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1.
Food Chem ; 371: 131115, 2022 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-34555710

RESUMO

The total antioxidant capacity (TAC) has become increasingly vital for evaluating antioxidant food quality in the field of healthcare. Herein, a convenient and sensitive method for TAC assay was proposed based on the absorbance difference of reaction systems between various antioxidants existed in food and Dex-FeMnzyme/oxTMB. Under the optimum condition, the limit of detection (LOD) of the colorimetric sensor was 1.17 µM with the linear concentration range from 1 µM to 30 µM. The analysis results demonstrated the excellent feasibility of practical application in fruit and vegetable food, which offered a new avenue for the establishment of colorimetric biosensors.


Assuntos
Antioxidantes , Frutas , Antioxidantes/análise , Colorimetria , Dextranos , Frutas/química , Oxirredução , Oxirredutases , Verduras
2.
Talanta ; 237: 122906, 2022 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-34736643

RESUMO

A rapid colorimetric method for detecting sodium benzoate in food products was established based on the d-amino acid oxidase (DAAO) and 2D metal organic framework (2D MOF) nanosheets mediated cascade enzyme reactions. Firstly, the synthesized 2D MOF nanosheets served as high efficient nanozyme with outstanding peroxidase-like catalytic activity and catalyzed the color reaction between H2O2 and 3, 3', 5, 5'- tetramethylbenzidine. Secondly, sodium benzoate as a competitive inhibitor of DAAO, could influence the production of H2O2 in DAAO mediated oxidation reaction. After a combination of those two reactions, this colorimetric quantitative method was constructed and validated for sodium benzoate determination with wide linear range (2.0-200.0 µM), low limit of detection (2.0 µM), high accuracy (recovery rate in 95.80-108.00%) and satisfied selectivity. Lastly, this method was utilized to analyze sodium benzoate concentration in juice, wine and vinegar by naked eyes.


Assuntos
Estruturas Metalorgânicas , Aminoácidos , Colorimetria , Peróxido de Hidrogênio , Oxirredutases , Benzoato de Sódio
3.
Talanta ; 237: 122957, 2022 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-34736682

RESUMO

The development of an efficient protein-inorganic nanohybrid with superior nanozyme activity for highly sensitive detection of glutathione (GSH) is essential for early diagnosis of human diseases. Herein, a rapid and highly sensitive colorimetric assay using self-assembled bovine serum albumin-hydrated manganese phosphate nanoflowers (MnPNF) as a biomimic oxidase is developed for GSH detection in human serum. The BSA can complex with Mn2+ to serve the nucleation center to produce MnPNF in the presence of phosphate-buffered saline (PBS). The morphology and surface characterization results show that the MnPNF is assembled with hierarchical nanoplates to form 500 nm nanoflowers. The oxidase-like activity of MnPNF is based on the redox reaction with 3,3',5,5'-tetramethylbenzidine. However, the addition of GSH can reduce MnPNF to Mn2+, and subsequently supresses the oxidase-like activity and a yellow color at 450 nm is observed in the presence of H2SO4. The MnPNF-based nanozyme exhibits excellent sensing ability toward GSH detection, and a good linear relationship between the change in absorbance at 450 nm and the added amounts of GSH at 50 nM-10 µM with low limits of detection of 20 and 26.6 nM in the PBS and diluted human serum, respectively, is observed. Moreover, the sensing probe shows a superior selectivity over the other 16 interferences, which drive the determination of GSH feasible in real human serum. Since the MnPNF can be simply prepared at room temperature and no functionalization is required, this assay can be used to design the highly efficient biomimic oxidase for effective sensing of GSH and other disease-related biomolecules in biological fluid samples.


