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Biochemical and Biophysical Research... Jun 2023Ascorbate (AsA) is a crucial antioxidant in plants, and its recycling is necessary for protecting cells from oxidative damage and imparting stress tolerance. The...
Ascorbate (AsA) is a crucial antioxidant in plants, and its recycling is necessary for protecting cells from oxidative damage and imparting stress tolerance. The monodehydroascorbate reductase (MDHAR) enzyme of the ascorbate-glutathione pathway plays a vital role in recycling AsA from monodehydroascorbate (MDHA) radical. Pennisetum glaucum (Pg), also known as pearl millet, is known to be more tolerant to abiotic stress than other food crops, such as rice. However, the contribution of MDHAR from this sessile plant to its unique stress tolerance mechanism is not well understood. In this study, we isolated a gene encoding the MDHAR enzyme from heat stress-adapted pearl millet and characterized it using enzyme kinetics, thermal stability assays, and crystal structure determination. Our results indicate that PgMDHAR is a more robust enzyme than its rice counterpart (Oryza sativa; Os). We solved the crystal structure of PgMDHAR at 1.8 Å and found that the enzyme has a more compact structure and greater stability than OsMDHAR. Using hybrid quantum mechanics and molecular mechanics calculations, we demonstrate that the structure of PgMDHAR contributes to increased stability towards bound FAD. Overall, the higher structural stability and affinity for NADH demonstrated by PgMDHAR are expected to impart improved stress tolerance. Our findings suggest that transgenic food crops expressing MDHAR from stress-adapted pearl millet may exhibit better tolerance to oxidative stress in the unpredictable climatic conditions prevalent today.
Topics: Pennisetum; Ascorbic Acid; Dehydroascorbic Acid
PubMed: 37119729
DOI: 10.1016/j.bbrc.2023.04.034 -
Journal of Agricultural and Food... May 2023Tricaine is a common anesthetic used in the long-distance transport of live fish. Recently, its negative impact on human health has aroused extensive concern. Thus,...
Tricaine is a common anesthetic used in the long-distance transport of live fish. Recently, its negative impact on human health has aroused extensive concern. Thus, rapid and reliable techniques for tricaine residue analysis are essential to ensuring the quality of aquatic products. Herein, a specific anti-tricaine monoclonal antibody (Mab) was prepared. Then, a sensitive and robust ratiometric fluorescence ELISA (RF-ELISA) was constructed for detecting tricaine based on two MnO nanoflake-mediated (MnO NFs) fluorogenic reactions. In the RF-ELISA protocol, MnO NFs with oxidase-like activity can trigger the formation of fluorescent 2,3-diaminophenazine (oxOPD) with an emissive peak at 570 nm from non-fluorescent -phenylenediamine (OPD), while ascorbic acid (AA) can decompose MnO NFs to lose their oxidase-mimicking activity, which is accompanied by the oxidation of AA into dehydroascorbic acid (DHAA). The subsequent reaction between the generated DHAA and OPD will result in the production of 3-(1,2-dihydroxy ethyl)furo[3,4-]quinoxalin-1(3H)-on (DFQ), which has a potent emission peak at 445 nm. By virtue of the alkaline phosphatase (ALP) labeled on the antibody, which can catalyze the production of AA from ascorbic acid 2-phosphate (AAP), the concentration of tricaine can be linked to the variation of the RF signal (F/F) via a competitive immunoreaction. After optimization, RF-ELISA displayed a detection limit (LOD) of 0.28 ng/mL toward tricaine (in buffer solution), which was 376-fold lower than that of the traditional colorimetric ELISA. For practical application, the LODs of RF-ELISA for tricaine detection in shrimp and tilapia samples were determined to be 2.8 and 5.6 ng/g, respectively. Recoveries for spiked shrimp and tilapia samples, as well as the validation data from LC-MS/MS, showed that RF-ELISA exhibited good accuracy, precision, and reliability. This RF-ELISA protocol opened up new ways for tricaine and other-target analyses in food safety detection.
Topics: Animals; Humans; Manganese Compounds; Oxides; Fluorescence; Reproducibility of Results; Chromatography, Liquid; Tandem Mass Spectrometry; Oxidoreductases; Enzyme-Linked Immunosorbent Assay; Coloring Agents; Limit of Detection
PubMed: 37057807
DOI: 10.1021/acs.jafc.3c00469 -
Biosensors Mar 2023In this study, a copper hexacyanoferrate nanoparticle with excellent oxidase-mimetic behaviour has been synthesized through a simple precipitation method. The...
