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1.
Mol Cell Biol ; 40(22)2020 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-32868290

RESUMO

Activating mutations in KEAP1-NRF2 are frequently found in tumors of the lung, esophagus, and liver, where they are associated with aggressive growth, resistance to cancer therapies, and low overall survival. Despite the fact that NRF2 is a validated driver of tumorigenesis and chemotherapeutic resistance, there are currently no approved drugs which can inhibit its activity. Therefore, there is an urgent clinical need to identify NRF2-selective cancer therapies. To this end, we developed a novel synthetic lethal assay, based on fluorescently labeled isogenic wild-type and Keap1 knockout cell lines, in order to screen for compounds which selectively kill cells in an NRF2-dependent manner. Through this approach, we identified three compounds based on the geldanamycin scaffold which display synthetic lethality with NRF2. Mechanistically, we show that products of NRF2 target genes metabolize the quinone-containing geldanamycin compounds into more potent HSP90 inhibitors, which enhances their cytotoxicity while simultaneously restricting the synthetic lethal effect to cells with aberrant NRF2 activity. As all three of the geldanamycin-derived compounds have been used in clinical trials, they represent ideal candidates for drug repositioning to target the currently untreatable NRF2 activity in cancer.


Assuntos
Antineoplásicos/farmacologia , Benzoquinonas/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Lactamas Macrocíclicas/farmacologia , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Fator 2 Relacionado a NF-E2/metabolismo , Animais , Antineoplásicos/química , Benzoquinonas/química , Benzoquinonas/metabolismo , Morte Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Feminino , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Lactamas Macrocíclicas/química , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Transplante de Neoplasias , Neoplasias/tratamento farmacológico , Neoplasias/genética , Proteína Oncogênica v-akt/antagonistas & inibidores , Oxirredução , Paclitaxel/farmacologia
2.
Postepy Biochem ; 66(2): 91-99, 2020 06 27.
Artigo em Polonês | MEDLINE | ID: mdl-32700506

RESUMO

Cytochromes bc1 and c b6f are part of respiratory or photosynthetic machinery. The main role of these enzymes is to build proton motive force across the bioenergetic membranes by coupling the proton translocations with electron transfer from the pool of membrane-soluble quinones to water-soluble redox proteins. Despite many years of research, the mechanism of quinol oxidation is not fully understood. It is assumed that unstable form of a partially oxidized quinol ­ semiquinone is an intermediate state of this process and that it is also a potential electron donor in the side reaction of superoxide generation. This semiquinone has remained experimentally elusive over years but recently a semiquinone interacting with the reduced iron-sulfur cluster was identified as a new state of the enzyme. The results indicate that semiquinone coupled to the iron-sulfur cluster is most probably an additional state that can prevent side reactions, including superoxide generation.


Assuntos
Benzoquinonas/química , Benzoquinonas/metabolismo , Elétrons , Citocromos/metabolismo , Transporte de Elétrons , Oxirredução , Superóxidos/metabolismo
3.
mBio ; 11(1)2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-32019804

RESUMO

In bacteria, the respiratory pathways that drive molecular transport and ATP synthesis include a variety of enzyme complexes that utilize different electron donors and acceptors. This property allows them to vary the efficiency of energy conservation and to generate different types of electrochemical gradients (H+ or Na+). We know little about the respiratory pathways in Bacteroides species, which are abundant in the human gut, and whether they have a simple or a branched pathway. Here, we combined genetics, enzyme activity measurements, and mammalian gut colonization assays to better understand the first committed step in respiration, the transfer of electrons from NADH to quinone. We found that a model gut Bacteroides species, Bacteroides fragilis, has all three types of putative NADH dehydrogenases that typically transfer electrons from the highly reducing molecule NADH to quinone. Analyses of NADH oxidation and quinone reduction in wild-type and deletion mutants showed that two of these enzymes, Na+-pumping NADH:quinone oxidoreductase (NQR) and NADH dehydrogenase II (NDH2), have NADH dehydrogenase activity, whereas H+-pumping NADH:ubiquinone oxidoreductase (NUO) does not. Under anaerobic conditions, NQR contributes more than 65% of the NADH:quinone oxidoreductase activity. When grown in rich medium, none of the single deletion mutants had a significant growth defect; however, the double Δnqr Δndh2 mutant, which lacked almost all NADH:quinone oxidoreductase activity, had a significantly increased doubling time. Despite unaltered in vitro growth, the single nqr deletion mutant was unable to competitively colonize the gnotobiotic mouse gut, confirming the importance of NQR to respiration in B. fragilis and the overall importance of respiration to this abundant gut symbiont.IMPORTANCE Bacteroides species are abundant in the human intestine and provide numerous beneficial properties to their hosts. The ability of Bacteroides species to convert host and dietary glycans and polysaccharides to energy is paramount to their success in the human gut. We know a great deal about the molecules that these bacteria extract from the human gut but much less about how they convert those molecules into energy. Here, we show that B. fragilis has a complex respiratory pathway with two different enzymes that transfer electrons from NADH to quinone and a third enzyme complex that may use an electron donor other than NADH. Although fermentation has generally been believed to be the main mechanism of energy generation in Bacteroides, we found that a mutant lacking one of the NADH:quinone oxidoreductases was unable to compete with the wild type in the mammalian gut, revealing the importance of respiration to these abundant gut symbionts.


