Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 41.020
Filtrar
1.
Methods Mol Biol ; 2554: 123-139, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36178624

RESUMO

Saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy is an established technique for detecting and characterizing the binding of small molecules, such as metabolites, to biological macromolecules like proteins and nucleic acids. STD NMR allows detection of binding in complex mixtures of potential ligands, which is often used for library screening in the pharmaceutical industry but may also be beneficial for binding studies with metabolite mixtures. The nature of the ligand is normally restricted to small molecules in terms of NMR spectroscopy, and the size of the macromolecule on the other side should be larger than 10-15 kDa. This technique is especially applicable to detecting binders of intermediate to low affinity with the dissociation constant (KD) above 1 µM. In this chapter, we focus on recent developments and the applications of STD NMR to studying interactions of natural products and metabolites, in particular. The reader is also referred to excellent reviews of the field and the literature cited therein. This chapter also provides a detailed experimental protocol for performing the STD NMR measurement based on the example of the subunit A of the Na+-transporting NADH/ubiquinone oxidoreductase (Na+-NQR) from V. cholerae interacting with its natural quinone substrate and inhibitors.


Assuntos
Produtos Biológicos , Ácidos Nucleicos , Vibrio cholerae , Misturas Complexas , Ligantes , Espectroscopia de Ressonância Magnética/métodos , NAD/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , Ácidos Nucleicos/metabolismo , Oxirredutases/metabolismo , Ligação Proteica , Proteínas/química , Ubiquinona/metabolismo , Vibrio cholerae/metabolismo
2.
Gene ; 850: 146926, 2023 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-36191825

RESUMO

Arsenic transforming bacterial strains belong to genus Pseudomonas sp.AK9 (KY569424), were isolated from the middle Gangetic plains of Bihar, India. The Pseudomonas sp. AK9 strains were able to transform toxic arsenite to a less toxic arsenate. In the present work, the presence of different arsenic resistance genes (aoxB, arsB, acr3 and aoxAB) were observed in isolated strain. Furthermore, the aoxB gene was amplified from genomic DNA of AK9, cloned in E.coli/DH5αcells, and sequenced. The BLASTn results and phylogenetic study of the aoxB gene showed 95.32 % and 90.07 % identity with the large subunit of aoxB gene of previous reported Thiomonas arsenivorans strain DSM16361 and Thiomonas arsenivorans strain b6, respectively. Further overhang primers were designed for amplifications of full length aoxB gene (∼1200 bp), and cloned in to the expression vector and host E.coli/BL21 cells. The GST-aoxB gene was expressed in BL21 cells, and a profound expression product of âˆ¼ 72 kDa was observed in SDS PAGE. The detection of a large subunit (aoxB) of arsenate oxidase protein in western blotting assay affirmed the expression of aoxB gene in recombinant E.coli/BL21 clone. Further, the recombinant E.coli/BL21cells showed increased growth than the normal E.coli/BL21 cells against As (III). Thus, this study showed the presence of aoxB gene in Pseudomonas sp. AK9 genome which regulates the resistant ability to arsenic toxicity.


Assuntos
Arsênio , Arsenitos , Oxirredutases , Arseniatos/metabolismo , Arsênio/toxicidade , Arsenitos/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Filogenia , Pseudomonas/genética , Pseudomonas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo
3.
Spectrochim Acta A Mol Biomol Spectrosc ; 285: 121900, 2023 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-36170775

RESUMO

A nanocomposite (UiO-66/Ce-MnO2) was fabricated by combining UiO-66 with cerium-doped manganese dioxide (Ce-MnO2) for colorimetric detecting vitamin C (Vc). Compared with traditional artificial enzymes, the as-synthesized UiO-66/Ce-MnO2 were simple to prepare and did not require the participation of other active substances. The doping of cerium increased the oxygen vacancies and the UiO-66 as a carrier improved the dispersibility. The formation of superoxide anion (O2-) and the inside Ce4+/Ce3+ and Mn4+/Mn3+ redox couples of UiO-66/Ce-MnO2 endowed UiO-66/Ce-MnO2 with a high catalytic capability, which could catalytically oxidize 3, 3', 5, 5'-tetramethylbenzidine (TMB) into oxidation state TMB (oxTMB) without H2O2, accompanying with color change and a prominent peak at 652 nm in UV-vis spectra. Based on the inhibitory effects of Vc on catalytic oxidation of TMB, detection of Vc can be achieved, exhibiting a linear relationship in the concentration of 1.13-17.01 µmol L-1 with a low detection limit of 65.82 nmol L-1. This system can also be detected by smartphone, the linear detection range is 12.47-22.67 µmol L-1. Vc contents in fruits and vegetables detected by the sensor were in good agreement with the 2, 4-Dinitrophenylhydrazine colorimetry method (P > 0.05), indicating a reliable sensor for Vc detection.


