Alu antisense RNA ameliorates methylglyoxal-induced human lens epithelial cell apoptosis by enhancing antioxidant defense

AIM:To determine whether an antisense RNA corresponding to the human Alu transposable element(Aluas RNA)can protect human lens epithelial cells(HLECs)from methylglyoxal-induced apoptosis.METHODS:Cell counting kit-8(CCK-8)and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assays were used to assess HLEC viability.HLEC viability/death was detected using a Calcein-AM/PI double staining kit;the annexin V-FITC method was used to detect HLEC apoptosis.The cytosolic reactive oxygen species(ROS)levels in HLECs were determined using a reactive species assay kit.The levels of malondialdehyde(MDA)and the antioxidant activities of total-superoxide dismutase(T-SOD)and glutathione peroxidase(GSH-Px)were assessed in HLECs using their respective kits.RT-q PCR and Western blotting were used to measure m RNA and protein expression levels of the genes.RESULTS:Aluas RNA rescued methylglyoxal-induced apoptosis in HLECs and ameliorated both the methylglyoxalinduced decrease in Bcl-2 m RNA and the methylglyoxalinduced increase in Bax m RNA.In addition,Aluas RNA inhibited the methylglyoxal-induced increase in Alu sense RNA expression.Aluas RNA inhibited the production of ROS induced by methylglyoxal,restored T-SOD and GSHPx activity,and moderated the increase in MDA content after treatment with methylglyoxal.Aluas RNA significantly restored the methylglyoxal-induced down-regulation of Nrf2 gene and antioxidant defense genes,including glutathione peroxidase,heme oxygenase 1,γ-glutamylcysteine synthetase and quinone oxidoreductase 1.Aluas RNA ameliorated methylglyoxal-induced increases of the m RNA and protein expression of Keap1 that is the negative regulator of Nrf2.CONCLUSION:Aluas RNA reduces apoptosis induced by methylglyoxal by enhancing antioxidant defense.

Receptor-like kinase OsASLRK regulates methylglyoxal response and content in rice

Receptor-like kinases(RLKs) are essential for plant abiotic stress responses. Methylglyoxal(MG) is a cellular metabolite that is often considered to be a stress signal molecule. However, limited information is available about the relationship between RLKs and MG. Here, we addressed the function of a receptor-like kinase, Os ASLRK, in the MG response and content in rice. A typical MG-responsive element(AAAAAAAA) exists in the promoter region of the OsASLRK gene. RTqPCR analysis indicated that the transcript level of OsASLRK was significantly increased by exogenous MG in a time-and dosage-dependent fashion. GUS staining also confirmed that the expression of Os ASLRK in rice root was enhanced by exogenous MG treatment. Genetic analysis suggested that the Osaslrk mutant displays increased sensitivity to MG and it showed higher endogenous MG content under exogenous MG treatments, while OsASLRK-overexpressing rice plants showed the opposite phenotypes. Diaminobenzidine(DAB) staining, scavenging enzyme activities and GSH content assays indicate that OsASLRK regulates MG sensitivity and content via the elevation of antioxidative enzyme activities and alleviation of membrane damage. Therefore, our results provide new evidence illustrating the roles that receptor-like kinase Os ASLRK plays in MG regulation in rice.

Pathogen-induced methylglyoxal negatively regulates rice bacterial blight resistance by inhibiting OsCDR1 protease activity

Xanthomonas oryzae pv.oryzae(Xoo)causes bacterial blight(BB),a globally devastating disease of rice(Oryza sativa)that is responsible for significant crop loss.Sugars and sugar metabolites are important for pathogen infection,providing energy and regulating events associated with defense responses;howev-er,the mechanisms by which they regulate such events in BB are unclear.As an inevitable sugar metabolite,methylglyoxal(MG)is involved in plant growth and responses to various abiotic stresses,but the underlying mechanisms remain enigmatic.Whether and how MG functions in plant biotic stress responses is almost completely unknown.Here,we report that the Xoo strain PXO99 induces OsWRKY62.1 to repress transcrip-tion of OsGLY Il genes by directly binding to their promoters,resulting in overaccumulation of MG.MG negatively regulates rice resistance against Pxo99:osglyll2 mutants with higher MG levels are more sus-ceptible to the pathogen,whereas OsGLYIl2-overexpressing plants with lower MG content show greater resistance than the wild type.Overexpression of OsGLYll2 to prevent excessive MG accumulation confers broad-spectrum resistance against the biotrophic bacterial pathogens Xoo and Xanthomonas oryzae pv.oryzicola and the necrotrophic fungal pathogen Rhizoctonia solani,which causes rice sheath blight.Further evidence shows that MG reduces rice resistance against PXO99 through CONSTITUTIVE DISEASE RESISTANCE 1(OsCDR1).MGmodifies the Arg97 residue of OsCDR1to inhibit its aspartic protease activ-ity,which is essential for OsCDR1-enhanced immunity.Taken together,these findings illustrate how Xoo promotes infection by hijacking a sugar metabolite in the host plant.