Assuntos
Bacteroides fragilis/enzimologia , Bacteroides fragilis/genética , Anaerobiose , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Benzoquinonas/metabolismo , Feminino , Vida Livre de Germes , Masculino , Redes e Vias Metabólicas , Camundongos , NAD/metabolismo , NADH Desidrogenase/genética , NADH Desidrogenase/metabolismo , Oxirredução , Quinona Redutases/genética , Quinona Redutases/metabolismo , Deleção de Sequência
4.
Food Chem ; 313: 126129, 2020 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-31935665

RESUMO

Thymoquinone is a chief phytochemical constituent of black cumin seed oil (BCSO) and shows strong bioactivity. It has a weak stability against environmental conditions like heat and light. Encapsulation process by Saccharomyces cerevisiae is a popular technique to preserve the bioactivity and increase the stability of functional bioactive compounds. In the current study, BCSO was encapsulated by both plasmolysed (PYC) and nonplasmolysed yeast cell (NPYC) and stability of thymoquinone and bioactive properties of all samples were evaluated. And also, some physicochemical, morphological and conformational characterizations were carried out for the encapsules. The results showed that thymoquinone concentration and its bioactivity were preserved better in PYC during storage compared to BCSO and NPYC. The highest degradation ratio of thymoquinone during storage for the BCSO was 96.78% while the lowest one was for the PYC sample (52.63%).


Assuntos
Benzoquinonas/química , Cápsulas/química , Nigella sativa/metabolismo , Óleos Vegetais/química , Saccharomyces cerevisiae/química , Benzoquinonas/metabolismo , Depuradores de Radicais Livres/química , Nigella sativa/química , Sementes/química , Sementes/metabolismo
5.
Biochim Biophys Acta Gen Subj ; 1864(3): 129514, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31911239

RESUMO

BACKGROUND: Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker is maintained as an epiphyte on citrus leaves until entering the plant tissue. During epiphytic survival, bacteria may encounter low water availability that challenges the infection process. Proteomics analyses of Xcc under saline stress, mimicking the conditions found during epiphytic survival, showed increased abundance of a putative NAD(P)H dehydrogenase encoded by XAC2229. METHODS: Expression levels of XAC2229 and a Xcc mutant in XAC2229 were analyzed in salt and oxidative stress and during plant-pathogen interaction. An Escherichia coli expressing XAC2229 was obtained, and the role of this protein in oxidative stress resistance and in reactive oxygen species production was studied. Finally, Xac2229 protein was purified, spectrophotometric and cofactor analyses were done and enzymatic activities determined. RESULTS: XAC2229 was expressed under salt stress and during plant-pathogen interaction. ΔXAC2229 mutant showed less number of cankers and impaired epiphytic survival than the wild type strain. ΔXAC2229 survived less in the presence of H2O2 and produced more reactive oxygen species and thiobarbituric acid-reactive substances than the wild type strain. Similar results were observed for E. coli expressing XAC2229. Xac2229 is a FAD containing flavoprotein, displays diaphorase activity with an optimum at pH 6.0 and has quinone reductase activity using NADPH as an electron donor. CONCLUSIONS: A FAD containing flavoprotein from Xcc is a new NADPH quinone reductase required for bacterial virulence, particularly in Xcc epiphytic survival on citrus leaves. GENERAL SIGNIFICANCE: A novel protein involved in the worldwide disease citrus canker was characterized.


