Analysis of hydroxylation and nitration products of d-phenylalanine for in vitro and in vivo radical determination using high-performance liquid chromatography and photodiode array detection

d-Phenylalanine is capable of trapping reactive oxygen species (ROS) and reactive nitrogen species (RNS) by forming three major hydroxylation (o-, m-, p-tyrosine) and two major nitration products (nitrophenylalanine, nitrotyrosine). Here, we show how a method for the analysis of these phenylalanine derivatives was established using isocratic HPLC (Nucleosil120, C18 column) coupled with photodiode array detection and validated for cell-free in vitro and in vivo determination of radical formation. An ideal separation was achieved using a mobile phase consisting of 5% acetonitrile, 50 mM KH₂PO₄, pH 3.0, a column temperature of 35 °C and a flow rate of 1.0 mL/min. Limits of detection were in the range of 5–100 nM. Linearity was given within 5 nM–100 μM (correlation coefficient >0.999). Retention times as well as peak heights exhibited a high precision (RSD: ≤0.1% and <1.5%, respectively). The feasibility of d-phenylalanine for ROS/RNS measurement was demonstrated in a cell-free in vitro assay using peroxynitrite and by analysis of brain samples of mice treated with the dopaminergic neurotoxin 6-hydroxydopamine.     

N-acetylcysteine prevents glucose/glucose oxidase-induced oxidative stress,mitochondrial damage and apoptosis in H9c2 cells

AimsHigh blood glucose may auto-oxidize and generate free radicals, which are proposed to induce apoptosis in cardiac cells. The aim of the present study was to investigate the cell damage induced by glucose/glucose oxidase-dependent oxidative stress and the protective effect of N-acetylcysteine (NAC) on H9c2 cardiac muscle cells.Main methodsH9c2 cells were exposed to 33 mM glucose (G) + 1.6 milliunits (mU) of glucose oxidase (GO) and termed G/GO. Cell apoptosis, generation of reactive oxygen species (ROS-super oxide anion and hydrogen peroxide) and reactive nitrogen species (RNS-peroxinitrite), and the change in mitochondrial membrane potential (ΔΨm) was studied using flow cytometry and confocal microscopy, and cytochrome c release was measured using confocal microscopy. The expression of Bcl-2, Bax and the activation of procaspase-9 was studied by western blot.Key findingsExposure of H9c2 cells to G/GO resulted in a significant increase in cellular apoptosis (P < 0.05) and the generation of ROS and RNS (P < 0.001). Further, G/GO treatment led to a decrease in ΔΨm, release of cytochrome c, decrease in Bcl-2, increase in Bax expression and the activation of procaspase-9. Treatment with NAC significantly decreased apoptosis (P < 0.05) and reduced the levels of ROS and RNS (P < 0.001). NAC was also able to normalize ΔΨm, inhibit cytochrome c release, increase Bcl-2 and decrease Bax expression and procaspase-9 activation.SignificanceOur studies suggest that NAC has antioxidative and antiapoptotic activity against G/GO-induced oxidative stress through the inhibition of mitochondrial damage in H9c2 cells.     

Antioxidant, α-amylase inhibitory and oxidative DNA damage protective property of Boerhaavia diffusa (Linn.) root

