição à Radiação , Transdução de Sinais
12.
Sci Rep ; 9(1): 18733, 2019 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-31822759

RESUMO

Potential application of magnetic nanoparticles as drug carriers in medical treatment requires prior determination of their effects on cells. In this work different spin labels and magnetic nanoparticles functionalized with spin labels as well as their interaction with yeast cells were investigated using electron spin resonance (ESR) method. ESR was demonstrated to be a suitable method for monitoring of magnetic core and attached spin labels. Particular emphasis was placed on characterization of endocytosis and redox processes running inside the cell, resulting in recombination of spin labels. Such data could only be obtained at reduced temperature of ESR measurements.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Nanopartículas de Magnetita/uso terapêutico , Óxidos N-Cíclicos/metabolismo , Portadores de Fármacos , Saccharomyces cerevisiae , Marcadores de Spin
13.
Molecules ; 24(15)2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31366105

RESUMO

The biological process, 3-O-galactosylation, is important in plant cells. To understand the mechanism of the reduction of flavonol antioxidative activity by 3-O-galactosylation, myricetin-3-O-galactoside (M3OGa) and myricetin aglycone were each incubated with 2 mol α,α-diphenyl-ß-picrylhydrazyl radical (DPPH•) and subsequently comparatively analyzed for radical adduct formation (RAF) products using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC-ESI-Q-TOF-MS) technology. The analyses revealed that M3OGa afforded an M3OGa-DPPH adduct (m/z 873.1573) and an M3OGa-M3OGa dimer (m/z 958.1620). Similarly, myricetin yielded a myricetin-DPPH adduct (m/z 711.1039) and a myricetin-myricetin dimer (m/z 634.0544). Subsequently, M3OGa and myricetin were compared using three redox-dependent antioxidant analyses, including DPPH•-trapping analysis, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide radical (PTIO•)-trapping analysis, and •O2 inhibition analysis. In the three analyses, M3OGa always possessed higher IC50 values than those of myricetin. Conclusively, M3OGa and its myricetin aglycone could trap the free radical via a chain reaction comprising of a propagation step and a termination step. At the propagation step, both M3OGa and myricetin could trap radicals through redox-dependent antioxidant pathways. The 3-O-galactosylation process, however, could limit these pathways; thus, M3OGa is an inferior antioxidant compared to its myricetin aglycone. Nevertheless, 3-O-galactosylation has a negligible effect on the termination step. This 3-O-galactosylation effect has provided novel evidence that the difference in the antioxidative activities of phytophenols exists at the propagation step rather than the termination step.


Assuntos
Flavonoides/química , Sequestradores de Radicais Livres/química , Galactosídeos/química , Superóxidos/química , Compostos de Bifenilo/antagonistas & inibidores , Óxidos N-Cíclicos/química , Óxidos N-Cíclicos/metabolismo , Dimerização , Flavonoides/metabolismo , Sequestradores de Radicais Livres/metabolismo , Radicais Livres , Galactosídeos/metabolismo , Glicosilação , Imidazóis/química , Imidazóis/metabolismo , Cinética , Oxirredução , Picratos/antagonistas & inibidores , Células Vegetais/química , Células Vegetais/metabolismo , Superóxidos/antagonistas & inibidores , Superóxidos/metabolismo
14.
J Phys Chem B ; 123(26): 5395-5404, 2019 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-31244099

RESUMO

The temperature-dependent structure and dynamics of two concentric solvent phases, the protein-associated domain (PAD) and the mesodomain, that surround the ethanolamine ammonia-lyase (EAL) protein from Salmonella typhimurium in frozen polycrystalline aqueous solution are addressed by using electron paramagnetic resonance spectroscopy of the paramagnetic nitroxide spin probe, TEMPOL, over the temperature ( T) range 190-265 K. Dimethyl sulfoxide (DMSO), added at 0.5, 2.0, and 4.0% v/v and present at the maximum freeze concentration at T ≤ 245 K, varies the volume of the interstitial aqueous DMSO mesodomain ( Vmeso) relative to a fixed PAD volume ( VPAD). The increase in Vmeso/ VPAD from 0.8 to 6.0 is quantified by the partitioning of TEMPOL between the two phases. As Vmeso/ VPAD is increased, the Arrhenius parameters for activated TEMPOL rotational motion in the mesodomain remain uniform, whereas the parameters for TEMPOL in the PAD show a progressive transformation toward the mesodomain values (higher mobility). An order-disorder transition (ODT) in the PAD is detected by the exclusion of TEMPOL from the PAD into the mesodomain. The ODT T value is systematically lowered by increased Vmeso/ VPAD (from 215 to 200 K), and PAD ordering kinks the mesodomain Arrhenius dependence. Thus there is reciprocity in PAD-mesodomain solvent coupling. The results are interpreted as a dominant influence of ice-boundary confinement on the PAD solvent structure and dynamics, which is transmitted through the mesodomain and which decreases with mesodomain volume at increased added DMSO. The systematic tuning of PAD and mesodomain solvent dynamics by the variation of added DMSO is an incisive approach for the resolution of contributions of protein-solvent dynamical coupling to EAL catalysis.