Effect of NOx and SO2 on the photooxidation of methylglyoxal:Implications in secondary aerosol formation

Methylglyoxal(CH3COCHO,MG),which is one of the most abundant α-dicarbonyl compounds in the atmosphere,has been reported as a major source of secondary organic aerosol(SOA).In this work,the reaction of MG with hydroxyl radicals was studied in a 500 L smog chamber at(293±3)K,atmospheric pressure,(18±2)%relative humidity,and under different NOx and SO2.Particle size distribution was measured by using a scanning mobility particle sizer(SMPS)and the results showed that the addition of SO2 can promote SOA formation,while different NOx concentrations have different influences on SOA production.High NOx suppressed the SOA formation,whereas the particle mass concentration,particle number concentration and particle geometric mean diameter increased with the increasing NOx concentration at low NOx concentration in the presence of SO2.In addition,the products of the OH-initiated oxidation of MG and the functional groups of the particle phase in the MG/OH/SO2 and MG/OH/NOx/SO2 reaction systems were detected by gas chromatography mass spectrometry(GC-MS)and attenuated total reflection fourier transformed infrared spectroscopy(ATR-FTIR)analysis.Two products,glyoxylic acid and oxalic acid,were detected by GC-MS.The mechanism of the reaction of MG and OH radicals that follows two main pathways,H atom abstraction and hydration,is proposed.Evidence is provided for the formation of organic nitrates and organic sulfate in particle phase from IR spectra.Incorporation of NOx and SO2 influence suggested that SOA formation from anthropogenic hydrocarbons may be more efficient in polluted environment.

Safety issues of methylglyoxal and potential scavengers

The health safety of methylglyoxal(MGO) has been recognized as a key issue owing to its ultra-high reactivity toward some key biomolecules such as amino acids, proteins, DNA, sulfhydryl-and basic nitrogencontaining compounds, including amino-bearing neurotransmitters. In this review, we have summarized the endoand exogenous sources of MGO and its accumulation inside the body due to high intake, abnormal glucose metabolism and or malfunctioning glyoxalases, and review the debate concerning the adverse functionality of MGO ingested from foods. Higher than normal concentrations of MGO in the circulatory system and tissues have been found to be closely associated with the production of advanced glycation end products(AGEs), increased oxidative stress, elevated inflammation and RAGE(AGE receptors) activity, which subsequently progresses to a pathological stage of human health, such as diabetes complications, cancer, cardiovascular and degenerative diseases. Having illustrated the mechanisms of MGO trapping in vivo, we advocate the development of efficient and efficacious MGO scavengers, either assisting or enhancing the activity of endogenous glyoxalases to facilitate MGO removal, or providing phytochemicals and functional foods containing them, or pharmaceuticals to irreversibly bind MGO and thus form MGO-complexes that are cleared from the body.

Sri Lankan black tea(Camellia sinensis L.)inhibits the methylglyoxal mediated protein glycation and potentiates its reversing activity in vitro

Objective:To evaluate inhibitory activity of methylglyoxal(MGO)mediated protein glycation and ability to potentiate its reversing activity and range of antioxidant properties of Sri Lankan low grown orthodox orange pekoe grade black tea.Methods:Freeze dried black tea brew(BTB)was used as the sample in this study.Antiglycation and glycation reversing activity was studied in bovine serum albumin(BSA)-MGO model.Antioxidant properties were studied using total polyphenolic content,total flavonoid content,2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid,1,1-diphenyl-2-picrylhydrazine and ferric reducing antioxidant power in vitro antioxidant assays.Results:The results demonstrated significant(P<0.05)and dose dependant inhibition of BSA-MGO glycation[IC_(50):(164.30±4.85)μg/mL],potentiating of its reversing activity[EC_(50):(235.39±5.37)μg/mL]and marked antioxidant properties[total polyphenolic content:(119.55±9.97)mg gallic acid equivalents/g BTB;total flavonoid content:(6.04±1.26)mg quercetin equivalents/g BTB;2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid,1,1-diphenyl-2-picrylhydrazine and ferric reducing antioxidant power:(3.29±0.06),(1.95±0.15)and(1.31±0.19)mmol Trolox equivalents/g BTB,respectively].No correlations were observed between antioxidant activity and BSA-MGO glycation.Conclusions:The novel properties observed for Sri Lankan orange pekoe grade black tea indicate its usefulness as a supplementary beverage in managing MGO and advanced glycation end products related diseases and ailments.