In this study, a copper hexacyanoferrate nanoparticle with excellent oxidase-mimetic behaviour has been synthesized through a simple precipitation method. The synthesized copper hexacyanoferrate nanoparticle has intrinsic oxidase-like activity, which can catalyze the chromogenic reaction of 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate) through an O reactive oxygen-species-participated process. On the other hand, K[Fe(CN)] can be reduced by ascorbic acid (AA) to produce K[Fe(CN)], thereby inhibiting the formation of the copper hexacyanoferrate nanoparticles. Furthermore, ascorbate oxidase (AAO) can catalyze the oxidation of AA to produce dehydroascorbic acid, which cannot reduce K[Fe(CN)]. Thus, a system for an AAO-regulated in situ formation of copper hexacyanoferrate nanoparticles was constructed by coupling a prepared copper hexacyanoferrate nanozyme with AA for the detection of AAO activity. This colorimetric sensing assay shows high sensitivity and selectivity for the detection of AAO activity (the limit of detection is 0.52 U/L) with a linear range of 1.1-35.7 U/L. Finally, the developed method was applied to detect the activity of AAO in normal human serum with a satisfactory sample spiked recovery (87.4-108.8%). In short, this study provides a good strategy for the construction of nanozyme-based multi-enzyme cascade-signal amplification assay.
Topics: Humans; Oxidoreductases; Ascorbate Oxidase; Copper; Colorimetry; Nanoparticles; Ascorbic Acid; Limit of Detection
PubMed: 36979556
DOI: 10.3390/bios13030344 -
Acta Pharmaceutica Sinica. B Mar 2023As a neurological disorder in the brain, epilepsy is not only associated with abnormal synchronized discharging of neurons, but also inseparable from non-neuronal...
As a neurological disorder in the brain, epilepsy is not only associated with abnormal synchronized discharging of neurons, but also inseparable from non-neuronal elements in the altered microenvironment. Anti-epileptic drugs (AEDs) merely focusing on neuronal circuits frequently turn out deficient, which is necessitating comprehensive strategies of medications to cover over-exciting neurons, activated glial cells, oxidative stress and chronic inflammation synchronously. Therefore, we would report the design of a polymeric micelle drug delivery system that was functioned with brain targeting and cerebral microenvironment modulation. In brief, reactive oxygen species (ROS)-sensitive phenylboronic ester was conjugated with poly-ethylene glycol (PEG) to form amphiphilic copolymers. Additionally, dehydroascorbic acid (DHAA), an analogue of glucose, was applied to target glucose transporter 1 (GLUT1) and facilitate micelle penetration across the blood‒brain barrier (BBB). A classic hydrophobic AED, lamotrigine (LTG), was encapsulated in the micelles self-assembly. When administrated and transferred across the BBB, ROS-scavenging polymers were expected to integrate anti-oxidation, anti-inflammation and neuro-electric modulation into one strategy. Moreover, micelles would alter LTG distribution with improved efficacy. Overall, the combined anti-epileptic therapy might provide effective opinions on how to maximize neuroprotection during early epileptogenesis.
PubMed: 36970212
DOI: 10.1016/j.apsb.2022.09.019 -
Fungal Biology Mar 2023Black spot rot caused by Alternaria alternata is one of the major postharvest disease of apple fruit during logistic. This study evaluated in vitro inhibitory effect of...
Black spot rot caused by Alternaria alternata is one of the major postharvest disease of apple fruit during logistic. This study evaluated in vitro inhibitory effect of 2-hydroxy-3-phenylpropanoic acid (PLA) at various concentrations on A. alternata and the possible mechanisms involved in its action. Results showed that different concentrations of PLA inhibited conidia germination and mycelial growth of A. alternata in vitro, and 1.0 g L was the lowest effective concentration to suppress A. alternata growth. Moreover, PLA significantly reduced relative conductivity and increased malondialdehyde and soluble protein contents. PLA also increased HO and dehydroascorbic acid contents, but reduced ascorbic acid content. Additionally, PLA treatment inhibited catalase, ascorbate peroxidase, monodehydroascorbate acid reductase, dehydroascorbic acid reductase and glutathione reductase activities, whereas promoted superoxide dismutase activity. All these findings suggest that the possible mechanisms involved in the inhibitory effect of PLA on A. alternata included damaging the cell membrane integrity to cause electrolyte leakage and destroying reactive oxygen species balance.