Assuntos
NAD(P)H Desidrogenase (Quinona)/metabolismo , Xanthomonas/enzimologia , Benzoquinonas/metabolismo , Citrus/metabolismo , Citrus/microbiologia , Peróxido de Hidrogênio/metabolismo , NAD(P)H Desidrogenase (Quinona)/genética , NADP/metabolismo , Estresse Oxidativo , Folhas de Planta/metabolismo , Estresse Salino/genética , Estresse Salino/fisiologia , Virulência , Xanthomonas/metabolismo , Xanthomonas/patogenicidade , Xanthomonas/fisiologia
6.
Life Sci ; 244: 117299, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31953157

RESUMO

AIMS: Notch signaling is highly implicated in several cancers and chemoresistance. Therefore, Notch-targeted therapies might be beneficial in enhancing chemotherapeutic effect and cancer regression. This study aimed to investigate implication of Notch in development and progression of solid Ehrlich carcinoma (SEC) and enhancement of anticancer effect of cisplatin (CIS) by addition of thymoquinone (TQ) and pentoxifylline (PTX) through modulation of Notch. MAIN METHODS: SEC was induced in mice as model for mammary carcinoma by s.c. injection of 1 × 106 Ehrlich cells into back of the mice. On 12th day, solid tumor was developed and mice were divided into seven groups; tumor control, early CIS (ECIS), ECIS + ETQ, ECIS + ETQ + EPTX, late CIS (LCIS), LCIS + LTQ, and LCIS + LTQ + LPTX. Early treatment was started on 12th day, whereas late treatment was begun on 19th day from tumor inoculation. At the endpoint, samples were collected for detection of Notch1, Hes1, Jagged1, ß-catenin, TNF-α, IL-6, IFN-γ, IL-2, VEGF, apoptosis, CD4, and CD8. KEY FINDINGS: Adding PTX and TQ to CIS significantly reduced Notch1, Hes1, Jagged1, ß-catenin, TNF-α, IL-6, IFN-γ, and VEGF with increment in IL-2, CD4, CD8, and apoptotic cells. Moreover, early treated groups showed remarkable attenuation in tumor growth and the relevant parameters compared to their counterpart later groups. SIGNIFICANCE: Addition of PTX with TQ to CIS showed a synergistic chemotherapeutic action and induced better oncostatic effect mainly through Notch suppression. Consequently, shutting Notch could be of great interest in promoting chemosensetivity and cancer control.


Assuntos
Benzoquinonas/farmacologia , Pentoxifilina/farmacologia , Receptores Notch/metabolismo , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Benzoquinonas/metabolismo , Carcinoma de Ehrlich/tratamento farmacológico , Carcinoma de Ehrlich/metabolismo , Cisplatino/metabolismo , Cisplatino/farmacologia , Feminino , Camundongos , Pentoxifilina/metabolismo , Receptor Notch1/metabolismo , Transdução de Sinais/efeitos dos fármacos
7.
Enzyme Microb Technol ; 132: 109414, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31731961

RESUMO

ß-glucosidase (BG) plays a key role in determining the efficiency of the enzymatic complex cellulase for the degradation of cellulose into sugars. It hydrolyses the cellobiose, an inhibitor of the enzymatic complex. Therefore, the immobilization of BG is a great challenge for the industrial application of cellulases. Cellulases usually contains a BG amount insufficient to avoid inhibition by cellobiose. Here the BG was immobilized by matrix assisted pulsed laser evaporation (MAPLE) technique. The frozen matrix was composed of water, water/m-DOPA and water/m-DOPA/quinone. The effect of the excipients on the final conformation of the enzyme after the MAPLE processing was determined. The enzyme secondary structure was studied by FTIR analysis. The catalytic performances of the deposited films were tested in the cellobiose hydrolysis reaction. The results demonstrate that the presence of the oxidized form of m-DOPA, the O-quinone form, can protect the protein native structure, with the laser inducing little or no damage. In fact, only the samples deposited from this target preserved the secondary structure of the polypeptide chain and allowed a complete hydrolysis of cellobiose for four consecutive runs, showing a high operational stability of the biocatalyst.


Assuntos
Benzoquinonas/metabolismo , Di-Hidroxifenilalanina/análogos & derivados , Di-Hidroxifenilalanina/metabolismo , beta-Glucosidase/metabolismo , Catálise , Celobiose/metabolismo , Celulase/metabolismo , Enzimas Imobilizadas/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Complexos Multienzimáticos , Quinonas/metabolismo , Temperatura
8.
Appl Microbiol Biotechnol ; 104(4): 1647-1660, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31853567

