NADPH oxidase 4(NOX4)as a biomarker and therapeutic target in neurodegenerative diseases

Diseases like Alzheimer’s and Parkinson’s diseases are defined by inflammation and the damage neurons undergo due to oxidative stress. A primary reactive oxygen species contributor in the central nervous system, NADPH oxidase 4, is viewed as a potential therapeutic touchstone and indicative marker for these ailments. This in-depth review brings to light distinct features of NADPH oxidase 4, responsible for generating superoxide and hydrogen peroxide, emphasizing its pivotal role in activating glial cells, inciting inflammation, and disturbing neuronal functions. Significantly, malfunctioning astrocytes, forming the majority in the central nervous system, play a part in advancing neurodegenerative diseases, due to their reactive oxygen species and inflammatory factor secretion. Our study reveals that aiming at NADPH oxidase 4 within astrocytes could be a viable treatment pathway to reduce oxidative damage and halt neurodegenerative processes. Adjusting NADPH oxidase 4 activity might influence the neuroinflammatory cytokine levels, including myeloperoxidase and osteopontin, offering better prospects for conditions like Alzheimer’s disease and Parkinson’s disease. This review sheds light on the role of NADPH oxidase 4 in neural degeneration, emphasizing its drug target potential, and paving the path for novel treatment approaches to combat these severe conditions.

Polyphenol Oxidase, Peroxidase and PhenylalanineAmmonium Lyase Induced in Postharvest Peach Fruitsby Inoculation with Pichia membranefaciensor Rhizopus stolonifer

Rhizopus rot of peach fruits could be significantly suppressed by Pichia membranefaciens. Polyphenol oxidase (PPO), peroxidase (POD) and phenylalanine ammonium-lyase (PAL) activities induced by inoculation with P. membrane faciens or R. stolonifer were studied in postharvest peach fruits. The activities of PPO and PAL in peaches increased significantly after being inoculated with P. membrane faciens + R. stolonifer by 24 h, the activities maintained at a high level throughout the experiment. Under the condition of infected with R. stolonifer alone, activity of PPO and PAL could also increased, but the levels were lower than those treated with P. membrane faciens+ R. stolonifer. However, fruits inoculaed with P. membrane-faciens + R. stolonifer or R. stolonifer alone did not stimulated POD activity. The results suggest that the activation of these defense enzymes is involved in the action of P. membrane faciens against R. stolonifer.

Type-B monoamine oxidase inhibitors in neurological diseases:clinical applications based on preclinical findings

Type-B monoamine oxidase inhibitors,encompassing selegiline,rasagiline,and safinamide,are available to treat Parkinson's disease.These drugs ameliorate motor symptoms and improve motor fluctuation in the advanced stages of the disease.There is also evidence suppo rting the benefit of type-B monoamine oxidase inhibitors on non-motor symptoms of Parkinson's disease,such as mood deflection,cognitive impairment,sleep disturbances,and fatigue.Preclinical studies indicate that type-B monoamine oxidase inhibitors hold a strong neuroprotective potential in Parkinson's disease and other neurodegenerative diseases for reducing oxidative stress and stimulating the production and release of neurotrophic factors,particularly glial cell line-derived neurotrophic factor,which suppo rt dopaminergic neurons.Besides,safinamide may interfere with neurodegenerative mechanisms,countera cting excessive glutamate overdrive in basal ganglia motor circuit and reducing death from excitotoxicity.Due to the dual mechanism of action,the new generation of type-B monoamine oxidase inhibitors,including safinamide,is gaining interest in other neurological pathologies,and many supporting preclinical studies are now available.The potential fields of application concern epilepsy,Duchenne muscular dystrophy,multiple scle rosis,and above all,ischemic brain injury.The purpose of this review is to investigate the preclinical and clinical pharmacology of selegiline,rasagiline,and safinamide in Parkinson's disease and beyond,focusing on possible future therapeutic applications.

Inhibitory effect of tartary buckwheat seedling extracts and associated flavonoid compounds on the polyphenol oxidase activity in potatoes(Solanum tuberosum L.)