Assuntos
Colorimetria , Glutationa , Humanos , Compostos Organometálicos , Oxirredução , Oxirredutases
4.
Anal Chem ; 93(45): 15150-15158, 2021 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-34738799

RESUMO

Mimicking enzyme specificity via construction of on-demand geometric structures on nanozymes is of great interest in recent years. Although building substrate-specific polymers on nanozymes has achieved great success, polymer-blocked active sites would inevitably lead to decreased activity of nanozymes. Here, we have developed three photoactive metal-organic framework (MOF)-based nanozymes (called 2D-TCPP, 3D-TCPP, and AD-TCPP), which have different geometric structures as well as unshielded active sites. Together with their structural variations and excellent photoresponsive oxidase-like activities, these photoactive nanozymes exhibit structure-dependent specificity for three kinds of substrates (typical oxidase substrates, organic pollutants, and antioxidants). Moreover, AD-TCPP and 3D-TCPP show potential applications for environmental protection and bioanalysis, respectively. This work offers a promising approach to the development of nanozymes with enzyme-like specificity.


Assuntos
Estruturas Metalorgânicas , Nanoestruturas , Catálise , Oxirredutases , Especificidade por Substrato
5.
Sensors (Basel) ; 21(22)2021 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-34833643

RESUMO

In recent years, cerium oxide (CeO2) nanoparticles (NPs) have drawn significant attention owing to their intrinsic enzyme mimetic properties, which make them powerful tools for biomolecular detection. In this work, we evaluated the effect of pyrophosphate (PPi) on the oxidase activity of CeO2 NPs. The presence of PPi was found to enhance the oxidase activity of CeO2 NPs, with enhanced colorimetric signals. This particular effect was then used for the colorimetric detection of target nucleic acids. Overall, the PPi-enhanced colorimetric signals of CeO2 NPs oxidase activity were suppressed by the presence of the target nucleic acids. Compared with previous studies using CeO2 NPs only, our proposed system significantly improved the signal change (ca. 200%), leading to more sensitive and reproducible colorimetric analysis of target nucleic acids. As a proof-of-concept study, the proposed system was successfully applied to the highly selective and sensitive detection of polymerase chain reaction products derived from Klebsiella pneumoniae. Our findings will benefit the rapid detection of nucleic acid biomarkers (e.g., pathogenic bacterial DNA or RNA) in point-of-care settings.


Assuntos
Cério , Nanopartículas , Ácidos Nucleicos , Colorimetria , Difosfatos , Oxirredutases
6.
J Mol Evol ; 89(9-10): 665-677, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34757471

RESUMO

Plant hormone cytokinins are important regulators of plant development, response to environmental stresses and interplay with other plant hormones. Cytokinin dehydrogenases (CKXs) are proteins responsible for the irreversible break-down of cytokinins to the adenine and aldehyde. Even though plant CKXs have been extensively studied, homologous proteins from other taxa remain mainly uncharacterised. Here we present our study on the molecular evolution and divergence of the CKX from bacteria, fungi, amoeba and viridiplantae. Although CKXs are present in eukaryotes and prokaryotes, they are missing in algae and metazoan taxa. The prevalent domain architecture consists of the FAD-binding and cytokinin binding domains, whereas some bacteria appear to have only cytokinin binding domain proteins. The CKXs play important role in the various aspects of plant life including control of plant development, response to biotic and abiotic stress, influence nutrition. Results of our study suggested that CKX originates from the FAD-linked C-terminal oxidase and has a defence-oriented function. The obtained results significantly extend the current understanding of the cytokinin dehydrogenases structure-function from the relationship to homologues from other taxa and provide a starting point baseline for their future functional characterization.


Assuntos
Citocininas , Regulação da Expressão Gênica de Plantas , Animais , Oxirredutases/genética , Oxirredutases/metabolismo , Estresse Fisiológico
7.
PLoS One ; 16(10): e0258610, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34648583