Boerhaavia diffusa Linn. of family Nyctaginaceae is a known traditional medicinal plant and has been used in the treatment of many free radical mediated diseases. Excessive formation of free radicals, either reactive oxygen species (ROS) or reactive nitrogen species (RNS) is responsible for damaging various biomolecules like DNA, lipids and proteins. The present investigation was initially carried out to explore the possible link between antioxidant, oxidative DNA damage protective and α-amylase inhibitory property of B. diffusa root extract and their bioactive fraction. Our results illustrated an enhanced DPPH radical scavenging activity/antioxidant power of methanol root extract (IC₅₀ < 250 μg/ml) than ethanol (IC₅₀ = 250 μg/ml) and aqueous extract (IC₅₀ > 250 μg/ml). In addition, the methanol root extract also showed better oxidative DNA damage protective activity and α-amylase inhibitory property than ethanol and aqueous root extract. Phytochemical screening of B. diffusa ethanol and methanol root extract showed the presence of saponins, alkaloids, flavonoids, glycosides and terpenoids in large amount. By means of repetitive preparatory TLC and HPLC the potent antioxidant and α-amylase inhibitory fraction was isolated from methanol root extract. Our results illustrated that DPPH radical scavenging activity (IC₅₀ < 250 μg/ml) and oxidative DNA damage protective and α-amylase inhibitory activity of the isolated/purified bioactive compound from methanol extract were significantly closer to that of crude extract, which in turn confirm that antioxidant and antidiabetic property of methanol root extract resides in this fraction and established a significant correlation between antioxidant and inhibitory α-amylase property of this bioactive fraction compound. These profound effects of B. diffusa methanol root extract and its purified fraction against oxidative plasmid DNA damage, antioxidant and α-amylase inhibitory activity may explain its extensive use in daily life and possible health benefits.     

Hemoglobin glutathionylation can occur through cysteine sulfenic acid intermediate: Electrospray ionization LTQ-Orbitrap hybrid mass spectrometry studies

Glutathionylated hemoglobin (Hb-SSG) is now recognized as a promising biomarker of systemic oxidative stress. Aim of this study is to gain a mechanistic insight into its formation. The ability of GSSG to form Hb-SSG through a thiol-disulfide exchange mechanism was firstly examined. For this purpose, GSSG (ranging from 0.23 to 230 μmol/g Hb, 15 μM–15 mM final concentrations) was incubated with 1 mM Hb and the relative content of Hb-SSG determined by direct infusion mass spectrometry (Orbitrap as analyzer). No detectable Hb-SSG was observed at a GSSG concentration range found in physiopathological conditions (0.13–0.23 μmol/g Hb). To reach a detectable Hb-SSG signal, the GSSG concentration was raised to 2.3 μmol/g Hb (0.5% relative abundance). The relative content of Hb-GSSG dose-dependently increased to 6% and 11% at 77 and 153 μmol/g Hb, respectively. The second step was to demonstrate whether Hb-SSG is formed through a sulfenic acid intermediate, a well-recognized mechanism of S-protein glutathionylation. Cys β93 sulfenic acid was found to be formed by oxidizing Hb with 1 mM H₂O₂, as demonstrated by direct infusion and LC–ESI-MS/MS experiments and using dimedone as derivatazing agent. When H₂O₂-treated Hb was incubated with physiological concentrations of GSH (9 μmol/g Hb), the corresponding Hb-SSG form was detected, reaching 15% of relative abundance. In summary, we here demonstrate that Hb glutathionylation can occur through a Cys sulfenic acid intermediate which is formed in oxidizing conditions. Hb glutathionylation is also mediated by a thiol-disulfide transfer mechanism, but this requires a concentration of GSSG which is far to be achieved in physiopathological conditions.     

Antioxidative effects of Panax notoginseng saponins in brain cells

Oxidative stress resulting from accumulation of reactive oxygen species (ROS) is involved in cell death associated with neurological disorders such as stroke, Alzheimer's disease and traumatic brain injury. Antioxidant compounds that improve endogenous antioxidant defenses have been proposed for neural protection. The purpose of this study was to investigate the potential protective effects of total saponin in leaves of Panax notoginseng (LPNS) on oxidative stress and cell death in brain cells in vitro. Lactate dehydrogenase (LDH) assay indicated that LPNS (5 μg/ml) reduced H₂O₂-induced cell death in primary rat cortical astrocytes (23 ± 8% reduction in LDH release vs. control). Similar protection was found in oxygen and glucose deprivation/reoxygenation induced SH-SY5Y (a human neuroblastoma cell line) cell damage (78 ± 7% reduction vs. control). The protective effects of LPNS in astrocytes were associated with attenuation of reactive oxygen species (ROS) accumulation. These effects involved activation of Nrf2 (nuclear translocation) and upregulation of downstream antioxidant systems including heme oxygenase-1 (HO-1) and glutathione S-transferase pi 1 (GSTP1). These results demonstrate for the first time that LPNS has antioxidative effects which may be neuroprotective in neurological disorders.     