RESUMO

More than two-third of known antibiotics are produced by actinomycetes of the genus Streptomyces. Unfortunately, the production rate from Streptomyces natural antibiotic is extremely slow and thus cannot satisfy industrial demand. In this study, the production of antibiotics by Streptomyces is enhanced by a "superplasmid" which including global regulatory factors afsR, cyclic adenosine receptor protein (CRP), RNA polymerase beta subunits (rpoB) with point mutation and acetyl coenzyme A carboxylase gene (accA2BE), these elements are controlled by the PermE* promoter and then transfer into Streptomyces coelicolor M145, Streptomyces mutabilis TRM45540, Streptomyces hygroscopicus XM201, and Streptomyces hygroscopicus ATCC29253 by conjugation to generate exconjugants. NMR, HPLC, and LC-MS analyses revealed that the superplasmid led to the overproduction of actinorhodin (101.90%), undecylprodigiosin (181.60%) in S. coelicolor M145:: pLQ003, of rapamycin (110%), hygrocin A (163.4%) in S. hygroscopicus ATCC29253:: pLQ003, and of actinomycin D (11.78%) in S. mutabilis TRM45540:: pLQ003, and also to the downregulation of geldanamycin in S. hygroscopicus XM201, but we found that mutant strains in mutant strains of S. hygroscopicus XM201 with regulatory factors inserted showed several peaks that were not found in wild-type strains. The results of the present work indicated that the regulator net working in Streptomyces was not uniform, the superplasmid we constructed possibly caused this overproduction and downregulation in different Streptomyces.


Assuntos
Antibacterianos/biossíntese , Engenharia Genética/métodos , Plasmídeos/genética , Streptomyces/genética , Acetil-CoA Carboxilase/genética , Bactérias/efeitos dos fármacos , Benzoquinonas/metabolismo , Dactinomicina/biossíntese , Lactamas Macrocíclicas/metabolismo , Mutação Puntual , Regiões Promotoras Genéticas
9.
Food Chem ; 302: 125275, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31442701

RESUMO

2,6-Dimethoxy-ρ-benzoquinone (DMBQ) is a potential anti-tumor substance found in the fermented wheat germ. In this study, ultrasound and Fe3O4 nanoparticles were used to improve the DMBQ yield. An artificial neural network (ANN) embedded separately with the back-propagation algorithm (BP), genetic algorithm (GA), particle swarm optimized algorithm (PSO), ant colony optimized algorithm (ACO), GA-ACO, GA-PSO and PSO-ACO, were used to establish the relationship between 11 factors and DMBQ yield. The robustness and generalization of PSO-ACO-ANN, which gave the minimum mean squared error and mean absolute percentage error for the training and test dataset, was superior to the others. Next, a modified Garson's algorithm and mixed partial derivatives algorithm indicated that the most influential paired-parameters were ultrasonic power and concentration of nanoparticles. Finally, the factors were optimized by six optimization algorithms, and confirmatory experimental results indicated that the optimum DMBQ yield was 0.213 ±â€¯0.007 mg/g, which was 161.2% higher than the control.


Assuntos
Algoritmos , Benzoquinonas/metabolismo , Fermentação/efeitos dos fármacos , Nanopartículas de Magnetita , Triticum/efeitos dos fármacos , Triticum/metabolismo , Ondas Ultrassônicas , Redes Neurais de Computação
10.
Pharmacol Rep ; 71(6): 1289-1292, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31693968

RESUMO

BACKGROUND: Methylphenidate (Ritalin®) is a psychostimulant used chronically to treat attention deficit hyperactivity disorder. Methylphenidate acts by preventing the reuptake of dopamine and norepinephrine, resulting in an increase in these neurotransmitters in the synaptic cleft. Excess dopamine can be autoxidized to a quinone that may lead to oxidative stress. The antioxidant, glutathione helps to protect the cell against quinones via conjugation reactions; however, depletion of glutathione may result from excess quinone formation. Chronic exposure to methylphenidate appears to sensitize dopaminergic neurons to the Parkinsonian toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). We hypothesized that oxidative stress caused by the autooxidation of the excess dopamine renders dopaminergic neurons within the nigrostriatal pathway to be more sensitive to MPTP. METHODS: To test this hypothesis, male mice received chronic low or high doses of MPH and were exposed to saline or MPTP following a 1-week washout. Quinone formation in the striatum was examined via dot blot, and striatal GSH was quantified using a glutathione assay. RESULTS: Indeed, quinone formation increased with increasing doses of methylphenidate. Additionally, methylphenidate dose-dependently resulted in a depletion of glutathione, which was further depleted following MPTP treatment. CONCLUSIONS: Thus, the increased sensitivity of dopamine neurons to MPTP toxicity following chronic methylphenidate exposure may be due to quinone production and subsequent depletion of glutathione.


