Superoxide Dismutase 1-mediated Production of Ethanol- and DNA-derived Radicals in Yeasts Challenged with Hydrogen Peroxide MOLECULAR INSIGHTS INTO THE GENOME INSTABILITY OF PEROXIREDOXIN-NULL STRAINS

Peroxiredoxins are receiving increasing attention as defenders against oxidative damage and sensors of hydrogen peroxide-mediated signaling events. In the yeast Saccharomyces cerevisiae, deletion of one or more isoforms of the peroxiredoxins is not lethal but compromises genome stability by mechanisms that remain under scrutiny. Here, we show that cytosolic peroxiredoxin-null cells (tsa1 Delta tsa2 Delta) are more resistant to hydrogen peroxide than wildtype (WT) cells and consume it faster under fermentative conditions. Also, tsa1 Delta tsa2 Delta cells produced higher yields of the 1-hydroxyethyl radical from oxidation of the glucose metabolite ethanol, as proved by spin-trapping experiments. A major role for Fenton chemistry in radical formation was excluded by comparing WT and tsa1 Delta tsa2 Delta cells with respect to their levels of total and chelatable metal ions and of radical produced in the presence of chelators. The main route for 1-hydroxyethyl radical formation was ascribed to the peroxidase activity of Cu, Zn-superoxide dismutase (Sod1), whose expression and activity increased similar to 5- and 2-fold, respectively, in tsa1 Delta tsa2 Delta compared with WT cells. Accordingly, overexpression of human Sod1 in WT yeasts led to increased 1-hydroxyethyl radical production. Relevantly, tsa1 Delta tsa2 Delta cells challenged with hydrogen peroxide contained higher levels of DNA-derived radicals and adducts as monitored by immuno-spin trapping and incorporation of (14)C from glucose into DNA, respectively. The results indicate that part of hydrogen peroxide consumption by tsa1 Delta tsa2 Delta cells is mediated by induced Sod1, which oxidizes ethanol to the 1-hydroxyethyl radical, which, in turn, leads to increased DNA damage. Overall, our studies provide a pathway to account for the hypermutability of peroxiredoxin-null strains.

Protoporphyrin IX Nanoparticle Carrier: Preparation, Optical Properties, and Singlet Oxygen Generation

The present study is focused on developing a nanoparticle carrier for the photosensitizer protoporphyrin IX for use in photodynamic therapy. The entrapment of protoporphyrin IX (Pp IX) in silica spheres was achieved by modification of Pp IX molecules with an organosilane reagent. The immobilized drug preserved its optical properties and the capacity to generate singlet oxygen, which was detected by a direct method from its characteristic phosphorescence decay curve at near-infrared and by a chemical method using 1,3-diphenylisobenzofuran to trap singlet oxygen. The lifetime of singlet oxygen when a suspension of Pp IX-loaded particles in acetonitrile was excited at 532 nm was determined as 52 mu s, which is in good agreement with the value determined for methylene blue in acetonitrile solution under the same conditions. The Pp IX-loaded silica particles have an efficiency of singlet oxygen generation (eta Delta) higher than the quantum yield of free porphyrins. This high efficiency of singlet oxygen generation was attributed to changes on the monomer-dimer equilibrium after photosentisizer immobilization.

Singlet oxygen reacts with 2 `,7 `-dichlorodihydrofluorescein and contributes to the formation of 2 `,7 `-dichlorofluorescein

There are controversial reports in the literature concerning the reactivity of singlet oxygen ((1)O(2)) with the redox probe 2`,7`-dichlorodihydrofluorescein (DCFH). By carefully preparing solutions in which (1)O(2) is quantitatively generated in the presence of DCFH, we were able to show that the formation rate of the fluorescent molecule derived from DCFH oxidation, which is 2`,7`-dichlorofluorescein (DCF), increases in D(2)O and decreases in sodium azide, proving the direct role of (1)O(2) in this process. We have also prepared solutions in which either (1)O(2) or dication (MB(center dot 2+)) and semi-reduced (MB(center dot)) radicals of the sensitizer and subsequently super-oxide radical (O(2)(center dot-)) are generated. The absence of any effect of SOD and catalase ruled out the DCFH oxidation by O(2)(center dot-), indicating that both (1)O(2) and MB(center dot 2+) react with DCFH. Although the formation of DCF was 1 order of magnitude larger in the presence of MB(center dot 2+) than in the presence of (1)O(2), considering the rate of spontaneous decays of these species in aqueous solution, we were able to conclude that the reactivity of (1)O(2) with DCFH is actually larger than that of MB(center dot 2+). We conclude that DCFH can continue to be used as a probe to monitor general redox misbalance induced in biologic systems by oxidizing radicals and (1)O(2).

