Removal Of Peroxide Research Articles

Low complex I content explains the low hydrogen peroxide production rate of heart mitochondria from the long‐lived pigeon,Columba livia

Across a range of vertebrate species, it is known that there is a negative association between maximum lifespan and mitochondrial hydrogen peroxide production. In this report, we investigate the underlying biochemical basis of the low hydrogen peroxide production rate of heart mitochondria from a long-lived species (pigeon) compared with a short-lived species with similar body mass (rat). The difference in hydrogen peroxide efflux rate was not explained by differences in either superoxide dismutase activity or hydrogen peroxide removal capacity. During succinate oxidation, the difference in hydrogen peroxide production rate between the species was localized to the DeltapH-sensitive superoxide producing site within complex I. Mitochondrial DeltapH was significantly lower in pigeon mitochondria compared with rat, but this difference in DeltapH was not great enough to explain the lower hydrogen peroxide production rate. As judged by mitochondrial flavin mononucleotide content and blue native polyacrylamide gel electrophoresis, pigeon mitochondria contained less complex I than rat mitochondria. Recalculation revealed that the rates of hydrogen peroxide production per molecule of complex I were the same in rat and pigeon. We conclude that mitochondria from the long-lived pigeon display low rates of hydrogen peroxide production because they have low levels of complex I.

Glucocorticoid Resistance in Lymphoma Cells Mediated by Hydrogen Peroxide Removal and Disregulation of MAPK Signaling

Glucocorticoid Resistance in Lymphoma Cells Mediated by Hydrogen Peroxide Removal and Disregulation of MAPK Signaling

Age-related changes in antioxidant enzymes related to hydrogen peroxide metabolism in rat inner ear

Oxidative stress is a pervasive factor in aging and has been implicated in noise-induced cochlear pathology. In this study, we measured the activities of two enzymes that catalyze the removal of hydrogen peroxide (H 2O 2), catalase and glutathione peroxidase (Gpx), in 3- and 24-month-old Fisher-344 rats, and reduced and oxidized glutathione in 3-, 12-, and 24-month-old rats. There was an increase in Gpx activity in vascular tissue (spiral ligament and stria vascularis), but no change in modiolar, sensory or vestibular tissue of the cochlea. The elevation in vascular tissue was age-related. We observed a significant elevation of catalase activity in vestibular tissue, a tendency for age-related elevation in the modiolus, but no change in vascular or sensory cochlear tissue. These findings suggest that increased Gpx activity in vascular cochlear tissue may be an age-related compensation for a decrease in glutathione and a decline in the redox state measured by the ratio of reduced to oxidized glutathione.

Cold Chemical Oxidation of Proteins

Various methods of protein footprinting use hydrogen peroxide as an oxidant. Its removal by various solid-phase desalting methods, catalase treatment, or freeze drying after the footprinting is critical to ensure no uncontrolled oxidation. Although catalase treatment removes hydrogen peroxide with little loss of protein or additional protein oxidation, we discovered that freeze drying or freezing of the protein in a peroxide solution does lead to protein oxidation. Interestingly, the oxidation is not a result of freeze or thaw processes but is dependent on the temperature and length of time for incubation. After 2 h, apomyoglobin undergoes almost-complete single oxidation at -80 degrees C and double oxidation at -15 degrees C. Minimal oxidation is observed at 4 and 22 degrees C, compared to oxidation at -80 or -15 degrees C. The concentration of hydrogen peroxide is critical; 75 mM (0.2%) is required to oxidize >50% of the protein at -15 degrees C and 100 mM (0.3%) is required at -80 degrees C. In addition to Met, approximately 5% of the tryptophan and tyrosine residues are oxidized, as well as lower amounts of His and Phe. Oxidation of Val 68 and Val 17 (a buried residue) also occurs, with the oxidation of Val 17 likely occurring by electron transfer from one of two of the oxidized aromatic residues that are in contact with Val 17. Here, we describe the need to remove the hydrogen peroxide prior to cold storage of proteins, and we also report some preliminary results pertaining to the mechanism of cold, solid-state oxidation.