Topics: Reactive Oxygen Species; Hydrogen Peroxide; Dehydroascorbic Acid; Cell Membrane; Alternaria; Polyesters
PubMed: 36906385
DOI: 10.1016/j.funbio.2023.02.001 -
Antioxidants (Basel, Switzerland) Jan 2023Vitamin C (ascorbic acid) is well known for its potent antioxidant properties, as it can neutralize ROS and free radicals, thereby protecting cellular elements from... (Review)
Review
Vitamin C (ascorbic acid) is well known for its potent antioxidant properties, as it can neutralize ROS and free radicals, thereby protecting cellular elements from oxidative stress. It predominantly exists as an ascorbate anion and after oxidation to dehydroascorbic acid and further breakdown, is removed from the cells. In nervous tissue, a progressive decrease in vitamin C level or its prolonged deficiency have been associated with an increased risk of disturbances in neurotransmission, leading to dysregulation in brain function. Therefore, understanding the regulatory function of vitamin C in antioxidant defence and identification of its molecular targets deserves more attention. One of the key signalling ions is calcium and a transient rise in its concentration is crucial for all neuronal processes. Extracellular Ca influx (through specific ion channels) or Ca release from intracellular stores (endoplasmic reticulum, mitochondria) are precisely controlled. Ca regulates the functioning of the CNS, including growth, development, myelin formation, synthesis of catecholamines, modulation of neurotransmission and antioxidant protection. A growing body of evidence indicates a unique role for vitamin C in these processes. In this short review, we focus on vitamin C in the regulation of calcium-involved pathways under physiological and stress conditions in the brain.
PubMed: 36829790
DOI: 10.3390/antiox12020231 -
Mikrochimica Acta Feb 2023Fumonisin B1 (FB1) is one of the important mycotoxins posing health risks in the area of food safety. A sensitive fluorescence ratio immunoassay has been...
Fluorescence ratio immunoassay for fumonisin B1 based on the oxidase characteristics of the growth of monodispersed 2-D MnO nanosheet on an individual gold nanoparticle (AuNP@MnO).
Fumonisin B1 (FB1) is one of the important mycotoxins posing health risks in the area of food safety. A sensitive fluorescence ratio immunoassay has been established for FB1 based on the growth of monodispersed 2-D MnO nanosheet on an individual gold nanoparticle (AuNP@MnO). FB1 competed with the coated FB1-BSA to bind the FB1 monoclonal antibody. After a washing step, alkaline phosphatase-labeled goat anti-mouse IgG (ALP-IgG) with high catalytic activity was combined with FB1 monoclonal antibody. ALP reacts with ascorbic acid 2-phosphate (AAP) to produce ascorbic acid (AA), which decomposes AuNP@MnO to dehydroascorbic acid (DHAA). O-Phenylenediamine dihydrochloride (OPD) is oxidized to yellow-fluorescent substrate of 2,3-diaminophenazine (DAP) (excitation, 423 nm; emission, 570 nm) by AuNP@MnO. Meanwhile, OPD can also be reduced to blue fluorescent substrate of OPDred (excitation, 350 nm; emission, 430 nm) by DHAA. The content of FB1 can be determined by fluorescence ratio of blue/yellow. The limit of detection (LOD) of the fluorescence ratio immunoassay for FB1 was 0.06 ng mL, and the linear range was from 0.25 to 60.00 ng mL. The effectiveness of the assay was verified in real maize samples, and satisfactory recoveries were attained. The correlation coefficient of these results between the fluorescence ratio immunoassay and commercial ELISA kit was 0.9999. This method provides a sensitive and selective tool for the detection of FB1 in maize samples.
Topics: Gold; Oxidoreductases; Manganese Compounds; Metal Nanoparticles; Oxides; Enzyme-Linked Immunosorbent Assay; Antibodies, Monoclonal; Immunoglobulin G
PubMed: 36790594
DOI: 10.1007/s00604-023-05675-5 -
Plants (Basel, Switzerland) Feb 2023The uplift of the Qinghai Tibet Plateau has led to a drastic change in the climate in Central Asia, from warm and rainy, to dry and less rainfall. , a rare evergreen...