Assuntos
Cério , Compostos de Manganês , Oxirredutases , Ácido Ascórbico/análise , Verduras , Óxidos , Frutas/química , Peróxido de Hidrogênio , Colorimetria/métodos , Limite de Detecção
4.
Spectrochim Acta A Mol Biomol Spectrosc ; 285: 121928, 2023 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-36191436

RESUMO

The low temperature solvothermal method synthesized Cr2O3 NPs has not only peroxidase activity, but also oxidase activity. Then, the oxidase activity of Cr2O3 NPs is effectively shielded by nanogel immobilization using three monomers acrylamide, NIPAAM (N-isopropylacrylamide) and MBA (N,N'-methylene bisacrylamide) in HEPES (4-(2-hydroxyerhyl)piperazine-1-erhanesulfonic acid) buffer. Ultimately, the enzymatic activity of Cr2O3@nanogel/Au is significantly enhanced after doping Au NPs by SERS (Surface Enhanced Raman Spectroscopy) evaluation. A SERS strategy was proposed for the detection of H2O2 by Cr2O3@nanogel/Au. The linear range was 10-8 mol·L-1-10-1 mol·L-1.


Assuntos
Ouro , Nanopartículas Metálicas , Ouro/química , Nanopartículas Metálicas/química , Peróxido de Hidrogênio , Nanogéis , Análise Espectral Raman/métodos , Peroxidases , Oxirredutases
5.
J Hazard Mater ; 443(Pt A): 130233, 2023 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-36308933

RESUMO

Ochratoxin A (OTA), which has strong hepatotoxicity and nephrotoxicity, can accumulate in the human body through the food chain; thus, the selective and effective detection of OTA is urgently required for food security. Nanozymes with hyperfine size and shape control have attracted attention because of their controllable structure and intrinsic activity. Herein, CuFe-bimetal coordinated N-doped carbon (Cu@Fe-NC) with morphology-driven peroxidase-mimicking activity was synthesized using Cu2O with specific polygonal cubes and fully exposed {111} crystalline facets as the template to produce a CuFe-bimetallic metal organic framework (MOF) and further treating CuFe-MOF with high-temperature pyrolysis. N-doping can confer electronegativity to exhibit high affinity, while the large surface area of the porous carbon support can facilitate rapid adsorption-desorption equilibrium. Using the peroxidase-mimicking Cu@Fe0.5-NC as a carrier, a versatile immunoassay for the detection of OTA was implemented based on the ratiometric fluorescence and the localized surface plasmon resonance peak shift, achieving a detection limit of 0.52 ng/L in the range of 0.001-10 µg/L. Therefore, the strategy of enhancing enzyme-mimicking activity using specific shapes and crystalline facets may open new avenues for food and environmental analysis.


Assuntos
Estruturas Metalorgânicas , Ocratoxinas , Humanos , Estruturas Metalorgânicas/química , Peroxidase , Colorimetria , Peroxidases/química , Oxirredutases , Carbono/química , Corantes
6.
J Environ Sci (China) ; 125: 712-722, 2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36375952