RESUMO

Leptocybe invasa Fisher et LaSalle is a global invasive pest that seriously damages Eucalyptus plants. Studying the genetic diversity, genetic structure and introgression hybridization of L. invasa in China is of great significance for clarifying the breeding strategy, future invasion and diffusion trends of L. invasa in China and developing scientific prevention and control measures. Genetic diversity and phylogenetic analyses of 320 L. invasa female adults from 14 geographic populations in China were conducted using 10 polymorphic microsatellite loci (SSRs) and mitochondrial DNA cytochrome oxidase I gene sequences (COIs). (1) The Bayesian phylogenetic tree and haplotype network diagram showed that only haplotype Hap3 existed in L. invasa lineage B in China, while haplotypes Hap1 and Hap2 existed in lineage A, among which haplotype Hap2 was found for the first time. The nucleotide and haplotype diversities of lineage A were higher than those of lineage B. (2) The SSR genetic diversity of the Wuzhou Guangxi, Ganzhou Jiangxi and Panzhihua Sichuan populations was higher than that of the other 11 populations, and the SSR genetic diversity of lineage A was higher than that of lineage B. (3) The AMOVA analysis of mitochondrial COI data showed that 75.55% of the variation was among populations, and 99.86% of the variation was between lineages, while the AMOVA analysis of nuclear SSR data showed that 35.26% of the variation was among populations, and 47.04% of the variation was between lineages. There were obvious differences in the sources of variation between the COI and SSR data. (4) The optimal K value of COI and SSR data in structure analysis was 2, and PCoA analysis also divided the dataset into two obvious categories. The UPMGA phylogenetic tree based on SSR data clustered 14 geographic species into two groups. The results of genetic structure analysis supported the existence of two lineages, A and B, in China. (5) Structural analysis showed that there was obvious introgressive hybridization in Wuzhou Guangxi, Ganzhou Jiangxi, Panzhihua Sichuan and other populations. These results suggest that lineage introgressive hybridization has occurred in the L. invasa population in China. The introgressive hybridization degree and genetic diversity of lineage A are obviously higher than those of lineage B. Lineage introgressive hybridization may be the driving force for further L. invasa invasion and diffusion in China in the future.


Assuntos
Himenópteros/classificação , Repetições de Microssatélites , Oxirredutases/genética , Animais , Teorema de Bayes , China , Feminino , Introgressão Genética , Variação Genética , Himenópteros/genética , Proteínas de Insetos/genética , Espécies Introduzidas , Controle de Pragas , Filogenia
8.
Int J Mol Sci ; 22(19)2021 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-34639193

RESUMO

Terminal respiratory oxidases are highly efficient molecular machines. These most important bioenergetic membrane enzymes transform the energy of chemical bonds released during the transfer of electrons along the respiratory chains of eukaryotes and prokaryotes from cytochromes or quinols to molecular oxygen into a transmembrane proton gradient. They participate in regulatory cascades and physiological anti-stress reactions in multicellular organisms. They also allow microorganisms to adapt to low-oxygen conditions, survive in chemically aggressive environments and acquire antibiotic resistance. To date, three-dimensional structures with atomic resolution of members of all major groups of terminal respiratory oxidases, heme-copper oxidases, and bd-type cytochromes, have been obtained. These groups of enzymes have different origins and a wide range of functional significance in cells. At the same time, all of them are united by a catalytic reaction of four-electron reduction in oxygen into water which proceeds without the formation and release of potentially dangerous ROS from active sites. The review analyzes recent structural and functional studies of oxygen reduction intermediates in the active sites of terminal respiratory oxidases, the features of catalytic cycles, and the properties of the active sites of these enzymes.


Assuntos
Oxirredutases/metabolismo , Bombas de Próton/metabolismo , Prótons , Catálise , Domínio Catalítico , Transporte de Elétrons , Oxirredutases/química , Bombas de Próton/química
9.
Plant Sci ; 312: 111043, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34620441

RESUMO

ζ-Carotene desaturase (ZDS) is one of the key enzymes regulating carotenoids biosynthesis and accumulation. Celery transgenic efficiency is low and it is difficult to obtain transgenic plants. The study on ZDS was limited in celery. Here, the AgZDS gene was cloned from celery and overexpressed in Arabidopsis thaliana and celery to verify its function. The AgZDS has typical characteristic of ZDS protein and is highly conserved in higher plants. Phylogenetic analysis showed that AgZDS has the closest evolutionary relationship with ZDSs from Solanum lycopersicum, Capsicum annuum and Tagetes erecta. Overexpression of AgZDS gene in A. thaliana and celery resulted in increased accumulations of lutein and ß-carotene and up-regulated the expression levels of the genes involved in carotenoids biosynthesis. The contents of lutein and ß-carotene in two lines, AtL1 and AgL5, were the highest in transgenic A. thaliana and celery, respectively. The relative expression levels of 5 genes (AtPDS, AtZISO, AtZEP, AtNCED3, and AtCCD4) were up-regulated compared to the wild type plants. The relative expression levels of most genes in carotenoids biosynthesis pathway, such as AgPDS, AgCRTISO1, and AgZISO, were up-regulated in transgenic celery plants. The antioxidant capacity of A. thaliana and photosynthetic capacity of celery were also enhanced. This research is the first report on the function of structure gene related to carotenoid biosynthesis in transgenic celery plants. The findings in this study demonstrated the roles of AgZDS in regulating carotenoids metabolism of celery, which laid a potential foundation for quality improvement of celery.