The uplift of the Qinghai Tibet Plateau has led to a drastic change in the climate in Central Asia, from warm and rainy, to dry and less rainfall. , a rare evergreen broad-leaved shrub distributed in the temperate desert region of Central Asia, has survived the drastic climate change in Central Asia caused by the uplift of the Qinghai-Tibet Plateau. Ascorbate oxidase (AO) regulates the redox status of the apoplast by catalyzing the oxidation of ascorbate acid to dehydroascorbic acid, and plays a key role in the adaptation of plants to environmental changes. Analyzing the evolution, environmental response, and biological functions of the AO family of is helpful for understanding how plant genome evolution responds to climate change in Central Asia. A total of 16 AOs were identified in , all of which contained the ascorbate oxidase domain, most of which contained transmembrane domain, and many were predicted to be localized in the apoplast. Segmental duplication and tandem duplication are the main factors driving the gene amplification of the AO gene family in . Gene expression analysis based on transcriptome data and fluorescence quantitative PCR, as well as enzyme activity measurements, showed that the expression levels of AO genes and total enzyme activity decreased under short-term osmotic stress and low-temperature stress, but the expression of some AO genes (, , and ) and total enzyme activity increased under 7 days of cold stress. and are targeted by . Further functional studies on AnAO5 showed that AnAO5 protein was localized in the apoplast. The expression of in yeast cells and the transient expression in tobacco enhanced the tolerance of yeast and tobacco to low-temperature stress, and the overexpression of enhanced the tolerance of Arabidopsis seedlings to cold stress. Our research provides important data for understanding the role of AOs in plant adaptation to environmental change.
PubMed: 36771760
DOI: 10.3390/plants12030677 -
Molecules (Basel, Switzerland) Jan 2023The identification of unstable metabolites of ellagitannins having ortho-quinone structures or reactive carbonyl groups is important to clarify the biosynthesis and...
The identification of unstable metabolites of ellagitannins having ortho-quinone structures or reactive carbonyl groups is important to clarify the biosynthesis and degradation of ellagitannins. Our previous studies on the degradation of vescalagin, a major ellagitannin of oak young leaves, suggested that the initial step of the degradation is regioselective oxidation to generate a putative quinone intermediate. However, this intermediate has not been identified yet. In this study, young leaves of were extracted with 80% acetonitrile containing 1,2-phenylenediamine to trap unstable ortho-quinone metabolites, and subsequent chromatographic separation afforded a phenazine derivative of the elusive quinone intermediate of vescalagin. In addition, phenylenediamine adducts of liquidambin and dehydroascorbic acid were obtained, which is significant because liquidambin is a possible biogenetic precursor of C-glycosidic ellagitannins and ascorbic acid participates in the production of another C-glycosidic ellagitannin in matured oak leaves.
Topics: Hydrolyzable Tannins; Quercus; Quinones
PubMed: 36770911
DOI: 10.3390/molecules28031246 -
Plant Physiology and Biochemistry : PPB Mar 2023S-nitrosylation, a post-translational modification (PTM) dependent on nitric oxide, is essential for plant development and environmental responsiveness. However, the...
S-nitrosylation, a post-translational modification (PTM) dependent on nitric oxide, is essential for plant development and environmental responsiveness. However, the function of S-nitrosylation of glutathione reductase (GR) in tomato (SlGR) under NaCl stress is yet uncertain. In this study, sodium nitroprusside (SNP), an exogenous NO donor, alleviated the growth inhibition of tomato under NaCl treatment, particularly at 100 μM. Following NaCl treatment, the transcripts, enzyme activity, and S-nitrosylated level of GR were increased. In vitro, the SlGR protein was able to be S-nitrosylated by S-nitrosoglutathione (GSNO), significantly increasing the activity of GR. SlGR overexpression transgenic tobacco plants exhibited enhanced germination rate, fresh weight, and increased root length in comparison to wild-type (WT) seedlings. The accumulation of reactive oxygen species (ROS) was lower, whereas the expression and activities of GR, ascorbate peroxidase (APX), superoxide dismutase (SOD), and catalase (CAT); the ratio of ascorbic acid/dehydroascorbic acid (AsA/DHA), reduced glutathione/oxidized glutathione (GSH/GSSG), total soluble sugar and proline contents; and the expression of stress-related genes were higher in SlGR overexpression transgenic plants in comparison to the WT plants following NaCl treatment. The accumulation of NO and S-nitrosylated levels of GR in transgenic plants was higher in comparison to WT plants following NaCl treatment. These results indicated that S-nitrosylation of GR played a significant role in salt tolerance by regulating the oxidative state.
Topics: Glutathione Reductase; Nicotiana; Solanum lycopersicum; Salt Tolerance; Sodium Chloride; Ascorbic Acid; Antioxidants; Glutathione; Oxidative Stress; Nitric Oxide; Seedlings
PubMed: 36764265
DOI: 10.1016/j.plaphy.2023.02.010