RESUMO

The sulfur-containing odor emitted from sludge composting could be controlled by sulfide oxidizing bacteria, yet mesophilic strains show inactivation during the thermophilic stage of composting. Aimed to investigate and characterize the thermotolerant bacterium that could oxidize sulfide into sulfate, a heterotrophic strain was isolated from sewage sludge composting and identified as Paenibacillus naphthalenovorans LYH-3. The effects of various environmental factors on sulfide oxidation capacities were studied to optimize the sulfate production, and the highest production rate (27.35% ± 0.86%) was obtained at pH 7.34, the rotation speed of 161.14 r/min, and the inoculation amount of 5.83% by employing Box-Behnken design. The results of serial sulfide substrates experiments indicated that strain LYH-3 could survive up to 400 mg/L of sulfide with the highest sulfide removal rate (88.79% ± 0.35%) obtained at 50 mg/L of sulfide. Growth kinetic analysis presented the maximum specific growth rate µm (0.5274 hr-1) after 22 hr cultivation at 50°C. The highest enzyme activities of sulfide quinone oxidoreductase (0.369 ± 0.052 U/mg) and sulfur dioxygenase (0.255 ± 0.014 U/mg) were both obtained at 40°C, and the highest enzyme activity of sulfite acceptor oxidoreductase (1.302 ± 0.035 U/mg) was assessed at 50°C. The results indicated that P. naphthalenovorans possessed a rapid growth rate and efficient sulfide oxidation capacities under thermophilic conditions, promising a potential application in controlling sulfur-containing odors during the thermophilic stage of sludge composting.


Assuntos
Compostagem , Paenibacillus , Esgotos/química , Sulfatos , Cinética , Sulfetos , Óxidos de Enxofre , Oxirredutases , Enxofre
7.
Enzyme Microb Technol ; 162: 110148, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36343469

RESUMO

Terpenoids and steroids are two large families of natural products with diverse biological activities. C3 oxidase and acetyltransferase are the key enzymes in the post-modification of terpenoids and steroids, while their discovery is of great importance for the efficient bioproduction and wide application of these natural products. In this review, we first explain the reaction mechanism of typical enzymes. Next, we summarize the current state-of-art strategies in the discovery of C3 oxidase and acetyltransferase, and discuss rational engineering of these enzymes with novel catalytic functions. Finally, we propose how to obtain an excellent biocatalyst of C3 oxidase and acetyltransferase by exploiting the discovery strategy and the reaction mechanism.


Assuntos
Produtos Biológicos , Terpenos , Engenharia Metabólica , Oxirredutases , Acetiltransferases , Esteroides
8.
Protein Expr Purif ; 202: 106187, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36216219

RESUMO

Recombinant expression and purification of proteins have become a staple of modern drug discovery as it enables more precise in vitro analyses of drug targets, which may help obtain biochemical and biophysical parameters of a known enzyme and even uncover unknown characteristics indicative of novel enzymatic functions. Such information is often necessary to prepare adequate screening assays and drug-discovery experiments in general. Toxoplasma gondii is an obligate protozoan parasite that is a member of the phylum Apicomplexa, can develop several neuro-degenerative symptoms and, in specific cases, certain death for human hosts. Its relict non-photosynthetic plastid, the apicoplast, harbours a unique de novo long-chain fatty acid synthesis pathway of a prokaryotic character, FASII. The FASII pathway shows plasticity and, is essential for many intracellular and membranal components, along with fatty acid uptake via salvaging from the host, therefore, its disruption causes parasite death. TgFabG, a FASII enzyme responsible for a single reduction step in the pathway, was recombinantly expressed, purified and biochemically and biophysically characterised in this study. The bioengineering hurdle of expressing the recombinant gene of a eukaryotic, signal peptide-containing protein in a prokaryotic system was overcome for the apicomplexan enzyme TgFabG, by truncating the N-terminal signal peptide. TgFabG was ultimately recombinantly produced in a plasmid expression vector from its 1131 base pair gene, purified as 260 and 272 amino acid proteins using a hexahistidine (6 × Histag) affinity chromatography and its biochemical (enzyme activity and kinetics) and biophysical characteristics were analysed in vitro.