Assuntos
Apium/genética , Apium/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Luteína/biossíntese , Oxirredutases/metabolismo , beta Caroteno/biossíntese , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Luteína/genética , Oxirredutases/genética , Plantas Geneticamente Modificadas , Verduras/genética , beta Caroteno/genética
10.
Orphanet J Rare Dis ; 16(1): 417, 2021 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-34627351

RESUMO

BACKGROUND: Biallelic variants in HSD3B7 cause 3ß-hydroxy-Δ5-C27-steroid oxidoreductase (HSD3B7) deficiency, a life-threatening but treatable liver disease. The goal of this study was to obtain detailed information on the correlation between the genotype and phenotype of HSD3B7 deficiency and to report on responses to primary bile acid therapy. METHODS: The medical records of a cohort of 39 unrelated patients with genetically and biochemically confirmed HSD3B7 deficiency were examined to determine whether there exist genotype-phenotype relationships in this bile acid synthesis disorder. RESULTS: In all, 34 of the 44 variants identified in HSD3B7 were novel. A total of 32 patients presented early with neonatal cholestasis, and 7 presented after 1-year of age with liver failure (n = 1), liver cirrhosis (n = 3), cholestasis (n = 1), renal cysts and abnormal liver biochemistries (n = 1), and coagulopathy from vitamin K1 deficiency and abnormal liver biochemistries (n = 1). Renal lesions, including renal cysts, renal stones, calcium deposition and renal enlargement were observed in 10 of 35 patients. Thirty-three patients were treated with oral chenodeoxycholic acid (CDCA) resulting in normalization of liver biochemistries in 24, while 2 showed a significant clinical improvement, and 7 underwent liver transplantation or died. Remarkably, renal lesions in 6 patients resolved after CDCA treatment, or liver transplantation. There were no significant correlations between genotype and clinical outcomes. CONCLUSIONS: In what is the largest cohort of patients with HSD3B7 deficiency thus far studied, renal lesions were a notable clinical feature of HSD3B7 deficiency and these were resolved with suppression of atypical bile acids by oral CDCA administration.


Assuntos
Colestase , Oxirredutases , 3-Hidroxiesteroide Desidrogenases , Ácidos e Sais Biliares , China , Humanos , Recém-Nascido
11.
Int J Mol Sci ; 22(19)2021 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-34638544

RESUMO

Oculocutaneous albinism type 3 (OCA3) is an autosomal recessive disorder caused by mutations in the TYRP1 gene. Tyrosinase-related protein 1 (Tyrp1) is involved in eumelanin synthesis, catalyzing the oxidation of 5,6-dihydroxyindole-2-carboxylic acid oxidase (DHICA) to 5,6-indolequinone-2-carboxylic acid (IQCA). Here, for the first time, four OCA3-causing mutations of Tyrp1, C30R, H215Y, D308N, and R326H, were investigated computationally to understand Tyrp1 protein stability and catalytic activity. Using the Tyrp1 crystal structure (PDB:5M8L), global mutagenesis was conducted to evaluate mutant protein stability. Consistent with the foldability parameter, C30R and H215Y should exhibit greater instability, and two other mutants, D308N and R326H, are expected to keep a native conformation. SDS-PAGE and Western blot analysis of the purified recombinant proteins confirmed that the foldability parameter correctly predicted the effect of mutations critical for protein stability. Further, the mutant variant structures were built and simulated for 100 ns to generate free energy landscapes and perform docking experiments. Free energy landscapes formed by Y362, N378, and T391 indicate that the binding clefts of C30R and H215Y mutants are larger than the wild-type Tyrp1. In docking simulations, the hydrogen bond and salt bridge interactions that stabilize DHICA in the active site remain similar among Tyrp1, D308N, and R326H. However, the strengths of these interactions and stability of the docked ligand may decrease proportionally to mutation severity due to the larger and less well-defined natures of the binding clefts in mutants. Mutational perturbations in mutants that are not unfolded may result in allosteric alterations to the active site, reducing the stability of protein-ligand interactions.