To improve the processing quality of potatoes,phosphate buffer extract(PBE),50%ethanol(E50),and aqueous extract(AE)of tartary buckwheat seedlings were evaluated for their ability to inhibit the enzymatic browning of potatoes.The results suggest that all extracts of tartary buckwheat seedlings exert significant inhibitory effects on the polyphenol oxidase(PPO)activity in potatoes.The relative concentrations required for a 50%reduction in the PPO activity(IC50)were 0.21,0.28 and 0.41 mg mL^-1,for PBE,E50 and AE,respectively.The strongest inhibitory activity was observed for PBE,followed by E50 and AE.Four flavone compounds in the PBE of tartary buckwheat seedlings(i.e.,rutin,kaempferol-3-O-rutinoside,quercetin,and kaempferol)were identified by high-performance liquid chromatography.These compounds were subsequently evaluated for their roles in the inhibition of PPO from potatoes using a model system.The results indicated that rutin exhibited the highest inhibition rate on the PPO of potato.A synergistic inhibitory effect was observed by mixing rutin,kaempferol-3-Orutinoside,quercetin,and proteins.The inhibitory patterns of rutin,kaempferol-3-O-rutinoside,and quercetin on the enzyme were noncompetitive and reversible,with inhibitory constants of 0.12,0.31,and 0.40 mg mL^-1,respectively.Flavonoids from tartary buckwheat seedlings may exhibit a common mechanism with phenolic compounds,involving the blockage of the reaction of oxygen with PPO leading to the inhibition of the enzymes involved in browning.Based on these results,extracts of tartary buckwheat seedlings can be used as potent natural inhibitors.

Potential of plant polyphenol oxidases in the decolorization and removal of textile and non-textile dyes

In this study an effort has been made to use plant polyphenol oxidases; potato （Solanum tuberosum） and brinjal （Solanum melongena）, for the treatment of various important dyes used in textile and other industries. The ammonium sulphate fractionated enzyme preparations were used to treat a number of dyes under various experimental conditions. Majority of the treated dyes were maximally decolorized at pH 3.0. Some of the dyes were quickly decolorized whereas others were marginally decolorized. The initial first hour was sufficient for the maximum decolorization of dyes. The rate of decolorization was quite slow on long treatment of dyes. Enhancement in the dye decolorization was noticed on increasing the concentration of enzymes. The complex mixtures of dyes were treated with both preparations of polyphenol oxidases in the buffers of varying pH values. Potato polyphenol oxidase was significantly more effective in decolorizing the dyes to higher extent as compared to the enzyme obtained from brinjal polyphenol oxidase. Decolorization of dyes and their mixtures, followed by the formation of an insoluble precipitate, which could be easily removed simply by centrifugation.

Effects of Terpinen-4-ol on Four Metabolic Enzymes and Polyphenol Oxidase (PPO) in Mythimna separta Walker

To study insecticidal mechanism of terpinen-4-ol, a main insecticidal composition in the essential oil of Sabina vulgaris, the 5th instar larvae of Mythimna separta, were investigated with terpinen-4-ol by topical application. The activities of phosphatase, glutathione S-transferase （GSTs）, cytochrome P450 （P450）, and polyphenol oxidase （PPO） of tested insects were determined in all poisoning stages, including exciting stage, convulsing stage, paralysis stage, and recover stage. The result showed that the activities of both acid phosphatase （ACP） and alkaline phosphatase （AKP） in treated insects were induced by terpinen-4-ol, but ACP was inhibited in paralysis stage. The activities of GSTs were inhibited in exciting stage, convulsing stage, and paralysis stage, but gradually recovered in recover stage. O-demethylase activity of cytochrome P450 was inhibited by terpinen-4-ol, and the inhibition rate in all poisoning stages were 26.27, 46.03, 80.24, and 90.22%, respectively. PPO activities were strongly inhibited by terpinen-4-ol both in vitro and in vivo. In conclusion, the activities of P450, GSTs, and PPO could have relation with toxicity of terpinen-4-ol against larvae of the Mythimna separta, but recover stage of the poisoning insects might be related to GSTs induced. As a new insecticide or synergist, terpinen- 4-ol has a potential value in field of insecticide resistance management.