Cholesterol Hydroperoxides Generate Singlet Molecular Oxygen [O(2) ((1)Delta(g))]: Near-IR Emission, (18)O-Labeled Hydroperoxides, and Mass Spectrometry

In mammalian membranes, cholesterol is concentrated in lipid rafts. The generation of cholesterol hydroperoxides (ChOOHs) and their decomposition products induces various types of cell damage. The decomposition of some organic hydroperoxides into peroxyl radicals is known to be a potential source of singlet molecular oxygen [O(2) ((1)Delta(g))] in biological systems. We report herein on evidence of the generation of O(2) ((1)Delta(g)) from ChOOH isomers in solution or in liposomes containing ChOOHs, which involves a cyclic mechanism from a linear tetraoxide intermediate originally proposed by Russell. Characteristic light emission at 1270 nm, corresponding to O(2) ((1)Delta(g)) monomolecular decay, was observed for each ChOOH isomer or in liposomes containing ChOOHs. Moreover, the presence of O(2) ((1)Delta(g)) was unequivocally demonstrated using the direct spectral characterization of near-infrared light emission. Using (18)O-labeled cholesterol hydroperoxide (Ch(18)O(18)OH), we observed the formation of (18)O-labeled O(2) ((1)Delta(g)) [(18)O(2) ((1)Delta(g))] by the chemical trapping of (18)O(2) ((1)Delta(g)) with 9,10-diphenylanthracene (DPA) and detected the corresponding (18)O-labeled DPA endoperoxide (DPA(18)O(18)O) and the (18)O-labeled products of the Russell mechanism using high-performance liquid chromatography coupled to tandem mass spectrometry. Photoemission properties and chemical trapping clearly demonstrate that the decomposition of Ch(18)O(18)OH generates (18)O(2) ((1)Delta(g)), which is consistent with the Russell mechanism and points to the involvement of O(2) ((1)Delta(g)) in cholesterol hydroperoxide-mediated cytotoxicity.

Nanoparticle Platform to Modulate Reaction Mechanism of Phenothiazine Photosensitizers

Herein, we report on the synthesis of photosensitizing nanoparticles in which the generation of different oxidizing species, i.e., singlet oxygen ((1)O(2)) or radicals, was modulated. Sol gel and surface chemistry were used to obtain nanoparticles with specific ratios of dimer to monomer species of phenothiazine photosensitizers (PSs). Due to competition between the reactions involving electron transfer within dimer species and energy transfer from monomer triplets to oxygen, the efficiency of (1)O(2) generation could be controlled. Nanoparticles with an excess of dimer have an (1)O(2) generation efficiency (S(Delta)) of 0.01 while those without dimer have a S, value of 0.4. Furthermore, we demonstrate that the PS properties of the nanoparticles are not subjected to interference from the external medium as is commonly the case for free PSs, i.e., PS ground and triplet states are not reduced by NADH and ascorbate, respectively, and singlet excited states are less suppressed by bromide. The modulated (1)O(2) generation and the PS protection from external interferences make this nanoparticle platform a promising tool to aid in performing mechanistic studies in biological systems. Also, it offers potential application in technological areas in which photo-induced processes take place.

The Effects of Physical Fitness and Body Composition on Oxygen Consumption and Heart Rate Recovery After High-Intensity Exercise

The aim of this study was to investigate the potential relationship between excess post-exercise oxygen consumption (EPOC), heart rate recovery (HRR) and their respective time constants (tvo(2) and t(HR)) and body composition and aerobic fitness (VO(2)max) variables after an anaerobic effort. 14 professional cyclists (age = 28.4 +/- 4.8 years, height = 176.0 +/- 6.7 cm, body mass = 74.4 +/- 8.1 kg, VO(2)max = 66.8 +/- 7.6 mL. kg(-1) . min(-1)) were recruited. Each athlete made 3 visits to the laboratory with 24h between each visit. During the first visit, a total and segmental body composition assessment was carried out. During the second, the athletes undertook an incremental test to determine VO(2)max. In the final visit, EPOC (15-min) and HRR were measured after an all-out 30s Wingate test. The results showed that EPOC is positively associated with % body fat (r = 0.64), total body fat (r = 0.73), fat-free mass (r = 0.61) and lower limb fat-free mass (r = 0.55) and negatively associated with HRR (r = - 0.53, p < 0.05 for all). HRR had a significant negative correlation with total body fat and % body fat (r = - 0.62, r = - 0.56 respectively, p < 0.05 for all). These findings indicate that VO(2)max does not influence HRR or EPOC after high-intensity exercise. Even in short-term exercise, the major metabolic disturbance due to higher muscle mass and total muscle mass may increase EPOC. However, body fat impedes HRR and delays recovery of oxygen consumption after effort in highly trained athletes.