Assuntos
Apicoplastos , Toxoplasma , Humanos , Apicoplastos/metabolismo , Toxoplasma/genética , Toxoplasma/metabolismo , Proteína de Transporte de Acila/metabolismo , Oxirredutases/metabolismo , Ácidos Graxos/metabolismo , Sinais Direcionadores de Proteínas , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo
9.
Urol Oncol ; 41(1): 50.e11-50.e17, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36319553

RESUMO

PURPOSE: The ability of 5α-reductase inhibitors (5ARI) to reduce the risk of new onset bladder cancer (BC) has been studied with variable results. Our objective was to conduct a retrospective cohort population-based study to evaluate the association between 5ARI use, BC diagnosis, and BC mortality. PATIENTS AND METHODS: We used routinely collected health care data from Ontario, Canada. Men ≥66 years of age with a prescription for a 5ARI were matched to non-5ARI users. Matching was done using a propensity score of selected covariates to make 96 different covariates comparable. We measured 5 additional baseline variables which may have impacted the risk of future BC diagnosis: prior cystoscopy, urine cytology, urinalysis, gross hematuria episodes, and transurethral resection of a bladder lesion. Only the first period of continuous usage of 5ARIs was considered. The prespecified at-risk period for outcomes started 1 year after initiating therapy and ended at the last date of 5ARI exposure + 1 year. RESULTS: We identified 93,197 men who initiated 5ARI therapy (52% dutasteride, and 48% finasteride) between 2003 and 2013 and matched them 1:1 to men who did not start a 5ARI. The median at-risk period for the 5ARI group was 1.68 years (interquartile range 1.00, 4.27). With adjustment for the variables related to prior BC investigations there was no significant difference in BC diagnosis (hazard ratio [HR] 1.05, 95% confidence interval [CI] 0.82-1.32) during the period of 0 to <2 years of 5ARI use; however, after ≥2 years of 5ARI use, the risk of BC diagnosis was significantly lower among the 5ARI group (HR 0.82, 95% CI 0.79-0.94). In a similarly adjusted model, BC mortality was lower among 5ARI users, but no longer statistically significant (HR 0.82, 95% CI 0.65, 1.02). When stratified by type of 5ARI, finasteride significantly reduced the risk of BC diagnosis after ≥2 years of continuous use (HR 0.86, 95% CI 0.76, 0.96); however, dutasteride did not (HR 0.92, 95% CI 0.83, 1.03). CONCLUSIONS: In a large cohort of men, the use of a 5ARI was associated with a significantly decreased the risk of BC diagnosis after more than 2 years of continuous therapy.


Assuntos
Hiperplasia Prostática , Neoplasias da Bexiga Urinária , Masculino , Humanos , Inibidores de 5-alfa Redutase/uso terapêutico , Dutasterida/uso terapêutico , Finasterida/uso terapêutico , Estudos Retrospectivos , Neoplasias da Bexiga Urinária/epidemiologia , Oxirredutases , Ontário/epidemiologia
10.
J Inorg Biochem ; 238: 112020, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36272837

RESUMO

Dehaloperoxidase (DHP) from the marine polychaete Amphitrite ornata is a multifunctional enzyme that possesses peroxidase, peroxygenase, oxidase and oxygenase activities. Herein, we investigated the reactivity of DHP B with bisphenol A (BPA) and related compounds (bisphenol E, bisphenol F, tetrachlorobisphenol A, 2,2'-biphenol, 3,3'-biphenol, 4,4'-biphenol, and 3,3'-dibromo-4,4'-biphenol). As a previously unknown substrate for DHP B, BPA (as a representative substrate) is an endocrine disruptor widely used in polycarbonate and epoxy resins, thus resulting in human exposure. Reactivity studies with these substrates were investigated using high performance liquid chromatography (HPLC), and their corresponding oxidation products were determined by mass spectrometry (GC-MS/ LC-MS). BPA undergoes oxidation in the presence of DHP B and hydrogen peroxide yielding two cleavage products (4-isopropenylphenol and 4-(2-hydroxypropan-2-yl)phenol), and oligomers with varying degrees of oxidation. 18O-labeling studies confirmed that the O-atom incorporated into the products was derived exclusively from water, consistent with substrate oxidation via a peroxidase-based mechanism. The X-ray crystal structures of DHP bound with bisphenol E (1.48 Å), bisphenol F (1.75 Å), 2,2'-biphenol (1.90 Å) and 3,3'-biphenol (1.30 Å) showed substrate binding sites are in the distal pocket of the heme cofactor, similar to other previously studied DHP substrates. Stopped-flow UV-visible spectroscopy was utilized to investigate the mechanistic details and enzyme oxidation states during substrate turnover, and a reaction mechanism is proposed. The data presented here strongly suggest that DHP B can catalyze the oxidation of bisphenols and biphenols, thus providing evidence of how infaunal invertebrates can contribute to the biotransformation of these marine pollutants.