Cloning of multicopper oxidase gene from <i>Ochrobactrum sp</i>. 531 and characterization of its alkaline laccase activity towards phenolic substrates

A 1602 bp fragment was cloned from a soil bacterium Ochrobactrum sp. 531. It contained an open reading frame (ORF) of 1092 bp which was identified as a multicopper oxidase (MCO) with potential laccase activity. After inserting the cloned gene into the expression vector pET23a, it was expressed in E. coli BL21(DE3)pLysS, and its product was purified to homogeneity through chromatography. The Ochrobactrum sp. 531 MCO, consisting of 533 amino acids with a molecular mass of 57.8 kDa, was quite stable in neutral pH and showed laccase-like activity oxidizing 2,6-dimethoxyphenol (DMP), 2,2’-azino-bis(3-ethylbe- nzthiazolinesulfonic acid) (ABTS), and syringaldazine (SGZ). The enzyme showed optimum activity towards DMP, ABTS, and SGZ at the pH 8.0, 3.6, and 7.5 respectively. Kinetic studies gave this enzyme Km, kcat and kcat//Km values of: 0.09 mM, 7.94 s–1, and 88.22 s–1?mM–1 for DMP;0.072 mM, 2.95 s–1, and 40.97 s–1.mM–1 for ABTS;and 0.015 mM, 2.4 s–1, and 160 s–1.mM–1 for SGZ. Our results demonstrate that Ochrobactrum sp. 531 MCO is a bacterial laccase which oxidized phenolic substrates DMP and SGZ effectively under alkaline conditions. These unusual properties make the enzyme an interesting biocatalyst in applications for which classical laccases are unsuitable.

Study on Enzymatic Kinetics of Nephelium lappaceum Polyphenol Oxidase

[Objective] This study aimed to investigate the enzymatic kinetics of polyphenol oxidase in peel of Nephelium lappaceum to explore the environmental factors affecting its catalytic activity. [ Method ] With N. lappaceum peel as the experimental material and catechol as substrate, effects of temperature, pH, four inhibitors （EDTA, Vc, sodium bisulfite and citric acid） and substrate concentration on the activity of polyphenol oxidase were investigated. [ Result] The optimal temperature for polyphenol oxidase in N. lappaceum peel was 40 %, and the optimal pH was 6.8. EDTA, Vc, sodium bisulfite and citric acid all showed ideal in- hibitory effects on the activity of polyphenol oxidase; specifically, EDTA had the strongest inhibitory effect. [ Conclusion] At low temperature, EDTA, Vc, sodi- um bisulflte and citric acid with certain concentrations can inhibit the catalytic activity of polyphenol oxidase in peel of N. lappaceum, extend the storage and trans- portation time of N. lappaceum fruit, and inhibit the browning. This study provides theoretical basis for the development and utilization of polyphenol oxidase in peel of N. lappaceum.

Natural variation in the cytochrome c oxidase subunit 5B OsCOX5B regulates seed vigor by altering energy production in rice

Seed vigor is a crucial trait for the direct seeding of rice.Here we examined the genetic regulation of seed vigor traits in rice,including germination index(GI)and germination potential(GP),using a genome-wide association study approach.One major quantitative trait locus,qGI6/qGP6,was identified simultaneously for both GI and GP.The candidate gene encoding the cytochrome c oxidase subunit 5B(OsCOX5B)was validated for qGI6/qGP6.The disruption of OsCOX5B caused the vigor traits to be significantly lower in Oscox5b mutants than in the japonica Nipponbare wild type(WT).Gene co-expression analysis revealed that OsCOX5B influences seed vigor mainly by modulating the tricarboxylic acid cycle process.The glucose levels were significantly higher while the pyruvic acid and adenosine triphosphate levels were significantly lower in Oscox5b mutants than in WT during seed germination.The elite haplotype of OsCOX5B facilitates seed vigor by increasing its expression during seed germination.Thus,we propose that OsCOX5B is a potential target for the breeding of rice varieties with enhanced seed vigor for direct seeding.