Redox-Optimized ROS Balance and the relationship between mitochondrial respiration and ROS

The Redox-Optimized ROS Balance [R-ORB] hypothesis postulates that the redox environment [RE] is the main intermediary between mitochondrial respiration and reactive oxygen species [ROS]. According to R-ORB, ROS emission levels will attain a minimum vs. RE when respiratory rate (VO₂) reaches a maximum following ADP stimulation, a tenet that we test herein in isolated heart mitochondria under forward electron transport [FET]. ROS emission increased two-fold as a function of changes in the RE (~ 400 to ~ 900 mV·mM) in state 4 respiration elicited by increasing glutamate/malate (G/M). In G/M energized mitochondria, ROS emission decreases two-fold for RE ~ 500 to ~ 300 mV·mM in state 3 respiration at increasing ADP. Stressed mitochondria released higher ROS, that was only weakly dependent on RE under state 3. As a function of VO₂, the ROS dependence on RE was strong between ~ 550 and ~ 350 mV·mM, when VO₂ is maximal, primarily due to changes in glutathione redox potential. A similar dependence was observed with stressed mitochondria, but over a significantly more oxidized RE and ~ 3-fold higher ROS emission overall, as compared with non-stressed controls. We conclude that under non-stressful conditions mitochondrial ROS efflux decreases when the RE becomes less reduced within a range in which VO₂ is maximal. These results agree with the R-ORB postulate that mitochondria minimize ROS emission as they maximize VO₂ and ATP synthesis. This relationship is altered quantitatively, but not qualitatively, by oxidative stress although stressed mitochondria exhibit diminished energetic performance and increased ROS release.     

Stable-isotope dilution GC–MS approach for nitrite quantification in human whole blood,erythrocytes, and plasma using pentafluorobenzyl bromide derivatization: Nitrite distribution in human blood

Previously, we reported on the usefulness of pentafluorobenzyl bromide (PFB-Br) for the simultaneous derivatization and quantitative determination of nitrite and nitrate in various biological fluids by GC–MS using their ¹⁵N-labelled analogues as internal standards. As nitrite may be distributed unevenly in plasma and blood cells, its quantification in whole blood rather than in plasma or serum may be the most appropriate approach to determine nitrite concentration in the circulation. So far, GC–MS methods based on PFB-Br derivatization failed to measure nitrite in whole blood and erythrocytes because of rapid nitrite loss by oxidation and other unknown reactions during derivatization. The present article reports optimized and validated procedures for sample preparation and nitrite derivatization which allow for reliable quantification of nitrite in human whole blood and erythrocytes. Essential measures for stabilizing nitrite in these samples include sample cooling (0–4 °C), hemoglobin (Hb) removal by precipitation with acetone and short derivatization of the Hb-free supernatant (5 min, 50 °C). Potassium ferricyanide (K₃Fe(CN)₆) is useful in preventing Hb-caused nitrite loss, however, this chemical is not absolutely required in the present method. Our results show that accurate GC–MS quantification of nitrite as PFB derivative is feasible virtually in every biological matrix with similar accuracy and precision. In EDTA-anticoagulated venous blood of 10 healthy young volunteers, endogenous nitrite concentration was measured to be 486 ± 280 nM in whole blood, 672 ± 496 nM in plasma (C_P), and 620 ± 350 nM in erythrocytes (C_E). The C_E-to-C_P ratio was 0.993 ± 0.188 indicating almost even distribution of endogenous nitrite between plasma and erythrocytes. By contrast, the major fraction of nitrite added to whole blood remained in plasma. The present GC–MS method is useful to investigate distribution and metabolism of endogenous and exogenous nitrite in blood compartments under basal conditions and during hyperemia.     

Postoperative endometrial carcinoma treated with external beam irradiation plus vaginal-cuff brachytherapy. Is there a dose relationship with G2 vaginal complications?