Assuntos
Antioxidantes/metabolismo , Benzoquinonas/metabolismo , Corpo Estriado/efeitos dos fármacos , Glutationa/metabolismo , Metilfenidato/farmacologia , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologia , Animais , Estimulantes do Sistema Nervoso Central/farmacologia , Corpo Estriado/metabolismo , Dopamina/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Masculino , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Substância Negra/efeitos dos fármacos , Substância Negra/metabolismo
11.
Acta Pharm ; 69(4): 563-584, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31639096

RESUMO

The impact of the selected entactogens and their o-quinone metabolites on the environment was explored in QSAR studies by the use of predicted molecular descriptors, ADMET properties and environmental toxicity parameters, i.e., acute toxicity in Tetrahymena pyriformis (TOX_ATTP) expressed as Th_pyr_pIGC50/mmol L-1, acute toxicity in Pimephales promelas, the fathead minnow (TOX_FHM) expressed as Minnow LC50/mg L-1, the acute toxicity in Daphnia magna (TOX_DM) expressed as Daphnia LC50/mg L-1 and bioconcentration factor (BCF). The formation of corresponding o-quinones via benzo-dioxo-lone ring, O-demethylenation was predicted as the main metabolic pathway for all entactogens except for 1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)propan-2-amine (DiFMDA). The least favourable ADMET profile was revealed for N-(1-(benzo[d][1,3]dioxol-5-yl)propan-2-yl)-O-methylhydroxylamine (MDMEO). QSAR studies revealed significant linear correlations between MlogP of entactogens and MlogP of o-quinone metabolites (R = 0.99), and Th_pyr_pIGC50/mmol L-1 (R = 0.94), also their MlogPs with Minnow_LC50/mg L-1 (R = 0.80 and R = 0.78), BCF (R = 0.86 and R = 0.82) and percentage of o-quinones' yields (R = 0.73 and R = 0.80). Entactogens were predicted as non-biodegradable molecules, whereas the majority of their o-quinones were biodegradable.


Assuntos
Fenetilaminas/metabolismo , Fenetilaminas/toxicidade , Animais , Benzoquinonas/metabolismo , Daphnia/efeitos dos fármacos , Ecotoxicologia/métodos , Relação Quantitativa Estrutura-Atividade , Tetrahymena pyriformis/efeitos dos fármacos
12.
Nutrients ; 11(10)2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31623257

RESUMO

Wheat germ is rich in quinones that exist as glycosides. In this study, we used Celluclast 1.5L to release the hydroxyquinones, which turn into benzoquinone, and prepared the water extract from enzyme-treated wheat germ (EWG). We investigated whether enzyme treatment altered the anti-inflammatory activity compared to the water extract of untreated wheat germ (UWG). UWG inhibited the production of inducible nitric oxide synthase (iNOS) and interleukin (IL)-12 and induced the production of IL-10 and heme oxygenase (HO)-1 in lipopolysaccharide (LPS)-stimulated macrophages. Enzyme treatment resulted in greater inhibition of iNOS and IL-10 and induction of HO-1 compared to UWG, possibly involving the modulation of nuclear factor (NF)-κB, activator protein 1 (AP-1) and nuclear factor erythroid 2-related factor (Nrf2). Mice fed UWG or EWG had decreased serum tumor necrosis factor (TNF)-α and increased serum IL-10 levels after intraperitoneal injection of LPS, with UWG being more effective for IL-10 and EWG more effective for TNF-α. Hepatic HO-1 gene was only expressed in mice fed EWG. We provide evidence that enzyme treatment is a useful biotechnology tool for extracting active compounds from wheat germ.


Assuntos
Anti-Inflamatórios/farmacologia , Benzoquinonas/farmacologia , Germinação , Glicosídeo Hidrolases/metabolismo , Mediadores da Inflamação/metabolismo , Macrófagos Peritoneais/efeitos dos fármacos , Sementes/metabolismo , Solventes/química , Triticum/metabolismo , Água/química , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/metabolismo , Benzoquinonas/química , Benzoquinonas/metabolismo , Células Cultivadas , Ciclo-Oxigenase 2/metabolismo , Citocinas/metabolismo , Heme Oxigenase-1/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Macrófagos Peritoneais/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos BALB C , Óxido Nítrico Sintase Tipo II/metabolismo , Sementes/crescimento & desenvolvimento , Transdução de Sinais , Triticum/crescimento & desenvolvimento
13.
Chem Biodivers ; 16(12): e1900503, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31660678

RESUMO

A series of 2,5-bis(alkyl/arylamino)-1,4-benzoquinones (1-12) were investigated in vitro for their potential to inhibit the activity of jack bean urease. Compounds 1-6, 8, 9, 11 and 12 effectively inhibited the jack bean urease activity by 90.8 % when tested at 5 µm, whereas 7 and 10 had relatively little effect. The IC50 for most compounds was in the nanomolar range (31.4 nm and 36.0 nm for 2 and 8, respectively). The mechanism of enzyme inhibition shown by 2 and 8 is typical of mixed-type inhibitors, whose affinity for the active site is over 6- and 2-fold higher (Ki =30.0 and 22.8 nm, for 2 and 8, respectively) than that of an allosteric site. Molecular docking studies revealed that both 2 and 8 establish hydrogen bonds with the amino acids residues Asp494, Met588, His593 and Ala636 in the active site of jack bean urease. These results indicate that such aminoquinones are useful leads for the development of more efficient urease inhibitors of wider utility.