Enhancing the Antitumor Activity of Adriamycin and Ionizing Radiation

Overexpression of manganese superoxide dismutase (MnSOD), when combined with certain chemicals that inhibit peroxide removal, increases cancer cell cytotoxicity. Elevating MnSOD levels in cells enhances the conversion of superoxide (O(2)(*-)) to hydrogen peroxide (H(2)O(2)), combined with inhibiting the removal of H(2)O(2), further increases H(2)O(2) levels, leading to increased cytotoxicity. We hypothesized that increasing endogenous O(2)(*-) production in cells that were pretreated with adenoviral MnSOD (AdMnSOD) plus 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) would lead to an increased level of intracellular H(2)O(2) accumulation and increased cell killing. The cytotoxic effects of Adriamycin or radiation, agents known to produce O(2)(*-), were determined in MDA-MB-231 breast cancer cells pretreated with AdMnSOD plus BCNU both in vitro and in vivo. In vitro, AdMnSOD plus BCNU sensitized cells to the cytotoxicity of Adriamycin or radiation. In vivo, AdMnSOD, BCNU, and Adriamycin or ionizing radiation inhibited tumor growth and prolonged survival. The results suggest that agents that produce O(2)(*-) in combination with AdMnSOD plus BCNU may represent a powerful new antitumor regimen against breast cancer.

Aliphatic Peptidyl Hydroperoxides as a Source of Secondary Oxidation in Hydroxyl Radical Protein Footprinting

Hydroxyl radical footprinting is a technique for studying protein structure and binding that entails oxidizing a protein system of interest with diffusing hydroxyl radicals, and then measuring the amount of oxidation of each amino acid. One important issue in hydroxyl radical footprinting is limiting amino acid oxidation by secondary oxidants to prevent uncontrolled oxidation, which can cause amino acids to appear more solvent accessible than they really are. Previous work suggested that hydrogen peroxide was the major secondary oxidant of concern in hydroxyl radical footprinting experiments; however, even after elimination of all hydrogen peroxide, some secondary oxidation was still detected. Evidence is presented for the formation of peptidyl hydroperoxides as the most abundant product upon oxidation of aliphatic amino acids. Both reverse phase liquid chromatography and catalase treatment were shown to be ineffective at eliminating peptidyl hydroperoxides. The ability of these peptidyl hydroperoxides to directly oxidize methionine is demonstrated, suggesting the value of methionine amide as an in situ protectant. Hydroxyl radical footprinting protocols require the use of an organic sulfide or similar peroxide scavenger in addition to removal of hydrogen peroxide to successfully eradicate all secondary oxidizing species and prevent uncontrolled oxidation of sulfur-containing residues.

Effects of postharvest ethanol vapor treatment on the ascorbate–glutathione cycle in broccoli florets

Broccoli ( Brassica oleracea L. Italica Group) senesces very rapidly at room temperature after harvest. Ethanol vapor treatment suppresses the senescence. Reactive oxygen species (ROS) are closely related with postharvest quality of horticultural crops, and the ascorbate–glutathione cycle plays a crucial role in controlling the level of ROS. The effects of postharvest ethanol vapor treatment on the cycle in broccoli florets were investigated to clarify the inhibition of senescence with ethanol. Broccoli branchlets were packed in a perforated polyethylene bag with or without a 3 g ethanol pad and stored at 20 °C in darkness. The ethanol concentrations in the atmosphere of the bag containing the ethanol pad were about 40 pmol mL −1. In control and ethanol-treated broccoli, the contents of l-ascorbic acid and reduced glutathione decreased during storage, but the decrease in control broccoli was more marked than that in ethanol-treated broccoli. The ascorbate peroxidase activities in ethanol-treated broccoli were constant during storage, whereas the activities in the control broccoli decreased almost linearly. The glutathione reductase activities in ethanol-treated broccoli were higher than those in the control at 5 d in storage (DIS). The dehydroascorbate reductase activities in ethanol-treated broccoli decreased gradually. On the other hand, the activities in the control broccoli decreased gradually until 3 DIS and then considerably. These results suggest that ethanol vapor treatment might suppress the decrease in reducing substances and enzyme activities associated with the ascorbate–glutathione cycle to reduce oxidative stress through effective removal of hydrogen peroxide, and the suppression might partly contribute to the suppression of senescence in ethanol vapor-treated broccoli.