Assuntos
Hemoglobinas , Peroxidases , Fenóis , Poliquetos , Hemoglobinas/química , Oxirredutases , Peroxidases/metabolismo , Poliquetos/enzimologia
11.
Food Chem ; 401: 134144, 2023 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-36108385

RESUMO

Ochratoxin A (OTA) is a powerful mycotoxin that can cause severe damage to human health, and its detection has attracted considerable attention in the field of food science. We present a robust and facile label-free colorimetric aptasensor for OTA detection using the aptamer-enhanced oxidase-like activity of MnO2 nanoflowers. The catalytic activities of the nanozymes could be improved by adsorption of the aptamers onto the MnO2 nanoflowers due to the increased affinity of the nanoflowers for the chromogenic substrate. The linear range for OTA detection varied from 0.05 to 33.35 ng/mL with a detection limit of 0.069 ng/mL. The limit of detection of the proposed strategy is equivalent to or even better than those of several previous methods. Moreover, the colorimetric aptasensor exhibited good specificity and stability for the analysis of OTA in wheat flour and red wine samples. Therefore, this method appears to have promising applications in the detection of mycotoxins.


Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Micotoxinas , Ocratoxinas , Humanos , Colorimetria/métodos , Compostos de Manganês , Oxirredutases , Farinha/análise , Compostos Cromogênicos , Limite de Detecção , Óxidos , Triticum , Ocratoxinas/análise , Micotoxinas/análise , Técnicas Biossensoriais/métodos
12.
Gene ; 849: 146906, 2023 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-36162526

RESUMO

NAC transcription factors (TFs) play an important role in the plant resistant response to biotic and abiotic stresses. However, the functions of the most NAC TFs are still unknown, especially in tomato. Here, we identified and functionally characterized an NAC TFs, SlNAP1, in tomato, and found that SlNAP1 was significantly induced by salt stress. Under 150 mM NaCl treatments, morphological indexes of SlNAP1 over-expressed (SlNAP1-OE) transgenic tomato lines were significantly better than the wild-type (WT) plants. The content of Na+ in leaves and roots of SlNAP1-OE transgenic plants decreased, while the K+ content in leaves, roots, and stems increased compared with WT plants. The expression of the salt stress-related genes (NHX1, HKT1;2 and SOS1) in SlNAP1-OE plants were also significantly up-regulated under salt stress. The SOD, POD and CAT activities and the expression level of antioxidant oxidase synthesis genes of SlNAP1-OE lines were significantly increased. In addition, the SlNAP1-OE lines accumulated less MDA, H2O2 and O2•-, improved antioxidant defense systems which contributed to increase salt tolerance. In summary, our data suggest that SlNAP1 positively regulates salt tolerance in tomato by regulating ion homeostasis and ROS metabolism.


Assuntos
Lycopersicon esculentum , Tolerância ao Sal , Tolerância ao Sal/genética , Lycopersicon esculentum/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Regulação da Expressão Gênica de Plantas , Antioxidantes , Peróxido de Hidrogênio/metabolismo , Cloreto de Sódio/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Homeostase , Oxirredutases/genética , Superóxido Dismutase/genética
13.
J Immunother Cancer ; 10(11)2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36323434