Manuka honey: an emerging natural food with medicinal use

The health value of honey is universally acknowledged from time immemorial.Manuka(Leptospermum scoparium)is a tree,indigenous to New Zealand and South East Australia,and from the myrtle family,Myrtaceae.The honey produced from its flowers is a uni-floral honey largely produced in New Zealand.It is becoming increasingly popular as a functional food,seen in the aisles of health stores as its displays superior nutritional and phytochemistry profile over other varieties of honey.Examining existing research databases revealed its biological properties ranging from anti-oxidant,anti-inflammatory,anti-bacterial,anti-viral,anti-biotic and wound healing to immune-stimulatory properties.Methylglyoxal is the unique compound in the honey responsible for some of its potent anti-microbial properties.Further,propolis another component of honey contains chiefly flavonoids(i.e.galangin,pinocembrin),phenolic acids and their esters that may also contribute to its immuno-stimulant properties.Recent findings of the biological roles have been discussed with emphasis on the underlying mechanisms.The hurdles associated in its development as a functional food and also nutraceutical with future scopes have also been mentioned.Relevant data published in MEDLINE,Cochrane library,and EMBASE in the past decade have been gathered to formulate this review.

Potassium-Induced Regulation of Cellular Antioxidant Defense and Improvement of Physiological Processes in Wheat under Water Deficit Condition

Drought is the most common form of abiotic stress that reduces plant growth and productivity.It causes plant injuries through elevated production of reactive oxygen species(ROS).Potassium(K)is a vital plant nutrient that notably ameliorates the detrimental effect of drought stress in the plant.A pot experiment was conducted at the Laboratory of Plant Stress Responses,Faculty of Agriculture,Kagawa University,Japan,under controlled environment of green house to explore the role of K in mitigating drought severity in wheat(Triticum asevitum L.)seedlings.Three days after germination,seedlings were exposed to three water regimes viz.,100,50,and 20%field capacity(FC)for 21 days.Potassium was adjusted in Hoagland nutrient solution at 0,6 and 12 mM concentration and applied to pot instead of normal water.Results show that,water deficit stress notably reduced plant growth,biomass accumulation,leaf relative water content(RWC)along with reduced photosynthetic pigments.Increased amount of biochemical stress markers viz.,malondialdehyde(MDA),hydrogen peroxide(H_(2)O_(2)),methylglyoxal(MG),proline(Pro)as well as an impaired antioxidant defense system were observed in drought affected wheat plants.On the contrary,K supplementation resulted in improvement of biochemical and physiological parameters that worked behind in improving growth and development of the wheat plants.In addition,enzymes of ascorbateglutathione(AsA-GSH)cycle were also enhanced by supplemented K that accelerated the ROS detoxification process in plant.Although glyoxalse system did not performed well till MG was detoxified might following another short stepped pathways.Our results revealed that drought stressed plants showed better performances in terms of biochemical and physiological attributes,antioxidant defense and glyoxalase system,as well as ROS detoxification due to K supplementation with better performance at 12 mM K added in 50%FC growing condition.

Effect of Italian Sour Cherry (<i>Prunus cerasus</i>L.) on the Formation of Advanced Glycation End Products and Lipid Peroxidation

Sweet and sour cherries contain several polyphenols that possess antioxidant and anti-inflammatory properties. Aim of this study was to investigate the effect of the maturity stage on phenol content and biological properties of extract of a local Morello-type of sour cherry (Prunus cerasus?L.), “visciola”. The study of total phenol content and total antioxidant potential was associated with the evaluation of the antioxidant property of extracts using a copper catalyzed human low density lipoproteins (LDL) oxidation as experimental model. Moreover, using albumin glycated by methylglyoxal, we evaluated the anti-glycation effect of fruit extract. The results demonstrated that fully ripened fruits exert higher antioxidant and anti-glycation properties when compared with partially ripened fruits. Information about the health-promoting components of “visciola” could lead to a better understanding and an increased consumption of these, including its use as functional food.