Study on Polyphenol Oxidase Activity and Total Phenol Content of Docynia indica

[Objectives] To study the polyphenol oxidase (PPO) activity and total phenol content of Docynia indica .[Methods] The tender branches and petioles of the medicinal and edible plant D. indica were used as experimental materials, and the annual changes of total phenol content and polyphenol oxidase (PPO) activity were analyzed.[Results] pH 7.0 was the optimum value of polyphenol oxidase activity in D. indica plants;the best substrate was catechol with the concentration of 0.15 mol/L;the optimum temperature was 25 ℃.[Conclusions] The total phenol content of D. indica plants was the lowest in April, May and September of each year. The polyphenol oxidase activity of D. indica plants at flowering and fruiting stage was significantly higher than that at pre-flowering stage.

Purification and Partial Characterization of Polyphenol Oxidase from Sapodilla Plum (<i>Achras sapota</i>)

The browning of fruits can be considered as an enzymatic oxidation which is believed to be one of the main causes of quality loss during post-harvest handling. The enzymes responsible for this are the oxidoreductases;the polyphenol oxidase (PPO) (monophenol, o-diphenol, oxygen oxidoreductase;EC 1.14.18.1) belongs to this group. This enzyme, which is found in the sapodilla plum (Achras sapota), was purified using a phenylsepharose and a SephacrylS-200 columns. The molecular weight of the purified enzyme was estimated to be about 66 kDa by gel filtration and 29 kDa by SDS-PAGE. A single protein band was found using the latter system (SDS-PAGE), which shows that the PPO of the pulp of the sapodilla plum may be composed of two protein subunits with similar molecular weight. The optimum pH was 7.0 and the optimum temperature 60℃. The most effective inhibitors tested were ascorbic acid, sodium metabisulfite and acetic acid.

Polyphenol Oxidase Inactivation by Microwave Oven and Its Effect on Phenolic Profile of Loquat (<i>Eriobotrya japonica</i>) Fruit

The objective of this research was investigated the effect of polyphenol oxidase microwave treatment on phenolic composition, antioxidant activity and microstructure of loquat fruit. Phenolic profile of methanolic extracts prepared from fresh, and microwave-treated samples were analyzed. Antioxidant activity was also evaluated by 2,2’-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS?+) and 1,1-diphenyl-2-picrylhydrazyl (DPPH+) methods. In addition, polyphenol oxidase inactivation was carried out using a response surface methodology to establish the optimal conditions of treatment. The phenolic content of fresh mesocarp was 311 ± 0.60 mg gallic acid equivalents (GAE)/100g dry weight (DW) and that of microwave-treated mesocarp was 1230 ± 0.36 mg GAE/100g DW. Total phenolic content of water/ methanol extract significantly increases after microwave treatment rather than methanolic extract of fresh loquat. Five glycoside phenolics were identified by HPLC-DAD-MS as 3-caffeoylquinic acid, 3-p-coumaroylquinic acid, 5-caffeoylquinic acid and quercetin-3-O-sambubioside. Methanolic extract of microwave-treated mesocarp showed higher antioxidant activity than that of fresh mesocarp. Thus, polyphenol oxidase inactivation by microwave energy preserved the integrity of phenolic compounds as well as antioxidant activity in mesocarp extracts prepared from loquat fruit. It was also noted that phenolics were more abundant in the microwaved samples than in the fresh samples.

Enhancing metformin-induced tumor metabolism destruction by glucose oxidase for triple-combination therapy

Despite decades of laboratory and clinical trials,breast cancer remains the main cause of cancer-related disease burden in women.Considering the metabolism destruction effect of metformin(Met)and cancer cell starvation induced by glucose oxidase(GOx),after their efficient delivery to tumor sites,GOx and Met may consume a large amount of glucose and produce sufficient hydrogen peroxide in situ.Herein,a pH-responsive epigallocatechin gallate(EGCG)-conjugated low-molecular-weight chitosan(LC-EGCG,LE)nanoparticle(Met–GOx/Fe@LE NPs)was constructed.The coordination between iron ions(Fe3+)and EGCG in this nanoplatform can enhance the efficacy of chemodynamic therapy via the Fenton reaction.Met–GOx/Fe@LE NPs allow GOx to retain its enzymatic activity while simultaneously improving its stability.Moreover,this pH-responsive nanoplatform presents controllable drug release behavior.An in vivo biodistribution study showed that the intracranial accumulation of GOx delivered by this nanoplatform was 3.6-fold higher than that of the free drug.The in vivo anticancer results indicated that this metabolism destruction/starvation/chemodynamic triple-combination therapy could induce increased apoptosis/death of tumor cells and reduce their proliferation.This triple-combination therapy approach is promising for efficient and targeted cancer treatment.