RESUMO

BACKGROUND: The use of immunotherapeutic vaccination in prostate cancer is a promising approach that likely requires the induction of functional, cytotoxic T cells . The experimental approach described here uses a well-studied adenovirus-poxvirus heterologous prime-boost regimen, in which the vectors encode a combination of prostate cancer antigens, with the booster dose delivered by either the intravenous or intramuscular (IM) route. This prime-boost regimen was investigated for antigen-specific CD8+ T cell induction. METHODS: The coding sequences for four antigens expressed in prostate cancer, 5T4, PSA, PAP, and STEAP1, were inserted into replication-incompetent chimpanzee adenovirus Oxford 1 (ChAdOx1) and into replication-deficient modified vaccinia Ankara (MVA). In four strains of mice, ChAdOx1 prime was delivered intramuscularly, with an MVA boost delivered by either IM or intravenous routes. Immune responses were measured in splenocytes using ELISpot, multiparameter flow cytometry, and a targeted in vivo killing assay. RESULTS: The prime-boost regimen was highly immunogenic, with intravenous administration of the boost resulting in a sixfold increase in the magnitude of antigen-specific T cells induced and increased in vivo killing relative to the intramuscular boosting route. Prostate-specific antigen (PSA)-specific responses were dominant in all mouse strains studied (C57BL/6, BALBc, CD-1 and HLA-A2 transgenic). CONCLUSION: This quadrivalent immunotherapeutic approach using four antigens expressed in prostate cancer induced high magnitude, functional CD8+ T cells in murine models. The data suggest that comparing the intravenous versus intramuscular boosting routes is worthy of investigation in humans.


Assuntos
Antígeno Prostático Específico , Neoplasias da Próstata , Masculino , Humanos , Camundongos , Animais , Camundongos Endogâmicos C57BL , Linfócitos T CD8-Positivos , Vírus Vaccinia , Neoplasias da Próstata/genética , Neoplasias da Próstata/terapia , Administração Intravenosa , Antígenos de Neoplasias , Oxirredutases
14.
Nat Commun ; 13(1): 7195, 2022 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-36418310

RESUMO

Various 4-alkylphenols can be easily obtained through reductive catalytic fractionation of lignocellulosic biomass. Selective dehydrogenation of 4-n-propylguaiacol results in the formation of isoeugenol, a valuable flavor and fragrance molecule and versatile precursor compound. Here we present the engineering of a bacterial eugenol oxidase to catalyze this reaction. Five mutations, identified from computational predictions, are first introduced to render the enzyme more thermostable. Other mutations are then added and analyzed to enhance chemoselectivity and activity. Structural insight demonstrates that the slow catalytic activity of an otherwise promising enzyme variant is due the formation of a slowly-decaying covalent substrate-flavin cofactor adduct that can be remedied by targeted residue changes. The final engineered variant comprises eight mutations, is thermostable, displays good activity and acts as a highly chemoselective 4-n-propylguaiacol oxidase. We lastly use our engineered biocatalyst in an illustrative preparative reaction at gram-scale. Our findings show that a natural enzyme can be redesigned into a tailored biocatalyst capable of valorizing lignin-based monophenols.


Assuntos
Lignina , Oxirredutases , Lignina/química , Oxirredutases/genética , Eugenol , Hidrolases
15.
Nitric Oxide ; 129: 102-109, 2022 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-36367524

RESUMO

Recent studies have identified skeletal muscle as a tissue compartment where nitrate and nitrite can be stored and utilized to potentially maintain nitric oxide (NO) homeostasis. Given its capacity to reduce nitrate and nitrite, the molybdopterin-containing enzyme, xanthine oxidoreductase (XOR) has been suggested as a key enzyme within skeletal muscle which catalytically reduces these N-oxides; however, there remains limited insight into the role of XOR in this process as well as how different conditions (e.g. health vs disease and rest vs exercise) may determine when and where, within skeletal muscle, XOR could serve as a significant source of NO. A key factor that determines the extent by which XOR may or may not contribute to NO generation in a biologically relevant manner is the biochemical landscape (e.g. oxygen tension, pH, isoform of XOR (XDH vs. XO) and substrate levels of the microenvironment in normal versus stressed skeletal muscle. As such, a critical focus of this review is the evaluation of the biochemical and physiologic data supporting the role of XOR within skeletal muscle for supplying nitrite and/or NO from endogenous and exogenous sources during pathophysiologic conditions and/or exercise stress.