Assuntos
Albinismo Oculocutâneo/genética , Melaninas/biossíntese , Melanócitos/metabolismo , Glicoproteínas de Membrana/genética , Oxirredutases/genética , Biologia Computacional , Humanos , Ligantes , Simulação de Acoplamento Molecular , Oxirredutases/metabolismo , Dobramento de Proteína , Estabilidade Proteica , Quinoxalinas/metabolismo
12.
Chem Biol Interact ; 349: 109680, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34606757

RESUMO

In living organisms most oxygen consumed is reduced to water via four-electron reduction. However, few percentages of oxygen are reduced by consecutive one electron mechanisms giving rise to superoxide anion radical, (O2•-), hydrogen peroxide (H2O2) and hydroxyl radical (HO•) and their derivatives collectively called reactive oxygen species (ROS). Nitric oxide (•NO) is produced at oxidation of arginine by nitric oxide synthase (NOS) or at reduction of nitrites by diverse reductases. Interaction of •NO with O2•- results in formation of peroxinitrite (ONOO-), a powerful oxidant. Additionally, H2O2 can interact with •NO resulting in HO• production. Nitric oxide and its derivatives are collectively called reactive nitrogen species (RNS) and together with ROS they form a group of so-called reactive oxygen/nitrogen species (RONS). Nonspecific effects of RONS are related to their interaction with various components of living organisms, whereas specific effects are based mainly on interaction with specific proteins containing [Fe-S]-clusters and thiol groups of cysteine residues. Most early ROS studies were mainly focused on their deleterious effects, whereas now more delicate mechanisms of their involvement in signaling and toxic processes are under inspection. Studies of RNS activities in biological systems started from their vasodilating effects which lead to discovery of activation of soluble guanylate cyclase. Interestingly, at low ROS and RNS concentrations signaling effects prevail, whereas at their high concentrations they affect biological systems inhibiting due to massive oxidation of cellular components.


Assuntos
Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Arginina/química , Arginina/metabolismo , Cisteína/química , Cisteína/metabolismo , Óxido Nítrico Sintase/metabolismo , Oxirredução , Oxirredutases/metabolismo , Plantas/química , Plantas/metabolismo , Espécies Reativas de Nitrogênio/química , Espécies Reativas de Oxigênio/química
13.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 50(4): 436-443, 2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34704411