Polyphenol Oxidase Activity and Inhibition in White Yam （Dioscorea Rotundata. Var. Laasirin） Chips as African Fries for Human Consumption

Polyphenol oxidase, a bi-functional enzyme, has been implicated in enzymatic browning of yam and other tubers in a negative way. The objective of this present study was to examine the activity of polyphenol oxidase in Dioscorea rotundata. Var. laasirin and the efficiency of heat and chemical treatments in inhibiting this enzyme. Crude Polyphenol Oxidase （PPO） of Dioscorea rotundata.Var. Laasirin was isolated and the kinetics studied using the lineweaver-burk plot. The activity of the enzyme was evaluated using spectrophotomeric method. Yam PPO catalyzes oxidation of various substrates with catechol being the most readily oxidized substrate. The Michaelis-Menten constant （Km） and maximum reaction velocity （Vmax） for yam PPO were 0.00037 and 0.3125 respectively. Inhibition data showed that the enzyme had least activity （71.70） when blanched at 95℃ for 7 mins with chemical treatment involving a combination of 0.5% Sodium metabisulphite （Food grade） and 0.5% Ascorbic acid （Food grade）. The activity was highest （83.02） when it was blanched at 95 ℃ for 7 rains. This study has shown that it is possible to inhibit polyphenol oxidase activity in white yam using the chemical pretreatments and processing conditions described in this study for possible adoption in the production of packaged frozen yam chips by food industries.

Stability and Refolding of Prophenol Oxidase Protein with 2-Propanol in Drosophila melanogaster

Phenol oxidase in Drosophila melanogaster occurs as folded phase precursors designated as prophenol oxidase A1 and A3, and prophenol oxidase is activated with alcohol, especially 2-propanol, within a few minutes as unfolded-phase in vitro. To clarify a common effect of alcohols on proteins and peptides, the extract containing prophenol oxidase protein was prepared. Phenol oxidase activity activated with 2-propanol has been maintained stable at least 24 hours remains as it is. Protein of prophenol oxidase was not denatured opposite hypnoses known as the instability of protein with alcohol. Activated prophenol oxidase with 2-propanol remain enzyme activity with no aggregation, stable, renaturation, and the refolding phenomena occurred around the active phase within the catalytic active center of prophenol oxidase protein in Drosophila melanogaster. This study is important to induce the wide range applications of the effect in many fields for rational drag design.

Polyphenol Oxidase Activity and Colour Changes of ‘Starking’ Apple Cubes Coated with Alginate and Dehydrated with Air

The objective was to study the effect of alginate coating on polyphenol oxidase (PPO) activity and colour of ‘Starking’ apple cubes during dehydration with hot air. Apple cubes were dehydrated at 20oC, 35oC, or 40oC, with a parallel airflow. Analysis of PPO activity, colour (L*, a*, b*) and dry matter were performed along the dehydration process at each temperature. All samples presented a peak in relative PPO activity in the beginning of the drying. Exponential models fitted well the experimental data after the peak. Cubes without coating presented lower PPO activity, suggesting lower browning, than coated samples throughout the dehydration process, for all temperatures. Better results for coated samples were obtained with a perpendicular airflow drying at 40oC, after dipping the whole apple in water at 60oC for 10 min. In order to prevent coated samples from browning, drying by perpendicular airflow preceded by a thermal treatment of the whole apple is required.

<i>In Silico</i>and <i>in Vitro</i>Approach for the Understanding of the Xanthine Oxidase Inhibitory Activity of Uruguayan Tannat Grape Pomace and Propolis Poliphenols