Assuntos
Nitritos , Xantina Oxidase , Nitratos , Oxirredutases , Xantina Desidrogenase , Óxido Nítrico , Músculo Esquelético
16.
Biomolecules ; 12(11)2022 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-36358916

RESUMO

LHON is a common blinding inherited optic neuropathy caused by mutations in mitochondrial genes. In this study, by using skin fibroblasts derived from LHON patients with the most common m.G11778A mutation and healthy objects, we performed proteomic analysis to document changes in molecular proteins, signaling pathways and cellular activities. Furthermore, the results were confirmed by functional studies. A total of 860 differential expression proteins were identified, containing 624 upregulated and 236 downregulated proteins. Bioinformatics analysis revealed increased glycolysis in LHON fibroblasts. A glycolysis stress test showed that ECAR (extra-cellular acidification rate) values increased, indicating an enhanced level of glycolysis in LHON fibroblasts. Downregulated proteins were mainly enriched in oxidoreductase activity. Cellular experiments verified high levels of ROS in LHON fibroblasts, indicating the presence of oxidative damage. KEGG analysis also showed the metabolic disturbance of fatty acid in LHON cells. This study provided a proteomic profile of skin fibroblasts derived from LHON patients bearing m.G11778A. Increased levels of glycolysis, decreased oxidoreductase activity and fatty acid metabolism could represent the in-depth mechanisms of mitochondrial dysfunction mediated by the mutation. The results provided further evidence that LHON fibroblast could be an alternative model for investigating the devastating disease.


Assuntos
Atrofia Óptica Hereditária de Leber , Humanos , Atrofia Óptica Hereditária de Leber/genética , Atrofia Óptica Hereditária de Leber/metabolismo , DNA Mitocondrial/genética , Proteômica , Oxirredutases/metabolismo , Mutação , Fibroblastos/metabolismo , Glicólise , Ácidos Graxos/metabolismo
17.
Biomolecules ; 12(11)2022 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-36359010

RESUMO

The study was to investigate the effect of canonical and noncanonical pyroptosis in apical periodontitis. Proteins' profiles of human apical periodontitis tissue were analyzed by label-free proteomics. Immunofluorescence was used to detect proteins related to pyroptosis in human apical periodontitis tissues and experimental apical periodontitis models. A dual experimental apical periodontitis model with both smaller (mandible) and larger (maxilla) bone lesions was established. THP-1-derived macrophages were stimulated with P. gingivalis lipopolysaccharide in vitro with or without the caspase-1/-4/-5 inhibitor Ac-FTDL-CMK. Propidium iodide staining, lactic dehydrogenase release and Western blot were applied to evaluate cell death and the protein expression. Caspase-1/-4/-5 were expressed in human apical periodontitis tissues. Caspase-1/-11 were involved in bone loss in experimental apical periodontitis. Caspase-1/-11 inhibitors reduced bone loss in larger lesions (maxilla) but accelerated bone loss in smaller lesions (mandible). Caspase-1/-4/-5 inhibitors also showed double-edged sword effects on propidium iodide staining and lactic dehydrogenase release in vitro. The expression of cleaved-caspase-1/-4/-5, mature interluekin-1ß and gasdermin D N-terminal domain increased in THP-1-derived macrophages after lipopolysaccharide stimulation but decreased after treatment with Ac-FTDL-CMK. Pyroptosis contributed to apical periodontitis and excited a double-edged sword effect in inducing bone loss in vivo and cell death in vitro.


Assuntos
Periodontite Periapical , Piroptose , Humanos , Piroptose/fisiologia , Lipopolissacarídeos/farmacologia , Propídio/farmacologia , Caspase 1/metabolismo , Caspases/metabolismo , Oxirredutases
18.
Analyst ; 147(23): 5355-5362, 2022 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-36373378

RESUMO

Due to their high catalytic activity, stability and low cost, nanozymes with oxidase-like activity have attracted widespread interest in the fields of analytical detection and colorimetric sensing. To further promote the catalytic activity and the sensitivity of dopamine (DA) sensing, herein, a mixed valence Ce-MOF (MVCM) with enhanced oxidase-like activity was synthesized by the dielectric barrier discharge (DBD) microplasma method. Compared with hydrothermal synthesis, the prepared MVCM synthesized using a microplasma showed a higher catalytic activity, which benefits from a low Ce3+/Ce4+ ratio. Due to the spontaneous redox properties of Ce3+/Ce4+ in a MVCM, the MVCM-based colorimetric sensing of dopamine was established and showed a limit of detection of 0.74 µM over a linear range of 5-100 µM with high selectivity and stability and has been applied for the detection of dopamine in sweat. The proposed study provides an effective synthesis method for nanozymes with enhanced activity and shows great promise in widespread analytical and sensing applications.