Oxidation of melatonin by AAPH-derived peroxyl radicals: Evidence of a pro-oxidant effect of melatonin

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Pancreas transplantation prevents cellular oxidative stress in kidneys of alloxan-induced diabetic rats

Purpose. Oxidative stress is one of the most important mechanisms to explain genesis of the complications in the chronic progression of diabetes. In this investigation we studied the effects of pancreas transplantation (PT) on the imbalance caused by excessive production of free oxygen radicals by antioxidant defenses of rats with serious chronic hyperglycemia induced by alloxan.Methods. Ninety inbred male Lewis rats were randomly distributed into three groups: NC-30 nondiabetic controls; DC-30 diabetic controls without any treatment; PT-30 diabetic rats undergoing syngeneic PT from normal donor Lewis rats. Each experimental group was then split into three subgroups of 10 animals for sacrifice after 1, 3, or 6 months. Clinical and laboratory parameters from all rats as well as lipid hydroperoxide (LPO) concentrations and renal tissue enzyme activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) were recorded for all rats.Results. Successful PT corrected clinical and laboratory alterations in diabetic rats with sustained normoglycemia throughout the study. A significant increase in LPO concentration and a marked reduction in SOD and CAT enzyme activity were observed in DC rats; there was no significant variation in renal tissue GSH-Px in this group. However, alterations in DC rats were completely restored from 1st month after PT; all evaluated enzyme levels did not significantly differ (P < .01) from those in NC controls.Conclusion. Successful PT controlled cellular oxidative stress in diabetic kidneys, which may prevent chronic lesions.

Predictors of first-year survival in patients with advanced COPD treated using long-term oxygen therapy

Little evidence-based guidance is available to aid clinicians in determining short-term prognoses in very severe COPD patients. Therefore, the present study was designed to provide a prospective assessment (1) of the mortality rates and (2) whether the baseline measurements may be determinants of 1-year mortality in hypoxemic COPD patients receiving long-term oxygen therapy (LTOT).Seventy-eight clinically stable patients with advanced COPD treated using LTOT were enrolled in a prospective cohort study. Outcome variable: first-year mortality. Baseline measurements: categorical variables: age (<60 or >= 60 years); gender; body mass index (<20 or >= 20 kg/m(2)); fat-free mass (FFM) index (<16 [men] and <15kg/m(2) [women]; baseline dyspnea index (BDI) (<= 3 or >3); and corticosteroid use. Continuous variables: smoking history; lung function; FFM; fat mass; hemoglobin; hematocrit; arterial blood gases; forearm muscle strength; St. George's Respiratory Questionnaire (SGRQ); and comorbidity score. By the end of 1-year of follow-up, 12 patients (15.4%) had died. Kaplan-Meier curves showed that BDI <= 3 was the only variable associated with higher mortality. Cox proportional hazards analysis revealed that tower PaO2 and SPO2, higher PaCO2 and SGRQ scores were associated with reduced survival. In the multivariate analysis, BDI remained predictive of mortality (hazard ratio [HR], 0.50; 95% confidence interval [CI], 0.31-0.81), as did PaO2 (HR, 0.49; 95% CI, 0.26-0.95). These data suggest that readily available parameters as dyspnea intensity and hypoxemia severity may be useful in predicting first-year survival rates in advanced COPD patients receiving LTOT (C) 2007 Elsevier Ltd. All rights reserved.