Lactic fermentation and antioxidant activity of Zingiberaceae plants in Taiwan

The present study evaluated functional properties of lactic-fermented ginger products. Three Zingiberaceae species were used as the substrate for fermentation using three lactic acid bacteria. The fermentation process ended 35–40 h after inoculation and reached a pH value of 3.5–4.0. Total antioxidant performances were 68–75%, and were best observed using Bifidobacteriumlongum as the starter in three ginger samples. DPPH scavenging was on average 70%, with free radical anion scavenging and peroxide removal effects of 30.6% and 43.7%, respectively. The product acceptance survey showed that the 100% fermented juice without a mixture with non-fermented ginger juice obtained the highest score in overall performance. The lactic-fermented Vanoverberghia and Hedychium ginger species retained an antioxidant activity and DPPH scavenging activity of on average 70%. This study may suggest a new way of ginger food processing with high functionality. Also, it may help to popularize the growing and processing of endemic ginger plants in Taiwan.

Redox Regulation of Protein Tyrosine Phosphatase 1B by Manipulation of Dietary Selenium Affects the Triglyceride Concentration in Rat Liver

Protein tyrosine phosphatase 1B (PTP1B) is a key enzyme in the counter-regulation of insulin signaling and in the stimulation of fatty acid synthesis. Selenium (Se), via the activities of glutathione peroxidase (GPx) and thioredoxin reductase (TrxR), is involved in the removal of H2O2 and organic peroxides, which are critical compounds in the modulation of PTP1B activity via glutathionylation. Our study with growing rats investigated how the manipulation of dietary Se concentration influences the regulation of PTP1B and lipogenic effects mediated by PTP1B. Weanling albino rats were divided into 3 groups of 10. The negative control group (NC) was fed a Se-deficient diet for 8 wk. Rats in groups Se75 and Se150 received diets supplemented with 75 or 150 μg Se/kg. Se supplementation of the rats strongly influenced expression and activity of the selenoenzymes cytosolic GPx, plasma GPx, phospholipidhydroperoxide GPx, and cytosolic TrxR, and liver PTP1B. Liver PTP1B activity was significantly higher in groups Se75 and Se150 than in the NC group and this was attributed to a lowered inhibition of the enzyme by glutathionylation. The increased liver PTP1B activity in groups Se75 and Se150 resulted in 1.1- and 1.4-fold higher liver triglyceride concentrations than in the NC rats. The upregulation of the sterol regulatory element binding protein-1c and of fatty acid synthase, 2 PTP1B targets, provided a possible explanation for the lipogenic effect of PTP1B due to the manipulation of dietary Se. We therefore conclude that redox-regulated proteins, such as PTP1B, represent important interfaces between dietary antioxidants such as Se and the regulation of metabolic processes.

Overexpression of Glucose-6-phosphate Dehydrogenase Extends the Life Span of Drosophila melanogaster

The redox state of tissues tends to become progressively more prooxidizing during the aging process. The hypothesis tested in this study was that enhancement of reductive capacity by overexpression of glucose-6-phosphate dehydrogenase (G6PD), a key enzyme for NADPH biosynthesis, could protect against oxidative stress and extend the life span of transgenic Drosophila melanogaster. Overexpression of G6PD was achieved by combining a UAS-G6PD responder transgene at one of four independent loci with either a broad expression (armadillo-GAL4, Tubulin-GAL4, C23-GAL4, and da-GAL4) or a neuronal driver (D42-GAL4 and Appl-GAL4). The mean life spans of G6PD overexpressor flies were extended, in comparison with driver and responder controls, as follows: armadillo-GAL4 (up to 38%), Tubulin-GAL4 (up to 29%), C23-GAL4 (up to 27%), da-GAL4 (up to 24%), D42-GAL4 (up to 18%), and Appl-GAL4 (up to 16%). The G6PD enzymatic activity was increased, as were the levels of NADPH, NADH, and the GSH/GSSG ratio. Resistance to experimental oxidative stress was enhanced. Furthermore, metabolic rates and fertility were essentially the same in G6PD overexpressors and control flies. Collectively, the results demonstrate that enhancement of the NADPH biosynthetic capability can extend the life span of a relatively long-lived strain of flies, which supports the oxidative stress hypothesis of aging.