Glutathione Production by <i>Yarrowia lipolytica</i>Showing Both Methyglyoxal Resistance and a High Activity of Glutathione Synthetase

Although Yarrowia lipolytica is an important host strain, there have so far been few studies on the production of glutathione by the strain. We therefore performed a study to obtain an improved strain of Y. lipolytica ATCC20688, which could produce a high yield of glutathione. First, the capability of glutathione production in the ATCC20688 strain was estimated. In comparison with other yeasts, the yield of this strain was higher than those in Pichia strains. Furthermore, this strain could produce glutathione by assimilating sodium oleate. We next performed mutation and gene cloning to improve the yield. After the yield of glutathione was improved in the isolated methylglyoxal-resistant mutant (MGR3), the glutathione synthetase gene was cloned into the MGR3 strain. By using this recombinant strain, we could reach the maximum yield and intracellular content of glutathione of 54 mg/L-medium and 30 mg/g-dry cell weight, respectively.

Analysis of Bactericidal Components in Japanese Honeys

It is known that the factors identified as contributing to the bactericidal activity of honeys are the high sugar concentration, hydrogen peroxide (H2O2) and low pH, and its bactericidal components depend on honey plant and natural environment. Manuka honey has been studied a lot about bactericidal effect. However, since Japanese honeys are mainly used as food, detailed analyzes of bactericidal components and its actions have not been reported. Therefore, we analyzed bactericidal components contained in nine Japanese honeys using Lucigenin-CL-HPLC. As our results, four species components of H2O2, glucose, fructose and methylglyoxal were detected from nine Japanese honeys. The H2O2 concentrations were 4.1 × 10-5 - 1.8 × 10-4 mol/L, the glucose concentrations were 1.4 - 2.8 mol/L, the fructose concentrations were 1.9 - 2.4 mol/L, the methylglyoxal concentrations were 4.0 × 10-3 - 1.6 × 10-2 mol/L. In this study, we confirmed that methylglyoxal is also contained in Japanese honeys, although it is a bactericidal component which is reported to be characteristic of manuka honey. It is considered that the further study of Japanese honeys is useful for more safe use, quality control, and clinical application.

Reactive carbonyl compounds(RCCs) cause aggregation and dysfunction of fibrinogen

Fibrinogen is a key protein involved in coagulation and its deposition on blood vessel walls plays an important role in the pathology of atherosclerosis.Although the causes of fibrinogen(fibrin)deposition have been studied in depth,little is known about the relationship between fibrinogen deposition and reactive carbonyl compounds(RCCs),compounds which are produced and released into the blood and react with plasma protein especially under conditions of oxidative stress and inflammation.Here,we investigated the effect of glycolaldehyde on the activity and deposition of fibrinogen compared with the common RCCs acrolein,methylglyoxal,glyoxal and malondialdehyde.At the same concentration(1 mmol/L),glycolaldehyde and acrolein had a stronger suppressive effect on fibrinogen activation than the other three RCCs.Fibrinogen aggregated when it was respectively incubated with glycolaldehyde and the other RCCs,as demonstrated by SDS-PAGE,electron microscopy and intrinsic fluorescence intensity measurements.Staining with Congo Red showed that glycolaldehyde-and acrolein-fibrinogen distinctly formed amyloid-like aggregations.Furthermore,the five RCCs,particularly glycolaldehyde and acrolein,delayed human plasma coagulation.Only glycolaldehyde showed a markedly suppressive effect on fibrinogenesis,none did the other four RCCs when their physiological blood concentrations were employyed,respectively.Taken together,it is glycolaldehyde that suppresses fibrinogenesis and induces protein aggregation most effectively,suggesting a putative pathological process for fibrinogen(fibrin)deposition in the blood.

Antiglycative and anti-inflammatory effects of lipophilized tyrosol derivatives

To expand the application of tyrosol,a series of lipophilized tyrosol derivatives were synthesized via esterification of tyrosol with fatty acids of different chain lengths.The antiglycative activity of tyrosol esters so prepared was subsequently examined in the bovine serumalbumin/glucose system.A quasi-parabolic shape was observed when the activity was plotted against alkyl chain length.Additionally,the anti-inflammatory effects of these derivatives were evaluated against methylglyoxalinduced inflammation in RAW264.7 cells.The same trend on anti-inflammatory activity was found as in the antiglycation study.The results showed that tyrosol esters with C12:0 and C14:0 were two most efficient ones among all the tested derivatives.Thus,some lipophilized tyrosol derivatives were stronger antiglycative and anti-inflammatory agents compared to the parent compound,tyrosol.