Assuntos
Benzoquinonas/química , Inibidores Enzimáticos/química , Urease/antagonistas & inibidores , Sítio Alostérico , Benzoquinonas/metabolismo , Benzoquinonas/farmacologia , Canavalia/enzimologia , Domínio Catalítico , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Cinética , Simulação de Acoplamento Molecular , Relação Estrutura-Atividade , Urease/metabolismo
14.
Proc Natl Acad Sci U S A ; 116(39): 19458-19463, 2019 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-31488720

RESUMO

Photosystem II (PSII), the light-driven water/plastoquinone photooxidoreductase, is of central importance in the planetary energy cycle. The product of the reaction, plastohydroquinone (PQH2), is released into the membrane from the QB site, where it is formed. A plastoquinone (PQ) from the membrane pool then binds into the QB site. Despite their functional importance, the thermodynamic properties of the PQ in the QB site, QB, in its different redox forms have received relatively little attention. Here we report the midpoint potentials (Em ) of QB in PSII from Thermosynechococcus elongatus using electron paramagnetic resonance (EPR) spectroscopy: Em QB/QB •- ≈ 90 mV, and Em QB •-/QBH2 ≈ 40 mV. These data allow the following conclusions: 1) The semiquinone, QB •-, is stabilized thermodynamically; 2) the resulting Em QB/QBH2 (∼65 mV) is lower than the Em PQ/PQH2 (∼117 mV), and the difference (ΔE ≈ 50 meV) represents the driving force for QBH2 release into the pool; 3) PQ is ∼50× more tightly bound than PQH2; and 4) the difference between the Em QB/QB •- measured here and the Em QA/QA •- from the literature is ∼234 meV, in principle corresponding to the driving force for electron transfer from QA •- to QB The pH dependence of the thermoluminescence associated with QB •- provided a functional estimate for this energy gap and gave a similar value (≥180 meV). These estimates are larger than the generally accepted value (∼70 meV), and this is discussed. The energetics of QB in PSII are comparable to those in the homologous purple bacterial reaction center.


Assuntos
Benzoquinonas/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Quinonas/metabolismo , Cianobactérias/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Transporte de Elétrons , Cinética , Luz , Oxirredução , Fotossíntese/fisiologia , Plastoquinona/análogos & derivados , Plastoquinona/metabolismo , Termodinâmica , Água/química
15.
J Toxicol Sci ; 44(8): 559-563, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31378767

RESUMO

Acetaminophen (Paracetamol, APAP) has been widely used for many decades as an analgesic and antipyretic agent but APAP overdose often causes acute adverse reactions, particularly liver damage. The metabolically oxidized form of APAP, N-acetyl-p-benzoquinone imine (NAPQI), is chemically reactive and binds covalently to proteins. Therefore, NAPQI is believed to be the key metabolite that causes hepatotoxicity, especially under conditions of glutathione depletion. Other APAP-induced adverse reactions, such as skin damage, are rare and remain poorly studied. Here, we report a case study of a male patient who presented with an acute swelling skin rash (without hepatotoxicity) caused by therapeutic doses of APAP. Plasma samples were collected at 17 hr after dosing (during the manifestation of symptoms) and at one month (after recovery) and were subjected to LC-MS analysis of NAPQI-adducts. A significant concentration of NAPQI-cysteine adduct (33 pmol/mL) was found together with low concentrations of NAPQI-N-acetylcysteine adduct (2.0 pmol/mL) and NAPQI-glutathione adduct (0.13 pmol/mL). However, the NAPQI-albumin adduct was below the detection limit (below 0.001% modification on albumin) despite a previous report of high concentrations of NAPQI-albumin adduct following acute liver injury. Therefore, the observed APAP-induced skin damage may have had a different cause from APAP-induced liver injury.