Intracellular antioxidant enzymes are not globally upregulated during hibernation in the major oxidative tissues of the 13-lined ground squirrel Spermophilus tridecemlineatus

Hibernating mammals exhibit oxidative stress resistance in brain, liver and other tissues. In many animals, cellular oxidative stress resistance is associated with enhanced expression of intracellular antioxidant enzymes. Intracellular antioxidant capacity may be upregulated during hibernation to protect against oxidative damage associated with the ischemia-reperfusion that occurs during transitions between torpor and arousal. We tested the hypothesis that the 13-lined ground squirrel ( Spermophilus tridecemlineatus), upregulates intracellular antioxidant enzymes in major oxidative tissues during hibernation. The two major intracellular isoforms of superoxide dismutase (MnSOD and CuZnSOD), which catalyze the first step in superoxide detoxification, were quantified in heart, brain and liver tissue using immunodetection and an in-gel activity assay. However, no differences in SOD protein expression or activity were found between active and hibernating squirrels. Measurements of glutathione peroxidase and glutathione reductase, which catalyze hydrogen peroxide removal, were not broadly upregulated during hibernation. The activity of catalase, which catalyzes an alternative hydrogen peroxide detoxification pathway, was higher in heart and brain of torpid squirrels, but lower in liver. Taken together, these data do not support the hypothesis that hibernation is associated with enhanced oxidative stress resistance due to an upregulation of intracellular antioxidant enzymes in the major oxidative tissues.

Hydrogen Peroxide Removal with Magnetically Responsive Saccharomyces cerevisiae Cells

Hydrogen peroxide (HP) is a promising chemical sanitizer for use in the food industry. Its residues have to be decomposed, usually using an enzyme process employing catalase. In order to offer an inexpensive biocatalyst and to simplify subsequent manipulation, we have prepared magnetically responsive alginate beads containing entrapped Saccharomyces cerevisiae cells and magnetite microparticles. Larger beads (2-3 mm in diameter) were prepared by dropping the mixture into calcium chloride solution, while microbeads (the diameter of majority of particles ranged between 50 and 100 microm) were prepared using the water in oil emulsification process. In general, microbeads enabled more efficient HP decomposition. The prepared microparticulate biocatalyst caused efficient decomposition of HP in water solutions (up to 2% concentration), leaving very low residual HP concentration after treatment (below 0.001% under appropriate conditions). The biocatalyst was stable; the same catalytic activity was observed after one month storage at 4 degrees C, and the microbeads could be used at least five times.

Hydrogen peroxide‐mediated nuclear factor κB activation in both liver and tumor cells during initial stages of hepatic metastasis

Various factors involved in tumor metastasis are regulated by the transcription factor nuclear factor kappaB (NF-kappaB). Because NF-kappaB activation may contribute to establishment of hepatic metastasis, its activation in liver cells and tumor cells was separately evaluated in a mouse model of hepatic metastasis. pNF-kappaB-Luc, a firefly luciferase-expressing plasmid DNA depending on the NF-kappaB activity, was injected into the tail vein of mice by the hydrodynamics-based procedure, a well-established method for gene transfer to BALB/c male mouse liver. The luciferase activity in the liver was significantly increased by an intraportal inoculation of murine adenocarcinoma colon26 cells, but not of peritoneal macrophages, suggesting that the NF-kappaB in liver cells is activated when tumor cells enter the hepatic circulation. Then, colon26 cells stably transfected with pNF-kappaB-Luc were inoculated. The firefly luciferase activity, an indicator of NF-kappaB activity in tumor cells, was significantly increased when colon26/NFkappaB-Luc cells were inoculated into the portal vein of BALB/c male mice. The NF-kappaB activation in both liver and tumor cells was significantly inhibited by injection of catalase derivatives, which have been reported to inhibit hepatic metastasis of tumor cells. These findings indicate for the first time that NF-kappaB, a key agent regulating the expression of various molecules involved in tumor metastasis, is activated in both liver and tumor cells during the initial stages of tumor metastasis through a hydrogen peroxide mediated pathway. Thus, the removal of hydrogen peroxide will be a promising approach to treating hepatic metastasis.