RESUMO

To explore the clinical features and long-term outcomes of patients with cblC type methylmalonic acidemia (MMA) carrying c.609G>A (p.W203X) mutation of gene. The clinical and laboratory findings of 720 patients with MMA carrying the c.609G>A mutation were retrospectively analyzed. There were 172 cases carrying homozygous mutations of c.609G>A (group A), 169 cases carrying compound heterozygous mutations of c.609G>A with c.482G>A (p.R161Q), c.80A>G or c.394C>T (p.R132X) (group B), and 379 cases carrying compound heterozygous mutations of c.609G>A with c.658_660delAAG(p.K220del), c.315A>Tor c.567dupT(p.I190fs13)(group C).The clinical manifestations, the level of blood acylcarnitine, homocysteine and urinary organic acid, and the therapeutic efficacy were compared among groups. Logistic regression was used to analyze the factors influencing the prognosis of patients. There were 306 patients (42.5%) detected from newborn screening, including 156 cases with disease onset; and 414 patients were not detected from the screening, among whom 10 cases were diagnosed by testing after the sibling confirmed, and the remaining 404 were clinical cases. In 560 patients with disease onset, the median onset age is (3 days to 20 years). The onset age of patients in group B was later than that in group A and group C (<0.01). Patients aged mostly manifested as vomiting, diarrhea, feeding difficulties and convulsions, while those year mostly manifested as movement disorders and mental retardation. Patients with renal disease all carried mutations of c.80A>G or c.482G>A, and patients with pulmonary hypertension all carried c.80A>G mutations. A total of 621 cases had long-term follow-up, 156 cases (25.1%) developed well, 433 cases (69.7%) had development delay and 32 cases (5.2%) died. The available data of 559 cases were analyzed by logistic regression, and the results showed that the neonatal screening, disease onset, age of onset and gene mutation site were significantly associated with the prognosis of patients (<0.05 or <0.01). The c.609G>A mutation in gene is associated with early-onset MMA, and most patients, clinical onset occurred within 1 month after birth. The neonatal screening and early treatment can improve the prognosis of patients,whereas clinical onset is unfavorable for prognosis. Patients with c.609G>A homozygous mutation have a worse prognosis than those with the compound heterozygous mutation of c.609G>A with other mutations.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos , Oxirredutases , Adolescente , Erros Inatos do Metabolismo dos Aminoácidos/genética , Criança , Pré-Escolar , Humanos , Lactente , Recém-Nascido , Mutação , Oxirredutases/genética , Estudos Retrospectivos , Adulto Jovem
14.
Molecules ; 26(20)2021 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-34684801

RESUMO

Halogenation often improves the bioactive properties of natural products and is used in pharmaceutical research for the generation of new potential drug leads. High regio- and stereospecificity, simple reaction conditions and straightforward downstream processing are the main advantages of halogenation using enzymatic biocatalysts compared to chemical synthetic approaches. The identification of new promiscuous halogenases for the modification of various natural products is of great interest in modern drug discovery. In this paper, we report the identification of a new promiscuous FAD-dependent halogenase, DklH, from Frankia alni ACN14a. The identified halogenase readily modifies various flavonoid compounds, including those with well-studied biological activities. This halogenase has been demonstrated to modify not only flavones and isoflavones, but also flavonols, flavanones and flavanonols. The structural requirements for DklH substrate recognition were determined using a feeding approach. The homology model of DklH and the mechanism of substrate recognition are also proposed in this paper.


Assuntos
Proteínas de Bactérias/metabolismo , Flavonoides/metabolismo , Halogenação , Oxirredutases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biocatálise , Descoberta de Drogas , Flavonoides/química , Frankia/enzimologia , Frankia/genética , Genes Bacterianos , Simulação de Acoplamento Molecular , Oxirredutases/química , Oxirredutases/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
15.
Sci Rep ; 11(1): 17976, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34504156

RESUMO

We present a computational case study of X-ray single-particle imaging of hydrated proteins on an example of 2-Nitrogenase-Iron protein covered with water layers of various thickness, using a start-to-end simulation platform and experimental parameters of the SPB/SFX instrument at the European X-ray Free-Electron Laser facility. The simulations identify an optimal thickness of the water layer at which the effective resolution for imaging the hydrated sample becomes significantly higher than for the non-hydrated sample. This effect is lost when the water layer becomes too thick. Even though the detailed results presented pertain to the specific sample studied, the trends which we identify should also hold in a general case. We expect these findings will guide future single-particle imaging experiments using hydrated proteins.


Assuntos
Lasers , Simulação de Dinâmica Molecular , Imagem Molecular/métodos , Oxirredutases/química , Oxirredutases/efeitos da radiação , Água/química , Difração de Raios X/instrumentação , Difração de Raios X/métodos , Raios X/efeitos adversos , Elétrons , Fótons
16.
Cells ; 10(9)2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34572080