AimTo analyse the possible relationship between the EQD2_(α/β=3Gy) at 2 cm³ of the vagina and late toxicity in vaginal-cuff-brachytherapy (VBT) after external-beam-irradiation (EBRT) for postoperative endometrial carcinoma (EC).Materials and methodsFrom 2014 to 2016, 62 postoperative EC patients were treated with EBRT + VBT. The median EBRT dose was 45 Gy (44 Gy–50.4 Gy). VBT involved a single 7 Gy dose. Toxicity was prospectively evaluated using the RTOG score for the rectum and bladder and the objective LENT-SOMA criteria for the vagina. EQD2_{(α/β = 3Gy)} at 2 cm³ of the most exposed part of the vagina was calculated by the sum of the EBRT + VBT dose. Statistics: Boxplot, Student’s t and Chi-square tests and ROC curves.ResultsMean follow-up: 39.2 months (15–68). Late toxicity: bladder:0 patient; rectum:2 patients-G1; Vagina: 26 patients-17G1, 9G2; median EQD2_(α/β=3Gy) at 2 cm³ in G0-G1 patients was 70.4 Gy(SD2.36), being 72.5 Gy(SD2.94) for G2p. The boxplot suggested a cut-point identifying the absence of G2: 100 % of G2p received >68 Gy, ROC curves showed an area under the curve of 0.72 (sensitivity of 1 and specificity of 0.15).ConclusionsDoses >68 Gy EQD2_(α/β=3Gy) at 2 cm³ to the most exposed area of the vagina were associated with late G2 vaginal toxicity in postoperative EC patients treated with EBRT + VBT suggesting a very good dose limit to eliminate the risk of G2 late toxicity. The specificity obtained indicates the need for prospective analyses.     

Increased levels of cell-free hemoglobin,oxidation markers,and the antioxidative heme scavenger α1-microglobulin in preeclampsia

Preeclampsia is a major cause of morbidity and mortality during pregnancy. To date, the pathogenesis of the disease is not fully understood. Recent studies show that preeclampsia is associated with overexpression of the hemoglobin genes α2 and γ and accumulation of the protein in the vascular lumen of the placenta. Hypothesizing that cell-free hemoglobin leaks from the placenta into the maternal circulation and contributes to the endothelial damage and symptoms by inducing oxidative stress, we analyzed fetal and adult hemoglobin (HbF, HbA), haptoglobin, oxidation markers, and the heme scavenger and antioxidant α₁-microglobulin in plasma, urine, and placenta in preeclamptic women (n = 28) and women with normal pregnancy (n = 27). The mean plasma concentrations of HbF, HbA, protein carbonyl groups, membrane peroxidation capacity, and α₁-microglobulin were significantly increased in preeclamptic women. The levels of total plasma Hb correlated strongly with the systolic blood pressure. The plasma haptoglobin concentrations of women with preeclampsia were significantly depressed. Increased amounts of α₁-microglobulin mRNA and protein were found in placenta from preeclamptic women, and the levels of plasma and placenta α₁-microglobulin correlated with the plasma Hb concentrations. The heme-degrading form t-α₁-microglobulin was significantly increased in urine in preeclampsia. These results support the idea that hemoglobin-induced oxidative stress is a pathogenic factor in preeclampsia.     

Zinc protects Ceratophyllum demersum L. (free-floating hydrophyte) against reactive oxygen species induced by cadmium

Evidence for Zn protection against Cd-induced reactive oxygen species in the free-floating hydrophyte Ceratophyllum demersum L. is presented in this paper. Metal treatments of 10 μmol/L Cd, 10 Cd μmol/L supplemented with Zn (10, 50, 100 and 200 μmol/L) and Zn-alone treatments of the same concentrations were used. Using 5,5 dimethyl pyrroline-N-oxide as the spin-probe, electron spin resonance spectra indicated a drastic increase in hydroxyl radicals (OH) in Cd-10 μmol/L treatments, which was closely correlating with the enhanced formation of hydrogen peroxide (H₂O₂) and generation of superoxide radical (O₂^?) triggered by the oxidation of NADPH. The supplementation of adding Zn (10–200 μmol/L) to the Cd-10 μmol/L treatments significantly decreased the production of free radicals especially by eliminating the precursors of OH through inhibition of NADPH oxidation. Cd-enhanced ROS production which substantially increased the oxidative products of proteins measured as carbonyls was effectively inhibited by Zn supplementation.     