Interactions of water stress and solar irradiance on the physiology and biochemistry of Ligustrum vulgare

We studied the interactive effects of water stress and solar irradiance on physiological and biochemical traits in Ligustrum vulgare L., with special emphasis on antioxidant enzymes and flavonoids. Water relations, photosynthetic performance, plant growth, activities of antioxidant enzymes and of phenylalanine-ammonia-lyase, and concentrations of nonstructural carbohydrates and phenylpropanoids were measured in plants growing in 12% (shade) or 100% (sun) sunlight and supplied with 100 or 40% of daily evapotranspiration-demand over a 4-week period. The mild water stress treatment caused leaf water potential and relative water content to decline on average by -0.22 MPa and 4.5%, respectively. In response to the water stress treatment, photosynthetic rates decreased more in sun plants than in shade plants, likely because of declines in photosystem II photochemistry, apparent maximum rate of carboxylation and apparent maximum electron transport rate coupled with significant reductions in stomatal conductance. Antioxidant enzymatic activities, which were much greater in sun leaves than in shade leaves under well-watered conditions, increased (particularly the enzymatic activities associated with hydrogen peroxide removal) in response to water stress only in shade leaves. Antioxidant phenylpropanoids, particularly quercetin and luteolin derivatives, markedly increased in response to full sunlight irrespective of water treatment; however, antioxidant phenylpropanoid concentrations increased in response to water stress only in shade leaves. We suggest that: (1) assimilated carbon in sun plants was used largely to support an effective antioxidant system capable of countering water-stress-induced oxidative damage--an example of cross tolerance; and (2) in shade plants, carbon was also diverted from growth to counter oxidative damage driven by the mild water-stress treatment. Both findings are consistent with the nearly exclusive distribution of L. vulgare in well-watered, partially shaded Mediterranean areas.

Studies on the activation of hydrogen peroxide for color removal in the presence of a new Cu(II)-polyampholyte heterogeneous catalyst

In this work we describe the application of a new non-soluble and non-porous complex with copper ion based on ethylene glycol diglycidyl ether (EGDE), methacrylic acid (MAA) and 2-methylimidazole (2MI) in the decolorization of an azo dye Methyl Orange (MO) as a model pollutant at room temperature.The complex with copper ion was studied by ESR and SEM and was tested as a heterogeneous catalyst for H2O2 activation. A possible mechanism of interaction involves the production of hydroxyl radicals (confirmed by ESR), dioxygen and water.The Cu(II)-polyampholyte/H2O2 system acted efficiently in the color removal of MO. The adsorption and oxidative degradation of the azo-based dye followed pseudo-first-order kinetic profiles, and the rate constant for degradation had a second-order dependence on copper ion content in the mixture.A removal of MO higher than 90% was achieved in 20min at pH 7.0, combining 0.8mM of complexed copper ions in the mixture with 24mM hydrogen peroxide.The dye adsorbed on the polyampholyte following a L4-type isotherm with 4.9μmolg−1 maximum loading capacity and 3.1μM dissociation constant for the first monolayer.

Increased Radioresistance of 32Dcl3 Murine Hematopoietic Progenitor Cells by Mitochondrial Targeting of a Catalase Transgene Product.