The use of food additives with xanthine oxidase (XO) inhibitory activity offers an alternative approach to hyperuricemic and gout disease treatment, and provides an example of antioxidant nutraceutics. The in vitro and in silico XO inhibitory activity of polyphenols from Uruguayan Tannat grape pomaces and propolis extracts was evaluated as well as the scavenging capacity of said compounds. When comparing propolis and grape pomace samples, the in vitro studies demonstrated that polyphenols extracted from propolis are more active as free radical scavengers than those from Tannat grape pomace. Both natural products effectively inhibited XO but the capacity of phenols present in GP is higher than the one present in P. The high content of anthocyanins in GP, absent in P, could account for this observation. In silico assays allowed us to determine relevant ligand-receptor interactions between polyphenols, from a database built with previously reported polyphenols from both natural products, and the active site of XO. The in silico results showed that compound (E)-isoprenylcaffeate from propolis was the best potential XO inhibitor displaying hydrophobic aromatic interaction between the conjugated ring of the caffeate moiety and polar interactions between hydroxyl groups from caffeate with the active site polar residues. Among grape pomaces, the Cyanidin-3-O-(6-(E)-p-coumaroyl)-glucoside was the best XO inhibitor;its moiety oxychromenyl being relevant to the docking stabilization. All these results lead us to propose Uruguayan propolis and Tannat grape pomace extracts as food additives as well as phytopharmaceuticals to decrease the uric acid levels in gout disease and to act against oxidative stress.

Anti-polyphenol oxidase mechanism of oligomeric procyanidins and its application on browning control of“Baiyu”loquat during storage

The harvested loquat fruits are prone to browning,leading to a decline in fruit quality.Inhibiting activity of polyphenol oxidase(PPO)is an important way to prevent browning.In this study,we evaluated the inhibitory impact of oligomeric procyanidins(OPC)on hydroxylation of monophenol and oxidation of o-diphenol catalyzed by PPO and effect of OPC on loquat fruits storage.The obtained results revealed that OPC could attenuate the activity of monophenolase and diphenolase.For monophenolase activity,the inhibition rate was observed to be dose-dependent with an IC50 of 11.6±3.4μg/mL for the steady-state rate.For diphenolase activity,OPC inhibited PPO activity(IC50=15.75±2.1μg/mL)with a reversible and competitive mechanism and quenched fluorescence of PPO by forming OPC-PPO complex with one binding site in a static procedure.In addition,the data obtained from storage assays revealed that OPC could enhance the quality of harvested loquat fruits by attenuating the PPO activity,augmenting the content of total phenolics and flavonoids,and simultaneously delaying the browning of loquats.In this research,we identified a potent PPO inhibitor and expanded its potential as a food preservative.

Activation of Prophenol Oxidase PHOX-S with Limited Proteolysis in Drosophila Melanogaster

Prophenol oxidase isoform A1 was isolated from Drosophila melanogaster （The sequence has been deposited in GenBank data base under accession number AB557586） PHOX-S strain, and its characteristics and activation mechanism were determined. The NH2-terminal region of PHOX-S A1 was determined to be comprised of 15 amino acids with the following sequence MTNMKMKMKAMMR. Comparison of an alignment in the known prophenol oxidase protein sequences from Drosophila melanogaster strains showed high homology in the copper-binding sequences at the Cu （A） site of the active center. Limited proteolysis takes place between Arg-50 and Val-51. Therefore, it is concluded that prophenol oxidase PHOX-S protein was evolved at the upstream, but no evolved at the central site in Drosophila melanogaster.

Immobilization and Properties of Polyphenol Oxidase

[Objectives]This study was conducted to investigate the effects of embedding conditions on activity and catalytic properties of immobilized polyphenol oxidase.[Methods]Polyphenol oxidase was immobilized in a polymer material by the embedding method,and the optimal immobilization conditions were obtained by single factor tests:CaCl_(2) concentration 2.0%,sodium alginate concentration 2.0%,immobilization time 2 h and mass ratio of enzyme to carrier 10 mg/100 g,under which the immobilized enzyme activity was 93.33 U/g.Under the above conditions,the properties of polyphenol oxidase immobilized by sodium alginate(A-PPO)and free polyphenol oxidase were studied.[Results]The thermostability of A-PPO was better than that of the free enzyme,but the pH stability of A-PPO was inhibited.The Michaelis constant K_(m) values of free polyphenol oxidase and A-PPO were 0.37 and 0.48 mmol/L,respectively,and the maximum reaction rate V_(max) values were 0.38 and 0.51 mmol/(L·g),respectively.[Conclusions]This study provides a theoretical basis for the study of the properties of polyphenol oxidase.