RESUMO

Embryonic stem cells (ESCs) are pluripotent cells with indefinite self-renewal ability and differentiation properties. To function properly and maintain genomic stability, ESCs need to be endowed with an efficient repair system as well as effective redox homeostasis. In this study, we investigated different aspects involved in ESCs' response to iron accumulation following stable knockdown of the ferritin heavy chain (FTH1) gene, which encodes for a major iron storage protein with ferroxidase activity. Experimental findings highlight unexpected and, to a certain extent, paradoxical results. If on one hand FTH1 silencing does not correlate with increased ROS production nor with changes in the redox status, strengthening the concept that hESCs are extremely resistant and, to a certain extent, even refractory to intracellular iron imbalance, on the other, the differentiation potential of hESCs seems to be affected and apoptosis is observed. Interestingly, we found that FTH1 silencing is accompanied by a significant activation of the nuclear factor (erythroid-derived-2)-like 2 (Nrf2) signaling pathway and pentose phosphate pathway (PPP), which crosstalk in driving hESCs antioxidant cascade events. These findings shed new light on how hESCs perform under oxidative stress, dissecting the molecular mechanisms through which Nrf2, in combination with PPP, counteracts oxidative injury triggered by FTH1 knockdown.


Assuntos
Ferritinas/genética , Células-Tronco Embrionárias Humanas/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Oxirredutases/genética , Elementos de Resposta Antioxidante , Apoptose , Diferenciação Celular , Células Cultivadas , Ferritinas/farmacologia , Inativação Gênica , Humanos , Oxirredução , Oxirredutases/metabolismo , Via de Pentose Fosfato , Transdução de Sinais
17.
Environ Toxicol Pharmacol ; 88: 103746, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34536620

RESUMO

Nonsteroidal anti-inflammatory drugs (NSAIDs) are a popular group of drugs used worldwide. These drugs are also available over the counter, which implies that their consumption is not strictly regulated. They are released through wastewater and feces and can have adverse effects on the environment. The present study aimed to evaluate the effect of two NSAIDs, diclofenac (DCF) and naproxen (NAP), and their mixture (DCF + NAP) on spring barley seedlings and ostracods Heterocypris incongruens. The tested drugs had a negative impact on bivalve ostracods and the studied plants. DCF was the most toxic toward ostracods, while spring barley seedlings were affected the most by NAP. The application of the tested compounds and their mixture resulted in a decrease in fresh weight yield and the content of photosynthetic pigments. In addition, an increase in H2O2 and proline content and changes in the activity of antioxidant enzymes (POD, APX, CAT, and SOD) were observed.


Assuntos
Anti-Inflamatórios não Esteroides/toxicidade , Crustáceos/efeitos dos fármacos , Diclofenaco/toxicidade , Hordeum/efeitos dos fármacos , Naproxeno/toxicidade , Plântula/efeitos dos fármacos , Animais , Carotenoides/metabolismo , Clorofila/metabolismo , Crustáceos/crescimento & desenvolvimento , Interações Medicamentosas , Hordeum/crescimento & desenvolvimento , Hordeum/metabolismo , Peróxido de Hidrogênio/metabolismo , Malondialdeído/metabolismo , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Prolina/metabolismo , Plântula/crescimento & desenvolvimento , Plântula/metabolismo
18.
Nat Chem Biol ; 17(11): 1178-1187, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34556860

RESUMO

Epitranscriptomic RNA modifications can regulate RNA activity; however, there remains a major gap in our understanding of the RNA chemistry present in biological systems. Here we develop RNA-mediated activity-based protein profiling (RNABPP), a chemoproteomic strategy that relies on metabolic RNA labeling, mRNA interactome capture and quantitative proteomics, to investigate RNA-modifying enzymes in human cells. RNABPP with 5-fluoropyrimidines allowed us to profile 5-methylcytidine (m5C) and 5-methyluridine (m5U) methyltransferases. Further, we uncover a new mechanism-based crosslink between 5-fluorouridine (5-FUrd)-modified RNA and the dihydrouridine synthase (DUS) homolog DUS3L. We investigate the mechanism of crosslinking and use quantitative nucleoside liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and 5-FUrd-based crosslinking and immunoprecipitation (CLIP) sequencing to map DUS3L-dependent dihydrouridine (DHU) modifications across the transcriptome. Finally, we show that DUS3L-knockout (KO) cells have compromised protein translation rates and impaired cellular proliferation. Taken together, our work provides a general approach for profiling RNA-modifying enzyme activity in living cells and reveals new pathways for epitranscriptomic RNA regulation.