Assuntos
Dimetil Sulfóxido/química , Etanolamina Amônia-Liase/química , Congelamento , Termodinâmica , Óxidos N-Cíclicos/química , Óxidos N-Cíclicos/metabolismo , Dimetil Sulfóxido/metabolismo , Etanolamina Amônia-Liase/metabolismo , Salmonella typhimurium/enzimologia , Soluções , Solventes/química , Marcadores de Spin , Água/química , Água/metabolismo
15.
Oxid Med Cell Longev ; 2019: 6373685, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31089411

RESUMO

The present study was directed to the development of EPR methodology for distinguishing cells with different proliferative activities, using "redox imaging." Three nitroxide radicals were used as redox sensors: (a) mito-TEMPO-cell-penetrating and localized mainly in the mitochondria; (b) methoxy-TEMPO-cell-penetrating and randomly distributed between the cytoplasm and the intracellular organelles; and (c) carboxy-PROXYL-nonpenetrating in living cells and evenly distributed in the extracellular environment. The experiments were conducted on eleven cell lines with different proliferative activities and oxidative capacities, confirmed by conventional analytical tests. The data suggest that cancer cells and noncancer cells are characterized by a completely different redox status. This can be analyzed by EPR spectroscopy using mito-TEMPO and methoxy-TEMPO, but not carboxy-PROXYL. The correlation analysis shows that the EPR signal intensity of mito-TEMPO in cell suspensions is closely related to the superoxide level. The described methodology allows the detection of overproduction of superoxide in living cells and their identification based on the intracellular redox status. The experimental data provide evidences about the role of superoxide and hydroperoxides in cell proliferation and malignancy.


Assuntos
Biomarcadores/metabolismo , Peróxido de Hidrogênio/metabolismo , Superóxidos/metabolismo , Antioxidantes/metabolismo , Linhagem Celular , Proliferação de Células , Óxidos N-Cíclicos/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Leucemia/metabolismo , Linfócitos/metabolismo , Óxidos de Nitrogênio/química , Óxidos de Nitrogênio/metabolismo , Oxirredução
16.
Appl Microbiol Biotechnol ; 103(15): 6195-6204, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31129741

RESUMO

Pseudomonas aeruginosa biofilm lifestyle exhibits multidrug resistance in chronic bacterial infections. Alternative antimicrobial compounds or combination drug therapies must be urgently developed. In this work, the antibiofilm effect of Ag nanoparticle (AgNP) combined with the quorum sensing inhibitor (QSI) 4-nitropyridine N-oxide (4NPO) on P. aeruginosa biofilms was investigated. The biofilm biomass of P. aeruginosa was considerably reduced by 1.56-50 mg/L AgNP. However, 4NPO enhanced the ability of AgNP to inhibit P. aeruginosa biofilm formation (P < 0.05). The combination of AgNP with 4NPO could continuously inhibit biofilm development after 12 h, and 50 mg/L AgNP combined with 6.25 mg/L 4NPO thoroughly suppressed biofilm growth. The expression levels of QS genes and exopolysaccharide genes of biofilm treated with the combination of AgNP with 4NPO (AgNP-4NPO combination) were lower than those treated with AgNP alone (P < 0.05). Additional extracellular proteins and polysaccharides were determined in the samples treated with AgNP-4NPO combination. Based on proteomic analysis, this result was attributed to cell rupture caused by antimicrobial agents and intracellular materials released. The combination of the two antimicrobial agents could weaken the swimming ability of bacterial cells by damaging bacterial flagella and blocking rhlA gene expression. Thus, AgNP combined with QSI showed stronger antibiofilm ability than AgNP alone. These results may contribute to the development of antimicrobial agents.


Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Óxidos N-Cíclicos/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Percepção de Quorum/efeitos dos fármacos , Prata/farmacologia , Sinergismo Farmacológico , Nanopartículas Metálicas , Pseudomonas aeruginosa/crescimento & desenvolvimento
17.
Chem Res Toxicol ; 32(3): 348-361, 2019 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-30817135

RESUMO

Enzymatic one-electron reduction of heterocyclic N-oxides can lead to the intracellular generation of reactive oxygen species via several different chemical pathways. These reactions may be relevant to hypoxia-selective anticancer drugs, antimicrobial agents, and unwanted toxicity of heterocylic nitrogen compounds.