Higher respiratory activity decreases mitochondrial reactive oxygen release and increases life span in Saccharomyces cerevisiae

Increased replicative longevity in Saccharomyces cerevisiae because of calorie restriction has been linked to enhanced mitochondrial respiratory activity. Here we have further investigated how mitochondrial respiration affects yeast life span. We found that calorie restriction by growth in low glucose increased respiration but decreased mitochondrial reactive oxygen species production relative to oxygen consumption. Calorie restriction also enhanced chronological life span. The beneficial effects of calorie restriction on mitochondrial respiration, reactive oxygen species release, and replicative and chronological life span could be mimicked by uncoupling agents such as dinitrophenol. Conversely, chronological life span decreased in cells treated with antimycin (which strongly increases mitochondrial reactive oxygen species generation) or in yeast mutants null for mitochondrial superoxide dismutase (which removes superoxide radicals) and for RTG2 (which participates in retrograde feedback signaling between mitochondria and the nucleus). These results suggest that yeast aging is linked to changes in mitochondrial metabolism and oxidative stress and that mild mitochondrial uncoupling can increase both chronological and replicative life span.

Insights into metal ion binding in phospholipases A(2): ultra high-resolution crystal structures of an acidic phospholipase A(2) in the Ca2+ free and bound states

The electrophile Ca2+ is an essential multifunctional co-factor in the phospholipase A(2) mediated hydrolysis of phospholipids. Crystal structures of an acidic phospholipase A(2) from the venom of Bothrops jararacussu have been determined both in the Ca2+ free and bound states at 0.97 and 1.60 angstrom resolutions, respectively. In the Ca2+ bound state, the Ca2+ ion is penta-coordinated by a distorted pyramidal cage of oxygen and nitrogen atoms that is significantly different to that observed in structures of other Group I/II phospholipases A(2). In the absence of Ca2+, a water molecule occupies the position of the Ca2+ ion and the side chain of Asp49 and the calcium-binding loop adopts a different conformation. (c) 2005 Elsevier SAS. All rights reserved.

Free radical scavenging activity of Pterogyne nitens Tul. (Fabaceae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Influence of oxygen flow on crystallization and morphology of PLZT thin films

PLZT thin films were prepared by a dip coating process using Pechini's method, also known as polymeric precursor method. The PLZT solution was obtained from a mixture of the individual cation solutions and the process to prepare each solution is based on metallic citrate polymerization. The viscosity of the PLZT solution was adjusted at 40 cP while the ionic concentration was adjusted at 0.1 M. PLZT solutions were deposited on silicon (100) and platinum coated silicon (100) substrates with withdrawal speed at 5 mm/min. The coated substrates were thermally treated with a heating rate of 1 degreesC/min up to 300 degreesC and 5 degreesC/min up to 650 degreesC in order to obtain homogeneous and cracks free films. The influence of oxygen flow on crystallization and morphology of PLZT (9/65/35) thin film is discussed. (C) 2002 Elsevier B.V. Ltd and Techna S.r.l. All rights reserved.

Control of retention and fatigue-free characteristics in CaBi4Ti4O15 thin films prepared by chemical method

Ferroelectric CaBi4Ti4O15 (CBTi144) thin films were deposited on Pt/Ti/SiO2/Si substrates by the polymeric precursor method. The films present a single phase of layered-structured perovskite with polar axis orientation after annealing at 700 degrees C for 2 h in static air and oxygen atmosphere. The a/b-axis orientation of the ferroelectric film is considered to be associated with the preferred orientation of the Pt bottom electrode. It is noted that the films annealed in static air showed good polarization fatigue characteristics at least up to 10(10) bipolar pulse cycles and excellent retention properties up to 10(4) s. on the other hand, oxygen atmosphere seems to be crucial in the decrease of both, fatigue and retention characteristics of the capacitors. Independently of the applied electric field, the retained switchable polarization approached a nearly steady-state value after a retention time of 10 s. (C) 2006 Elsevier B.V. All rights reserved.

REACTIVE OXYGEN GENERATION BY AZOMETHINE-H - A NEW ANTIMALARIAL DRUG

Superoxide radical (O-2(-)) is a free radical that may be involved in various toxic processes. Cu-Zn superoxide dismutase catalyzes the dismutation of the superoxide free radical and protects cells from oxidative damage. A rat bioassay validated for the identification of the toxic effects of azomethine H revealed increased serum activities of amylase, alanine transaminase, and alkaline phosphatase. The lipoperoxide and bilirubin concentrations were also increased in animals that received azomethine H (1 g/kg) from ascorbic or hydrochloric acid solutions. Azomethine H increased Cu-Zn superoxide dismutase activity. This elevation of Cu-Zn superoxide dismutase activity was highest on the 7th day and was at levels comparable with those of control rats from day 60 onwards. Superoxide is an important intermediate in the action and toxicity of azomethine H.