Mitochondrial localization of the radioprotective MnSOD transgene in hematopoietic cells dismutates irradiation induced superoxide to hydrogen peroxide which is converted to water and oxygen by catalase or glutathione peroxide. Increased concentration of hydrogen peroxide can be toxic. We hypothesized, that increased mitochondrial localized catalase to remove hydrogen peroxide would further increase radioresistance. The human catalase transgene was cloned into a pSVZeo plasmid. To localize the transgene product catalase to the mitochondria, the mitochondrial localization sequence of MnSOD was cloned and attached to the catalase transgene (mt-catalase), then cloned into a pSVZeo plasmid. The plasmids were electroporated into murine hematopoietic cell line 32Dcl3 and 32Dcl3 MnSOD transgene overexpressing clonal cell line 2C6 and subclones of each expressing the non-targeted catalase or mt-catalase selected by growing the cells in zeomycin. The clonal cell lines were shown to express either catalase or mt-catalase by RT-PCR using specific primers. Catalase biochemical activity was determined and 32D-cat and 32D-mt-cat cells had increased catalase activity (595.7 ± 15.3 or 603.3 ± 3.0 μM, respectively) compared to 539.7 ± 3.7 μM (p = 0.0288 or 0.0002, respectively) for 32Dcl3. Compared to the 32Dcl3 cells, 2C6 cells had decreased catalase activity of 205.0 ± 10.0 compared to 539.7 ± 3.7 (p < 0.0001). Catalase activity was increased in 2C6-cat and 2C6-mt-cat (333.3 ± 12.7 and 467.0 ± 1.0, respectively) compared to 205.0 ± 1.0 for 2C6 (p <0.001). Western analysis confirmed the differences in catalase activity. Cells from 32Dcl3, 32D-cat, 32D-mt-catalase, and subclones of 2C6 cells were irradiated to doses ranging from 0 to 8 Gy, plated in methylcellulose, incubated at 37° C for seven days, and colonies of greater that 50 cells counted. The data was analyzed by linear quadratic and single-hit, multi-target models. The 32D-mt-cat cells were more radioresistant than 32D-cat cells by an increased shoulder on the survival curve (n = 10.3 ± 0.5 or 5.9 ± 0.2, respectively, p = 0.0025). Both 32D-mt-cat and 32D-cat were more resistant compared to 32Dcl3 cells (n = 2.9 ± 1.1, p = 0.0196 or 0.0479). Cells from the 2C6 transfected with mt-catalase, but not catalase, showed increased radioresistance increasing the Do from 0.979 ± 0.1Gy for 2C6 to 1.171 ± 0.1 Gy for 2C6-mCat cells. To determine if increased catalase activity altered antioxidant status, levels of glutathione (GSH) and glutathione peroxidase (GPX) were measured. There was no significant change in GSH between cell lines. However, there were increased levels of GPX in 32D-cat and 32D-mt-cat of 260.4 ± 24.6 or 257.1 ± 17.1 μM, respectively, compared to 105.5 ± 1.6 μM (p = 0.0005 or 0.0134, respectively). Cells from 2C6, 2C6-cat or 2C6-mt-cat had decreased GPX activity to 46.7 ± 1.3, 39.1 ± 0.9 or 44.1 ± 1.5μM, respectively, compared to 105.5 ± 1.6μM for 32Dcl3 (p<0.0001). Thus, overexpression of both MnSOD and mt-catalase transgene provides superior radioprotection compared to one alone.

Radioresistance Induced by MnSOD Overexpression in 32DCl 3Murine Hematopoietic Progenitor Cells is Further Increased by Localization of a Catalase Transgene Product to the Mitochondria

Mitochondrial localization of the radioprotective MnSOD transgene in hematopoietic cells dismutates irradiation induced superoxide to hydrogen peroxide which is converted to water and oxygen by catalase or glutathione peroxide. Increased concentration of hydrogen peroxide can be toxic. We hypothesized, that increased mitochondrial localized catalase to remove hydrogen peroxide would further increase radioresistance.