Assuntos
Acetaminofen/efeitos adversos , Acetilcisteína/sangue , Benzoquinonas/efeitos adversos , Benzoquinonas/sangue , Cisteína/sangue , Edema/induzido quimicamente , Exantema/induzido quimicamente , Glutationa/sangue , Iminas/efeitos adversos , Iminas/sangue , Dermatopatias/induzido quimicamente , Acetilcisteína/metabolismo , Doença Aguda , Adulto , Benzoquinonas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas , Cromatografia Líquida , Cisteína/metabolismo , Glutationa/metabolismo , Humanos , Iminas/metabolismo , Masculino , Ligação Proteica , Albumina Sérica/metabolismo , Espectrometria de Massas em Tandem
16.
Appl Microbiol Biotechnol ; 103(18): 7741-7750, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31372705

RESUMO

2-Chloro-4-nitrophenol (2C4NP) is the most common chlorinated nitrophenol pollutant, and its environmental fate is of great concern. Cupriavidus sp. CNP-8, a Gram-negative bacterium, has been reported to degrade 2C4NP via the 1,2,4-benzenetriol (BT) pathway, significantly different from the (chloro)hydroquinone pathways reported in all other Gram-negative 2C4NP-utilizers. Herein, the BT pathway of the catabolism of 2C4NP in this strain was characterized at the molecular, biochemical, and genetic levels. The hnp gene cluster was suspected to be involved in the catabolism of 2C4NP because the hnp genes are significantly upregulated in the 2C4NP-induced strain CNP-8 compared to the uninduced strain. HnpAB, a two-component FAD-dependent monooxygenase, catalyzes the conversion of 2C4NP to BT via chloro-1,4-benzoquinone, with a Km of 2.7 ± 1.1 µΜ and a kcat/Km of 0.17 ± 0.03 µΜ-1 min-1. hnpA is necessary for strain CNP-8 to utilize 2C4NP in vivo. HnpC, a BT 1,2-dioxygenase, was proved to catalyze BT ring-cleavage with formation of maleylacetate by HPLC-MS analysis. Phylogenetic analysis indicated that HnpA likely has different evolutionary origin compared to other functionally identified 2C4NP monooxygenases. To our knowledge, this is the first report revealing the catabolic mechanism of 2C4NP via the BT pathway in a Gram-negative bacterium, increasing our knowledge of the catabolic diversity for microbial 2C4NP degradation at the molecular and biochemical level.


Assuntos
Proteínas de Bactérias/metabolismo , Cupriavidus/enzimologia , Hidroquinonas/metabolismo , Oxigenases de Função Mista/metabolismo , Nitrofenóis/metabolismo , Proteínas de Bactérias/genética , Benzoquinonas/metabolismo , Biodegradação Ambiental , Cupriavidus/genética , Redes e Vias Metabólicas , Oxigenases de Função Mista/genética , Família Multigênica , Filogenia
17.
Int J Biol Macromol ; 140: 614-620, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31446103

RESUMO

Type I collagen is a vital structural component of the extracellular matrix providing the connective tissues with biomechanical support. One of the interesting properties of collagen is to self-associate into fibrils. The present work aims to direct the self-assembly of collagen through different molecular forces, which are tuned on the addition of thymoquinone a well-known phytochemical. A change in relative viscosity and stress of collagen-thymoquinone blends influenced the interfibrillar aggregates around its hydration shell. Further, secondary structural integrity was studied via cotton curve effect, and vibrational frequency shifts showed a characteristic interaction of thymoquinone at the N-terminal residues of the triple helix. Finally, the spontaneous self-association of fibrils was tracked by calculating the rate of fibril growth kinetics, which potentially decreased with increase in thymoquinone concentration. The fibrils were eventually visualized under the high resolution-scanning microscope showing morphological variations. Therefore, such a protein-phytochemical interaction may tend to play with the hydration network of collagen and covalently interact with its imino acid residues. It may be speculated that such an inhibitory process portrayed by thymoquinone may have a fortune in the targeted and sustainable delivery to the site of action for certain diseases, which includes collagen accumulation. Moreover, its directed assembly could be utilized for designing templates as in manipulating the collagen as a nanoporous membrane to make nanofibers and further tuned by small molecules for nanoparticle synthesis application.


Assuntos
Benzoquinonas/metabolismo , Colágeno Tipo I/metabolismo , Animais , Matriz Extracelular/metabolismo , Cinética , Simulação de Dinâmica Molecular , Ratos , Ratos Wistar
18.
Appl Microbiol Biotechnol ; 103(20): 8545-8557, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31468089