Ozone-induced oxidative modification of plasma fibrin-stabilizing factor

The plasma fibrin-stabilizing factor (pFXIII) function is to maintain a hemostasis by the fibrin clot stabilization. The conversion of pFXIII to the active form of the enzyme (FXIIIа) is a multistage process. Ozone-induced oxidation of pFXIII has been investigated at different stages of its enzyme activation. The biochemical results point to a decrease of an enzymatic activity of FXIIIа depending largely on the stage of the pFXIII conversion into FXIIIа at which oxidation was carried out. UV-, FTIR- and Raman spectroscopy demonstrated that chemical transformation of cyclic, NH, SH and S–S groups mainly determines the oxidation of amino acid residues of pFXIII polypeptide chains. Conversion of pFXIII to FXIIIa proved to increase protein sensitivity to oxidation in the order: pFXIII < pFXIII activated by thrombin < pFXIII in the presence of calcium ions < FXIIIa. The dynamic light scattering data indicate that the three-dimensional structure of pFXIII becomes loosened due to oxidative modification. ESR spectroscopy data also point to conformational changes of the fibrin-stabilizing factor under oxidation. Taking into account these new findings it seems reasonable to assume that the inhibitory/carrier FXIII-B subunits can serve as scavengers of ROS. Hypothetically, this mechanism could help to protect the key amino acid residues of the FXIII-A subunits responsible for the enzymatic function of FXIIIa.     

Promising role of ferulic acid,atorvastatin and their combination in ameliorating high fat diet-induced stress in mice

AimsThe present study evaluated a comparative and combined hepatoprotective effect of atorvastatin (AS) and ferulic acid (F) against high fat diet (HFD) induced oxidative stress in terms of hyperlipidemia, anti-oxidative status, lipid peroxidation and inflammation.Main methodsMale Swiss albino mice were given a diet containing high fat (H) (23.9% wt/wt), supplemented with AS (10 mg/kg) or F (100 mg/kg) and both (10 and 100 mg/kg) for 8 weeks. The control mice (C) were fed with normal diet.Key findingsThe H mice exhibited increased body weight; hyperlipidemia; serum level of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6); hepatic lipid profile; lipid accumulation; reactive oxygen species (ROS) of hepatocytes, lipid peroxidation and liver antioxidant capacity was decreased. Immunofluorescent and Western blot assay revealed activation of nuclear factor kappa B (NF-κB) signaling pathway. The addition of F or AS and both in the diet significantly counteracted HFD induced body weight gain; hyperlipidemia; TNF-α, IL-6; hepatic lipid profile; fatty infiltration; NF-κB signaling pathway; ROS; lipid peroxidation and moreover elevated levels of hepatic antioxidant enzymes activity were observed.SignificanceSimultaneous treatment with AS, F and their combination protected against HFD induced weight gain and oxidative stress. The protection may be attributed to the hypolipidemic and free radical scavenging activity of AS or F and their combination. This study illustrates that AS and F have relatively similar hypolipidemic, antioxidative, anti-inflammatory actions and the AS + F combination along with HFD has shown outstanding effects as compared to other treated groups.     

Quercetin in vesicular delivery systems: Evaluation in combating arsenic-induced acute liver toxicity associated gene expression in rat model