Double Mutants Deficient in Cytosolic and Thylakoid Ascorbate Peroxidase Reveal a Complex Mode of Interaction between Reactive Oxygen Species, Plant Development, and Response to Abiotic Stresses

Reactive oxygen species (ROS) play a key signaling role in plants and are controlled in cells by a complex network of ROS metabolizing enzymes found in several different cellular compartments. To study how different ROS signals, generated in different cellular compartments, are integrated in cells, we generated a double mutant lacking thylakoid ascorbate peroxidase (tylapx) and cytosolic ascorbate peroxidase1 (apx1). Our analysis suggests that two different signals are generated in plants lacking cytosolic APX1 or tylAPX. The lack of a chloroplastic hydrogen peroxide removal enzyme triggers a specific signal in cells that results in enhanced tolerance to heat stress, whereas the lack of a cytosolic hydrogen peroxide removal enzyme triggers a different signal, which results in stunted growth and enhanced sensitivity to oxidative stress. When the two signals are coactivated in cells (i.e. tylapx/apx1), a new response is detected, suggesting that the integration of the two different signals results in a new signal that manifests in late flowering, low protein oxidation during light stress, and enhanced accumulation of anthocyanins. Our results demonstrate a high degree of plasticity in ROS signaling in Arabidopsis (Arabidopsis thaliana) and suggest the existence of redundant pathways for ROS protection that compensate for the lack of classical ROS removal enzymes such as cytosolic and chloroplastic APXs. Further investigation of the enhanced heat tolerance in plants lacking tylAPX, using mutants deficient in chloroplast-to-nuclei retrograde signaling, suggests the existence of a chloroplast-generated stress signal that enhances basal thermotolerance in plants.

Regulation of Ionizing Radiation-induced Apoptosis by Mitochondrial NADP+-dependent Isocitrate Dehydrogenase

Ionizing radiation induces the production of reactive oxygen species, which play an important causative role in apoptotic cell death. By supplying NADPH for antioxidant systems, we recently demonstrated that the control of mitochondrial redox balance and the cellular defense against oxidative damage are some of the primary functions of mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDPm). In this study, we demonstrate that modulation of IDPm activity in U937 cells regulates ionizing radiation-induced apoptosis. When we examined the regulatory role of IDPm against ionizing radiation-induced apoptosis in U937 cells transfected with the cDNA for mouse IDPm in sense and antisense orientations, a clear inverse relationship was observed between the amount of IDPm expressed in target cells and their susceptibility to apoptosis. Upon exposure to 2 gray gamma-irradiation, there was a distinct difference between the IDPm transfectant cells in regard to the morphological evidence of apoptosis, DNA fragmentation, cellular redox status, oxidative damage to cells, mitochondrial function, and the modulation of apoptotic marker proteins. In addition, transfection of HeLa cells with an IDPm small interfering RNA decreased the activity of IDPm, enhancing the susceptibility of radiation-induced apoptosis. Taken together, these results indicate that IDPm may play an important role in regulating the apoptosis induced by ionizing radiation, and the effect of IDPm small interfering RNA on HeLa cells offers the possibility of developing a modifier of radiation therapy.

Dark production of reactive oxygen species in photosystem II membrane particles at elevated temperature: EPR spin-trapping study

In our study, EPR spin-trapping technique was employed to study dark production of two reactive oxygen species, hydroxyl radicals (OH ) and singlet oxygen ( 1O 2), in spinach photosystem II (PSII) membrane particles exposed to elevated temperature (47 °C). Production of OH , evaluated as EMPO-OH adduct EPR signal, was suppressed by the enzymatic removal of hydrogen peroxide and by the addition of iron chelator desferal, whereas externally added hydrogen peroxide enhanced OH production. These observations reveal that OH is presumably produced by metal-mediated reduction of hydrogen peroxide in a Fenton-type reaction. Increase in pH above physiological values significantly stimulated the formation of OH , whereas the presence of chloride and calcium ions had the opposite effect. Based on our results it is proposed that the formation of OH is linked to the thermal disassembly of water-splitting manganese complex on PSII donor side. Singlet oxygen production, followed as the formation of nitroxyl radical TEMPO, was not affected by OH scavengers. This finding indicates that the production of these two species was independent and that the production of 1O 2 is not closely linked to PSII donor side.