RESUMO

Salmonella Typhimurium, a common Gram-negative foodborne pathogen, threatens public health and hinders the development of the food industry. In this study, we evaluated the antibiofilm activity of coenzyme Q0 (CoQ0) against S. Typhimurium. Besides, the inhibition of the S. Typhimurium's adhesion to and invasion of Caco-2 cells and its survival and replication in RAW 264.7 cells by CoQ0 were also explored. The minimum inhibitory concentrations and minimal bactericidal concentrations of CoQ0 against Salmonella were both 100-400 µg/mL. Salmonella Typhimurium biofilm formation was effectively inhibited by subinhibitory concentrations (SICs) of CoQ0. The CoQ0-affected biofilm morphology was observed with light microscopy and field-emission scanning electron microscopy. CoQ0 at SICs reduced the swimming motility and quorum sensing of S. Typhimurium and repressed the transcription of critical virulence-related genes. CoQ0 at SICs also clearly reduced the adhesion of S. Typhimurium to and its invasion of Caco-2 cells and reduced its survival and replication within RAW 264.7 macrophage cells. These findings suggest that CoQ0 has strong antibiofilm activity and can be used as an anti-infectious agent against Salmonella.


Assuntos
Antibacterianos/metabolismo , Aderência Bacteriana/efeitos dos fármacos , Benzoquinonas/metabolismo , Biofilmes/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Viabilidade Microbiana/efeitos dos fármacos , Salmonella typhimurium/efeitos dos fármacos , Animais , Células CACO-2 , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Células RAW 264.7
19.
Fungal Biol ; 123(8): 601-610, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31345414

RESUMO

Oosporein was first identified from the insect pathogen Beauveria bassiana >50 y ago. Here, we investigate the insecticidal, anti-feedant and immunomodulation effects of oosporein produced by Beauveria caledonica on the forestry pest Hylobius abietis and model insect Galleria mellonella. We report a novel feedback induction mechanism regulating oosporein production in B. caledonica; exogenous oosporein induces the expression of the oosporein cluster, leading to increased abundance of oosporein biosynthetic enzymes, as shown by label-free quantitative proteomics. Oosporein did not have an anti-feedant effect on H. abietis adults - on the contrary, insects exposed to oosporein-treated food fed more than those exposed to untreated food only. Injected oosporein did not kill insect larvae but increased susceptibility of H. abietis to a subsequent infection. Oosporein did not act as a contact toxin on H. abietis adults and G. mellonella larvae at the concentrations tested. Therefore, it appears that oosporein promotes infection rather than directly killing insects; this could be mediated both by a reduction in haemocyte numbers and by alterations to the humoral immune system. This work makes a case for future research into the potential use of B. caledonica as a biocontrol agent through combinations with oosporein or with enhanced production of oosporein.


Assuntos
Beauveria/metabolismo , Benzoquinonas/metabolismo , Benzoquinonas/toxicidade , Inseticidas/metabolismo , Inseticidas/toxicidade , Gorgulhos/microbiologia , Animais , Beauveria/química , Beauveria/patogenicidade , Vias Biossintéticas , Comportamento Alimentar/efeitos dos fármacos , Feminino , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Masculino , Virulência , Gorgulhos/fisiologia
20.
Mikrochim Acta ; 186(8): 527, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31297616

RESUMO

A fluorometric assay is described for the determination of the herbicide atrazine. The assay is based on the use of tyrosinase and fluorescent nitrogen-doped graphene quantum dots (N-GQDs). The N-GQDs were synthesized via one-pot hydrothermal reaction starting from citric acid and ammonia. Their fluorescence excitation and emission maxima are at 355 and 435 nm, and the quantum yield is 18%. Tyrosinase catalyzes the oxidation of dopamine to form dopaquinone which reduces the fluorescence of the N-GQDs through a dynamic quenching process. On addition of atrazine, the catalytic activity of tyrosinase is inhibited. This leads to less formation of dopaquinone and less reduction of fluorescence. The assay has a linear response in the 2.5-100 ng·mL-1 atrazine concentration range, and the detection limit is 1.2 ng·mL-1. The assay was applied to the determination of atrazine in spiked environmental water samples. Graphical abstract Schematic presentation of the fluorometric assay of atrazine detection based on tyrosinase-induced fluorescence (FL) quenching effect on the nitrogen-doped graphene quantum dots (N-GQDs) and inhibitory effect of atrazine on tyrosinase.


Assuntos
Atrazina/análise , Fluorometria/métodos , Grafite/química , Monofenol Mono-Oxigenase/metabolismo , Pontos Quânticos/química , Atrazina/química , Atrazina/metabolismo , Benzoquinonas/química , Benzoquinonas/metabolismo , Di-Hidroxifenilalanina/análogos & derivados , Di-Hidroxifenilalanina/química , Di-Hidroxifenilalanina/metabolismo , Dopamina/química , Dopamina/metabolismo , Água Doce/análise , Limite de Detecção , Monofenol Mono-Oxigenase/antagonistas & inibidores , Nitrogênio/química
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