Assuntos
Óxidos N-Cíclicos/metabolismo , Oxigênio/metabolismo , Pró-Fármacos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Aerobiose , Anaerobiose , Óxidos N-Cíclicos/química , Hipóxia , Oxigênio/química , Pró-Fármacos/química
18.
ACS Chem Biol ; 14(2): 192-197, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30702848

RESUMO

Clinical investigation of the fatty acid amide hydrolase (FAAH) inhibitor BIA 10-2474 resulted in serious adverse neurological events. Structurally unrelated FAAH inhibitors tested in humans have not presented safety concerns, suggesting that BIA 10-2474 has off-target activities. A recent activity-based protein profiling (ABPP) study revealed that BIA 10-2474 and one of its major metabolites inhibit multiple members of the serine hydrolase class to which FAAH belongs. Here, we extend these studies by performing a proteome-wide analysis of covalent targets of BIA 10-2474 metabolites. Using alkynylated probes for click chemistry-ABPP in human cells, we show that des-methylated metabolites of BIA 10-2474 covalently modify the conserved catalytic cysteine in aldehyde dehydrogenases, including ALDH2, which has been implicated in protecting the brain from oxidative stress-related damage. These findings indicate that BIA 10-2474 and its metabolites have the potential to inhibit multiple mechanistically distinct enzyme classes involved in nervous system function.


Assuntos
Amidoidrolases/antagonistas & inibidores , Óxidos N-Cíclicos/farmacologia , Inibidores Enzimáticos/farmacologia , Piridinas/farmacologia , Aldeído-Desidrogenase Mitocondrial/metabolismo , Área Sob a Curva , Linhagem Celular Tumoral , Cromatografia Líquida , Química Click , Óxidos N-Cíclicos/metabolismo , Óxidos N-Cíclicos/farmacocinética , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacocinética , Células HEK293 , Humanos , Espectrometria de Massas , Piridinas/metabolismo , Piridinas/farmacocinética
19.
Life Sci ; 221: 65-71, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30738867

RESUMO

AIMS: Oxidative stress induced by diabetes mellitus (DM) is considered as one of the main causes of infertility in diabetic patients. The aim of the present study was to assess the effect of Tempol - as a synthetic antioxidant- on the testis oxidative stress and sperm parameters in type 2 diabetic (T2D) rats. MAIN METHODS: Twenty male Wistar rats were divided into 4 groups. Control groups (C) and diabetic groups (D); the control and diabetic groups received Tempol (100 mg/kg) for one month. Sperm parameters and oxidative stress biomarkers were evaluated in testicular tissue. KEY FINDINGS: The results demonstrated that administration of Tempol in diabetic rats improved sperm motility and viability and decreased the count of abnormal sperms. Also Tempol decreased the fasting blood sugar (FBS) and lipid peroxidation (LPO). In addition, Tempol significantly increased total antioxidant capacity (TAC) levels in testis tissue of T2D rats. Histopathological changes were also improved in the diabetic treated group. SIGNIFICANCE: Taken together, the results indicated that Tempol improved fertility parameters in a diabetic rat through reducing oxidative stress.


Assuntos
Óxidos N-Cíclicos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Testículo/efeitos dos fármacos , Animais , Antioxidantes , Catalase , Óxidos N-Cíclicos/farmacologia , Diabetes Mellitus Experimental/metabolismo , Glutationa Peroxidase , Peroxidação de Lipídeos , Masculino , Estresse Oxidativo/fisiologia , Ratos , Ratos Wistar , Contagem de Espermatozoides , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Marcadores de Spin , Superóxido Dismutase , Testículo/metabolismo
20.
Biochim Biophys Acta Mol Basis Dis ; 1865(6): 1152-1159, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30684639

RESUMO

The nitrone spin trap 5,5­dimethyl­1­pyrroline N­oxide (DMPO) dampens endotoxin-induced and TLR4-driven priming of macrophages, but the mechanism remains unknown. The available information suggests a direct binding of DMPO to the TIR domain, which is shared between TLRs. However, TLR2-TIR domain is the only TLR that have been crystallized. Our in silico data show that DMPO binds to four specific residues in the BB-loop within the TLR2-TIR domain. Our functional analysis using hTLR2.6-expressing HEKs cells showed that DMPO can block zymosan-triggered-TLR2-mediated NF-κB activation. However, DMPO did not affect the overall TLR2-MyD88 protein-protein interaction. DMPO binds to the BB-loop in the TIR-domain and dampens downstream signaling without affecting the overall TIR-MyD88 interaction. These data encourage the use of DMPO-derivatives as potential mechanism-based inhibitors of TLR-triggered inflammation.


Assuntos
Óxidos N-Cíclicos/metabolismo , Inflamação/metabolismo , Óxidos de Nitrogênio/metabolismo , Transdução de Sinais , Marcadores de Spin , Receptor 2 Toll-Like/metabolismo , Animais , Óxidos N-Cíclicos/química , Células HEK293 , Humanos , Inflamação/imunologia , Camundongos , Simulação de Dinâmica Molecular , Fator 88 de Diferenciação Mieloide/química , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/química , NF-kappa B/metabolismo , Óxidos de Nitrogênio/química , Ligação Proteica , Domínios Proteicos , Células RAW 264.7 , Receptor 2 Toll-Like/antagonistas & inibidores , Receptor 2 Toll-Like/química
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