Assuntos
Oxirredutases/metabolismo , RNA/metabolismo , Linhagem Celular , Humanos
19.
Front Immunol ; 12: 726615, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34512659

RESUMO

Toxoplasma gondii (T. gondii) is an intracellular parasitic protozoan that can cause serious public health problems. However, there is no effectively preventive or therapeutic strategy available for human and animals. In the present study, we developed a DNA vaccine encoding T. gondii oxidoreductase from short-chain dehydrogenase/reductase family (TgSDRO-pVAX1) and then entrapped in chitosan and poly lactic-co-glycolic acid (PLGA) to improve the efficacy. When encapsulated in chitosan (TgSDRO-pVAX1/CS nanospheres) and PLGA (TgSDRO-pVAX1/PLGA nanospheres), adequate plasmids were loaded and released stably. Before animal immunizations, the DNA vaccine was transfected into HEK 293-T cells and examined by western blotting and laser confocal microscopy. Th1/Th2 cellular and humoral immunity was induced in immunized mice, accompanied by modulated secretion of antibodies and cytokines, promoted the maturation and MHC expression of dendritic cells, and enhanced the percentages of CD4+ and CD8+ T lymphocytes. Immunization with TgSDRO-pVAX1/CS and TgSDRO-pVAX1/PLGA nanospheres conferred significant immunity with lower parasite burden in the mice model of acute toxoplasmosis. Furthermore, our results also lent credit to the idea that TgSDRO-pVAX1/CS and TgSDRO-pVAX1/PLGA nanospheres are substitutes for each other. In general, the current study proposed that TgSDRO-pVAX1 with chitosan or PLGA as the delivery vehicle is a promising vaccine candidate against acute toxoplasmosis.


Assuntos
Antígenos de Protozoários/administração & dosagem , Quitosana/administração & dosagem , Nanosferas/administração & dosagem , Oxirredutases/administração & dosagem , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/administração & dosagem , Proteínas de Protozoários/administração & dosagem , Vacinas Protozoárias/administração & dosagem , Toxoplasmose Animal/prevenção & controle , Vacinas de DNA/administração & dosagem , Animais , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/genética , Células Dendríticas/imunologia , Feminino , Células HEK293 , Humanos , Imunoglobulina G/imunologia , Linfócitos/imunologia , Camundongos Endogâmicos BALB C , Oxirredutases/genética , Plasmídeos , Proteínas de Protozoários/genética , Ratos Sprague-Dawley , Toxoplasma/imunologia
20.
Elife ; 102021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34499029

RESUMO

Although much progress has been made in cancer treatment, the molecular mechanisms underlying cancer radioresistance (RR) as well as the biological signatures of radioresistant cancer cells still need to be clarified. In this regard, we discovered that breast, bladder, lung, neuroglioma, and prostate 6 Gy X-ray resistant cancer cells were characterized by an increase of lipid droplet (LD) number and that the cells containing highest LDs showed the highest clonogenic potential after irradiation. Moreover, we observed that LD content was tightly connected with the iron metabolism and in particular with the presence of the ferritin heavy chain (FTH1). In fact, breast and lung cancer cells silenced for the FTH1 gene showed a reduction in the LD numbers and, by consequence, became radiosensitive. FTH1 overexpression as well as iron-chelating treatment by Deferoxamine were able to restore the LD amount and RR. Overall, these results provide evidence of a novel mechanism behind RR in which LDs and FTH1 are tightly connected to each other, a synergistic effect that might be worth deeply investigating in order to make cancer cells more radiosensitive and improve the efficacy of radiation treatments.


Assuntos
Ferritinas/metabolismo , Gotículas Lipídicas/efeitos da radiação , Neoplasias/metabolismo , Neoplasias/radioterapia , Oxirredutases/metabolismo , Linhagem Celular Tumoral , Ferritinas/genética , Humanos , Gotículas Lipídicas/metabolismo , Neoplasias/genética , Oxirredutases/genética , Tolerância a Radiação , Raios X
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