Assuntos
Colorimetria , Dopamina , Colorimetria/métodos , Oxirredutases , Catálise , Oxirredução
19.
Cancer Biomark ; 35(3): 331-342, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36373310

RESUMO

BACKGROUND: Sinonasal mucosal melanoma (SNMM) is a lethal malignancy with poor prognosis. Treatment outcomes of SNMM are poor. Novel prognostic or progression markers are needed to help adjust therapy. METHODS: RNA-seq was used to analyze the mRNA expression of tumor tissues and normal nasal mucosa from primary SNMM patients (n= 3). Real-time fluorescent quantitative PCR (qRT-PCR) was used to validate the results of RNA-seq (n= 3), while protein expression was analyzed by immunohistochemistry (IHC, n= 31) and western blotting (n= 3). Retrospective studies were designed to determine the clinical parameters and the total survival rate, and correlation between the protein expression levels of the most significant key genes and prognosis was analyzed. RESULTS: In total, 668 genes were upregulated and 869 genes were downregulated in SNMM (fold change ⩾ 2, adjusted p value < 0.01). Both mRNA and protein expression levels of the key genes in SNMM tumor tissues were higher than those in the normal control nasal mucosal tissues. The expression rates of TYRP1, ABCB5, and MMP17 in 31 primary SNMM cases were 90.32%, 80.65%, and 64.52%, respectively. In addition, age, typical symptoms, and AJCC stage were related to overall survival rate of patients with SNMM (p< 0.05). Furthermore, the expression of ABCB5 was age-related (p= 0.002). Compared with individuals with negative ABCB5 expression, those with positive expression exhibited significantly poor overall survival (p= 0.02). CONCLUSION: The expression levels of TYRP1, ABCB5, and MMP17 were significantly upregulated in SNMM tissues, and the expression of ABCB5 was related to poor prognosis in SNMM. Thus, ABCB5 may serve as a progression marker and can predict unfavorable prognosis in patients with SNMM.


Assuntos
Metaloproteinase 17 da Matriz , Melanoma , Humanos , Estudos Retrospectivos , Melanoma/genética , Sequenciamento Completo do Exoma , RNA Mensageiro , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Glicoproteínas de Membrana , Oxirredutases
20.
Int J Mol Sci ; 23(21)2022 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-36362382

RESUMO

Pyranose oxidase (POx, glucose 2-oxidase; EC 1.1.3.10, pyranose:oxygen 2-oxidoreductase) is an FAD-dependent oxidoreductase and a member of the auxiliary activity (AA) enzymes (subfamily AA3_4) in the CAZy database. Despite the general interest in fungal POxs, only a few bacterial POxs have been studied so far. Here, we report the biochemical characterization of a POx from Streptomyces canus (ScPOx), the sequence of which is positioned in a separate, hitherto unexplored clade of the POx phylogenetic tree. Kinetic analyses revealed that ScPOx uses monosaccharide sugars (such as d-glucose, d-xylose, d-galactose) as its electron-donor substrates, albeit with low catalytic efficiencies. Interestingly, various C- and O-glycosides (such as puerarin) were oxidized by ScPOx as well. Some of these glycosides are characteristic substrates for the recently described FAD-dependent C-glycoside 3-oxidase from Microbacterium trichothecenolyticum. Here, we show that FAD-dependent C-glycoside 3-oxidases and pyranose oxidases are enzymes belonging to the same sequence space.


Assuntos
Flavina-Adenina Dinucleotídeo , Oxirredutases , Filogenia , Oxirredutases/genética , Oxirredutases/metabolismo , Monossacarídeos , Cinética , Bactérias/metabolismo , Glicosídeos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...