Arsenic, the environmental toxicant causes oxidative damage to liver and produces hepatic fibrosis. The theme of our study was to evaluate the therapeutic efficacy of liposomal and nanocapsulated herbal polyphenolic antioxidant Quercetin (QC) in combating arsenic induced hepatic oxidative stress, fibrosis associated upregulation of its gene expression and plasma TGF ß (transforming growth factor ß) in rat model.A single dose of Arsenic (sodium arsenite-NaAsO₂, 13 mg/kg b.wt) in oral route causes the generation of reactive oxygen species (ROS), arsenic accumulation in liver, hepatotoxicity and decrease in hepatic plasma membrane microviscosity and antioxidant enzyme levels in liver. Arsenic causes fibrosis associated elevation of its gene expression in liver, plasma TGF ß (from normal value 75.2 ± 8.67 ng/ml to 196.2 ± 12.07 ng/ml) and release of cytochrome c in cytoplasm. Among the two vesicular delivery systems formulated with QC, polylactide nanocapsules showed a promising result compared to liposomal delivery system in controlling arsenic induced alteration of those parameters. A single dose of 0.5 ml of nanocapsulated QC suspension (QC 2.71 mg/kg b.wt) when injected to rats 1 h after arsenic administration orally protects liver from arsenic induced deterioration of antioxidant levels as well as oxidative stress associated gene expression of liver. Histopathological examination also confirmed the pathological improvement in liver. Nanocapsulated plant origin flavonoidal compound may be a potent formulation in combating arsenic induced upregulation of gene expression of liver fibrosis through a complete protection against oxidative attack in hepatic cells of rat liver.     

A pH control strategy for increased β-carotene production during batch fermentation by recombinant industrial wine yeast

The effects of different pH values, ranging from 4.0 to 7.0, on cell growth and β-carotene production by recombinant industrial wine yeast Saccharomyces cerevisiae T73-63 in a synthetic grape juice medium was investigated. Based on the kinetic analysis of the batch fermentation process, a two-stage pH control strategy was developed in which the pH was maintained at 7.0 for the first 24 h and then shifted to 5.0 after 24 h. Using this strategy, the highest β-carotene production (50.39 mg/l) and the formation rate (1.40 mg/l/h) were increased by 19.1% and 18.6%, respectively, compared to the maximum values of constant pH fermentation. The oxidative stress during β-carotene production was also determined in terms of the catalase (CAT) and superoxide dismutase (SOD) activities. Oxidative stress appears to be induced by the lowering of pH as indicated by the increase in activities of CAT and SOD due to pH shift from pH 7.0 to pH 5.0. Pre-treating cells with ascorbic acid (an antioxidative agent) reversed the improvement of β-carotene production while addition of H₂O₂ enhanced it. Considering that induction of oxidative stress is associated with increased β-carotene production, it was concluded that the enhancement of β-carotene production by the low-pH strategy involved the induction of oxidative stress.     

Nutritionally rich marine proteins from fresh herring by-products for human consumption

Marine oils and proteins are valuable compounds, but under unfavorable conditions these components are easily oxidized and enzymatically degraded. We have designed an industrial process for producing high quality human grade proteins from fresh herring by-products. Two different processes were tested in semi-industrial scale: (i) thermal extraction to separate oil and proteins and (ii) enzymatic hydrolysis with different commercial proteases to produce fish protein hydrolysates (FPH) and separate oil. Both stick water from thermal extraction and fish protein hydrolysates, after hydrolysis, are nutritionally rich fractions and yielded approx. 18 and 25% of dry material in these fractions respectively. Neither season nor enzymes used influenced the color of hydrolysate powders, but time and temperature of the processes are important tools for controlling the color. Fishing seasons did not influence bitterness of hydrolysates and stick water samples, but both the process conditions and applied enzymes played an important role for the formation of bitterness. Insoluble fractions after both processes had significantly higher protein efficiency ratio: 3.08 and 2.93 compared to soluble fractions: 2.62 and 2.76 after hydrolysis and thermal extraction respectively. Both stick water and FPH showed antioxidative activity against both iron (15 μM of Fe³⁺) and hemoglobin (80 ppm) induced oxidation. Herring proteins (1.25 mg/ml) were able to reduce iron induced oxidation by 50–70%, while Hb induced oxidation was reduced by 70–80% using 4 mg/ml proteins concentration.     

Design,solid phase synthesis and evaluation of cationic ferrocenoyl peptide bioconjugates as potential antioxidant enzyme mimics

Synthetic C-terminal amidated cationic ferrocenoyl peptide bioconjugates Fc-Orn-Orn-Orn (1) and Fc-Tyr-Orn-Orn-Orn (2) were rationally designed as superoxide dismutase (SOD) mimics based on the structure of the iron SOD from Escherichia coli. Ferrocenoyl peptide bioconjugates 1, 2 and ferrrocenecarboxylic acid (4) were subsequently evaluated as SOD mimics and as inhibitors of peroxynitrite-mediated tyrosine nitration. Due to their cationic character, ferrocenoyl peptide bioconjugates 1 and 2 exerted an acceptable SOD activity (EC₅₀ = 575 μM and 310 μM, respectively) in comparison with 4 (EC₅₀ = 1.4 mM). The C-terminal amidated cationic peptide Ac-Tyr-Orn-Orn-Orn (3), designed as marker of peroxynitrite, was used to evaluate the inhibitory activity of 1 and 4 towards peroxynitrite-mediated tyrosine nitration. Both compounds proved to inhibit the nitration especially the cationic ferrocenoyl peptide bioconjugates 1. The ferrocene moiety of conjugate 2 displayed a strong inhibitory activity of peroxynitrite-mediated nitration of the neighboring tyrosine.     

Improved tolerance toward low temperature in banana (Musa AAA Group Cavendish Williams)

To further understand the physiological mechanisms of cold-tolerance in banana plants, the responses of four introducing cultivars (cv.) W811 (via long-term cold adaptation), PB, BJ10 and BJ11 to low-temperature stress (LT) were investigated. LT caused increased malondialdehyde (MDA) content, elevated contents of hydrogen peroxide (H₂O₂) and superoxide radical (O₂⁻), and decreased photochemical efficiency (F_v/F_m) and net photosynthetic rate (P_n) in the leaves of four banana cultivars, but cv. W811 showed better LT tolerance than the other three cultivars. After 72 h of LT, four key antioxidative enzymes in the four cultivars showed different responses. Compared to controls, superoxide dismutase (SOD) activities in the four cultivars showed a significant decrease and W811 had the smallest amount of decrease. Catalase (CAT) activities showed a significant decrease. Peroxidase (POD) activities kept relatively higher activities and showed no significant changes (P > 0.05) in W811, BJ10 and BJ11 whereas that in PB showed a significant increase (P < 0.001). Ascorbate peroxidase (APX) activities in W811 and PB showed no significant changes (P > 0.05). Our results showed that higher cold-tolerance in cv. W811 may correlate with the long-term cold adaptation of the antioxidative enzymes such as SOD, POD and APX that alleviate oxidative stress caused by LT.     

Salivary DNA,lipid, and protein oxidation in nonsmokers with periodontal disease

Reactive oxygen species (ROS) are implicated in the destruction of the periodontium during periodontitis. The imbalance in oxidant activity may be a key factor.The aim of this paper is to determine whether periodontitis is associated with increased oxidative damage to DNA, lipids, and proteins and modification of total antioxidant capacity (TAC) in saliva. Saliva was collected from 58 periodontitis patients and 234 healthy controls, all nonsmokers. Periodontal disease status was characterized using the Community Periodontal Index of Treatment Needs (CPITN). Assays for 8-OHdG (ELISA), 8-epi-PGF2α (ELISA), and total protein carbonyls (ELISA), and oxy-blotting (Western)/mass spectrometry were performed to quantify oxidative damage to nucleic acids, lipids, total and individual proteins, respectively, in whole nonstimulated saliva. Salivary TAC was measured by inhibition of ABTS oxidation by metmyoglobin. We observed (i) significantly higher levels of 8-OHdG, 8-epi-PGF2α, and carbonylated proteins in saliva of periodontal patients as compared with controls (P = 0.0003, < 0.0001 and < 0.0001); (ii) 8-OHdG, 8-epi-PGF2α, and carbonylated proteins were independently negatively associated with CPITN (P = 0.004, 0.02, and < 0.0001); (iii) a positive correlation between salivary TAC and periodontal disease status in the study group (P < 0.0001); and (iv) specific oxidation of transferrin, human IgG1 heavy chain fragment, and salivary amylase in periodontitis. Periodontal disease is associated with increased oxidative modification of salivary DNA, lipids, and proteins. Augmented salivary total antioxidant capacity may represent an adaptive response to oxidative stress. Salivary amylase, transferrin, and human IgG1 heavy chain fragments are particularly prone to enhanced oxidation in periodontitis.